Mechanical Changes of MSCs and Fibroblasts Figure 5. Multiple particle tracking microrheology. The ensemble averaged mean squared displacements (,,r2(Dt)..) of 100 nm particles embedded in the cytoplasm of TCM-treated MSCs (A) and 3T3 fibroblasts (B) were evaluated from 0–3 hours. For both cell lines, treatment with TCM reduced the rate of cytoplasmic particle transport in a time-dependent manner. Fluorescent image of 100 nm particles (green) in the cytoplasm of a MSC, which was fixed and stained with phalloidin (red) and DAPI (blue) (C). The phase angle, d = arctan (G0(v))/G9(v)), was used to characterize the viscoelastic nature of the cytoplasm over the course of the experiment (D). The viscoelastic nature of MSCs and 3T3 fibroblasts were similar initially and 3 hours after TCM-treatment; however, MSCs responded much more rapidly to TCM with a 4-fold reduction in d within 60 minutes. (scale bar = 10 mm) doi:10.1371/journal.pone.0033248.g005 factor a (TNF-a), whereas serum-free media and 3T3 conditioned media had no effect on cytokine production [44]. In contrast, tumor cells exchange paracrine signals with normal fibroblasts, which have been shown to promote matrix remodeling and tumor cell invasiveness [41]. One way this occurs is through tumor cell secretion of soluble factors, such as IL-1, FGF-2, PDGF, and/or TGF- b, which induce hepatocyte growth factor (HGF) secretion from fibroblasts. HGF then binds with its cognate receptor, c- MET, which is expressed by normal and cancer stem cells [45]. The HGF/c-MET signaling pathway triggers cell growth and angiogenesis, which are important mechanisms of cancer devel- opment, normal growth, and wound healing [41]. We hypothesize that several of the growth factors and chemokines in TCM, such as VEGF, PDGF, TGF- b1, directly stimulate changes in MSCs and fibroblasts; however, other factors in TCM, such as IL-1b, colony-stimulating factor (CSF), and TNF-a are more potent mediators of changes in MSCs due to autocrine signaling pathways including SDF-1, MCP-1, and Gro- b, which they activate rapidly. Interestingly, a more detailed analysis of MSC migration to individual growth factors and chemokines found that TNF-a stimulation was necessary for MSC migration towards all chemokines tested, but had little effect on the migration of MSCs toward growth factors [23]. Due to the complicated nature of the autocrine and paracrine signaling pathways involved in the recruitment of cells to tumors, we focused our study on characterizing the effects of a cocktail of these factors in TCM on MSC and fibroblast behavior. The molecular response of MSCs to various soluble factors has been well characterized, but the mechanical properties that govern their migration are yet to be fully investigated. MPTM was used to analyze the real-time changes in live-cell mechanics. Previous MPT studies have demonstrated not only that induced pluripotent stem cells differ with respect to intracellular rheology from their parental fibroblasts line [46], but also that fibroblasts stiffen both during migration and in response to activation of RhoA, Cdc42, or Rac1, with the largest response to RhoA [25,42]. This technique revealed cytoskeletal stiffening after TCM treatment larger than previously documented, completely changing the intracellular mechanical phenotype, three-fold faster in MSCs (Fig. 5, 6). Analysis of expression of RhoA in untreated MSCs and fibroblasts revealed two-fold higher expression in MSCs (Fig. 8A), which could account for more rapid cytoskeletal stiffening of MSCs. RhoA can act by Rho Kinase (ROCK) to induce stress fiber formation and focal adhesions or to crosslink actin filaments via the downstream effector a-actinin [47]. In MSCs, inhibition of ROCK has been shown to increase cell motility, an effect which is increased in presence of the RhoA activator lysophosphatidic acid (LPA) [48]. In fibroblasts, equivalent stiffening to that induced by TCM was found by inhibition of ROCK before RhoA activation [25]. Moreover, this increase in elastic character is in good agreement with in vitro experiments analyzing actin polymerization and crosslinking dynamics [49]. A simplified schematic of this process can be seen in Figure 8B. PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33248