Mechanical Changes of MSCs and Fibroblasts Figure 4. Changes in the distribution and strength of focal adhesions. Confocal micrographs (A) of 24-hour CM- (left) and TCM- (right) treated MSCs stained with Phalloidin (F-actin, red), and anti-vinculin (green). The vinculin to actin ratios quantified from confocal images (B) demonstrated a reduced concentration of focal adhesion proteins in TCM-treated MSCs. Epifluorescent microscopy was used to further investigate the effect of TCM on focal adhesion distribution in MSCs and 3T3 fibroblasts (C). CM-treated MSCs displayed a brush-like pattern of focal adhesions; whereas, focal adhesion on TCM-treated cells appeared as points at the end of cytoskeletal extensions. TCM-treatment had no effect on the pattern of focal adhesions on 3T3 cells. A centrifuge-based adhesion assay was used to determine the effects of TCM-treatment on the adhesion of MSCs and 3T3 fibroblasts (D). TCM treatment resulted in a reduced fraction of adherent cells in a dose-dependent manner for both cell type (left), but significantly more for MSCs (right). (scale bars = 10 mm) doi:10.1371/journal.pone.0033248.g004 CM and TCM, though more significantly toward TCM (Fig. 7C). Statistical analysis also revealed synergistic effects of TCM pre- treatment and migration toward TCM for both cell types, suggesting that pre-treatment not only increases motility but also causes a preferential migration towards chemotactic gradients. Changes in Gene Expression Associated with Altered Mechanical Response To understand genotypic changes resulting in altered mechan- ical responses, we performed qRT-PCR to investigate the expression of the Rho GTPases RhoA, Rac1, and Cdc42 that modulate the actin cytoskeleton (Fig. 8A). We found that after TCM treatment, both cell types express significantly more Rac1 (p,.05) as compared to their respective controls. Furthermore, ANOVA revealed that MSCs expressed significantly more RhoA overall (p,.0001). Moreover, it showed a significant interaction effect between cell type and TCM treatment for Cdc42 (p,.005), suggesting that this molecule is largely responsible for the altered mechanical response. Discussion In this study, TCM isolated from 4T1 metastatic breast cancer cells was used to mimic the chemotactic factors released by tumors. Previous studies have demonstrated that TCM is a potent pro-migratory cocktail of growth factors and chemokines [35,36] (for a detailed list of acronyms see Table S1). Literature on 4T1 tumor cell conditioned media shows that it contains soluble growth factors including platelet derived growth factor (PDGF), transforming growth factor (TGF-b1), and vascular endothelial growth factor (VEGF) [37], as well as cytokines, chemokines, acute phase proteins and proteases. In two separate studies [38,39], researchers examined the gene expression profile of 4T1 tumor cells in comparison to less metastatic cell lines, finding a variety of up-regulated pro-migratory soluble factors including (a) growth factors angiopoietin 2, VEGF-C, insulin-like growth factor 2, (b) chemokines CCL5, CCL7, CXCL1, CXCL16, CSF2, CSF3, (c) interleukins 1a and 23, and (d) matrix metalloproteases 13,3,9. Yet this list is not comprehensive, as the complexity of TCM continues to yield novel pro-migratory soluble factors [36] in addition to unique miRNA secretomes [40]. Some of these growth factors, such as PDGF, TGF- b1, and VEGF, have been shown to directly induce mechanical and migratory changes in MSCs and fibroblasts [23,24,35,41,42,43]. However, the effects of other soluble factors present in TCM are often much more difficult to determine. Menon et al. found that MSC exposure to tumor cell conditioned media up-regulates mRNA levels of 104 genes, including genes for stromal cell-derived factor 1 (SDF-1/CXCL12), monocyte chemotactic protein 1 (MCP-1/CCL2), and growth-regulated protein b (Gro-b/ CXCL2), which are all potent chemokines that act in an autocrine fashion to further stimulate MSC migration [35]. In the same study, Menon et al. also found that TCM altered the organization of cytoskeletal actin, resulting in increased polarity and directional migration [35]. In another study, soluble factors (identified as extracellular matrix peptides) in LLC1 tumor cell conditioned media induced bone marrow derived cell secretion of pro- inflammatory cytokines, including IL-1b, IL-6, and tumor necrosis PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33248