Three-Dimensional Cell Culture Models of Hepatocellular Carcinoma - a Review.

INTRODUCTION: Three-dimensional (3D) cell culture studies are becoming extremely common because of their capability to mimic tumor architecture, such as cell-cell and cell-ECM interactions, more efficiently than 2D monolayer systems. These interactions have important roles in defining the tumor cell behaviors, such as proliferation, differentiation, and most importantly, tumor drug response. OBJECTIVE: This review aims to provide an overview of the methods for 3D tumor spheroid formation to model human tumors, specifically concentrated on studies using hepatocellular carcinoma (HCC) cells. METHOD: We obtained information from previously published articles. In this review, there is discussion of the scaffold and non-scaffold-based approaches, including hanging drop, bioreactors and 3D bioprinting. RESULTS AND CONCLUSION: The mimicking of the tumor microenvironment (TME) as tumor spheroids could provide a valuable platform for studying tumor biology. Multicellular tumor spheroids are self-assembled cultures of mixed cells (tumor and stromal cells) organized in a 3D arrangement. These spheroids closely mimic the main features of human solid tumors, such as structural organization, central hypoxia, and overall oxygen and nutrient gradients. Hepatocellular carcinoma (HCC) is the most common liver malignancy, and most difficult to overcome because of its drug resistance and tumor heterogeneity. In order to mimic this highly heterogeneous environment, 3D cell culture systems are needed.

A Novel Function for KLF4 in Modulating the De-differentiation of EpCAM-/CD133- nonStem Cells into EpCAM+/CD133+ Liver Cancer Stem Cells in HCC Cell Line HuH7.

The complex and heterogeneous nature of hepatocellular carcinoma (HCC) hampers the identification of effective therapeutic strategies. Cancer stem cells (CSCs) represent a fraction of cells within tumors with the ability to self-renew and differentiate, and thus significantly contribute to the formation and maintenance of heterogeneous tumor mass. Increasing evidence indicates high plasticity in tumor cells, suggesting that non-CSCs could acquire stem cell properties through de-differentiation or reprogramming processes. In this paper, we reveal KLF4 as a transcription factor that can induce a CSC-like phenotype in non-CSCs through upregulating the EpCAM and E-CAD expression. Our studies indicated that KLF4 could directly bind to the promoter of EpCAM and increase the number of EpCAM+/CD133+ liver cancer stem cells (LCSCs) in the HuH7 HCC cell line. When KLF4 was overexpressed in EpCAM-/CD133- non-stem cells, the expressions of hepatic stem/progenitor cell genes such as CK19, EpCAM and LGR5 were significantly increased. KLF4 overexpressing non-stem cells exhibited greater cell viability upon sorafenib treatment, while the cell migration and invasion capabilities of these cells were suppressed. Importantly, we detected an increased membranous expression and colocalization of ß-CAT, E-CAD and EpCAM in the KLF4-overexpressing EpCAM-/CD133- non-stem cells, suggesting that this complex might be required for the cancer stem cell phenotype. Moreover, our in vivo xenograft studies demonstrated that with a KLF4 overexpression, EpCAM-/CD133- non-stem cells attained an in vivo tumor forming ability comparable to EpCAM+/CD133+ LCSCs, and the tumor specimens from KLF4-overexpressing xenografts had increased levels of both the KLF4 and EpCAM proteins. Additionally, we identified a correlation between the KLF4 and EpCAM protein expressions in human HCC tissues independent of the tumor stage and differentiation status. Collectively, our data suggest a novel function for KLF4 in modulating the de-differentiation of tumor cells and the induction of EpCAM+/CD133+ LCSCs in HuH7 HCC cells.