De Novo Emergence of Mesenchymal Stem-Like CD105+ Cancer Cells by Cytotoxic Agents in Human Hepatocellular Carcinoma12

BACKGROUND Cancer stem cells (CSCs) are considered a pivotal target for the eradication of hepatocellular carcinoma (HCC). Recently, we reported that the CSC markers epithelial cell adhesion molecule (EpCAM) and CD90 are expressed independently in primary HCCs and cell lines, and CD90+ cells share features of metastatic vascular endothelial cells and express the vascular endothelial marker CD105, a co-receptor of transforming growth factor-beta. METHODS The EpCAM+ cell lines HuH1 and HuH7 were treated with 5-fluorouracil (5-FU) or epirubicin in vitro. Gene and protein expression levels were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and fluorescence-activated cell sorting, respectively. The expression of CD105 in primary HCC was evaluated by immunohistochemistry. The relationship of CD105 expression status and HCC prognosis was evaluated using 85 surgically resected HCC tissues by Kaplan-Meier survival analysis. RESULTS 5-FU or epirubicin treatment resulted in the generation of CD90+ and CD105+ cells in vitro in HuH1 and HuH7 cells, which originally contain no CD90+ or CD105+ cells. This phenomenon was validated by qRT-PCR analysis with activation of the epithelial-mesenchymal transition (EMT) program regulators Snail family zinc finger 1 (SNAI1) and SNAI2. Immunohistochemical analysis indicated that CD105+ cells were morphologically identical to vascular endothelial cells in untreated primary HCCs. However, surgically resected specimens after transcatheter arterial chemoembolization clearly indicated that CD105+ cancer cells survived at the peripheral edge of the tumor. Kaplan-Meier survival analysis indicated that HCCs expressing CD105 showed poor prognosis after surgery with statistical significance. CONCLUSIONS Taken together, our data highlight the role of CD105+ HCC cells with activation of the EMT program generated de novo after cytotoxic therapy on the prognosis of HCC patients.