Sa1710 Suppression of Tumor Progression in Patients With Advanced Stage of Hepatocellular Carcinoma by M1 Macrophage Induction

Abstract

Background: Overcrowding always lead to the shedding of live cells to maintain homeostasis in epithelial cell sheets. Recently, some reports have shown that overcrowded cancer cells can be basally extruded by their neighbors, and then form metastases. However, this interesting phenomenon needs to be further clarified, and the exact mechanisms driving the extrusion of crowded cancer cell sheets remain elusive. We have previously confirmed that BVES, a novel adhesion molecule regulating tight junction formation, could inhibit liver cancer cell extrusion in a simply and skillfully designed 2D cell culture model, as cancer cells are always basally extruded, in this study, we performed a 3D cell culture model to further confirm the inhibition role of BVES in liver cancer cell extrusion. Methods: We designed a 3D cell culture model, after centrifuge, 5×10 6 cells were resuspended by 120μl 10% sucrose solutions, and quickly mixed with equal volume of 0.5% hydrogel solution( BeaverNanoTM ), the cells were then seeded in a Class Bottom Cell Culture Dish(NEST), and allowed to grow for six days. Then, cells were fixed with 4 % paraformaldehyde, incubated with primary antibody and secondary antibody, observed under a confocal microscopy. Extracted cell number were also investigated after enhanced or silenced expression of BVES. RhoA agonist U-46619 and inhibitor C3 enzyme were used to verify the role of RhoA/Rho-kinase pathway in BVES inhibited cancer cell extrusion. Results: Cells successfully grown to form an overcrowded cell mass in the 3D cell culture model, the number of extruded SK-Hep-1 cells, a high metastasis potential cell line, was significantly more than Huh7 cells with low metastasis potential, the normal liver cell line LO2 cells nearly had no cell extruded. Meanwhile, the extruded cells had decreased expression of BVES. Overexpression of BVES inhibited cells extrusion in the 3D cell culture model and increased RhoA activity in SK-Hep-1 cells, while stable knockdown of BVES remarkably promoted cells extrusion and decreased RhoA activity in Huh7 cells. U-46619 increased Huh7 cells extrusion while treated with C3 enzyme significantly diminished cells extrusion. Conclusion: These results suggest that in crowding liver cancer, BVES inactivate RhoA signaling pathway to decrease liver cancer cells extrusion, subsequently may inhibits tumor metastasis. (This study is supported by the National Natural Science Foundation of China NO:81472311 and the Fundamental Research Funds for the Central Universities 2014ZHYX020)