Abstract 5789: Characterization of mixed-cell spheroid as an in vitro model of hepatocellular carcinoma for epithelial-to-mesenchymal transition and chemoresistance
Authors: Kuh Hyo-Jeong , Khawar Iftikhar Ali 
Published: July 2017

Interaction between cancer cells and stromal components in the tumor microenvironment is well known for their significant roles in tumor progression and subsequent treatment failure. Hepatic stellate cells (HSCs), as a predominant cell type in the microenvironment of hepatocellular carcinoma (HCC), are involved in creating desmoplastic and chemoresistance-inducing microenvironment. HSCs secrete various paracrine factors that modify the HCC tumor microenvironment leading to tumor growth, drug resistance and metastasis via promoting epithelial-to-mesenchymal transition (EMT). Although several studies using 2D co-culture system have shown bidirectional cross-talk between cancer cells and HSCs, data obtained remain limited in their clinical relevance due to lack of in vivo tumor-like characteristics.

We developed a mixed-cell spheroid model which recapitulates direct 3D tumor-HSCs interactions in paracrine and contact-mediated manners. Huh-7, human HCC cells, were mixed co-cultured with LX-2 cells, immortalized human HSC at 1:3 ratio using liquid overlay technique in 96-well plates. Electron microscopy was used to examine subcellular structural changes. Cellular distribution within spheroids was observed by labeling LX-2 cells with fluorescent tracer. The expression of pro-fibrotic and EMT markers was detected by immunohistochemistry or immunofluorescence staining on paraffin embedded sections. Invasion ability was determined using migration assay into 3D matrigel matrix. Dose-response curve was obtained by using APH assay and Ki-67 detection.

Mixed co-culture of Huh-7 and LX-2 cells showed a spontaneous self-organization forming highly compact mixed-cell spheroids with well-defined contour. Heterotypic cell-to-cell contact increased as shown by gap junction and desmosomes in the mixed-cell spheroids. LX-2 cells were uniformly distributed within the spheroids. Expression of fibroblast-associated factors such as α-SMA, collagen I, TGF-β and CTGF showed similar patterns of distribution to that of LX-2 cells, but at an elevated level in the mixed-cell spheroids compared to that of cancer cell-alone spheroids. An increased expression of EMT-related factors was observed along with enhanced invasion into 3D matrix. Differential drug sensitivity was shown; mixed-cell spheroids were sensitive to sorafenib but not to other agents including oxaliplatin, gemcitabine, and 5-FU. Based on these results, the mixed cell-spheroids of HCC cells-HSCs may be proposed as a useful 3D model for in vitro therapeutic screening of targets and agents as well as for the study of malignant progression in HCC.

Citation Format: Hyo-Jeong Kuh, Iftikhar Ali Khawar. Characterization of mixed-cell spheroid as an in vitro model of hepatocellular carcinoma for epithelial-to-mesenchymal transition and chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5789. doi:10.1158/1538-7445.AM2017-5789