Abstract 1017: Wnt signalings contributes to the maintenance of cancer stem cell-like populations in Huh7 hepatocellular carcinoma cell line: Role of hepatic stellate cells

Abstract

Abstract BACKGROUND: Cancer stem cells play crucial roles in cancer development and progression. The function and survival of stem cells appears to be regulated by local microenvironment or niche. In many tumors, tumor cells actively recruit a variety type of cells including myofibroblasts and create a tumor niche that participates in the process of cancer progression, chemo-resistance, and metastasis. During liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibroblasts and the resulting fibrosis is associated with the development of hepatocellular carcinoma (HCC). However, the possible contribution of HSCs to tumor-stroma interactions is totally unknown. Therefore, our AIM is to investigate precise role of HSCs on interactions with tumor cells, especially with cancer stem cells. METHODS: Hepatoma cell line Huh7 was used in this study. FACS analysis was used to evaluate stem cell markers such as side population (SP) and CD133. Each subpopulation was isolated and stem cell-like properties were evaluated by in vivo tumorigenesity and in vitro sphere-forming activity. To evaluate the role of HSCs on tumor-stromal interactions, Huh7 cells (at lower dish) and immortalized HSC line hTERT HSCs (at upper dish) were co-cultured in Transwell culture dishes. In some experiments, Huh7 and hTERT HSCs were mix-cultured in the upper dish and Huh7 cells in the lower dish was analyzed by FACS. RESULTS: We identified a small percentage (approximately 0.6%) of Huh7 cells as a side population (SP) relative to the main population (MP) cells by Hoechst 33342-based FACS profiles. In SP subpopulation, CD133-positive cells were enriched as compared with MP subpopulation (60.3% in total cells, 81.3% in SP, and 58.0% in MP). Only CD133-positive SP cells, but not CD133-negative SP cells nor CD133-positive MP cells, displayed tumor forming capacity and sphere forming activity, suggesting that distinct CD133-positive SP subpopulation have stem cell-like properties in Huh7 cells. The SP subpopulation of Huh7 is increased after the co-culture with hTERT HSCs for 4 days (7.6 hold), suggesting that HSCs maintains self-renewal of cancer stem cells in Huh7. CD133-positive cells were not increased after the co-culture. An increased number of SP subpopulation was attenuated by the treatment with Wnt antagonist DKK-1. The SP subpopulation in the lower dish was further increased when cancer cells and HSCs were mix-cultured in the upper dish (13.5 hold v.s. single culture). CONCLUSION: CD133-positive SP cells are a distinct cancer stem cell population in Huh7 cells. HSCs play an important role in supporting cancer stem cells in the tumor niche via Wnt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1017.