Extended Data Fig. 10: Fibroblast-derived MMP3 promotes epithelial growth in vitro.

a) FACS plots showing gating strategy for isolation of GFP-transduced human fibroblasts isolated from patient breast tissue in forward and side scatter, singlets gate, and GFP gate. b) Representative images of cocultures after 5 days of seeding. 4000 primary mammary epithelial cells (NonCarrier 32) were cultured alone (No Fibroblasts) or with 1×10⁵ primary mammary fibroblasts (NonCarrier 33 or BRCA1^+/mut 19) transduced with lentivirus to express GFP only (+GFP) or GFP and MMP3 ( + MMP3) in Matrigel for 5 days. Fibroblasts are distinguished from epithelial spheres (GFP-negative) with GFP fluorescence. Scale bar = 400 μm. c) Quantification of spheres after 5 days. Values are represented as mean ± SD from 3 separate experiments with 3 triplicate wells per experiment. P values were determined by unpaired two-tailed t-tests. d) Mean values of sphere counts pooled from 3 separate experiments from (c) and Fig. 5c. Values are represented as mean ± SD. Statistical significance between all groups was determined with a one-way ANOVA test. e) 10×10⁵ FACS-isolated epithelial cells from 4 patient samples were seeded in Matrigel and treated with 0.5 µg/mL or 1 μg/mL recombinant MMP3 and spheres were counted after 5 and 10 days. Bar chart values are represented as mean ± SD from triplicates from three separate experiments. P values were determined using unpaired two-tailed t-tests. Representative bright field images of mammospheres after 10 days of culture are shown on the right (scale bar = 400 μm). f) Bar graph depicting fold change in sphere count after 10 days of culture with human recombinant MMP3 compared to control (dotted red line). Values are displayed as mean ± SD from 15 independent experiments (5 different patient samples with 3 separate experiments each). P values were determined using unpaired two-tailed t-tests. g) Primary human breast fibroblasts isolated by FACS from patient sample “NonCarrier 27” were transduced to express mouse MMP3 (mMMP3) and GFP or GFP only. qPCR analysis was performed on transduced fibroblasts in two separate trials with three replicates per group. Amplification plot is shown with the difference in the normalized reporter value of the experimental reaction minus the normalized reporter value generated by the instrument (ΔRn) on the y-axis and the cycle number on the x-axis.