Microenvironment and Immunology Snail1-Expressing Fibroblasts in the Tumor Microenvironment Display Mechanical Properties That Support Metastasis Jelena Stanisavljevic 1 , Jordina Loubat-Casanovas 1 , Mercedes Herrera 2 , Tom as Luque 3,4 , Ra  ul Pe ~ na 1 , Ana Lluch 5,6 , Joan Albanell 7,8,9 ,F elix Bonilla 2 , Ana Rovira 7,8 , Cristina Pe ~ na 2 , Daniel Navajas 3,4,12 , Federico Rojo 7,10,11 , Antonio García de Herreros 1,9 , and Josep Baulida 1 Abstract Crosstalk between tumor and stromal cells in the tumor micro- environment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-asso- ciated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/aSMA– dependent mechanism that sustains oriented tumor cell migra- tion and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFb, including RhoA acti- vation, aSMA-positive stress fibers, increased fibronectin fibrillo- genesis, 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 fibro- blasts. Pathologically, human breast cancers with Snail1 þ CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involve- ment 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. Ó2014 AACR. Introduction Myofibroblasts are activated fibroblasts that remodel connec- tive tissues in processes, such as development and wound healing (1, 2). They typically contain contractile aSMA (smooth muscle alpha actin)-positive stress fibers linked to and required for the formation of supermature integrin focal contacts, named fibro- nexus. Fibronexus transmits intracellular tensional forces to extra- cellular fibronectin molecules, allowing their assemblage into fibers (3). Extracellular fibronectin fibers facilitate and guide the polymerization of other molecules, such as thrombospondin-1, perostin, tenascin C (4), fibrillin, and collagen (5), into the extracellular matrix (ECM). aSMA-positive stress fibers also connect intercellular cadherin junctions that permit them to withstand mechanical stress between neighbor cells; indeed, adherens junctions of cultured myofibroblasts are significantly larger than those of aSMA-neg- ative fibroblasts (6). The ECM architecture of connective tissues and the myofibroblast phenotype, including nuclei (7) and cell shapes (3), ultimately depend on an intraextracellular tensional dialog mediated by these specialized cell–substrate and cell–cell structures. Cancer-associated fibroblasts (CAF) are a heterogeneous population of activated fibroblasts whose activity in the stroma associates with tumor progression and malignancy. CAFs pro- duce paracrine growth factors, proteolytic enzymes, and ECM components, and contribute to generate a desmoplastic response (fibrillar network deposition) around cancer cells (8) similar to that at the granulation tissue of wounds. Thus, CAF activity perturbs not only the biochemical but also the biomechanical homeostasis of the tumor microenvironment; these perturbances are sensed by tumor cells and ultimately affect their behavior (9). In breast cancer, mechanical proper- ties of the stroma, such as stiffness (10) and fiber alignment (11), force progression of the disease. In fact, the presence of dense and aligned collagen fibers around human breast carci- nomas is a prognostic signature for poor survival (12, 13). CAFs are permanently activated by a TGFb autocrine loop (14), and 1 Programa de Recerca en C ancer, Institut Hospital del Mar d'Investiga- cions M ediques, Barcelona, Spain. 2 Department of Medical Oncology, Puerta de Hierro Majadahonda University Hospital, Majadahonda, Madrid, Spain. 3 Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, Spain. 4 Institute for Bioengineering of Catalo- nia, Barcelona, Spain. 5 Department of Oncology and Hematology, Hospital Clínico Universitario, Valencia, Spain. 6 Department of Medi- cine, Valencia Central University, Valencia, Spain. 7 Molecular Thera- peutics and Biomarkers in Cancer Laboratory, Institut Hospital del Mar d'Investigacions M ediques, Hospital del Mar, Barcelona, Spain. 8 Med- ical Oncology Department, Hospital del Mar, Barcelona, Spain. 9 Depar- tament de Ci  encies Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain. 10 Department of Pathology, IIS-Fundaci  on Jim enez Díaz, Madrid, Spain. 11 Department of Pathology, Hospital del Mar, Barcelona, Spain. 12 Ciber Enfermedades Respiratorias (CIBERES), 07110-Bunyola, Spain. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Josep Baulida, IMIM, C/Dr. Aiguader, 88, 08003, Bar- celona, Spain. Phone: 34-3-316-0436; Fax: 34-3-316-0410. E-mail: jbaulida@imim.es doi: 10.1158/0008-5472.CAN-14-1903 Ó2014 American Association for Cancer Research. Cancer Research Cancer Res; 75(2) January 15, 2015 284 on April 13, 2017. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst December 8, 2014; DOI: 10.1158/0008-5472.CAN-14-1903