3D-Hepatocellular Carcinoma (3D-HCC) Models of Diffused and Aggregated Spheroids using a 96-Pillar/Well Plate

Abstract Background: Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related mortality worldwide. The genetic and physiological complexity of HCC is a major barrier to the study of tumorigenesis and identification of therapeutic targets. The three-dimensional (3D) culture of cancer cells within an extracellular matrix (ECM) provides an in vitro tumor model that best recapitulates in vivo tumor pathophysiology.Methods: In the current study, we cultured cells and made spheroids by aggregating cells or diffusing cells in Matrigel attached on the tip of the 96-pillar plate. According to the initial cell position in Matrigel, two 3D-HCC cancer in-vitro models (diffused spheroid model and aggregated spheroid model) were established. These two models were applied to drug sensitivity assays to identify drug sensitivity changes. The protein expression and cytokine activation related to the drug resistance and maintenance of the physiological properties of HCC cells were analyzed in both models. This 3D culture method not only maintained the phenotype of the tumor but allowed easy high-throughput screening (HTS) due to its 96-array plate configuration.Results: The Aggregated Spheroid Model (ASM) showed higher expression of cancer markers associated with proliferation, tight junctions formation and epithelial cell identity in HCC cells, as well as cytokine factors associated with immune cell recruitment/activation, ECM regulation, cancer interaction, and angiogenesis regulation.Conclusions: Overall, the proposed ASM better recapitulates the tumor microenvironment, demonstrating the involvement of the ECM/tight junctions in the cell-to-cell signaling processes. This provides more instructive data for in vitro screening of tumor cells.