Mechanical Changes of MSCs and Fibroblasts Figure 8. Alterations in Gene Expression. Changes in expression of small Rho GTPases RhoA, Rac1, and Cdc42 before and after prolonged treatment with TCM (A). A simplified diagram of short-term response of cells to treatment with TCM by actin polymerization and crosslinking (B). After 24 hour exposure to TCM, alterations in morphology and adhesion can be explained by differential gene expression in MSCs and fibroblasts (C). doi:10.1371/journal.pone.0033248.g008 Two unique obstacles in MSC homing as compared to fibroblasts include extravasation from the bone marrow to enter circulation and intravasation into the site of inflammation [54]. The differential cellular elongation could aid as cells squeeze through the basement membrane. Since undifferentiated MSCs have a more deformable nucleus, they are better suited for this task [55]. Our results indicate that MSC nuclei elongate in response to TCM (Fig. 3D). In contrast to fibroblasts, MSCs have the ability to squeeze through 3 mm pore transwell inserts, which are approx- imately 3-fold smaller than the cell diameter (Fig. 7A). The up- regulation of Cdc42 may also prove beneficial in this process, as it is critical for nuclear translocation [56]. These results elucidate a fundamental difference in the recruitment of fibroblasts and MSCs to tumors by utilizing a unique set of mechanical changes to overcome their extensive physiological barriers. Furthermore, this new knowledge may be used in the development of novel therapeutics. Understanding the mechanisms of MSC homing can be used for more successful tumor targeting. Further studies can be focused on discovering the complex interactions of Rho GTPases and their effectors which modulate these cell-specific mechanical changes. Materials and Methods Materials Cellgro IMDM, RPMI 1640, DMEM, L-glutamine, sodium bicarbonate, PBS, and penicillin-streptomycin were purchased from Mediatech. FBS was purchased from Atlanta Biologicals and Type I, II collagenase from Worthington Biochemicals. Rat anti-mouse PE-Sca1, PerCP-CD45, PE-CD29, and APC-CD11b, and red blood cell lysis buffer were purchased from Biolegend. Rhodamine-Phalloidin, rabbit vinculin monoclonal antibody, Alexa Fluor 488 anti-rabbit IgG, FITC-conjugated mouse anti- alpha tubulin, and Fluospheres carboxylate-modified 100 nm particles (F8801) were purchased from Invitrogen. Cy3-SMA was purchased from Sigma. PDS-1000 Biolistic Helium Particle Injection, 1800 psi rupture discs, and macrocarriers were purchased from BioRad. Primers for gene analysis were obtained from IDT. All other reagents were purchased from VWR or Sigma-Alrich unless otherwise specified. Cell Culture Swiss 3T3 fibroblasts and 4T1 mammary carcinoma cells were purchased from American Type Cell Culture and cultured according to manufacturer’s protocol. MSCs were isolated from murine bone marrow and cultured in MSC growth media (IMDM supplemented with 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin). Bone marrow was extracted from the femurs and tibias of 8–10 week old Balb/C mice (Charles River Laboratories, Wilmington, MA) by crushing the bones in FBS solution (1 mg/ml Type I collagenase in 30% FBS and 70% PBS), filtering the cell suspension using a 70-mm cell strainer, and centrifuging at 20006 g for 10 minutes. The bone marrow cells were resuspended in MSC growth media and seeded at 36106 cells/cm 2 . After 24 hours, non-adherent cells were removed, and cells were cultured in MSC growth media (replaced 3 times per week). Differentiation was induced with either adipogenic or osteogenic media per standard protocols [33]. See PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33248