Characterization Of A Stem-like Population In Hepatocellular Carcinoma Mhcc97 Cells

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Despite advances in the detection and treatment of HCC, the mortality rate remains high because the majority of HCC patients present at an advanced stage or with metastasis for which most potentially curative therapies have limited efficacy. Hence, understanding the mechanism underlying carcinogenesis and metastasis formation is essential for the management of liver malignancies.It is presumed that an accumulation of genetic alterations contributes to processes which are critical for multistep hepatocarcinogenesis and metastasis formation. Therefore only cells which have a high proliferative potential and a long life span, such as stem cells, would have the ability to accumulate the necessary mutations to transform into cancer cells. There is growing evidence that cancerous tissues, including HCC, are heterogeneous and may include long-lived stem-like cancer cells. Stem-like cancer cells are responsible for tumor formation and progression as they can perpetuate themselves through self-renewal and to generate mature cells of a particular lineage through differentiation. Stem-like cancer cells have been identified and isolated from leukemia, breast cancer , glioblastoma , prostate cancer , gastric cancer , lung cancer and colon cancer . The side population (SP) phenotype, first described in hematopoietic malignancies, is defined as the population of cells that have the blockable ability to efflux the nucleic acid dye Hoechst 33342 via ATP-binding cassette transporters such as ABCG2/BCRP1. The SP is thought to be enriched for stem-like cells in several normal human tissues, cancers and cell lines, and thus may be useful for the identification and isolation of stem-like cancer cells and the mechanism of tumor progression.Up to now, the whole mechanism underlying hepatocarcinogenesis has not been clearly documented. The identification of stem-like cancer cells and elucidation of the hierarchy in HCC cells might contribute to the understanding of hepatocarcinogenesis, metastasis formation and the exploration of novel therapeutic approaches. In the present study, we report the identification and isolation of SP cells phenotype in human HCC cell lines that demonstrate specific characteristics of stem-like cancer cells when compared with MP cells, such as quiescence, elevated chemo-resistance, increased tumorigenicity, higher actin polymerization ability and increased migration capacity towards the chemokine CXCL12, whose receptor CXCR4 has been linked to HCC dissemination and poor prognosis. This data demonstrates that the SP in human HCC contains stem-like cells, and may provide novel diagnostic tools and strategies for controlling this malignancy.Part 1. Isolations of SP and MP cells from human HCC cell lines.Object: To identify and isolate the SP and MP cells from human HCC cell lines.Methods: Sigle cells suspensions were stained with Hoechst 33342 dye with proper concentration. After being washed and filtered, cells were then analysis and sorting using a flow cytometer. Results: Using flow cytometry, we were able to identify and successfully isolate populations of SP and MP cells from the three human HCC cell lines. The SP gate was defined as the region where cells were absent in the presence of verapamil, an agent which blocks the efflux of Hoechst 33342. The three cell lines, MHCC97, Huh-7 and HepG2, contained 2.64±0.31%, 0.32±0.09% and 0.09±0.03% SP cells respectively. Since MHCC97 contained the highest percentage of SP cells further experiments were performed using this cell line. The purities of sorted SP and MP cells were both higher than 97%. Unstained SP cells could also be observed under fluorescence microscope. Conclusion: Fluorescence Activated Cell Sorting is a feasible method to isolate SP and MP cells from HCC cell lines.Part 2. The biological characteristics of SP and MP cells of MHCC97 cell line.Object: To analyze the biological characteristics of SP and MP cells of MHCC97 cell line. Methods: Sorted SP or MP cells were restained and reanalyzed by after cultured in vitro for two weeks to measure their self-renewal abilities. Sorted SP or MP cells were stained with Heochst33342 and Pyronin Y to examine their cell cycle status. The expressions of AFP, CK19 or ABCG2/BCRP1 were tested by Real-time PCR. Sorted SP or MP cells were cultured with chemotherapeutic agent for 72 hours. Then the cell viability was examined by an MTS-based Cell Titer 96 Aqueous One Solution Cell Proliferation Assay. Sorted SP or MP cells were subcutaneously injected into nude mice under anesthesia. Mice were inoculated with 1x105 or 1x106 non-sorted cells, 1x103 or 1x104 SP cells, or 1x103 or 1x104 MP cells (five mice per group). Tumor growth was monitored every week. Cryostat sections of subcutaneous tumors were subjected to hematoxylin and eosin staining. Results: When cultured in vitro, SP cells generated both SP and MP cells with a SP fraction size (4.82±0.32%) that was larger than in the original population, whereas MP cells produced predominantly MP cells. Cells cycle analysis indicated that 13.03±2.51%, 63.90±1.95% and 23.07±2.60% of the SP cells were in the G0, G1 and S/G2/M phase, respectively. In MP cells, 0.73±0.31%, 27.63±5.59% and 71.63±5.62% cells were in the G0, G1 and S/G2/M phase, respectively. Real-time PCR results indicated that AFP, CK19 and ABCG2/BCRP1expreesion were (1.81±0.15)-fold, (1.61±0.13)-fold and (1.85±0.13)–fold up-regulated in SP relative to MP cells. SP cells exhibited a viability rate of 76.6±5.6% compared with 36.8±3.6% after 72 h incubation with Doxrubicin and the SP percentage increased to (5.2±0.36) %. Following 72 h of Methotrexate treatment, cell viability was 59.8±6.2% in SP cells and 39.1±4.8% in MP cells. Transfer of 1x105 or 1x106 unsorted MHCC97 cells led to tumor formation in nude mice. Transplantation of 1x103 SP or 1x104 SP cells produced tumors in 60% or 80% of the mice. In contrast, transplantation of 1x103 MP cells consistently failed to form tumors, while 1x104 MP cells showed tumor formation in only 20% of the mice. histological analysis of SP or MP origin tumors showed similarities to primary tumors. Conclusion: MHCC97 SP cells regenerated both SP and MP cells and remained quiescent. They display a potential ability to differentiate into hepatocytes and cholangiocytes under appropriate conditions. SP cells were more resistant to anticancer drugs than MP cells and showed significantly higher tumorigenicity than MP cells. These data indicated that SP cells contained the stem-like characteristics and played a key role in tumorigenicity of HCC.Part 3. The metastasis capacities of SP and MP cells of MHCC97 cell line.Object: To examine the metastasis abilities of MHCC97 SP and MP cells through their chemotaxis to CXCL12. Methods: Human CXCL12 were added to cell suspensions and aliquots were taken at the indicated times. The relative F-actin index was determined as the ratio of the F-actin level of SP or MP cells treated with CXCL12 to SP or MP cells treated with PBS. Using Transwell system to measuer the relative migrating index of SP and MP cells, which was determined as the ratio of the number of migrated cells in each group to the number of migrated cells in total cell group. Results: Sorted SP cells showed (1.27±0.17)-fold, (1.43±0.16)-fold, and (1.29±0.13)-fold F-actin staining intensity at the studied time points. The relative migrating index of SP cells (1.56±0.10%) was higher than in untreated SP cells (1.05±0.07%), and MP cells (1.16±0.08%). Conclusion: These data suggest that CXCL12 treatment increased the extent of actin polymerization in SP cells more than MP cells at the studied time points, and CXCL12 could induce higher chemotaxis of SP cells than MP cells.In conclusion, we isolated a tumorigenic SP cell population by sorting the MHCC97 cell line and propose that these SP cells exhibit stem-like characteristics. Our data suggests that the SP cells show tumorigenic potential, chemo-resistance and migratory ability, which might lead to relapse and metastasis formation, which may have important clinical implications in HCC treatment. Further studies on the identification and characterization of SP cells using clinical HCC specimens will contribute to the understanding of the mechanism underlying hepatocarcinogenesis and metastasis formation and aid the development of effective therapeutic strategies.