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Objective To investigate the cause of differences in confluent growth between hepatic(L02) and hepatoma carcinoma(HCCLM3) cells by comparing responses of the two cells to different substrate stiffness (0.5, 4 kPa and glass). MethodsThe real-time photomicrography, immunofluorescence staining, flow cytometry, and Western Blotting techniques were respectively employed to observe the morphological characteristics, the cytoskeleton conformation and the distribution of E-cad of confluent L02 and HCCLM3 cells on different substrates, and test the changes in expression of E-cad, Integrinβ1 and p-Src. Results (1) Confluent L02 cells displayed a round or cubic shape, while HCCLM3 cells showed a polygon shape. The morphology of HCCLM3 cells were spread and polarized more obviously than that of L02 cells. With the increase of substrate stiffness, the variation of L02 cells with time was smaller than that of HCCLM3 cells. (2) The cytoskeleton of confluent L02 cells showed a ring-like conformation under the cortex, and E-cad was located at the cell-cell contact sites. However, the ring-like cytoskeleton of HCCLM3 cells was incomplete and distributed radially along the basement, while E-cad was dispersed in cytoplasm. (3) As the substrate stiffness increased, expression of E-cadherin in both L02 and HCCLM3 cells was significantly decreased (P<0.01), while the level of p-Src and integrinβ1 was increased significantly, with greater changes in HCCLM3 cells than in L02 cells. Conclusions The assembling of cortical ring-like cytoskeleton was positively correlated with the location of E-cad at the cell-cell contact sites. The substrate stiffness showed a more obvious impact on the balanced regulation between cadherin and integrin mediated adhesion system of hepatocarcinoma cells than that of hepatic cells.
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Objective To investigate the effects of substrate stiffness on the adhesion, spreading and migration of hepatocellular carcinoma cells as well as the regulation of cytoskeleton assembly and integrinβ1 expression, and to explore the role of substrate mechanical properties in the metastasis of hepatocellular carcinoma cells. Methods The polyarcylamide gel with different stiffness was achieved by varying the relative ratio of acrylamide to bis acrylamide. The substrate surface was cross linked with extracellular matrix molecules for cell adhesion. The adhesion, spreading and migration of hepatocellular carcinoma cells on substrates with different stiffness were recorded by phase contrast microscope and made quantitative analysis by Image J software. The cytoskeleton assembly on substrates with different stiffness was detected by immunofluorences assay, and the expression of integrinβ1on different substrates was measured by flow cytometer. Results The rigid substrate enhanced the adhesion and spreading of hepatocellular carcinoma cells in shortened time. Neither the soft (1.1 kPa) nor over rigid (glass) substrate facilitated the migration of hepatocellular carcinoma cells, and the maximum migration velocity was found on the substrate with moderate stiffness（10.7 kPa）. The rigid substrate could promote cytoskeleton assembly and integrinβ1 expression. Conclusions The effects of substrate stiffness on adhesion, spreading and migration of hepatocellular carcinoma cells are regulated by the cytoskeleton assembly and integrin expression.
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Objective To investigate the cause of tumor cell migration by comparing the effect of substrate stiffness on hepatic and hepatoma carcinoma cell migration so as to understand the invasive characteristics of tumor cells. Methods Immunofluorescence staining, morphological analysis and transwell were employed to observe the morphological characteristics of HCCLM3 and L02 cells on different substrates and test their migration characteristics with the quantitative analysis. Results (1) The migration rate and net translocation of HCCLM3 and L02 cells on 4 kPa substrate was higher than those both on 0.5 kPa(most soft one) and on glass (the hardest one) substrates, and L02 cells also displayed higher migration efficiency than HCCLM3 cells on such substrates. (2) The mean squared displacement of HCCLM3 and L02 cells on different substrates showed consistent tendency, and the directional persistence of L02 cells on the softer substrate was significantly higher than that of HCCLM3 cells. (3) In 0.5 and 1 mg/mL three dimensional collagen environment, the number of invasive cells of HCCLM3 was remarkably more than that of L02 cells. After adding MMPs inhibitor GM6001 (40 μg/mL), the number of invasive cells was notably increased in HCCLM3 cells, but notably decreased in L02 cells. Conclusions (1) In two dimensional comparatively soft environment, L02 cells displayed an efficient migration due to its higher directional persistence. (2) In three-dimensional collagen environment, the invasion efficiency of HCCLM3 cells was significantly higher due to the various modes of migration adaptation to the microenvironment.