Experimental Investigation On Invasion And Metastasis Potentials Of Hepatocellular Carcinoma Cells By Using Three-dimensional Culture Model

Hepatocellular carcinoma (HCC) is a highly malignant tumor characterized by rapid progression, poor prognosis, and frequent tumor recurrence. The high potentiality of migration and invasion of HCC is closely correlated with the interactions between tumor cells and the extracellular microenvironment that consists of stromal cells (immune cells, inflammatory cells, vascular cells and fibroblasts), extracellular matrix (ECM) proteins, and soluble factors such as cytokines and growth factors. The regular two-dimensional (2D) cell culture system, in which only very little ECM is involved, fails to provide a well-defined extracellular microenvironment for HCC functional research. Thus, new functionally relevant three-dimensional (3D) cell culture models have been established by using ECM-like biomatrices, such as collagen I (rat tail collagen) and reconstituted ECM (Matrigel), etc.. The 3D cell culture models provide a well-defined in vitro microenvironment for studying the interactions of cell-ECM and cell-cell.HAb18G/CD147, a member of CD147 family, is highly enriched on the surface of HCC cells. Previous reports have shown that HAb18G/CD147 has important functions in HCC progression, migration and invasion by using 2D cell culture system.Aims of the study: (1) to establish 3D HCC cell culture model; (2) to investigate the effects of microenvironment on morphological characteristics of both HCC cells and fibroblasts under 3D cell culture condition; (3) to investigate the effects of HAb18G/CD147 on synthesis of MMPs and expressions of focal adhesion molecules in 3D HCC cells culture model, and to further elucidate the function of HAb18G/CD147 in HCC adhesion, invasion and metastasis.Our study consists three parts. Firstly, we established 3D HCC cell culture model by using reconstituted Matrigel. And then, we investigated the expression of HAb18G/CD147, synthesis of MMPs, the expressions of focal adhesion molecules, and the distribution of cytoskeleton protein in 3D HCC cell monoculture model. Lastly, we investigated the effects of HAb18G/CD147 on synthesis of MMPs and expressions of focal adhesion molecules in 3D HCC cells and fibroblasts co-culture model.To mimic local tissue environment in vivo, HCC cells were resuspended in reconstituted Matrigel to establish 3D cell culture model. Under 3D HCC cell monoculture condition, ECM ultimately determined the phenotypic characteristics of HHC cells, enhanced expression of HAb18G/CD147 together with high expressions of MMPs and adhesion molecules, changed the distributions of cytoskeleton and increased the adhesion and invasion potentials of HCC cells. In 3D cell co-culture model, the overexpression of HAb18G/CD147 increased MMPs (MMP2 and MMP9) production, and was obviously accompanied with enhanced expressions of paxillin, FAK and p-FAK. All the results suggest that HAb18G/CD147 plays an important role in HCC adhesion, invasion and metastasis mainly via modulating synthesis of MMPs and activating integrin signal pathways in fibroblasts and tumor cells themselves under the 3D co-culture conditions.