crc colonization  (ATCC)


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    Name:
    Fusobacterium nucleatum subsp nucleatum VPI 4351 1210
    Description:

    Catalog Number:
    23726
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    Structured Review

    ATCC crc colonization
    Localization of F. nucleatum to Established <t>CRC</t> Tumors Requires <t>Fap2</t>

    https://www.bioz.com/result/crc colonization/product/ATCC
    Average 90 stars, based on 48 article reviews
    Price from $9.99 to $1999.99
    crc colonization - by Bioz Stars, 2020-09
    90/100 stars

    Images

    1) Product Images from "Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc"

    Article Title: Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.07.006

    Localization of F. nucleatum to Established CRC Tumors Requires Fap2
    Figure Legend Snippet: Localization of F. nucleatum to Established CRC Tumors Requires Fap2

    Techniques Used:

    Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment
    Figure Legend Snippet: Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment

    Techniques Used: Binding Assay

    Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment
    Figure Legend Snippet: Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment

    Techniques Used: Binding Assay

    2) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    3) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    4) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    5) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    6) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    7) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    8) Product Images from "Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii. Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii"

    Article Title: Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii. Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.444

    radD and cmpA expression: WT Fusobacterium nucleatum was grown in Columbia broth for 25 hr. Cell samples were collected every 3 hr and (a) OD 600 was measured, as well as (b) radD and cmpA expression by qRT ‐ PCR . Gene expression was normalized to rpoB and compare to the first time point. The dashed line was added to aid in the comparison. (c) radD and cmpA expression were also measured in cells from Fn biofilm grown overnight in SHI ‐ FSMS . Gene expression was normalized to rpoB and compare to planktonic cells grown under the same conditions. Each value represents means and standard deviation of at least three independent experiments. To aid with visualization, the dashed line represents no change
    Figure Legend Snippet: radD and cmpA expression: WT Fusobacterium nucleatum was grown in Columbia broth for 25 hr. Cell samples were collected every 3 hr and (a) OD 600 was measured, as well as (b) radD and cmpA expression by qRT ‐ PCR . Gene expression was normalized to rpoB and compare to the first time point. The dashed line was added to aid in the comparison. (c) radD and cmpA expression were also measured in cells from Fn biofilm grown overnight in SHI ‐ FSMS . Gene expression was normalized to rpoB and compare to planktonic cells grown under the same conditions. Each value represents means and standard deviation of at least three independent experiments. To aid with visualization, the dashed line represents no change

    Techniques Used: Expressing, Quantitative RT-PCR, Standard Deviation

    Quantitative coaggregation of (a) wild‐type ( WT ) Streptococcus gordonii strain V288 and the WT Fusobacterium nucleatum strain ATCC 23726, or the radD mutant derivative, at different phases of growth. (b) Quantitative coaggregation of WT S. gordonii strains ( DL 1, V288, ATCC 10558, and ATCC 51656) with WT F. nucleatum strain ATCC 23726, or the radD mutant derivative at exponential growth. Data represent means and standard deviation of percent coaggregation of at least three independent experiments. * p
    Figure Legend Snippet: Quantitative coaggregation of (a) wild‐type ( WT ) Streptococcus gordonii strain V288 and the WT Fusobacterium nucleatum strain ATCC 23726, or the radD mutant derivative, at different phases of growth. (b) Quantitative coaggregation of WT S. gordonii strains ( DL 1, V288, ATCC 10558, and ATCC 51656) with WT F. nucleatum strain ATCC 23726, or the radD mutant derivative at exponential growth. Data represent means and standard deviation of percent coaggregation of at least three independent experiments. * p

    Techniques Used: Mutagenesis, Standard Deviation

    Quantitative coaggregation of wild‐type Streptococcus gordonii strain (a) V288, (b) ATCC 10558, (c) DL 1, and (d) ATCC 51656 with Fusobacterium nucleatum strains: Wild‐type ( WT ) and the mutant derivatives: radD , cmpA , and radD cmpA double mutant. Data represent means and standard deviation of percent coaggregation of at least three independent experiments. * p
    Figure Legend Snippet: Quantitative coaggregation of wild‐type Streptococcus gordonii strain (a) V288, (b) ATCC 10558, (c) DL 1, and (d) ATCC 51656 with Fusobacterium nucleatum strains: Wild‐type ( WT ) and the mutant derivatives: radD , cmpA , and radD cmpA double mutant. Data represent means and standard deviation of percent coaggregation of at least three independent experiments. * p

    Techniques Used: Mutagenesis, Standard Deviation

    (a) Fusobacterium nucleatum radD cmpA double mutant (strain BL 83) was constructed by insertion of the inactivation plasmid pBPL 9 into cmpA (Fn1554) in a radD::catP mutant background. Black arrows indicate the location of primers used for mutant construction and analysis. (b) Confirmation of plasmid insertion into cmpA by PCR analysis of the cmpA mutant (m) with wild‐type (WT) as the control
    Figure Legend Snippet: (a) Fusobacterium nucleatum radD cmpA double mutant (strain BL 83) was constructed by insertion of the inactivation plasmid pBPL 9 into cmpA (Fn1554) in a radD::catP mutant background. Black arrows indicate the location of primers used for mutant construction and analysis. (b) Confirmation of plasmid insertion into cmpA by PCR analysis of the cmpA mutant (m) with wild‐type (WT) as the control

    Techniques Used: Mutagenesis, Construct, Plasmid Preparation, Polymerase Chain Reaction

    Streptococcus gordonii and Fusobacterium nucleatum dual‐species biofilm: (a) Confocal laser scanning microscopy of syto9‐stained dual‐species biofilm after overnight incubation. S. gordonii (Sg) cells constitutively express mCherry from their chromosome and appear orange/yellow on the images. Wild‐type ( WT ) F. nucleatum (Fn) and its mutant derivatives ( radD , cmpA , and radD cmpA double mutant) are strained by syto9‐only and appear green on the images. (b) The presence of the Fn mutant strains in the Sg‐Fn dual‐species biofilm is displayed as the percentage of Fn cells normalized to the number of attached Sg cells/well, compared to that measured with WT Fn. Cellular ratios were determined by measuring DNA concentration by qPCR , targeting the F. nucleatum gene fomA and the S. gordonii gene srtA . At least three independent experiments were performed per strain combination. Each value represents means and standard deviation of at least three independent experiments. * p
    Figure Legend Snippet: Streptococcus gordonii and Fusobacterium nucleatum dual‐species biofilm: (a) Confocal laser scanning microscopy of syto9‐stained dual‐species biofilm after overnight incubation. S. gordonii (Sg) cells constitutively express mCherry from their chromosome and appear orange/yellow on the images. Wild‐type ( WT ) F. nucleatum (Fn) and its mutant derivatives ( radD , cmpA , and radD cmpA double mutant) are strained by syto9‐only and appear green on the images. (b) The presence of the Fn mutant strains in the Sg‐Fn dual‐species biofilm is displayed as the percentage of Fn cells normalized to the number of attached Sg cells/well, compared to that measured with WT Fn. Cellular ratios were determined by measuring DNA concentration by qPCR , targeting the F. nucleatum gene fomA and the S. gordonii gene srtA . At least three independent experiments were performed per strain combination. Each value represents means and standard deviation of at least three independent experiments. * p

    Techniques Used: Confocal Laser Scanning Microscopy, Staining, Incubation, Mutagenesis, Concentration Assay, Real-time Polymerase Chain Reaction, Standard Deviation

    Quantitative coaggregation of wild‐type ( WT ) Streptococcus gordonii strain V288 with Fusobacterium nucleatum ATCC 23726 WT strain and OMP mutant derivatives ( radD , Fn254, cmpA , Fn1893, Fn2047, and aim1 ). Data represent means and standard deviation of percent coaggregation of at least three independent experiments. * p
    Figure Legend Snippet: Quantitative coaggregation of wild‐type ( WT ) Streptococcus gordonii strain V288 with Fusobacterium nucleatum ATCC 23726 WT strain and OMP mutant derivatives ( radD , Fn254, cmpA , Fn1893, Fn2047, and aim1 ). Data represent means and standard deviation of percent coaggregation of at least three independent experiments. * p

    Techniques Used: Mutagenesis, Standard Deviation

    9) Product Images from "Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc"

    Article Title: Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.07.006

    Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment
    Figure Legend Snippet: Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment

    Techniques Used: Binding Assay

    Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment
    Figure Legend Snippet: Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment

    Techniques Used: Binding Assay

    10) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    11) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    12) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    13) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    14) Product Images from "Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc"

    Article Title: Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.07.006

    Localization of F. nucleatum to Established CRC Tumors Requires Fap2
    Figure Legend Snippet: Localization of F. nucleatum to Established CRC Tumors Requires Fap2

    Techniques Used:

    Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment
    Figure Legend Snippet: Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment

    Techniques Used: Binding Assay

    Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment
    Figure Legend Snippet: Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment

    Techniques Used: Binding Assay

    15) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    16) Product Images from "Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc"

    Article Title: Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.07.006

    Localization of F. nucleatum to Established CRC Tumors Requires Fap2
    Figure Legend Snippet: Localization of F. nucleatum to Established CRC Tumors Requires Fap2

    Techniques Used:

    Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment
    Figure Legend Snippet: Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment

    Techniques Used: Binding Assay

    Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment
    Figure Legend Snippet: Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment

    Techniques Used: Binding Assay

    17) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    18) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    19) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    20) Product Images from "Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc"

    Article Title: Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.07.006

    Localization of F. nucleatum to Established CRC Tumors Requires Fap2
    Figure Legend Snippet: Localization of F. nucleatum to Established CRC Tumors Requires Fap2

    Techniques Used:

    Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment
    Figure Legend Snippet: Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment

    Techniques Used: Binding Assay

    Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment
    Figure Legend Snippet: Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment

    Techniques Used: Binding Assay

    21) Product Images from "Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc"

    Article Title: Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.07.006

    Localization of F. nucleatum to Established CRC Tumors Requires Fap2
    Figure Legend Snippet: Localization of F. nucleatum to Established CRC Tumors Requires Fap2

    Techniques Used:

    Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment
    Figure Legend Snippet: Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment

    Techniques Used: Binding Assay

    Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment
    Figure Legend Snippet: Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment

    Techniques Used: Binding Assay

    22) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    23) Product Images from "Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc"

    Article Title: Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.07.006

    Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment
    Figure Legend Snippet: Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment

    Techniques Used: Binding Assay

    Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment
    Figure Legend Snippet: Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment

    Techniques Used: Binding Assay

    24) Product Images from "Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc"

    Article Title: Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2016.07.006

    Localization of F. nucleatum to Established CRC Tumors Requires Fap2
    Figure Legend Snippet: Localization of F. nucleatum to Established CRC Tumors Requires Fap2

    Techniques Used:

    Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment
    Figure Legend Snippet: Fap2 Binding to GalNAc in Human CRC Mediates F. nucleatum Adenocarcinoma Enrichment

    Techniques Used: Binding Assay

    Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment
    Figure Legend Snippet: Fap2-Dependent Gal-GalNAc Binding Mediates F. nucleatum CRC Attachment

    Techniques Used: Binding Assay

    25) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    26) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    27) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    28) Product Images from "Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression"

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16967-2

    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Figure Legend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.
    Figure Legend Snippet: AT3 mammary tumor acceleration by F. nucleatum is immune-mediated. a 1 × 10 6 AT3 cells were injected to the mammary fat pad of 6–7-week-old female C57BL/6 mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested subjected to flow cytometry and abundance of NK cells, CD4+ T cells and CD8+ T cells was determined. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value. n.s.: non-significant. ** p = 0.00216, * p = 0.0152, two-tailed Mann–Whitney (each symbol represents the mean of two technical replicates, n = 6 per group). b 1 × 10 5 AT3 or GFP-expressing AT3 cells were injected to the mammary fat pad of 6–7-week-old female SCID-beige mice. When tumor volume reached 500 mm 3 , mice were IV injected with PBS vehicle or with 5 × 10 7 F. nucleatum ATCC 23726 (726). At day 8 post injection, mice were sacrificed, tumors were harvested and weighed in an analytic weigh. Boxplot whiskers represent extrema, box bounds represent upper and lower quartiles, and center-line represents the median value of two independent experiments. n.s.: non-significant. n = 8 per vehicle group and n = 10 per F. nucleatum -infected group. c Representative picture and densitometry analysis of gelatin zymography of conditioned medium prepared from mouse mammary tumor cell line AT3 or from the human breast cancer cell line MDA-MB-231 that were co-cultured or not, with F. nucleatum strains ATCC 25586 or ATCC 23726. Bars indicate mean ± SEM of six independent experiments. * p = 0.031 two-tailed paired Wilcoxon. Note that mouse MMP-9 is larger than that of human. Source data are provided as a Source data file.

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Infection, Zymography, Multiple Displacement Amplification, Cell Culture

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p
    Figure Legend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Techniques Used: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p
    Figure Legend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Techniques Used: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification

    29) Product Images from "Fusobacterium nucleatum Outer Membrane Proteins Fap2 and RadD Induce Cell Death in Human Lymphocytes ▿"

    Article Title: Fusobacterium nucleatum Outer Membrane Proteins Fap2 and RadD Induce Cell Death in Human Lymphocytes ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00567-10

    Transcriptional analysis of fap2 from F. nucleatum PK1594. Relative cDNA abundance of fap2 was determined for cells grown in broth culture to logarithmic or stationary phase, on agar plates, or in the presence of Jurkat cells (4-h incubation) to logarithmic phase. n = 3. ND, not determined; *, P ≤ 0.01 compared to logarithmic phase results.
    Figure Legend Snippet: Transcriptional analysis of fap2 from F. nucleatum PK1594. Relative cDNA abundance of fap2 was determined for cells grown in broth culture to logarithmic or stationary phase, on agar plates, or in the presence of Jurkat cells (4-h incubation) to logarithmic phase. n = 3. ND, not determined; *, P ≤ 0.01 compared to logarithmic phase results.

    Techniques Used: Incubation

    Transcription of fap2 and its downstream ORF, Fn1448, in wild-type F. nucleatum and the mutant derivative lacking fap2 . F. nucleatum cells were grown to mid-exponential-growth phase to ensure fap2 expression. fap2 , but not Fn1448, expression in the mutant was significantly different ( P ≤ 0.01) from that in the wild type. n = 3.
    Figure Legend Snippet: Transcription of fap2 and its downstream ORF, Fn1448, in wild-type F. nucleatum and the mutant derivative lacking fap2 . F. nucleatum cells were grown to mid-exponential-growth phase to ensure fap2 expression. fap2 , but not Fn1448, expression in the mutant was significantly different ( P ≤ 0.01) from that in the wild type. n = 3.

    Techniques Used: Mutagenesis, Expressing

    Related Articles

    Mutagenesis:

    Article Title: Characterization of Fusobacterium nucleatum ATCC 23726 adhesins involved in strain-specific attachment to Porphyromonas gingivalis
    Article Snippet: .. For F. nucleatum ATCC 23726 and its mutant derivatives, a portion of the Fusobacterium -specific fomA gene was amplified with Fn-F (forward) 5′-AGTTGCTCCAGCTTGGAGACCAAAT-3′ and Fn-R (reverse) 5′-AAGTTTACTTTTGTTAAAGTTTGTAATCTTCC-3′ primers. ..

    Amplification:

    Article Title: Characterization of Fusobacterium nucleatum ATCC 23726 adhesins involved in strain-specific attachment to Porphyromonas gingivalis
    Article Snippet: .. For F. nucleatum ATCC 23726 and its mutant derivatives, a portion of the Fusobacterium -specific fomA gene was amplified with Fn-F (forward) 5′-AGTTGCTCCAGCTTGGAGACCAAAT-3′ and Fn-R (reverse) 5′-AAGTTTACTTTTGTTAAAGTTTGTAATCTTCC-3′ primers. ..

    other:

    Article Title: Insights into the Evolution of Host Association through the Isolation and Characterization of a Novel Human Periodontal Pathobiont, Desulfobulbus oralis
    Article Snippet: CFS from a type strain of F. nucleatum (ATCC 23726) also supported the growth of D. oralis , indicating its physiological dependence is not restricted to the strain it coenriched with, although we have not extended that analysis to additional strains or species.

    Binding Assay:

    Article Title: Characterization of Fusobacterium nucleatum ATCC 23726 adhesins involved in strain-specific attachment to Porphyromonas gingivalis
    Article Snippet: .. Autoaggregation of bacteria and coaggregation of F. nucleatum ATCC 23726 with P. gingivalis displays a strain-specific profile Qualitative and quantitative autoaggregation and coaggregation assays revealed a strain-dependent binding profile of F. nucleatum ATCC 23726 with the seven different strains of P. gingivalis tested in this study. .. Robust interactions were observed with P. gingivalis strains 4612 (62%±8% coaggregation), T22 (54%±8% coaggregation) binding and ATCC 33277 (65%±17% coaggregation; ).

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    ATCC wild type fap2 expressing f nucleatum atcc 23726
    Wild Type Fap2 Expressing F Nucleatum Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wild type fap2 expressing f nucleatum atcc 23726/product/ATCC
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    84
    ATCC fap2 expressing f nucleatum atcc 23726
    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 <t>Fap2-deficient</t> F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.
    Fap2 Expressing F Nucleatum Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Journal: Nature Communications

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    doi: 10.1038/s41467-020-16967-2

    Figure Lengend Snippet: F. nucleatum ATCC 23726 accelerates tumor growth and progression of lung metastasis. a Experimental scheme: AT3 breast cancer cell line was injected to the mammary fat pad of 6–7-week-old C57BL/6 mice. When tumor size reached 500 mm 3 , PBS vehicle (V), 5 × 10 7 F. nucleatum ATCC 23726 only (726), 5 × 10 7 F. nucleatum ATCC 23726 with metronidazole (726+MTZ.), metronidazole only (MTZ.), or 5 × 10 7 Fap2-deficient F. nucleatum K50 were injected into the tail vein. On days 2–3 post inoculation, mice were injected twice a day with metronidazole (M) or with PBS vehicle (V). On days 4–7 post inoculation, mice were injected once a day with metronidazole or with PBS. Eight days after inoculation, mice were sacrificed, and tumor and lungs harvested. Breast cancer tissues were weighed, and lung metastases were counted. b Tumor weights normalized as the percentage compared with the average tumor weight in the PBS group (vehicle control) in each experiment (set as 100% tumor weight). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 3.48 × 10 −5 , ** p = 0.002. Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 21, MTZ. n = 17, K50 n = 7, 726 n = 19, 726+MTZ. n = 12. c Representative tumors post-harvest. d Representative image of lung metastases. Scale bar, 500 µm in the large image, 250 µm in the indicated inset. e Lung metastasis rank [number of metastases per lung area (pixel 2 )] calculated as percentage compared with the average metastasis rank in the vehicle control group of each experiment (set as 100% metastasis rank). Each symbol represents one mouse. Presented results are of three independent experiments. **** p = 4.08 × 10 −5 , * p = 0.0177, n.s.: non-significant, Holm’s correction, one-tailed Mann–Whitney. Vehicle n = 20, MTZ. n = 17, K50 n = 7, 726 n = 18, 726+met n = 12. f Images taken by fluorescence binocular of lung metastases in four mice implanted with GFP-expressing AT3 cells as described above. Two tumor-bearing mice were inoculated with F. nucleatum and two were treated by PBS vehicle. Scale bar, 1000 µm. Source data are provided as a Source data file.

    Article Snippet: Intravascularly inoculated Fap2-expressing F. nucleatum ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria are impaired in tumor colonization.

    Techniques: Injection, Mouse Assay, One-tailed Test, MANN-WHITNEY, Fluorescence, Expressing

    F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Journal: Nature Communications

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    doi: 10.1038/s41467-020-16967-2

    Figure Lengend Snippet: F. nucleatum colonizes mammary cancer in a Fap2-mediated mechanism. a Orthotropic 4T1 BALB/c mouse mammary cancer model. When reaching 500 mm 3 size, 5 × 10 7 F. nucleatum ATCC 23726 were IV injected. Twenty-four hours post inoculation, breast cancer and normal tissue from an adjacent mammary were harvested. b Representative images and sum of fluorescence intensity of binding of FITC-labeled PNA (green) to tumor and normal tissue stained with Hoechst dye (blue). Each paired symbols represents one mouse ( n = 7), ** p = 0.0078, one-tailed, Wilcoxon matched-paired signed rank test. c Relative F. nucleatum gDNA abundance (2 −ΔCt ) in breast tissue from tumor-free mice (No tumor, n = 3), and in tumor and matching normal tissue of 4T1 tumor-bearing mice ( n = 5) all inoculated with F. nucleatum ATCC 23726. * p = 0.0358 Holm-corrected one-tailed Mann–Whitney test. * p = 0.0313, one-tailed, Wilcoxon matched-paired signed rank test (for paired normal adjacent-tumor samples). d Orthotropic AT3 C57BL/6 mouse mammary cancer model. When reaching 500 mm 3 , mice were inoculated with 5 × 10 7 F. nucleatum ATCC 23726 (726; blue), 5 × 10 7 Fap2 mutant K50 (MUT K50; red) or with 5 × 10 7 P. gingivalis (Pg; black). Twenty-four hours later, normal mammary and breast cancer tissue were harvested from each mice and bacteria quantified. e Bacterial abundance (CFU/g tissue), and relative bacterial gDNA abundance (2 −ΔCt ) in tumor and normal tissue of AT3 tumor-bearing mice inoculated with P. gingivalis ( Pg , n = 9), K50 ( n = 12), or F. nucleatum ATCC 23726 (726, n = 12). Each symbol represents one mouse. * p = 0.0156 one-tailed, Wilcoxon matched-paired signed rank test. *** p = 0.0006, ** p = 0.001598. Holm-corrected one-tailed Mann–Whitney test (left panel). **** p

    Article Snippet: Intravascularly inoculated Fap2-expressing F. nucleatum ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria are impaired in tumor colonization.

    Techniques: Injection, Fluorescence, Binding Assay, Labeling, Staining, One-tailed Test, Mouse Assay, MANN-WHITNEY, Mutagenesis

    Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Journal: Nature Communications

    Article Title: Breast cancer colonization by Fusobacterium nucleatum accelerates tumor growth and metastatic progression

    doi: 10.1038/s41467-020-16967-2

    Figure Lengend Snippet: Attachment of F. nucleatum ATCC 23726 to breast cancer is Fap2-mediated and Gal-GalNAc dependent. a Representative images (left panels, scale bar 20 µm) and quantitative analysis (Fn/mm 2 ) (right panel) of binding of Cy3-labeled (white) WT F. nucleatum ATCC 23726 (726) and of its Cy5-labeled (red) Fap2-inactivated isogenic mutant K50, to Hoechst-stained (blue) human breast cancer TMAs (HBre-Duc060CS-01 and BR1006). Results in the right panel are classified by tissue type (normal n = 35, benign n = 7, malignant n = 64) and bacterial strain (K50, 726). Each core is measured twice, once for each of the strains in the appropriate tissue type (as demonstrated in the pictures). When an individual contributed a single core to the experiment, the two observations are shown as circles (●); when she contributed two cores, these were to the normal type and the malignant type, and the four observations are depicted as plus signs (+). The six classes were compared against each other in pairs: the exact two-tailed Wilcoxon test was used for pairs that were from the same core, the exact two-tailed Mann–Whitney test for independent samples. Where the same hypothesis was tested on more than one pair, and the individual pairs were mutually independent, the corresponding p -values were combined by Fisher’s method. Wherever the pairs were not independent, the multiple comparisons were adjusted for using the Holm modification of the Bonferroni correction. **** p

    Article Snippet: Intravascularly inoculated Fap2-expressing F. nucleatum ATCC 23726 specifically colonize mice mammary tumors, whereas Fap2-deficient bacteria are impaired in tumor colonization.

    Techniques: Binding Assay, Labeling, Mutagenesis, Staining, Two Tailed Test, MANN-WHITNEY, Modification