The plasticity of the stem cells in the hepatocarcinogenesis and metastasis: study in vitro and in vivo

Recent studies had indicated the importance of the stem cells (SC) in systemic and solid cancers, including in primary liver cancers (PLC). The SC can contribute in the tumor-stromal cross talk in the cancer microenvironment. However their flexibility in the progression and the spread of the PLC is still unclear. In this study, we isolated and characterized a cells population with SC characteristic from paired neoplastic and non-neoplastic tissues from a total 15 patients of hepatocellular carcinoma (HCC, n=10), cholangiocarcinoma (CC, n=3) and non-tumoral liver (n=2). The cells were maintained in selective media and characterized by flow cytometry, RT-PCR, anchorage-independent assay and in vitro cells-directed trans-differentiation using inducer media. In vivo xenograft assay was performed by subcutaneous and orthotopic inoculation of primary cells from HCC and CC in male adult athymic nude and NOD/SCID mice, respectively. Isolated cells were negative for the expression of CD34, CD45, CD117 and CD133, but positive for CD90 and CD44. They expressed mesenchymal SC and pluripotency factors and had the ability to clone after low density plating in 3D matrix. After induction into insulin-secreting cells, they showed gene up-regulations of pancreatic cells markers somatostatin and gastric inhibitory protein, showing potency to trans-differentiate to endodermal lineage. Under adipogenic stimulation, they differentiate into adipocytes as confirmed by lipid droplets accumulation in the cytoplasm and high expression of adipocytes regulator PPARG, detected by nile red lipid staining and RT-PCR, respectively. Under osteogenic condition, the cells became positive of alkaline phosphatase staining and showed up-regulation of bone specific genes such as bone sialoprotein and osteopontin. In vivo assay in nude mice, alpha fetoprotein and albumin in primary cells were noticed 4 months after injection, albeit no visible tumor nodules were detected. Interestingly, by orthotopic injection in NOD/SCID mice, tumor nodules in the liver and tumor metastasis on lung were observed, indicating the capacity of the SC to enter into circulation. Further analysis on the xenograft cells showed a mixed population between human and mouse cells as confirmed by RT-PCR and DNA sequencing. The expression of liverspecific markers were also identified in metastatic site. Our data provide clear evidence of the plasticity of the SC-like cells isolated from HCC and CC in the process of hepatocarcinogenesis and metastasis. Their trans-differentiation flexibility may 804A AASLD ABSTRACTS HEPATOLOGY, October, 2012 induce a switch from normal to cancerous microenvironment. It supports the importance of the presence of the SC in human PLC. Disclosures: The following people have nothing to disclose: Caecilia H. Sukowati, Beatrice Anfuso, Lory S. Croc\ue9, Claudio Tiribell