Substrate Stiffness Mediated Metastasis Like Phenotype of Colon Cancer Cells is Independent of Cell to Gel Adhesion
Substrate Stiffness Mediated Metastasis Like Phenotype of Colon Cancer Cells is Independent of Cell to Gel Adhesion
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DOI：
10.1007/s12195-014-0345-8
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发表时间：
2014-12-01
影响因子：
2.8
通讯作者：
Saif, M. Taher A.
中科院分区：
文献类型：
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作者：
Ali, M. Yakut;Saif, M. Taher A.
越来越多的实验证据表明，细胞可以感觉到并响应于它们所粘附的基底的机械刚度。当在具有适当机械刚度（20-47 kPa）的细胞外基质（ECM）包被的聚丙烯酰胺（PA）水凝胶上培养时，人结肠癌（HCT-8）细胞可以在体外表现出解离的、转移样表型（MLP），但在非常硬（3.6 GPa）的聚苯乙烯基底上不表现出这种表型。在这项研究中，我们提出了这样一个问题：细胞-细胞粘附分子是否发生了类似的形态学转变，即，E-钙粘蛋白包被的PA凝胶，如果是这样，肌动蛋白细胞骨架和粘着斑与ECM介导的凝胶反应如何比较。首先，我们在特定机械刚度（20 kPa）的E-钙粘蛋白包被的PA凝胶和非常硬的玻璃（类似于70 GPa）基底上培养HCT-8细胞。有趣的是，HCT-8细胞在培养的第六天仅在20 kPa凝胶上（而不是在硬玻璃上）显示出明显的分离的体外MLP;略早于对照（ECM/纤连蛋白包被的20 kPa凝胶）。药理学试剂blebbistatin对E-钙粘蛋白包被的凝胶的MLP的完全抑制暗示了MLP中非肌肉肌球蛋白II活性的参与。共聚焦激光扫描显微镜和定量图像分析结果表明，肌动蛋白的细胞骨架结构特征不同的凝胶表面附近的E-钙粘蛋白和纤连蛋白包被的凝胶类似的刚度解离前。相反，在两种情况下，在解离的细胞中观察到相同的皮质肌动蛋白结构。总之，这些结果表明，HCT-8细胞在PA凝胶上的MLP不依赖于2D体外培养中细胞与凝胶的粘附。
Growing experimental evidences suggest that cells can feel and respond to the mechanical stiffness of the substrate on which they adhere. Human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on extra-cellular matrix (ECM) coated polyacrylamide (PA) hydrogels with appropriate mechanical stiffness (20-47 kPa), but not on very stiff (3.6 GPa) polystyrene substrates. In this study, we ask the question whether similar morphological transition occurs on cell-cell adhesion molecule, i.e., E-cadherin coated PA gels and if so, how the actin cytoskeleton and focal adhesions compare with ECM mediated response on gels. First, we culture the HCT-8 cells on E-cadherin coated PA gels of specific mechanical stiffness (20 kPa) and very stiff glass (similar to 70 GPa) substrates. Interestingly, HCT-8 cells show the distinct dissociative in vitro MLP on 20 kPa gel only (not on stiff glass) on sixth day of culture; slightly earlier than the control (ECM/fibronectin coated 20 kPa gels). The complete inhibition of MLP on E-cadherin coated gels by pharmacological agent, blebbistatin, implicates the involvement of non-muscle myosin II activity in MLP. Confocal laser scanning microscopy and quantitative image analysis results suggest that the actin cytoskeletal architecture was characteristically different near the gel surface of E-cadherin and fibronectin coated gels of similar stiffness before dissociation. Conversely, identical cortical actin only structure was observed in the dissociated cells in both cases. Overall, these results suggest that MLP of HCT-8 cells on PA gels is independent of cell to gel adhesion in 2D in vitro culture.