Microvesicles released from tumor cells disrupt epithelial cell morphology and contractility

Abstract

During tumor progression, cancer cells interact and communicate with non-malignant cells within their local microenvironment. Microvesicles (MV) derived from human cancer cells play an important role in mediating this communication. Another critical aspect of cancer progression involves widespread ECM remodeling, which occur both at the primary and metastatic sites. ECM remodeling and reorganization within the tumor microenvironment is generally attributed to fibroblasts. Here, using MCF10a cells, a well-characterized breast epithelial cell line that exhibits a non-malignant epithelial phenotype, and MVs shed by aggressive MDA-MB-231 carcinoma cells, we show that non-malignant epithelial cells can participate in ECM reorganization of 3D collagen matrices following their treatment with cancer cell-derived MVs. In addition, MVs trigger several changes in epithelial cells under 3D culture conditions. Furthermore, we show that this ECM reorganization is associated with an increase in cellular traction force following MV treatment, higher acto-myosin contractility, and higher FAK activity. Overall, our findings suggest that MVs derived from tumor cells can contribute to ECM reorganization occurring within the tumor microenvironment by enhancing the contractility of non-malignant epithelial cells.

Introduction

Early in tumor progression, extensive extracellular matrix (ECM) remodeling occurs (Butcher et al., 2009, Levental et al., 2009, Provenzano et al., 2008). Reorganization of the ECM is dependent on cell-mediated collagen bundling as well as deposition of new ECM components (Even-Ram and Yamada, 2005, Friedl and Wolf, 2010). Additionally, cells can reorganize collagen into parallel fibers radiating perpendicularly to the tumor to facilitate invasion (Provenzano et al., 2006). Interestingly, ECM remodeling and reorganization is observed in the early stages of tumor progression, and even adjacent non-tumoral sites show signs of ECM remodeling (Theret et al., 2001, Zhu et al., 1995). While fibroblasts are often described as mediating these changes (Cukierman and Bassi, 2010, Dumont et al., 2013, Mao et al., 2013), healthy epithelial and myoepithelial cells residing near the premalignant lesion could also contribute to these changes and respond to signals from cancer cells.

There is now a large body of evidence that shows that cancer cells can interact and communicate with healthy cells present within the local microenvironment (Al-Nedawi et al., 2008, Al-Nedawi et al., 2009, Brucher and Jamall, 2014). Interestingly, cells have the ability to release extracellular vesicles that appear to play an important role in the transfer of signaling proteins and cargo to other cells (Al-Nedawi et al., 2008, Al-Nedawi et al., 2009). There are two known types of extracellular vesicles, namely the smaller exosomes (<100 nm) and larger microvesicles (MVs) (Santana et al., 2014). While exosomes are produced by a wide variety of cell types, production of significant quantities of MVs appears to be limited to tumor cells (Santana et al., 2014). MVs shed by tumor cells can contain ECM components, growth factor receptors, cytoskeletal proteins and signaling molecules as cargo (Antonyak et al., 2011, Grange et al., 2011). When these MVs interact with non-tumorigenic cells, they can promote transformation-like characteristics such as increased cell growth, survival, and migration (Antonyak et al., 2011, Webber et al., 2010). Of note, recent evidence suggests that tumor-shed extracellular vesicles can prime the pre metastatic niche (Costa-Silva et al., 2015, Grange et al., 2011, Peinado et al., 2012), suggesting a role for extracellular vesicles in modifying the local microenvironment (Costa-Silva et al., 2015). However, it remains unclear whether MVs can contribute to the local ECM remodeling that occurs during the establishment of the pre-metastatic niche.

In the work reported here, we investigated the effect of MVs on non-malignant epithelial cell-mediated matrix reorganization within in vitro 3D collagen scaffolds. MCF10a epithelial cells were cultured in the presence of MVs collected from highly aggressive MDA-MB-231 carcinoma cells. Notably, the MCF10a cells cultured in 3D collagen scaffolds showed altered cell morphology and increased ECM reorganization following their treatment with MVs. In addition, 2D traction force microscopy measurements reveal that MCF10a cells generate more traction when they are cultured in the presence of MVs. Correspondingly, we observe a MV-mediated increase in both focal adhesion kinase (FAK) and myosin light chain phosphorylation. Overall, our results indicate that MVs shed by tumor cells can induce phenotypic changes in non-malignant epithelial cells, resulting in increased contractility and modifications to the ECM in the local microenvironment.