Fig. 2. The profile of breast tumor gene expression is affected by SNAI2 deficiency in a non-cell-autonomous manner.

(A) Detection of SNAI2 in Snai2 WT^ErbB2 and Snai2 KO^ErbB2 tumors by western blot. Mouse embryonic fibroblasts (MEFs) were used as a positive control for SNAI2 expression. (B) QPCR quantification of Snai2 in the epithelial and stromal components from Snai2 WT^ErbB2+ and Snai2 KO^ErbB2+ tumors. Snai2 RNA is not detected in Snai2 WT^ErbB2+ mice by QPCR after EpCAM⁺ cell sorting of the epithelial component, yet it is found in the stromal CD140+ cells. (C) SNAI2 protein in western blots of CAFs from mouse breast tumors. (D) Scheme representing the study design. Tumors from Snai2 WT^ErbB2 and Snai2 KO^ErbB2 mice were analyzed, both from nulliparous (ERBB2+ expression: WT N=41 and KO N=33) and parous (ERBB2++ expression) females in which the tumors developed after two pregnancies (WT, N=33, and KO, N=22). (E) The 602 gene expression signature allows unsupervised classification of the tumors from Snai2 WT^ErbB2 nulliparous and parous mice. (F) The signature does not permit the clustering of Snai2 KO^ErbB2 nulliparous and parous mice. (G) GSEA analysis: representation of the enrichment of the EMT pathway in tumors from Snai2 WT^ErbB2+ relative to the tumors from Snai2 KO^ErbB2+ nulliparous mice (above), and in tumors Snai2 KO^ErbB2++ relative to the tumors from Snai2 WT^ErbB2++ parous mice (below). (H) Conditioned medium (CM) from cultured human normal fibroblasts (hNFs) or human cancer-associated fibroblasts (hCAFs) was used to induce MDA-MB-231 cell migration (N=3). (I) Representative western blot of normal hCAFs and hCAFs in which SNAI2 was depleted by specific siRNAs, probed for SNAI2. (J) Migration of MDA-MB-231 cells in the presence of CM from control or SNAI2 depleted hCAFs (N=3). (K) Migration of BT474 cells using CM from control or SNAI2 depleted hCAFs (N=3). Statistical significance in panels I, K was evaluated using an unpaired t-test. In E and F red and blue color represent over-expressed and down-expressed genes, respectively.