Influence of Tgf-B on Mechanobiology of Human Breast Cancer Cells

Abstract

Breast cancers are characterized by extensive modifications to mechanical properties of the tissue via increased ECM production and vascularization. TGF-B is a multi-functional cytokine that is implicated in cancer invasion and metastasis. Cellular response to TGF-B is context dependent that can be either tumor suppressive or promoter and is based on differential regulation of target genes through activation of downstream canonical SMAD function or non-canonical pathways. Multiple investigations into the TGF-B response have focused on the biochemical signaling associated with TGF-B in breast cancers. However, the role of TGF-B in modulating mechanical properties of cells has not been explored in detail. In the present study, we used tools in atomic force microscopy and traction force microscopy to quantify the mechanical properties of breast cancer cells of varying invasive potential, in response to TGF- and linked them to the underlying cytoskeletal remodeling. Indentation experiments using atomic force microscopy show reduction in the Young’s moduli of breast cancer cells after treatment with TGF-B. This implies increased deformability of the cell that can aid in migration through tight spaces. TGF-B mediated cell softening shows a temporal response with low invasive cells taking 48 hours to show softening as compared to cells with higher invasive potential which show softening after 24 hours treatment. This temporal response has not been seen earlier. We used high resolution imaging to link the mechanical changes to cytoskeletal architecture and showed the presence of a highly disoriented cytoskeleton in cells treated with TGF-. The disorientation in the actin cytoskeleton correlated with increased cell deformability. Characterization of the viscoelastic properties of cells using stress relaxation experiments showed increased fluid-like behavior of the cells after exposure to TGF-. These changes were reversed in the presence of TGF-RI receptor inhibitor; these studies clearly showed that TGF- acts as a modulator of cell stiffness in human breast cancer cells. We compared the most commonly used traction computation methods using simulated and experimental approaches and showed the robustness of the regularized- Fourier Transform Traction Cytometry (Reg-FTTC) method even in the presence of displacement noise. Contrary to literature we showed the emergence of varying values of optimal regularization parameter in our simulations and experimental measurements. Node-by node comparisons of tractions between different methods confirmed the stability of Reg-FTTC; comparison of traction outputs showed significant differences of upto 92% between the methods. Finally, we showed the effect of TGF- on cell contractility using traction force microscopy. Our results showed reduction in strain energies of cells after TGF- treatment in both MCF-7 and MDA-MB-231 cells. This reduction was seen only on softer substrates but not on stiffer ones; these indicated a substrate stiffness dependent response following treatment with TGF-B. In summary, this work highlights the changes in the mechanics of cells due to TGF-B at two different levels; first, based on the cell mechanical properties, and second, on the cell-substrate interactions. Mechanical changes quantified in this study may help link the phenotypic changes which are traditionally associated with TGF-B in human breast cancers.