Supplemental Figure 1. Representative Images from 4 Patients with ER+ Breast Cancer Stained

Supplemental Figure 1.Representative images from 4 patients with ER+ breast cancer stained CK8/18 (red, vector red staining) and vimentin (DAB, brown staining). Tumor cells (yellow arrow heads) are vimentin negative, CK8/18 positive and morphologically distinct from stroma cells, which are CK8/18 negative and vimentin positive. Scale bar = 100 m

Supplemental Figure 2. (A) Representative flow cytometry plots demonstrates that our fibroblast isolation protocol produced highly enriched fibroblast populations from both ER+ breast cancer associated tissue (A) and (B) normal breast tissue. Individual patient samples contain both CD146pos and CD146neg fibroblasts. CD326 (epithelial), CD31 (endothelial and most pericyte), NG2 (all pericytes and some fibroblasts), FSP1 (most fibroblasts, not pericytes) and CD146 were stained in together. Vimentin (most fibroblasts and pericytes) was a separate single stain. Gating in these panels are on the negative population, which was determined, using the fluorescence minus one controls.

Supplemental Figure 3. (A) Flow cytometry of isolated stromal population from patient matched normal (N = 4) and ER+ breast cancer associated tissue (N = 4) demonstrate significant enrichment for fibroblasts (FSP1+ and VIM+). (B) Gene expression data for vimentin of human tumor stroma fibroblast clone TS10-33 (CD146neg) and TS10-25 (CD146pos) fibroblasts compared to HS5 (CD146neg) and HS27a (CD146pos) fibroblasts. (C) Representative images of clonal fibroblasts isolated from normal breast tissue. (D) Representative images of clonal fibroblasts isolated from ER+ breast cancer associated tissue. Scale bar = 100 m.

Supplemental Figure 4.(A-B) MNCs co-cultured with CD146pos fibroblasts (HS27a) or primary cancer associated stroma form significantly more cobblestone colonies (yellow circles) compared to CD146neg fibroblasts (HS5) or primary cancer associated stroma. (B) Frequency of cobblestone area-forming cells in HS27a (CD146+) vs. HS5 (CD146-) co-cultures and in primary CD146+ and CD146- tumor stroma co-cultures. (C) Gene expression hierarchical cluster analysis demonstrates that primary CD146+ tumor stroma is highly similar to normal HS27a fibroblasts and primary CD146- tumor stroma is similar to normal HS5 fibroblasts. Scale bars: 20 m. Cobblestone formation was quantified using 20X images and a formation was considered 12 or more closely embedded cells. N = 4 co-cultures per group. ****p < 0.0001.

Supplemental Figure 5. (A) MCF-7 cells were co-cultured with our primary derived CAF fibroblasts TS10-25 (CD146pos) and TS10-33 (CD146neg) for five days and harvested for immunofluorescence staining for ER (red) and CK18 (green). Results show decreased ER expression in MCF-7 co-cultured with CD146neg fibroblasts and sustained ER expression in MCF-7 cells co-cultured with CD146neg fibroblasts. Scale bar = 100 M. (B) UCD12 cells are ER+ primary tumor cells generated at the University of Colorado from a patient derived tissue. Live cell imaging of UCD12/CAF co-cultures demonstrate that CD146pos fibroblasts promote UCD12 proliferation in response to estrogen treatment and decreased proliferation in response to tamoxifen treatment as measured by live cell imaging. In contrast, influence from CD146neg fibroblasts results in loss of estrogen and tamoxifen responsiveness. (C) SRB total protein analysis in ER+ T47D cells measuring proliferation demonstrates that influence from CD146pos conditioned media renders T47D cells estrogen and tamoxifen responsive, whereas influence from CD146neg conditioned media renders them unresponsive. (D) Representative images from ER+ T47D cell lines grown in conditioned media from CD146pos (HS27a) or CD146neg (HS5) fibroblasts and stained for CK8/18 (green) and ER (red) show decreased ER staining intensity. *p < 0.05; **p < 0.01; ****p < 0.0001, Scale bar = 50 M.

Supplemental Figure 6. (A) Representative images of immunohistochemical staining for the tumor cell marker cytokeratin 8/18 (vector red staining) and fibroblast/stroma marker vimentin (DAB, brown staining) showing dense tumor regions in vehicle treated MCF-7/CD146pos tumors and both tamoxifen treated and vehicle treated MCF-7/CD146neg tumors. MCF-7/CD146pos tumors treated with tamoxifen display less tumor density. Scale bar = 100 M. (B) Western blot analysis of MCF-7 cells demonstrates that EGFR is expressed at low levels in the presence of MCF-7 CM and CM from CD146pos (HS27) or CD146neg (HS5) fibroblasts. (C)Our CAF influenced gene signature is not predictive for recurrence-free survival in patients who were not treated with tamoxifen, demonstrating that the signature is specific to tamoxifen response. (D) Inclusion of 37 genes predicted in literature to be involved in estrogen responsiveness and/or tamoxifen resistance are not predictive of recurrence-free survival, demonstrating that our CAF influenced signature is unique. (E-F) Splitting estrogen receptor (ER) expression into high and low expression along the median in our tamoxifen treated training (E) and validation (F) set is not predictive of recurrence-free survival, demonstrating that the predictive power of our CAF influenced signature is not due to the correlation between CD146 expression and ER Allred scores. (G) Splitting ER expression into high and low expression along the median in our untreated patient training set is not predictive of recurrence-free survival. Significance is considered p < 0.05.

Supplemental Table 1.Gene expression analysis in CD146pos (HS27a and our primary CAFs) and CD146neg (HS5 and our primary CAFs) demonstrates that all four cell types express high levels of genes associated with activated fibroblasts. Affymetrix gene analysis with a signal range of 0 to 13.

Supplemental Table 2.RNAseq analysis of MCF-7 cells after co-culture with CD146pos (HS27a) fibroblasts reveals increased expression of genes identified in literature to be associated with tamoxifen responsiveness in ER+ breast cancer, as compared to expression in MCF-7 cells co-cultured with CD146neg (HS5) fibroblasts. Genes in bold are included in the gene signature we developed as predictive for recurrence-free survival.

Supplemental Table 3.RNAseq analysis of MCF-7 cells after co-culture with CD146neg (HS5) fibroblasts reveals increased expression of genes identified in literature to be associated with tamoxifen resistance in ER+ breast cancer, as compared to expression in MCF-7 cells co-cultured with CD146pos (HS27a) fibroblasts. Genes in bold are included in the gene signature we developed as predictive for recurrence-free survival.