Mechanical Changes of MSCs and Fibroblasts Figure 3. MSCs reorganize their cytoskeleton in response to tumor-secreted soluble factors. (A) Confocal micrographs of CM and TCM- treated MSCs (a–c) and 3T3 fibroblasts (d–e) stained with Phalloidin (F-actin, red), anti-a-tubulin (microtubules, green), and DAPI (nucleus, blue). The shape and cytoskeletal organization of CM-treated MSCs (a) and CM- (d) and TCM- (e) treated Swiss 3T3 fibroblasts were similar (24 hours after CM or TCM addition); whereas, TCM-treated MSCs were elongated with extended cytoskeletal filaments (b–c). MSC elongation increased between 12- (b) and 24- (c) hours, indicating that cytoskeletal changes may be progressive. Cytoskeletal parameters (B–D) were determined by analysis of confocal images with a custom MATLAB routine. The cell (B) and nuclear (D) shape factors were used to characterize the circularity of an elliptical outline of the cell or nucleus, respectively, with a shape factor of 1 indicating a perfect circle. The stress fiber factor (C) was used to characterize the density of actin stress fibers per cell area. Cytoskeletal changes observed in TCM-treated MSCs (b–c) were confirmed using the cytoskeletal parameters (B–D), which indicated dramatic reductions in cell and nuclear shape factors and stress fiber densities. (scale bars = 10 mm) doi:10.1371/journal.pone.0033248.g003 Cytoskeletal Stiffening in Response to TCM MPT was used to characterize the immediate effects of TCM (30 minutes to 3 hours) on the intracellular mechanical properties of MSCs (Fig. 5A, Fig. 6A–E) and 3T3 fibroblasts (Fig. 5B, Fig. 6F– J). Corresponding morphological changes were not seen in immunostained fixed MSCs until 12–24 hours after TCM treatment and were never seen in 3T3 fibroblasts (Fig. 3–4). For MPT studies, 100 nm probe particles, with diameters more than 100-fold smaller than the cells, were injected into the cytoplasm (Fig. 5C), and their mobility, characterized by the ,,Dr2(Dt).. (Fig. 5A–B), was used to determine intracellular rheology. Initially, the amplitude and logarithmic slope of the ,,Dr2(Dt).. for MSCs and 3T3 fibroblasts were comparable, indicating similarity in their microscopic mechanical properties (Fig. 6A,F). Within 30 minutes, TCM treatment reduced the ,,Dr2(Dt).. for MSCs up to 20-fold; however, similar changes were not seen in 3T3 fibroblasts until 2–3 hours after TCM treatment. The ratio of viscous to elastic character, or phase angle (d), of control cells was 55u,d,70u, indicating that initially the cytoplasm behaved more like a viscous liquid than an elastic solid. One hour after TCM treatment, the cytoplasm of MSCs had dramatically stiffened with 10u,d,15u. This effect was not seen in 3T3 fibroblasts until 3 hours after TCM treatment. MPTM was also used to quantify the rheological properties of the cytoplasm, which is highly viscoelastic in part to its crowded nature. The frequency-dependent viscoelasticity of MSCs changed dramatical- ly after TCM treatment (Fig. 6A–E). In fact, the cytoplasm became highly elastic with G9,400 dyn/cm2 (Fig. 6B–E). TCM Rapidly Increases In Vitro Cell Migration Previous studies documented increased MSC migration toward chemotactic factors after 18–24 hours [23,24,35]. Our multiple particle tracking studies revealed dramatic changes in intracellular mechanics within 1 hour. To determine if these mechanical changes were correlated with increased mobility, the migration of control and treated cells toward CM or TCM was measured hourly for 3 hours using transwell inserts with 3 mm (Fig. 7A) or 8 mm (Fig. 7B) pores. Without chemotactic factors present, few MSCs and 3T3 fibroblasts migrated through 3 mm pores; however, when TCM was added, there was a significant increase in MSC migration within 3 hours (p,0.01, Fig. 7A). MSC migration through 8 mm pores was almost always significantly greater than 3T3 fibroblast migration (Fig. 7B–C)). MSCs also responded more rapidly to TCM, with increased MSC migration within 2 hours (p,0.001) and increased 3T3 fibroblast migration within 3 hours (p,0.01). To determine if mechanical changes exhibited by MSCs in the first hour were indicative of a more migratory phenotype, we pre-treated both cell types for one hour with TCM before seeding in 8 mm transwell inserts. Pre-treatment with TCM increased MSC and 3T3 fibroblast migration toward PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33248