Osteogenesis and adipogenesis were induced in passage 4 MSCs using standard protocols [33]. After 4 week incubation in either adipogenic media or osteogenic media, MSCs differentiated into adipocytes (Fig. 1B) and osteoblasts (Fig. 1C) as verified by histological staining and reverse transcriptase PCR [34]. Morphological Changes after Incubation with Tumor Cell-Secreted Soluble Factors To investigate the response of MSCs to tumor cell-conditioned media (TCM), cells were incubated in serum-free TCM or control media (CM) for 24 hours. MSCs underwent a morphological change from a cobblestone appearance to an elongated phenotype (Fig. 2A–B). In comparison, Swiss 3T3 fibroblasts (Fig. 2C–D) and primary isolated kidney fibroblasts (Fig. 2E–F) had similar initial morphologies, but did not undergo a morphological change upon treatment with TCM. A 3D sandwich culture model was used to determine if tumor cells could stimulate elongation through a Figure 2. Tumor-secreted soluble factors alter MSC morphol- ogy. Brightfield images of murine MSCs (isolated from Balb/C mouse bone marrow; A–B), Swiss 3T3 fibroblasts (C–D), and primary fibroblasts (isolated from Balb/C mouse kidney; E–F) incubated for 24 hours in control media (CM, top) or tumor cell-conditioned media (TCM, bottom), then fixed in methanol, and stained with crystal violet. MSCs elongated dramatically in response to TCM (A–B); whereas, immortal- ized (C–D) and primary (E–F) fibroblasts did not respond to TCM treatment. A ‘sandwich’ co-culture model was used to study the migratory behavior of CM-DiL-labeled MSCs through a layer of polymerized collagen toward a monolayer of MSCs (G) or 4T1 tumor cells (H). Interestingly, MSCs elongated toward the tumor cells (H) but not toward the MSCs. (scale bar = 100 mm) doi:10.1371/journal.pone.0033248.g002 Mechanical Changes of MSCs and Fibroblasts viscoelastic collagen gel. Moreover, MSCs elongated through the collagen gel toward a monolayer of 4T1 tumor cells (Fig. 2H) but did not elongate towards a monolayer of MSCs (Fig. 2G). Cytoskeletal Changes after TCM Treatment We next examined the changes in cytoskeletal arrangement necessary to produce the elongated phenotype. To accomplish this, CM and TCM treated cells were fixed and stained for filamentous actin, microtubules, and nuclei (Fig. 3A). Cytoskeletal parameters were quantified from maximum intensity projections of confocal image stacks (3 images per stack) using a custom- written MATLAB algorithm. First, the cell elongation factor was quantified using a ratio of the minor to major cell axis (Fig. 3B). For control MSCs and 3T3 fibroblasts, the cell shape factors were near unity, indicating similarity in their native cell shape. In the presence of TCM, MSCs elongated by ,30% after 12 hours (p,0.05) and ,75% after 24 hours (p,0.001); whereas, the elongation factor of 3T3 fibroblasts remained near unity. We then quantified the stress fiber factor, which is the density of stress fibers normalized to cell area (Fig. 3C). The stress fiber factor of TCM- treated 3T3 fibroblasts remained constant; whereas, the stress fiber factor of MSCs was reduced more than 25% within 12 hours. Though this could in part be due to control cells being flatter than TCM treated cells, the use of maximum intensity projections minimizes this possibility, suggesting the changes are in fact due to decreases in actin stress fiber density. Upon stimulation with TCM, the nuclei of MSCs elongated dramatically (Fig. 3A). A nuclear shape factor was used to characterize nuclear elongation (Fig. 3D), with a value of one indicating the nucleus is perfectly round. Initially, MSCs and 3T3 fibroblasts had nuclear shape factors of approximately 0.75; however, upon incubation with TCM for 12 hours, MSC nuclei were elongated by approximately 13% (Fig. 3D). Nuclear elongation was never seen in 3T3 fibroblasts and no further elongation was observed in MSCs from 12–24 hours (Fig. 3D). TCM Alters the Distribution and Strength of MSC Focal Adhesions The cytoskeleton is linked to the extracellular environment via focal adhesion complexes, which are important regulators of cell motility. To determine the effects of TCM on cell adhesion, control and TCM-treated MSCs and 3T3 fibroblasts (treated for 24 hours) were fixed and stained with FITC anti-vinculin, a focal adhesion marker, and Rhodamine-Phalloidin, which stains filamentous actin (Fig. 4A,C). TCM treatment dramatically affected the overall number and morphology of focal adhesions on MSCs (Fig. 4A–C); however, it had no apparent effect on 3T3 fibroblast focal adhesions (Fig. 4C). For MSCs, the ratio of vinculin to actin was quantified from confocal images using a MATLAB routine (Fig. 4A,B). Twenty-four hours after TCM addition, the vinculin to actin ratio was reduced by 50% (Fig. 4B), and focal adhesions no longer displayed their characteristic brush- like pattern (Fig. 4B–C) but were instead localized at the tips of elongated cells (Fig. 4A,C). A centrifuge-based adhesion assay was used to determine if changes in vinculin expression were correlated with changes in cell adhesion (Fig. 4D). MSCs incubated for 24 hours with 80% or 100% TCM were significantly less adhesive, with a 36% or 54% reduction in the adherent cell fraction, respectively. Incubation with 100% TCM also reduced 3T3 fibroblast adhesion by 24%. Changes in the adherent fraction were only significant at high TCM concentration ($80%), indicating that the adhesive response to tumor-secreted soluble factors would be most important near the tumor. PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33248