Michael Ng

UNLABELLED: Increasing evidence has revealed the importance of cancer stem cells (CSCs) in carcinogenesis. Although liver CSCs have been identified in hepatocellular carcinoma (HCC) cell lines, no data have shown the presence of these cells in human settings. The present study was designed to delineate CSCs serially from HCC cell lines, human liver cancer specimens to blood samples, using CD90 as a potential marker. The number of CD90(+) cells increased with the tumorigenicity of HCC cell lines. CD45(-)CD90(+) cells were detected in all the tumor specimens, but not in the normal, cirrhotic, and parallel nontumorous livers. In addition, CD45(-)CD90(+) cells were detectable in 90% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis. A significant positive correlation between the number of CD45(-)CD90(+) cells in the tumor tissues and the number of CD45(-)CD90(+) cells in the blood samples was identified. CD90(+) cells sorted from cell lines and CD45(-)CD90(+) cells from the tumor tissues and blood samples of liver cancer patients generated tumor nodules in immunodeficient mice. Serial transplantation of CD90(+) cells from tumor xenografts generated tumor nodules in a second and subsequently third batch of immunodeficient mice. Treatment of CD90(+) CSCs with anti-human CD44 antibody induced cell apoptosis in a dose-dependent manner. CONCLUSION: Identification of CD45(-)CD90(+) CSCs in both tumor tissues and circulation suggests that CD45(-)CD90(+) could be used as a marker for human liver cancer and as a target for the diagnosis and therapy of this malignancy.

OBJECTIVE: To investigate whether circulating cancer stem cells (CSCs) of hepatocellular carcinoma (HCC) can predict its recurrence after hepatectomy. BACKGROUND: HCC recurrence frequently occurs within the first year after hepatectomy, probably due to circulating tumor cells that have been shed from the primary tumor before hepatectomy. Because CSCs are more likely to initiate tumor growth than mature cancer cells, a high level of circulating CSCs may be a hint for HCC recurrence. METHODS: Multicolor flow cytometry was used to detect the number of circulating CSCs (CD45CD90CD44) in the peripheral circulation of 82 HCC patients 1 day before hepatectomy. The patients were monitored by CT or MRI for recurrence every 3 months. RESULTS: Forty-one (50%) patients had recurrence after a median follow-up period of 13.2 months (range, 1.3-57.1 months). Patients with recurrence had a higher median level of circulating CSCs than patients without recurrence (0.02% vs. 0.01%; P < 0.0001). Circulating CSCs > 0.01% predicted intrahepatic recurrence (relative risk 3.54; 95% CI, 1.41-8.88; P = 0.007) and extrahepatic recurrence (relative risk 10.15; 95% CI, 3-34.4; P = 0.0002). Patients with >0.01% circulating CSCs had a lower 2-year recurrence-free survival rate (22.7% vs. 64.2%; P < 0.0001) and overall survival rate (58.5% vs. 94.1%; P = 0.0005) than patients with ≤0.01% circulating CSCs. On multivariable analysis, circulating CSCs > 0.01%, tumor stage and tumor size were independent factors predicting recurrence-free survival. CONCLUSIONS: Circulating CSCs predicted posthepatectomy HCC recurrence with high accuracy. They may be the target of eradication in the prevention of posthepatectomy HCC metastasis and recurrence.

PURPOSE: The goals of the present study were to investigate the mechanism of hypoxia-mediated chemoresistance in liver cancer cells and tumorigenic hepatic progenitor (oval) cells and to determine whether disrupting an Akt/hypoxia-inducible factor-1alpha (HIF-1alpha)/platelet-derived growth factor (PDGF)-BB autocrine loop can enhance chemotherapeutic efficacy in hypoxia. EXPERIMENTAL DESIGN: Five hepatocellular carcinoma (HCC) cell lines and two hepatic progenitor cell lines were treated in vitro with cisplatin under both normoxic and hypoxic conditions. To generate ischemic hypoxia for tumor cells in vivo, hepatic artery ligation was applied to an orthotopic HCC model. Cisplatin and YC1, which is a HIF-1alpha inhibitor, were administered by portal vein and intratumoral injections, respectively. RESULTS: Cell viability was higher under hypoxic than normoxic conditions. HIF-1alpha and Akt were up-regulated under hypoxic conditions, forming an autocrine signaling loop with PDGF-BB. Akt/HIF-1alpha/PDGF-BB signaling regulated Akt to confer cisplatin resistance to HCC cell lines in vitro. This autocrine signaling loop also contributed to chemoresistance in the tumorigenic hepatic progenitor cell line PIL2 under hypoxic conditions but not in the nontumorigenic cell line PIL4. In an orthotopic HCC model, combining blockade of HIF-1alpha activity with ischemic hypoxia significantly enhanced the efficacy of chemotherapy, leading to suppression of tumor growth and prolongation of animal survival. CONCLUSION: Blockade of Akt/HIF-1alpha/PDGF-BB autocrine signaling could enhance the chemosensitivity of liver cancer cells and tumorigenic hepatic progenitor cells under hypoxic conditions and thus provide an effective therapeutic strategy for HCC.

BACKGROUND: Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90(+) liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90(+) cells sorted from tumor (CD90(+)CSCs) with parallel non-tumorous liver tissues (CD90(+)NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CD90(+) cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90(+) cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90(+)CSCs and CD90(+)NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90(+)CSCs and CD90(+)NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90(+)CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90(+)CSCs compared to CD90(+)NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90(+)CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90(+)CSCs in liver tumor tissues. CONCLUSIONS/SIGNIFICANCE: The identified genes, such as GPC3 that are distinctly expressed in liver CD90(+)CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells.