Journal: bioRxiv

Article Title: Carbon ion irradiation plus CTLA4 blockade elicits therapeutic immune responses in a murine tumor model

doi: 10.1101/2022.07.22.500608

Figure Lengend Snippet: (A) Treatment schedule: C57BL/6 mice were bilaterally challenged by time-shifted injections (s.c.) with 4 × 10 5 EO771 cells. When tumors had established, mice were randomized into treatment groups based on the volume of in-field (iF) and out-of-field (oF) tumors. Then, the iF tumor was irradiated with 3.08 Gy carbon ions on three consecutive days. On day 12, 15, and 18, mice received i.p. injections of blocking antibodies against PD-L1 or CTLA4. Control mice were injected with IgG isotype controls. (B) Individual growth curves of iF and oF tumors of untreated control mice or mice treated as indicated. (C) Kaplan-Meier survival analysis. In addition, complete response (CR) rates for each treatment group are shown. Significance was determined using a log-rank (Mantel-Cox) test with Holm-Bonferroni correction.

Article Snippet: EO771 cells were purchased from TEBU-Bio (Offenbach, Germany) and were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% heat-inactivated FCS, 10 mmol/L HEPES (Thermo Fisher Scientific), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Thermo Fisher Scientific).

Techniques: Irradiation, Blocking Assay, Injection

Journal: bioRxiv

Article Title: Carbon ion irradiation plus CTLA4 blockade elicits therapeutic immune responses in a murine tumor model

doi: 10.1101/2022.07.22.500608

Figure Lengend Snippet: (A) Percentage of successful tumor engraftment in untreated mice or in mice whose EO771 tumors had regressed upon treatment with photon or carbon ion RT, respectively combined with CTLA4 blockade. Numbers of tumor rejecting mice per animals engrafted are depicted above the columns. (B) INF-γ ELISpot assay with splenocytes from cured mice and naïve control animals co-cultured o/n with EO771 target cells. (C) Specificity of the T cell response was assessed by IFN-γ ELISpot assays performed after co-culture of splenocytes with syngenic tumor cells lines B16F10 and RMA in comparison to EO771 cells. Significance was determined by one-way ANOVA with post-hoc Turkey’s test.

Article Snippet: EO771 cells were purchased from TEBU-Bio (Offenbach, Germany) and were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% heat-inactivated FCS, 10 mmol/L HEPES (Thermo Fisher Scientific), 100 U/ml Penicillin, and 100 μg/ml Streptomycin (Thermo Fisher Scientific).

Techniques: Enzyme-linked Immunospot, Cell Culture, Co-Culture Assay

Journal: Oncogene

Article Title: A CCL8 gradient drives breast cancer cell dissemination

doi: 10.1038/onc.2016.161

Figure Lengend Snippet: Ccl8 expression in tumors, stroma and peripheral tissues. (a) Ccl8 levels of EO771 tumors developed in wt (n=6) and Ccl8KO (n=4) mice. Tumor volumes in all cases analyzed ranged between 200mm 3 –300mm 3 . (b) Expression of Ccl8 in the stroma of EO771 tumors growing in wt mice. Right panel shows in higher magnification of the area marked in the left panel by a blue square. (c) Serum Ccl8 levels in relation to tumor volume in different EO771 breast cancer – bearing mice. Ep, epithelium, St, fibroblastic stroma. p<0.05, Pearson’s correlation (d) Tumoral Ccl8 (n=6) and Ccl8 in various organs from tumor free (n=4) and mice bearing EO771 tumors (n=5) (200mm 3 –300mm 3 ). *, p<0.05 Student’s t-test

Article Snippet: EO771 cells, prior to mice inoculation were tested for viral pathogens and mycoplasma contamination (IDEXX Bioresearch, Columbia, MO).

Techniques: Expressing

Journal: Oncogene

Article Title: A CCL8 gradient drives breast cancer cell dissemination

doi: 10.1038/onc.2016.161

Figure Lengend Snippet: Effect of Ccl8 inhibition in EO771 tumors in vivo . (a) EO771 tumor onset in wt mice following administration of a neutralizing antibody for CCL8. (b) Inhibition of mCcl8-induced chemoattraction by a neutralizing antibody for CCL8 in RAW 264.7 macrophages in transwells. (c) EO771 tumor onset in wt and Ccl8KO mice (24). (d) Histology of EO771 tumors in wt and Ccl8KO mice. Genotypes are shown in white insets. The upper panel corresponds to images from the core of the tumor while the lower panel from the margins. Lower right graph indicates cellularity expressed as epithelial cells per high power optic field. (n=3 per genotype). Ep, epithelium, FS, fibroblastic stroma, AC, antipocytic stroma. *, (p<0.05,Student’s t-test). (e) Van Gieson staining of EO771 tumors in control and Ccl8KO mice. (f) Vimentin expression (brown staining) close to tumor margins in EO771 tumors that developed in wt and Ccl8KO mice. Right panel shows in higher magnification of the area marked in the left panel by a black square.

Article Snippet: EO771 cells, prior to mice inoculation were tested for viral pathogens and mycoplasma contamination (IDEXX Bioresearch, Columbia, MO).

Techniques: Inhibition, In Vivo, Staining, Control, Expressing

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Plac1 expression and lentivirus-mediated reduction of Plac1 in EO771 cells. ( a ) EO771 mouse mammary tumor cells expressed high levels of Plac1 in comparison to mouse placenta. ( b ) EO771 cells were transduced with lentiviruses expressing crambled RNA (Scr) or four Plac1 shRNAs designated sh81, sh187, sh300 and sh490; sh490 inhibited RNA expression >98%, and these cells were designated EO771/shPlac1. ( c ) EO771/Scr and EO771/shPlac1 cells were grown as monolayers, and the number of viable cells were quantified by sulforhodamine B staining. Shown is the mean ± S.D. of triplicate analysis of three samples. The growth of EO771/shPlac1 cells differed significantly ( P < 0.001) from EO771/Scr cells by the two-sided Student’s t test. ( d ) qRT-PCR analysis of immune cell-related gene expression downregulated in EO771/shPlac1 cells. Shown is the mean ± S.D. of triplicate analysis of three samples. Significant differences between EO771/Scr and EO771/shPlac1 cells were obtained for CD274 ( P < 0.01), Plac1 ( P < 0.01), Cxcl1 ( P < 0.001), Ccl5 ( P < 0.001) and Lif ( P < 0.001) using the two-tailed Student’s t test; values for Ccl7 were not significantly different ( P > 0.05). ( e ) Heatmap of gene expression as determined by Affymetrix microarray analysis of EO771/Scr (Ctl) and EO771/shPlac1 (sh) cells. Shown are immune cell-related transcripts (Table ) representing ≥3.0-fold change in expression.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Comparison, Transduction, RNA Expression, Staining, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Microarray

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Growth of EO771/Scr and EO771/shPlac1 cells in syngeneic and SCID mice. ( a ) Syngeneic C57BL/6 mice or ( b ) SCID mice at five weeks of age, were inoculated in the mammary gland with 1 × 10 6 cells. Tumor size was measured by calipers in two dimensions. Tumor growth for EO771/Scr and EO771/shPlac1 cells in syngeneic mice differed significantly (P = 0.040) by the unpaired Student’s t test. There was no significant difference (P > 0.05) in tumor growth between the two cell lines in SCID mice. Shown is the mean ± SD, N = 5 per group. ( c ) H&E staining and Plac1 IHC in isografts of EO771/Scr and EO771/shPlac1 cells. Magnification 400X.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Staining

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Growth of EO771 cells in syngeneic mice following treatment with a Cxcr 2 antagonist. ( a ) Syngeneic 57BL/6 mice were inoculated in the mammary gland with 1 × 10 6 at five weeks of age, and injected i.p. daily with vehicle (blue) or 2 mg/kg (red) or 20 mg/kg (green) SB225002 beginning 11 days after cell inoculation. SB225002 completely suppressed tumor growth after 14 days. Differences between vehicle- and 2 mg/kg SB225002-treated mice were not significantly different (P = 0.145); differences between vehicle- and 20 mg/kg SB225002-treated mice were significantly different (P = 0.005) by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N = 5 per group. ( b ) Immune gene expression in tumors 17 days after treatment with 20 mg/kg SB225002. Shown is the relative expression in control and SB225002-treated mice in comparison to their changes in EO771/shPlac1 cells (Table ). ( c ) FACS analysis of immune cell tumor infiltrates in isografts after treatment with vehicle or SB225002 as in ( b ). SB225002 treatment reduced the percentage of immune cell tumor infiltrates of CD11b + /Gr-1 + myeloid-derived suppressor cells ( MDSC ) and Foxp3 + /CD25 + T cells ( Treg ), and increased the percentages of CD8 + /CD4 + T cells ( T) , CD3 + /NK1.1 + NK cells ( NK ) and F4/80 + /CD80/86 + macrophages ( Mϕ ) and CD11c + /CD80/86 + dendritic cells ( DC ). Numbers in parentheses ( ) represent the percentages of each cell population. ( d ) Bar graph represents the mean±SD of the percent distribution of immune cell tumor infiltrates as in ( c ); P values were determined by the unpaired two-tailed Student’s t test, N = 4 per group. ( e ) CD8 + T cell infiltration determined by IHC in tumor isografts from vehicle-treated ( EO771/Ctl ) and SB225002-treated ( EO771/SB ) mice. Infiltration of CD8 + T cells increased after treatment with 20 mg/kg SB225002. Magnification 600X. ( f ) Macrophage ( F4/80 ) and Treg cell ( Foxp3 ) infiltration, Plac1 expression and apoptosis by cleaved caspase-3 expression ( Caspase ) in tumor isografts from vehicle-treated ( EO771/Ctl ) and SB225002-treated ( EO771/SB ) mice. Infiltration of macrophages and Treg cells were reduced and apoptosis was increased after treatment with 20 mg/kg SB225002. Magnification 400X

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Injection, Two Tailed Test, Gene Expression, Expressing, Control, Comparison, Derivative Assay

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Lentivirus-mediated reduction of Cxcl1 in EO771 cells. ( a ) EO771 cells were transduced with lentiviruses expressing scrambled RNA (Scr) or three Cxcl1 shRNAs designated sh118, sh174, sh218; sh174 inhibited RNA expression >99% (EO771/shCxcl1). ( b ) EO771/Scr and EO771/shCxcl1 cells were grown as monolayers, and the number of viable cells were determined by sulforhodamine B staining. Shown is the mean ± S.D. of triplicate analysis from three samples, which were significantly different ( P < 0.001) by the two-tailed Student’s t test. ( c ) Growth of EO771/Scr and EO771/shCxcl1 cells in syngeneic mice. Mice at five weeks of age were inoculated in the mammary gland with 1x10 6 cells, and tumor size was measured by calipers in two dimensions. Differences in tumor growth between EO771/Scr and EO771/shCxcl1 cells were significantly different (P = 0.006) by the unpaired two-tailed Student’s t test; N = 5. ( d ) qRT-PCR analysis of genes downregulated in EO771/shCxcl1 cells. Shown is the mean ± SD of triplicate analysis of 3 samples. Significant differences between EO771/Scr and EO771/shCxcl1 cells were obtained for Plau ( P < 0.02), C3 ( P < 0.01), Ly6a ( P < 0.01), Ccl7 ( P < 0.001) and Il23a ( P < 0.01) by the two-sided Student’s t test; differences for CD68 were not significantly different ( P > 0.05).

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Transduction, Expressing, RNA Expression, Staining, Two Tailed Test, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Expression of immune-related genes in E0771/shCxcl1 cells. Shown are ≥3-fold changes in gene expression with a raw score ≥300 in EO771/shCxcl1 or E0771/Scr cells.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Gene expression common to EO771/shPlac1 and EO771/shCxcl1 cells. Shown is the ratio between EO771/shPlac1 or EO771/shCxcl1 cells to EO771/Scr control cells for genes with ≥3.0-fold changes in expression and a raw score ≥300.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Gene Expression, Control, Expressing

Journal: Scientific Reports

Article Title: Plac1 Is a Key Regulator of the Inflammatory Response and Immune Tolerance In Mammary Tumorigenesis

doi: 10.1038/s41598-018-24022-w

Figure Lengend Snippet: Cxcl1 rescue of EO771/sh490 cells. ( a ) EO771/Scr and EO771/sh490 cells expressing eGFP were transduced with a lentivirus expressing Cxcl1 and mCherry, and selected for 35 days in 3.5 mg/ml G418. The merged photo shows cells co-expressing eGFP and mCherry (yellow). Magnification 200X. ( b ) qRT-PCR for Plac1 and Cxcl1 in EO771/Scr, EO771/sh490 and EO771/sh490/Cxcl1 cells. Shown is the mean ± S.D. of triplicate determinations.( c ) EO771/sh490/Cxcl1 cells were grown in 96-well plates at an initial density of 5,000 cells per well in media supplemented with 3.5 mg/ml G418. Cell density was determined by sulforhodamine B staining. Shown is the mean ± SD of triplicate determinations. ( d ) Syngeneic C57BL/6 mice were inoculated in the mammary gland with 1 × 10 6 at five weeks of age. There was a significant difference in the growth EO771/sh490 cells (P = 0.021) and EO771/sh490/Cxcl1 cells (P = 0.034) vs. EO771/Scr cells by the unpaired two-tailed Student’s t test. Shown is the mean ± SD, N=6 per group.

Article Snippet: EO771 cells at an inoculum of 1 × 10 6 cells/0.1 ml were injected into the no. 4 mammary gland of C57BL/6 or SCID mice (Taconic), and tumor growth was monitored daily.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Cancers

Article Title: Conversion of Osteoclasts into Bone-Protective, Tumor-Suppressing Cells

doi: 10.3390/cancers13225593

Figure Lengend Snippet: Vicious interactions between differentiated RAW264.7 osteoclasts and EO771 mammary tumor cells. CN = control, RANKL CM = differentiated osteoclast-derived conditioned medium (treated with 10 ng/mL RANKL and 2 ng/mL M-CSF for 2 days), and EO CM = EO771 tumor cell-derived CM. * p < 0.05 and ** p < 0.01. ( A – C ) Elevation of MTT-based viability, transwell invasion, and scratch-based migration of EO771 cells by RANKL CM. ( D ) Elevation of PTHrP in RANKL CM. ( E ) Increase in the number of TRAP-positive RAW264.7 osteoclasts by EO CM. ( F ) Elevation of cathepsin K and NFATc1 in RANKL-stimulated RAW264.7 osteoclasts in response to EO CM. ( G ) Increase in the size and weight of mammary tumors in C57BL/6 female mice.

Article Snippet: On day 1, C57BL/6 female mice (8 mice per group, ~8 weeks, Envigo RMS, Inc., Indianapolis, IN, USA) received a subcutaneous injection of EO771 cells (3.0 × 10 5 cells in 50 μL PBS) to the mammary fat pad in the mouse model of a mammary tumor [ ].

Techniques: Control, Derivative Assay, Migration

Journal: Cancers

Article Title: Conversion of Osteoclasts into Bone-Protective, Tumor-Suppressing Cells

doi: 10.3390/cancers13225593

Figure Lengend Snippet: Conversion of RAW264.7 pre-osteoclasts into iTS cells by the treatment with BML284. CN = control, CM = conditioned medium, RAW = RAW264.7 osteoclasts, MC3T3 = MC3T3 osteoblasts, MSC = mesenchymal stem cells, A5 = MLO-A5 osteocytes, 231 = MDA-MB-231 breast cancer cells, EO = EO771 mammary tumor cells, and 4T1.2 = 4T1.2 mammary tumor cells. ** p < 0.01 vs. CN, while ## p < 0.01 vs. A5 CM. ( A – D ) MTT-based viability of EO771 mammary tumor cells in response to a chemically treated conditioned medium, derived from MLO-A5 osteocytes, MSCs, MC3T3 osteoblasts, and RAW264.7 osteoclasts, respectively. NS = NSC228155 (EGF activator), RC = RCGD423 (JAK/STAT activator), m3 = m-3M3FBS (phospholipase C activator), CW = CW008 (PKA activator), OA = OAC2 (Oct4 activator), YS = YS49 (PI3K activator), and BM = BML284 (Wnt activator). ( E , F ) Tumor selectivity of the inhibitory action of RAW CM, examined tumor selectivity of the inhibitory action using 3 tumor cell lines (MDA-MB-231 breast cancer cell line using 3 tumor cell lines (MDA-MB-231 breast cancer cell line, EO771 mammary tumor cell line, and 4T1.2 mammary tumor cell line), and KTB6 human breast epithelial cells. ( G ) Reduction in PTHrP in BM CM.

Article Snippet: On day 1, C57BL/6 female mice (8 mice per group, ~8 weeks, Envigo RMS, Inc., Indianapolis, IN, USA) received a subcutaneous injection of EO771 cells (3.0 × 10 5 cells in 50 μL PBS) to the mammary fat pad in the mouse model of a mammary tumor [ ].

Techniques: Control, Derivative Assay

Journal: Cancers

Article Title: Conversion of Osteoclasts into Bone-Protective, Tumor-Suppressing Cells

doi: 10.3390/cancers13225593

Figure Lengend Snippet: Tumor-suppressive effects of BM CM. CN = control, CM = RAW264.7 osteoclasts-derived conditioned medium, no-BM = treated without BML284, BM = BML284 treated. ** p < 0.01. ( A – C ) Suppression of EdU-based proliferation, transwell invasion, and scratch-based migration of EO771 tumor cells by BM CM. ( D ) Downregulation of Lrp5, Runx2, and TGFβ in EO771 tumor cells by BM CM. ( E ) Shrinkage of human breast-cancer-tissue fragments by BM CM for 3 days.

Article Snippet: On day 1, C57BL/6 female mice (8 mice per group, ~8 weeks, Envigo RMS, Inc., Indianapolis, IN, USA) received a subcutaneous injection of EO771 cells (3.0 × 10 5 cells in 50 μL PBS) to the mammary fat pad in the mouse model of a mammary tumor [ ].

Techniques: Control, Derivative Assay, Migration

Journal: Cancers

Article Title: Conversion of Osteoclasts into Bone-Protective, Tumor-Suppressing Cells

doi: 10.3390/cancers13225593

Figure Lengend Snippet: Inhibitory action of Hsp90ab1 and Eno1. Hab1 = Hsp90ab1, Haa1 = Hsp90aa1, siHab1 = Hsp90ab1 siRNA, CN = control, and BM CM = BML284-treated RAW264.7-derived CM. ** p < 0.01. ( A ) Expression of Lrp5, Runx2, and TGFβ in response to Hsp90ab1 and Hsp90aa1in EO771 cells. ( B ) Elevation of MTT-based viability of EO771 cells in response to Hsp90ab1 siRNA-treated, RAW276.4-derived CM. ( C ) Expression of Lrp5, Runx2, and TGFβ in response to Hsp90ab1 siRNA-treated, RAW276.4-derived CM. ( D ) Co-immunoprecipitation of LAP (latency-associated peptide) TGFβ by Hsp90ab1 in BM CM, Eno1 by Hsp90ab1, Hsp90ab1 by Eno1, and CD44 by Eno1 in EO771 cells. ( E , F ) Suppression of Eno1-mediated inhibition of the proliferation of EO771 cells by RNA silencing of CD44. ( G ) Suppression of Eno1-mediated downregulation of Lrp5, Runx2, and TGFβ in EO771 cells by RNA silencing of CD44. ( H , I ) Suppression of Hab1-mediated inhibition of the proliferation, and downregulation of Lrp5, Runx2, and TGFβ of EO771 cells by RNA silencing of CD44. ( J ) Schematic illustration of the possible regulatory mechanism with BML284-treated RAW264.7 osteoclast-derived CM (BM CM).

Article Snippet: On day 1, C57BL/6 female mice (8 mice per group, ~8 weeks, Envigo RMS, Inc., Indianapolis, IN, USA) received a subcutaneous injection of EO771 cells (3.0 × 10 5 cells in 50 μL PBS) to the mammary fat pad in the mouse model of a mammary tumor [ ].

Techniques: Control, Derivative Assay, Expressing, Immunoprecipitation, Inhibition

Journal: JNCI Journal of the National Cancer Institute

Article Title: Effects of Obesity on Transcriptomic Changes and Cancer Hallmarks in Estrogen Receptor–Positive Breast Cancer

doi: 10.1093/jnci/dju158

Figure Lengend Snippet: Effect of obesity on breast cancer progression and tumor growth in orthotopic/syngeneic mice. A ) Representative in vivo bioluminescent imaging of tumors performed 4 weeks after orthotopic/syngeneic allografting of EO771-FG12 cells into female transgenic lean and obese mice negative for the MMTV- TGFα transgene (n = 7 mice per group). B ) Representative in vivo bioluminescent imaging of tumors performed 4 weeks after orthotopic/syngeneic allografting of EO771-FG12 cells into female a/a lean mice (n = 7), A y /a obese mice (n = 6), A y /a obese mice treated with metformin (300mg/kg daily; n = 6) and A y /a obese mice treated with everolimus (4mg/kg daily; n = 8). C ) Representative images of syngeneic allografted tumors harvested from randomized lean, obese, metformin-treated, and everolimus-treated obese female mice (scale bars represent 5mm; left panel ). A bar graph illustrates the mean tumor weights from the same experiment (n = 6–8 mice per group; right panel ). Statistical significance was calculated by one-way analysis of variance. D ) Phospho-protein levels of members of the AKT/mTOR signaling pathway from syngeneic allografted tumor lysates from obese mice plotted relative to those from lean mice (n = 5). E ) Western blot analysis of total and phospho-AKT (Ser473), total and phospho-mTOR (Ser2448), and total and phospho-p70S6K (Thr389). Error bars in panels ( B ) and ( D ) represent 95% confidence intervals.

Article Snippet: Orthotopic/syngeneic allografting experiments were performed by injecting EO771-FG12 cells in Matrigel (BD Bioscience, San Jose CA) into the left fourth mammary fat pad.

Techniques: In Vivo, Imaging, Transgenic Assay, Western Blot

Journal: JNCI Journal of the National Cancer Institute

Article Title: Effects of Obesity on Transcriptomic Changes and Cancer Hallmarks in Estrogen Receptor–Positive Breast Cancer

doi: 10.1093/jnci/dju158

Figure Lengend Snippet: Effect of adipocytes on breast cancer cell proliferation and viability. A ) MCF7 cell proliferation after having been cultured alone, in coculture with 3T3-L1 fibroblasts, or in coculture with 3T3-L1 mature adipocytes for 4 days. B ) EO771 cell proliferation after having been cultured alone or in coculture with 3T3-L1 mature adipocytes for 3 days with high glucose Dulbecco’s modified Eagle medium and 10% bovine calf serum. C ) Cell viability of T47D BC cells after incubation with undifferentiated 3T3-L1 pre-adipocyte-conditioned media or with differentiated 3T3-L1 mature adipocyte-conditioned media. D ) MCF7 cell proliferation after metformin treatment cultured in coculture with 3T3-L1 mature adipocytes for 4 days. E ) Adipokine profile array of conditioned media supernatants from 3T3-L1 mature adipocytes cultured for 24 hours relative to 3T3-L1 pre-adipocytes (n = 3 composite; left panel ). Adipokines of conditioned media supernatants from 3T3-L1 mature adipocytes cultured with metformin for 24 hours relative to nontreated 3T3-L1 adipocytes (n = 3; right panel ). Asterisks represent statistical significance (* P <.05, ** P <.01, *** P <.001), one-way analysis of variance. F ) MCF7 cell proliferation after being cocultured with 3T3-L1 mature adipocytes for 4 days alone or with TIMP-1 neutralizing antibody (2 μg/mL; R&D Systems). G ) MCF7 cell proliferation after being cultured for 4 days alone or with human TIMP-1 (100ng/mL; Millipore, Billerica, MA). H ) Representative photomicrographs of invasion assay of MCF7 cells, which were cultured for 4 days alone or with TIMP-1 (100ng/mL). Images were captured at ×4 magnification. Error bars in panels ( A – G ) represent 95% confidence intervals (n ≥ 3). Statistical significances were calculated by one-way analysis of variance for panels ( A ) and ( D ) and by two-tailed t test for panels ( C ), ( F ), and ( G ).

Article Snippet: Orthotopic/syngeneic allografting experiments were performed by injecting EO771-FG12 cells in Matrigel (BD Bioscience, San Jose CA) into the left fourth mammary fat pad.

Techniques: Cell Culture, Modification, Incubation, Invasion Assay, Two Tailed Test