Separation, Identification And Characterization Of CD133~+CD24~+Hepatic Cancer Stem Cells

Background and objective：Hepatocellular carcinoma (HCC) is the fifth most prevalentmalignant cancer, and the third most common cause of cancer-related deaths worldwide. Itis reported that approximately one million cases of HCC are diagnosed annually, witharound600,000of these cases resulting in patient death. The incidence of HCC is expectedto increase globally, and with a5-year survival rate of below5%.Various treatments can be employed for HCC management according to the clinicalliver cancer staging system. Hepatic resection is a common therapy for HCC, particularlyfor noncirrhotic patients. It is also a first-line treatment for patients with early HCC. But thetumor recurrence is still a problem after resection, and the recurrence rate remains high afteroperation, indicating the incomplete eradication of tumor cells in patients. Orthotopic livertransplantation (OLT) has become a promising therapeutic option for cirrhotic patients withunresectable HCC, but these treatments are limited by the advanced stage when the diseaseis usually diagnosed and the shortage of liver grafts available. Local ablative strategy is abetter treatment of choice for the management of early-stage HCC with small tumor. Inspite of technological advances in treatment interventions, the survival outcome of HCCpatients at present is still unsatisfactory. Chemotherapy either by TACE or systemictreatment is also available, due to the highly chemotherapy-resistant nature of the disease,however, response rates are low.The exact mechanism of tumor recurrence remains vague due to a limitedunderstanding of hepatocarcinogenesis. It is suspected that tumor relapse is likely to becaused by cancer stem cells (CSCs) that are resistant to current treatments. Based on theCSC theory, it is believed that CSCs only represent a minority number of the entire tumor mass (generally less than5%of the total tumor). However, the percentage of CSCs isolatedfrom hepatocellular cancer cell lines or tissue is more than50%. So it is not likely that allthe separated from HCC cell lines are CSCs by CSC markers-CD133. And CSCs in othercancer types have also been characterized by more than one marker. For this reason, tobetter characterize the CSC population in HCC, the identification of other markers,expressed along with CD133, is much required and was also our purpose for this study.Methods:1．Fluorescence activated cell sorting was used to assess cancer stem cell markerexpression in hepatocellular cancer cell lines, and identify and isolate CD133+CD24+cells;2．Detection of clonogenic ability and tumorsphere formation ability of sorted cells;3．CD133and CD24expression was analyzed by RT-PCR;4．CD133and CD24expression was analyzed by Western blot;5. Tumor formation ability of the sorted cell was determined in Balb/C mice in vivo;6．CD133and CD24expression was detection in HCC tissue specimens by HE andIHC stainning;7．Cell viability was analysed by MTT array.Results: We identified and isolated a small population hepatic cancer cells co-expressionCD133and CD24surface markers from human liver cell lines Huh-7. The sorted cellspossessed property as follows:1. CD133and CD24were co-expressed in hepatocellular cancer cell line2. Increased spheroid formation ability and higher clonogenic ability inCD133+CD24+cells3. Compared to CD133-CD24-cells, and CD24were preferentially co-expressed inCD133+CD24+cells, RT-PCR and western blot analyses revealed that the CD133and CD24gene expression of sorted cells was in line with their phenotype in RNA lever and proteinlevel.4. The sorted CD133+CD24+cells from Huh-7cells had a stronger tumorigenicity. 5. Significant expression of CD133and CD24in HCC tissue specimens fromxenograft mice model and the tumor was very similar with the primary cancer.6. The percentage of G0and G1phrase was more higher in CD133+CD24+cells.7. CD133+CD24+cells from Huh-7cells were highly resistant to chemotherapeuticagent lobaplatin, and the IC50was9.9μg/mL.In a word, CD133+CD24+cells possessed a greater colony-forming efficiency, higherproliferative output, and greater ability to form tumor in vivo, which was identical to theoriginal tumor, and had features of hepatic cancer stem cells. CD133and CD24double-positive cells were more resistant to chemotherapeutic agents.Conclusions:1. Hepatocellular cancer cell line Huh-7expressed cancer stem cell marker CD133and CD24.2. CD133+CD24+Huh-7cells possessed cancer stem cell propersity, includingself-renewal, differentiation, increased spheroid formation ability, higher clonogenic ability,and a stronger tumorigenicity in SCID mice.3. Compared to CD133-CD24-Huh-7cells，CD133+CD24+Huh-7cells werehighly resistant to chemotherapeutic agent lobaplatin。Taken together, Our results suggest that hepatic cancer stem cell could be preciselydefined by co-expression of CD133and CD24cell surface markers. The identification oftumorigenic liver CSCs could provide new insight into the HCC tumorigenic process andpossibly bear great therapeutic implications.Innovations: We demonstrated that CD133and CD24could served as hepatic cancerstem markers, we can better characterize the CSC population in HCC by combinationCD133with CD24.