Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: Human skin fibroblasts were transfected with non-targeting siRNA (siCtrl) or Col3 siRNA (siCol3). a , b After 96h, the efficiency and specificity of knockdown were confirmed by qPCR for COL3A1 and COL1A1 . c , d Representative Second Harmonic Generation (SHG) images of collagen within human fibroblast-derived matrices (FDMs) prepared from fibroblast/ECM units treated with control and Col3-targeted siRNA. Collagen matrices were analyzed for ( e ) total collagen amount and ( f ) collagen alignment. CT-FIRE was used to quantify ( g ) collagen fiber number, ( h ) straightness, ( i ) width, and ( j ) length from SHG images. Data from representative experiments are presented (5 individual experiments performed with similar results). Error bars show mean ± SD ( n = 5). Statistical significance was determined by unpaired Student t tests: * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bar = 25 µm.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330- 01; Southern Biotech, Birmingham, AL), Col1 (PA5-95137; ThermoFisher Scientific), and cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Transfection, Knockdown, Derivative Assay, Control

Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: Human breast CAFs were transfected with non-targeting siRNA (siCtrl) or Col3 siRNA (siCol3). a , b After 5 days, the efficiency and specificity of knockdown were confirmed by qPCR for COL3A1 and COL1A1 . c , d Representative second harmonic generation (SHG) images of collagen within human CAF-derived matrices (FDMs) prepared from fibroblast/ECM units treated with control and Col3-targeted siRNA. SHG obtained images of collagen matrices were analyzed for ( e ) total collagen amount and ( f ) collagen alignment. Data represents individual FDMs from 3 independent experiments. g , h Representative images of FDMs prepared from breast CAFs cultured on with rhCol1- or rhCol3-coated substrata at 8 days. Collagen matrices were analyzed for ( i ) total collagen amount and ( j ) collagen alignment. Data represents individual FDMs from 2 independent experiments. Error bars show mean ± SD. Significance determined with Student’s unpaired t tests: * p < 0.05; *** p < 0.001; **** p < 0.0001. Scale bar = 25 µm.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330- 01; Southern Biotech, Birmingham, AL), Col1 (PA5-95137; ThermoFisher Scientific), and cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Transfection, Knockdown, Derivative Assay, Control, Cell Culture

Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: Three breast cancer cell lines, MCF-7, MDA-MB-453 and MDA-MB-231 were cultured on collagen matrices produced by control fibroblasts (siCtrl) or Col3-deficient fibroblasts (siCol3). Cells were fixed and stained with Ki67 (a cell proliferation marker), or active caspase 3 (a cell apoptosis marker), and DAPI (a nuclear marker, blue). a – c Quantification of the percentages of Ki67 (green), and ( d – f ) active caspase 3 (green) positive cells. Error bars show mean ± SD (n = 3 for Ki67, n = 4 for active caspase 3). Significance determined by paired Student’s t tests: * p < 0.05; ** p < 0.01; *** p < 0.001. Insets show representative images of immunoreactivity for ( a – c ) Ki67 and ( d – f ) Active caspase 3 in siCtrl (left image) and siCol3 (right image) treated cultures above respective quantitative data. Scale bar = 100μm.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330- 01; Southern Biotech, Birmingham, AL), Col1 (PA5-95137; ThermoFisher Scientific), and cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Cell Culture, Produced, Control, Staining, Marker

Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: a Hematoxylin-Eosin images were used to identify invasive and non-invasive regions within the same human triple-negative breast cancer (TNBC) biopsy . b A representative image of a serial section of this human TNBC biopsy that was immunolabeled for Col1 (Red), Col3 (Green), and stained with the nuclear stain DRAQ5 (Blue). c , d Second harmonic generation microscopy (SHG, white; Cytokeratin, magenta; DRAQ5/nuclei, blue) images were combined with ( e , f ) confocal fluorescent imaging (Col1, red; Col3, green). g SHG images were used to measure fiber alignment (FFT aspect ratio). The positive immunoreactivity of ( h ) Col1 and ( i ) Col3 integrated density of in both invasive and non-invasive regions, j and their ratio, was analyzed using ImageJ. n = 23 tumors. Significance determined by paired Student’s t tests: ** p < 0.01, **** p < 0.0001. Scale bar = 4mm.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330- 01; Southern Biotech, Birmingham, AL), Col1 (PA5-95137; ThermoFisher Scientific), and cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Immunolabeling, Staining, Microscopy, Imaging

Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: a Kaplan–Meier survival curves showing that Col3:Col1 high patients have significantly better prognosis than Col1:Col3 high patients in terms of disease-free survival (DFS) and progression-free survival (PFS) across the entire TCGA BRCA patient cohort ( p < 0.05 ). b Patients with Col3:Col1 high basal tumors demonstrated significantly better DFS, PFS, disease-specific survival (DSS), and overall survival (OS) compared to those with Col1:Col3 high basal tumors ( p < 0.05).

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330- 01; Southern Biotech, Birmingham, AL), Col1 (PA5-95137; ThermoFisher Scientific), and cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques:

Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: a – c MCF-7 cells were grown in 3D-cultures supplemented with human recombinant Col1, Col3 or 50:50 Col1+Col3 for 10 days. Images were taken with confocal microscopy of nuclei (DRAQ5, magenta), E-cadherin (cyan, shown in inset only) and fibrillar collagen (SHG, white) on max projection images. Mid-sections of the images showed the apparition of lumens (insets). d – f Spheroids (green outlines) and single cells (red outlines) were identified using ImageJ. g Quantification of the area of single cells normalized to the total nuclei area. h Integrated density of collagen fibers was analyzed by ImageJ and normalized to the total nuclei area. i Lumens were counted and normalized to the total spheroids. Error bars show mean ± SD ( n = 3). Data from three independent experiments. Significance determined by one-way AVOVAs followed by Tukey post-hoc tests: * p < 0.05; ** p < 0.01. Scale bar =100 µm.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330- 01; Southern Biotech, Birmingham, AL), Col1 (PA5-95137; ThermoFisher Scientific), and cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Recombinant, Confocal Microscopy

Journal: NPJ Breast Cancer

Article Title: Prognostic and therapeutic implications of tumor-restrictive type III collagen in the breast cancer microenvironment

doi: 10.1038/s41523-024-00690-y

Figure Lengend Snippet: a RNA was collected from 4T1 tumors in Col3 +/+ and Col3 +/− mice and analyzed for Col1a1 and Col3a1 expression. Fold changes to Col3 +/+ D0 samples were used to generate Col1:Col3 ratios. b Tumor volume 14 days after marginal resection in Col3 +/+ and Col3 +/− mice. c Hydrogels were prepared with murine TNBC-like breast cancer cells (4T1) and supplemented with 100 μg of human recombinant Col3 or vehicle (Ctrl) prior to injection into mammary fat pads of BALB/c mice and harvested after 28 days. Tumor sections were stained for DAPI (nuclei, blue) and d Ki67 (proliferating nuclei, red) or e active caspase 3 (apoptotic cells, red). Scale bar = 50 µm. d , e Staining was quantified as % Ki67 positive nuclei or total active caspase 3 staining (integrated density). f Pulmonary metastatic burden was quantified from ( g , h ) H&E stained lung sections with overlay showing identified metastases (schematic of workflow of quantification of metastatic burden and additional representative images can be found in Supplementary Fig. ). Scale bar = 3mm. For tumor growth, 2-way ANOVA followed by Sidak’s post-hoc test was performed; for Ki67, active caspase 3, and metastasis, unpaired Student’ t tests were utilized. * p < 0.05; ** p < 0.01; *** p < 0.001 for all graphs.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330- 01; Southern Biotech, Birmingham, AL), Col1 (PA5-95137; ThermoFisher Scientific), and cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Expressing, Recombinant, Injection, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: USP10 Targeted Self-Deliverable siRNA to Prevent Scarring in the Cornea

doi: 10.1016/j.omtn.2020.07.032

Figure Lengend Snippet: Immunohistochemical Analysis of Collagen III after Wounding (A–C) Frozen sections of corneas 6 weeks after wounding were immunostained for collagen III (green), DAPI (blue). (A) UnWnd, (B) Wnd-NTC with magnified inset, and (C) Wnd-US09 with magnified inset. Scale bar, 0.5 mm. (D) Compared to UnWnd, Wnd-NTC demonstrated a 276.2-fold increase in collagen III immunostaining (p < 0.01), which was reduced by 71.7% (p < 0.05) with US09 treatment. The comparison between UnWnd and Wnd-US09 was not significant. (E) By qPCR, days 1 and 2 combined, compared to UnWnd, Wnd-NTC demonstrated a 4.26-fold increase in USP10 gene expression (p < 0.001). Compared to Wnd-NTC, USP10 expression with Wnd-US09 treatment was reduced by 56.8% (p < 0.01). N = 4 rabbits per condition. At 6 weeks, compared to UnWnd, Wnd-NTC demonstrated a 35.7-fold increase in USP10 gene expression (p < 0.05). Compared to Wnd-NTC, USP10 expression with Wnd-US09 treatment was reduced by 91.2% (p < 0.05). N = 6 rabbits per condition.

Article Snippet: On the next day sections were rehydrated in PBS for 15 min, treated with blocking buffer (10% normal goat serum in PBS, Jackson ImmunoResearch Laboratories) for 20 min, and then incubated with primary antibodies (FN-EDA [Sigma, F6140], collagen III [Novus Biologicals, NBP105119B], α-SMA [Sigma, C6198], CD45 Thermo Fisher Scientific, MA5-28392]) at 1:250 for 1 h in a moist chamber at room temperature (RT).

Techniques: Immunohistochemical staining, Immunostaining, Comparison, Gene Expression, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: USP10 Targeted Self-Deliverable siRNA to Prevent Scarring in the Cornea

doi: 10.1016/j.omtn.2020.07.032

Figure Lengend Snippet: Immunohistochemical Analysis of α-SMA, Cell Proliferation, and Thickness after Wounding (A–C) Frozen sections of corneas 6 weeks after wounding were immunostained for α-SMA (green), DAPI (blue). (A) UnWnd, (B) Wnd-NTC with magnified inset, and (C) Wnd-US09 with magnified inset. Scale bar, 0.5 mm. (D) Compared to UnWnd, Wnd-NTC demonstrated a 5.77-fold increase in α-SMA immunostaining (p < 0.05). Compared to Wnd-NTC, α-SMA immunostaining after Wnd-US09 treatment was reduced by 83.6% (p < 0.05). The comparison between UnWnd and Wnd-US09 was not significant. (E and F) Cell proliferation was analyzed by the object counter plugin in ImageJ software. “Inside the wound” is denoted by the anterior cornea demarcated by the collagen III scar. “Outside the wound” is the posterior cornea beneath the scar. These counts were normalized by the total area of each portion to generate a nuclei density measurement. (E) Compared to UnWnd, Wnd-NTC demonstrated a 1.61-fold increase in cell proliferation (p < 0.01). Compared to Wnd-NTC, cell proliferation after Wnd-US09 treatment was reduced by 29.9% (p < 0.05). The comparison between UnWnd and Wnd-US09 was not significant. (F) Cell proliferation below the scar, in the stroma down to the endothelium. All relationships were not significant. (G) Corneal thickness was measured at pixel resolution in these thresholded images as the distance across the nonzero region, and thickness is averaged across the entire cornea. Wnd-NTC trended toward a slight decrease in thickness (p = 0.05). Wnd-US09 treatment restored corneal thickness to non-wounded parameters. N = 6 rabbits per condition.

Article Snippet: On the next day sections were rehydrated in PBS for 15 min, treated with blocking buffer (10% normal goat serum in PBS, Jackson ImmunoResearch Laboratories) for 20 min, and then incubated with primary antibodies (FN-EDA [Sigma, F6140], collagen III [Novus Biologicals, NBP105119B], α-SMA [Sigma, C6198], CD45 Thermo Fisher Scientific, MA5-28392]) at 1:250 for 1 h in a moist chamber at room temperature (RT).

Techniques: Immunohistochemical staining, Immunostaining, Comparison, Software

Journal: Research Square

Article Title: Tumor-restrictive type III collagen in the breast cancer microenvironment: prognostic and therapeutic implications

doi: 10.21203/rs.3.rs-2631314/v1

Figure Lengend Snippet: Human skin fibroblasts were transfected with non-targeting siRNA (siCtrl) or Col3 siRNA (siCol3). After 96h, the efficiency and specificity of knockdown were confirmed by qPCR for col3a1 and col1a1 (A and B). Representative Second Harmonic Generation (SHG) images of collagen matrices from human fibroblast-derived matrices (FDM) prepared from fibroblast/ECM units treated with control and Col3-targeted siRNA (C and D). Collagen matrices were analyzed for total collagen amount (E) and collagen alignment (F). CT-FIRE was used to quantify collagen fiber number (G) straightness (H), width (I), and length (J) from SHG images. Data from representative experiments are presented (5 individual experiments performed with similar results). Error bars show mean ± SD (n = 5). Student unpaired t-tests: (*,p< 0.05; **,p < 0.01; ****, p<0.0001). Scale bar = 25 μm.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330-01; Southern Biotech, Birmingham, AL), Col1 (PA5-9513; ThermoFisher Scientific), and Cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Transfection, Knockdown, Derivative Assay, Control

Journal: Research Square

Article Title: Tumor-restrictive type III collagen in the breast cancer microenvironment: prognostic and therapeutic implications

doi: 10.21203/rs.3.rs-2631314/v1

Figure Lengend Snippet: Three breast cancer cell lines, MCF-7, MDA-MB-453 and MDA-MB-231 were cultured on collagen matrices produced by control fibroblasts (siCtrl) or Col3-deficient fibroblasts (siCol3). Cells were fixed and stained with Ki67, a cell proliferation marker or active caspase 3, a cell apoptosis marker. Quantification of the percentages of Ki67 (A, B, C), and active caspase 3 positive cells (D, E, F). Error bars show mean±SD (n = 3 for ki67, n = 4 for active caspase 3). Paired Student t-tests: (*,p<0.05; **, p<0.01; ***,p<0.001).

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330-01; Southern Biotech, Birmingham, AL), Col1 (PA5-9513; ThermoFisher Scientific), and Cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Cell Culture, Produced, Control, Staining, Marker

Journal: Research Square

Article Title: Tumor-restrictive type III collagen in the breast cancer microenvironment: prognostic and therapeutic implications

doi: 10.21203/rs.3.rs-2631314/v1

Figure Lengend Snippet: Hematoxylin-Eosin images were used to identify invasive and noninvasive regions within the same human triple-negative breast cancer (TNBC) biopsy. (A-B) A representative image of a human TNBC biopsy was immunolabeled for Col1 (Red), Col3 (Green), and stained with the nuclear stain DRAQ5 (Blue) (B). Second harmonic generation microscopy (SHG, white; Cytokeratin, magenta; DRAQ5/nuclei, blue; C-D) images were combined with confocal fluorescent imaging (Col1, red; Col3, green; E-F). SHG images were used to measure fiber alignment (FFT aspect ratio, G). The positive immunoreactivity of Col1 and Col3 integrated density of in both invasive and noninvasive regions, and their ratio, was analyzed using ImageJ (H-J). N=23 tumors. Paired Student t-tests: (**p<0.01, ****p<0.0001). Scale bar = 4mm.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330-01; Southern Biotech, Birmingham, AL), Col1 (PA5-9513; ThermoFisher Scientific), and Cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Immunolabeling, Staining, Microscopy, Imaging

Journal: Research Square

Article Title: Tumor-restrictive type III collagen in the breast cancer microenvironment: prognostic and therapeutic implications

doi: 10.21203/rs.3.rs-2631314/v1

Figure Lengend Snippet: A. Kaplan-Meier survival curves showing that Col3:Col1 high patients have significantly better prognosis than Col1:Col3 high patients in terms of DFS and PFS across the entire TCGA BRCA patient cohort (p<0.05). B. Patients with Col3:Col1 high basal tumors demonstrated significantly better DFS, PFS, DSS, and OS compared to those with Col1:Col3 high basal tumors (p<0.05).

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330-01; Southern Biotech, Birmingham, AL), Col1 (PA5-9513; ThermoFisher Scientific), and Cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques:

Journal: Research Square

Article Title: Tumor-restrictive type III collagen in the breast cancer microenvironment: prognostic and therapeutic implications

doi: 10.21203/rs.3.rs-2631314/v1

Figure Lengend Snippet: MCF-7 cells were grown in 3D-culture consisting of Matrigel supplemented with human recombinant Col1, Col3 or 50:50 Col1+Col3 for 10 days. Images were taken with confocal microscopy of nuclei (DRAQ5, magenta), E-cadherin (cyan, shown in inset only) and fibrillar collagen (SHG, white) on max projection images. Mid-sections of the images showed the apparition of lumens (insets) (A-C). Spheroids (green outlines) and single cells (red outlines) were identified using ImageJ (D-F). Quantification of the area of single cells normalized to the total nuclei area (G). Integrated density of collagen fibers was analyzed by ImageJ and normalized to the total nuclei area (H). Lumens were counted and normalized total spheroids (I). Error bars show mean ± SD (n = 3). Data from three independent experiments. One-way AVONAs followed by Tukey post-hoc tests: (*, p<0.05; **,p<0.01,). Scale bar= 100μm.

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330-01; Southern Biotech, Birmingham, AL), Col1 (PA5-9513; ThermoFisher Scientific), and Cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Recombinant, Confocal Microscopy

Journal: Research Square

Article Title: Tumor-restrictive type III collagen in the breast cancer microenvironment: prognostic and therapeutic implications

doi: 10.21203/rs.3.rs-2631314/v1

Figure Lengend Snippet: RNA was collected from 4T1 tumors in Col3 +/+ and Col3 +/− mice and analyzed for Col1 and Col3 expression. Fold changes to Col3 +/+ D0 samples were used to generate Col1:Col3 ratios (A). Tumor volume 14 days after marginal resection in Col3 +/+ and Col3 +/− mice (B). Hydrogels were prepared with murine TNBC-like breast cancer cells (4T1) and supplemented with 100 μg of human recombinant Col3 or acetic acid (Ctrl) prior to injection into mammary fat pads of BalbC mice and harvested after 28 days (C). Tumor sections were stained for DAPI (nuclei, blue) and Ki67 (proliferating nuclei, red, D) or active caspase 3 (apoptotic cells, red, E). Scale bar = 50 μm. Staining was quantified as % Ki67 positive nuclei (D) or total active caspase 3 staining (integrated density, E). Lung sections were stained with H&E (F-G) and gross pulmonary metastasis was quantified (H). Scale bar = 3mm. For tumor growth, 2-way ANOVA followed by Sidak’s post-hoc test; for Ki67, active caspase 3, and metastasis, unpaired student t-tests: (*,p<0.05; **,p<0.01).

Article Snippet: Sections were incubated with antibodies directed against Col3 (1330-01; Southern Biotech, Birmingham, AL), Col1 (PA5-9513; ThermoFisher Scientific), and Cytokeratin (M351501-2; Agilent, Santa Clara, Ca), followed by Alexa Fluor secondary antibodies (ThermoFisher Scientific) and DRAQ5.

Techniques: Expressing, Recombinant, Injection, Staining