Journal: bioRxiv

Article Title: Stromal NRG1 in luminal breast cancer defines pro-fibrotic and migratory cancer-associated fibroblasts

doi: 10.1101/2020.04.06.026971

Figure Lengend Snippet: A , expression of NRG1 in breast cancer subtypes (PAM50) extracted from METABRIC and TCGA datasets. Boxes indicate mean +/- quartiles and minimum and maximum values are represented by bars. Only statistically significant comparisons with luminal subtypes are depicted. ANOVA multiple comparison test (*** P < 0.001). B , expression of NRG1 in the epithelial and stromal compartment in 4 laser-capture microdissected (LCM) breast cancer datasets (GSE10797, n = 28; GSE14548, n = 14; GSE35019, n = 53; and GSE83591, n = 39). Boxes indicate mean +/- quartiles and minimum and maximum values in bars. Two-tailed paired Student’s t-test (* P < 0.05; ** P < 0.01; *** P < 0.001). C , viability of T47D (black) and MCF7 (grey) measured 72h after treatment with HER3 mAb lumretuzumab or pertuzumab at indicated doses. Values represent median of 3 independent experiments (n = 5). U-Mann Whitney two-tailed test was applied. No significant differences were observed. D , ectopic NRG-1β (50ng/mL) was added to T47D and MCF7 cell lines and viability quantified. The untreated control was set to 100% (not shown) and used to normalize the results from conditions where cells had been preincubated for 1 hour with lumretuzumab (Lum) or pertuzumab (Pert) at 10µg/mL. Two-tailed unpaired Student’s t-test (* P < 0.05; ** P <0.01, *** P <0.001) comparing each treatment with untreated condition. Bars represent average of two independent experiments +/- s.e.m. E , representative Western blot showing total levels and phosphorylation of HER3 and downstream effectors AKT and ERK1/2, 5min after addition of NRG-1β (50ng/ml). Some samples were either pre-incubated with mAbs lumretuzumab (Lum) or pertuzumab (Pert) at 10µg/ml for one hour.

Article Snippet: Prior to addition of CAF-CM or human recombinant NRG-1β (4711, BioCat), cells were pre-treated with either lumretuzumab or pertuzumab (10μg/ml) for 30min in low serum media (1% FCS).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Western Blot, Incubation

Journal: bioRxiv

Article Title: Stromal NRG1 in luminal breast cancer defines pro-fibrotic and migratory cancer-associated fibroblasts

doi: 10.1101/2020.04.06.026971

Figure Lengend Snippet: A , heatmap representing relative phosphorylation values of HER3, AKT and ERK1/2 in T47D and MCF7 cells after addition of CAF conditioned media (CM) for 5min with or without pre-incubation with lumretuzumab (Lum) (10µg/ml). Values represent median of three technical replicates and three biological replicates. Color intensities are ranked per each antibody (red = maximum, blue = minimum). B , relative proliferation of T47D and MCF7 cancer cells after 72h with different CAF-CM with lumretuzumab (red squares) or untreated (black circles). Dots represent mean +/- s.e.m of three independent replicates (n = 4). P values were determined by two-tailed U-Mann Whitney test for each CM (* P < 0.01; ** P < 0.001; *** P < 0.0005; **** P <0.0001). C , percentage of closure in a scratch assay of T47D or MCF7 cancer cells, after 21h of treatment with 10μg/ml lumretuzumab (red) or untreated (black), and with conditioned media (CM) of indicated CAFs. DMEM-F12 1% FCS was used as negative control and NRG-1β (50ng/ml) as positive control. Box plots correspond to the mean and s.e.m. of 2 independent experiments (n = 6 technical replicates). Two-tailed U-Mann Whitney test for each CM (* P < 0.01; **P < 0.001; *** P < 0.0005; **** P <0.0001).

Article Snippet: Prior to addition of CAF-CM or human recombinant NRG-1β (4711, BioCat), cells were pre-treated with either lumretuzumab or pertuzumab (10μg/ml) for 30min in low serum media (1% FCS).

Techniques: Incubation, Two Tailed Test, MANN-WHITNEY, Wound Healing Assay, Negative Control, Positive Control