Liver-derived mesenchymal stem cells promote epithelial-mesenchymal transition in a hepatocellular carcinoma spheroid coculture model

Background and Aims: Mesenchymal stem cells (MSC) play a highly debated role in cancer. Potent anti- and pro-tumour properties have been shown in vitro and in vivo depending on the type of tumour studied and the origin and priming of the MSC used. Here, we investigate the interaction between liver-derived MSC and hepatocellular carcinoma (HCC), with a focus on cancer cell proliferation, invasiveness and gene expression. We postulate that their shared hepatic origin can lead to unique insights on the potential implication of MSC in carcinogenesis. Method: We established a 3D coculture spheroid model by combining Human Adult Liver Progenitor Cells (HALPC), a population of liver-derived mesenchymal stem cells described by our team, and one of three human hepatocellular carcinoma cell lines (HuH7, HepG2, and Hep3B). Cancer cell proliferation was studied through spheroid growth follow-up, chemiluminescent measurement of ATP, and Ki67 immunostaining using light sheet fluorescent microscopy. Cancer cell invasiveness was investigated using transwell and spheroid invasion assays. RNA sequencing was conducted on HALPC and cancer cells from control and coculture spheroids separated by fluorescence-activated cell sorting. Results: HCC 3D coculture with HALPC resulted in a dose-dependent inhibition of spheroid growth. At 25 days post-formation, control spheroids were up to 70 times larger in volume than 50:50 coculture spheroids. This correlated with lower total ATP contents in coculture spheroids at several time points, indicative of lower amounts of cells per spheroids. The inhibition of cancer cell proliferation was then confirmed by a reduction of up to 66% in Ki67-positive cells in coculture conditions (p = 4.6x10-5), using light sheet microscopy. Invasion studies demonstrated the induction of an invasive phenotype in the cancer cell lines when co-cultured with HALPC. Comprehensive transcriptomic analysis showed massive changes in expression levels, with 2002 and 3601 up- or downregulated genes in HALPC and cancer cells, respectively (| logFC | > 1 ; p adj. < 0.05). In cancer cells, gene set enrichment analysis revealed dysregulation of several cancer hallmarks gene sets, with significant positive enrichment of the epithelial-mesenchymal transition (EMT) gene set (Normalized Enrichment score: + 2.7 ; p adj. 1.1x10-18). Conclusion: We showed in a 3D coculture model that liver-derived MSC decreased the proliferation and induced the invasiveness of several human HCC cell lines. This was associated with the induction of an EMT phenotype at the transcriptomic level. These findings provide evidence for a potential role of organ resident MSC in HCC progression via EMT. Ongoing work will confirm the acquisition of the EMT phenotype at the protein level and validate the obtained results in vivo, using an orthotopic xenogeneic bioluminescent mouse model currently being established in our laboratory.