Differential Mechanical Response of Mesenchymal Stem Cells and Fibroblasts to Tumor-Secreted Soluble Factors Daniel J. McGrail1, Deepraj Ghosh1, Nhat D. Quach2, Michelle R. Dawson1,3* 1 School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia, United States of America, 2 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, United States of America, 3 The Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia, United States of America Abstract The progression of neoplastic malignancies is a complex process resulting not only from the accumulation of mutations within tumor cells, but also modulation of the tumor microenvironment. Recent advances have shown that the recruitment and subsequent heterotypic interactions of stromal cells—including fibroblasts and bone marrow-derived mesenchymal stem cells (MSCs)—are crucial for carcinogenesis. Though extensive work has been done analyzing the signals that recruit these cells, the governing mechanical properties have not been fully investigated. Here, we report that despite their initial similarities, MSCs respond not only faster but also more dramatically to pro-migratory tumor-secreted soluble factors. Utilizing multiple particle tracking microrheology to probe the cytoskeletal mechanical properties, we show that MSCs stiffen completely within one hour, three times faster than fibroblasts. In addition, unlike fibroblasts, MSCs exposed to tumor-secreted soluble factors display a functionally different phenotype characterized by morphological elongation, decreased actin stress fiber density, and decreased adhesion. Quantitative real-time PCR indicates these phenomena occur based on differential expression of small GTPases RhoA and Cdc42, but not Rac1. These findings demonstrate a fundamental difference in the recruitment of fibroblasts and MSCs. Citation: McGrail DJ, Ghosh D, Quach ND, Dawson MR (2012) Differential Mechanical Response of Mesenchymal Stem Cells and Fibroblasts to Tumor-Secreted Soluble Factors. PLoS ONE 7(3): e33248. doi:10.1371/journal.pone.0033248 Editor: Laurent Kreplak, Dalhousie University, Canada Received August 16, 2011; Accepted February 13, 2012; Published March 16, 2012 Copyright: 2012 McGrail et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for this work was provided by the National Science Foundation (1032527) and the Georgia Cancer Coalition (GCC-276). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mdawson@gatech.edu Introduction The microenvironment in a solid tumor develops under the constant influence of inflammatory mediators [1]. These mole- cules, which include a milieu of cytokines, chemokines, and growth factors, are important targets for recruitment of a variety of cells such as leukocytes, macrophages, monocytes, fibroblasts and mesenchymal stem cells (MSCs) [2]. Previous literature on MSCs and fibroblasts suggests functional similarity as indicated both by global gene expression [3] as well as immunosuppression in allogeneic transplantation [4]. In the tumor stroma both can also form activated cancer-associated fibroblasts (CAFs) or myofibro- blasts [2,5], though MSCs can additionally differentiate into pericyte progenitor cells (PPCs) [6]. Increased numbers of myofibroblasts in the wound bed and in other sites of chronic inflammation have also been associated with MSC progenitors [2]. Notably, both cell types also aid in tumor growth and metastasis via autocrine and paracrine signaling [7,8]. In light of this, recent studies have begun to investigate these cells not only as alternative targets for anti-cancer therapy [9], but also for use as targeted gene-delivery vehicles [10]. For the latter approach, MSCs have shown greater promise than fibroblasts [11]. This may be in part because fibroblasts are recruited locally to form activated CAFs [12], whereas MSCs derived from the bone marrow must natively home through circulation to distal tumor sites. Consequentially, the therapeutic use of systemically infused MSCs to tumors has been investigated in breast [13], colon [14], ovarian carcinomas [15], gliomas [16], and Kaposi’s sarcomas [17]. Despite this potential, extended ex vivo culture reduces homing capacity of MSCs [18] and the majority of systemically infused MSCs become trapped in the lungs [5,19,20]. To overcome this, breast cancer cell-conditioned media [7], hypoxic preconditioning [21,22], and treatment with individual chemokines or growth factors [23,24] have all been investigated to increase MSC mobility; however, the effects of inflammatory mediators on the microscopic mechanical properties of MSCs have not been fully elucidated. The mechanical properties within the cell are largely regulated by the actin cytoskeleton, a complex network of interconnected actin filaments and regulating proteins [25,26]. Dynamic changes in the organization of the cytoskeleton transform cell shape and generate mechanical forces required for numerous cellular processes, including adhesion, migration, division, molecular transport, and differentiation [27]. Cytoskeletal effector proteins coordinate these changes by polymerization, depolymerization, cross-linking, and bundling of actin filaments into actin stress fibers, lamellipod extensions, and actin networks. Parallel bundles of actin filaments provide tensile strength and strong contractile activity, whereas cross-linked bundles of actin filaments increase intracellular elasticity [27]. Chemical and physical stimuli have been shown to alter cell shape and cytoskeletal organization by activating cytsokeletal mediators, including RhoA, Rac1, and Cdc42 [28,29,30]. Recent studies have highlighted the importance of mechanical [31] and chemical [32] cues on MSC fate: in these PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33248