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Video S4. MDA-MB231 cells on 3D-ECM generated by TGFβ-treated KO MEFs.

Video S6. Ela-Myc cells on 3D-ECM generated by TGFβ-treated Control MEFs.

Supplemental Methods

Supplementary Figure S1. TGF beta remodels the ECM generated by mesenchymal stem and 1BR3G cells in a Snail1-dependent manner. Supplementary Figure S2. Cell Commitment is Modulated by Fibroblast-derived Matrices in a Snail1-dependent Manner. Supplementary Figure S3. TGF beta controls focal contacts in a Snail1-dependent manner. Supplementary Figure S4. TGF beta regulates N-cadherin intercellular contacts in a Snail1-dependent manner. Supplementary Figure S5. Alignment of MEFs bo-cultured with CAFs. Supplementary Table S1. Baseline Characteristics According to Snail1 Expression in the Stroma Supplementary Table S2. Overall Survival Analysis in Patients with Snail1 Expression in Stroma Supplementary Table S3. Antibodies Used for Protein Analysis

Video S5. Ela-Myc cells on 3D-ECM generated by Control MEFs.

Video S7. Ela-Myc cells on 3D-ECM generated by KO MEFs.

Video S1. MDA-MB231 cells on 3D-ECM generated by Control MEFs.

Video S3. MDA-MB231 cells on 3D-ECM generated by KO MEFs.

Video S2. MDA-MB231 cells on 3D-ECM generated by TGFβ-treated Control MEFs.

Video S8. Ela-Myc cells on 3D-ECM generated by TGFβ-treated KO MEFs.

Crosstalk between tumor and stromal cells in the tumor microenvironment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-associated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/αSMA–dependent mechanism that sustains oriented tumor cell migration and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFβ, including RhoA activation, αSMA-positive stress fibers, increased fibronectin fibrillogenesis, and production of a stiff ECM with oriented fibers. Snail1 expression in human tumor–derived CAF was associated with an ability to organize the ECM. In coculture, a relatively smaller number of Snail1-expressing CAF were capable of imposing an anisotropic ECM architecture, compared with nonactivated fibroblasts. Pathologically, human breast cancers with Snail1+ CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involvement in driving an ordered ECM was further confirmed in wound-healing experiments in mice, with Snail1 depletion preventing the anisotropic organization of granulation tissue and delaying wound healing. Overall, our results showed that inhibiting Snail1 function in CAF could prevent tumor-driven ECM reorganization and cancer invasion. Cancer Res; 75(2); 284–95. ©2014 AACR.