173PCancer stem-like phenotypes including immune surveillance and its responsible genes in induced liver cancer stem-like cells. (24th November 2019) - Tsunedomi, R
Abstract: Background: Cancer stem cells are thought to play important roles in carcinogenesis, recurrence, metastasis, and therapy-resistance. We have successfully induced cancer stem-like sphere cells (CSLCs) from hepatoma cell lines using a unique medium. Methods: The human hepatoma cell line SK-HEP-1 was used as parental cells for CSLC induction. To assess the chemoresistance and liver metastasis, MTS assay and splenic injection were performed, respectively. RNA-seq analysis followed by gene set enrichment analysis (GSEA) was performed with cell line derivatives and clinical specimens of hepatoma. Protein expressions were conducted by western blotting, flow-cytometry, or ELISA. Knock-out experiments was accomplished by CRISPR-Cas9 genome-editing. Following rescue experiments were performed with an expression vector. Results: The obtained CSLCs showed increased metastatic potentials and resistance to anti-cancer drugs including molecularly targeted drugs for liver cancer. Regarding immune resistance, CSLCs showed increased proportion of positive cells for PD-L1 and PD-L2. In contrast to decreased membrane-bound MICA/MICB, increased soluble MICA in the medium were observed in CSLCs compared to those from the parental cells. As a result of integrated analysis with RNA-seq, a RAB3B was identified as an up-regulated gene in both CSLCs and poor prognostic liver cancers. Mono-allelic RAB3B knock-out cells showed altered sphere formation, significantly lower chemoresistance, and metastatic potential than that of parental cells, and those were rescued by the complementation of RAB3B. The knock-out cells also showed decreased PD-L1 and PD-L2 expressions in sphere induction medium than those in cells cultured with normal medium. GSEA following RNA-seq with the derivative cells revealed that negative enrichment of G2M checkpoint and positive enrichment of interferon response in CSLCs. Conclusions: Our induced CSLCs showed characteristic phenotypes such as metastatic ability, chemo- and immune-resistances. The up-regulation of RAB3B may plays important roles in CSLCs. Legal entity responsible for the study: Ryouichi TSUNEDOMI. Funding: Japan Society for the Promotion of Science. Disclosure: All authors have declared no conflicts of interest.