| BackgroundIt is always a focus in research on the carcinogenesis, development, invasion, metastasis and therapeutics of cancer. Generally it has been believed that tumor cells are mutated from mature cells and have homogeneous potential of proliferation. All tumor cells have the abilities to proliferate infinitely. Accordingly, it is an accepted standard for the curative effect in clinical tumor therapy that tumor volume could grown downwards or even vanish. However, some tumors recurred or even distantly metastasized and could not been cured completely after tumor removal by surgery or some others treatment and had been stable for some periods.Recent researches had discovered that there were some stem-like subpopulations in tumor tissues which had the potential to proliferate infinitely and played much crucial role in initiating carcinogenesis and development, while the other cells would die after transitory differentiation. It was thought that the tumor growth resulted from the proliferation of some tumor stem cells with special surface markers. For this reason, the tumor treatment must aim directly at the tumor stem cells but not at the bulk of tumor cells with little ability to proliferate and differentiate. This theory might bring about some revolutionary in tumor therapeutics.Under the guide of this theory, scientists believed that hepatocellular carcinoma possibly also originated from the hepatoma stem cells. There were be great importance to study on isolation, culture, identification and the basic characteristics of them. However, the key point of the research on cancer stem cells was how to establish the special surface markers of them. However, it was hope to achieve an efficient sorting scheme of cancer stem cells by detecting the surface markers with some directing antigens, which would save a lots of time and research funds. Based on the departed research on cancer stem cells, we found that there might be an efficient method to isolate cancer stem cells from cancer tissue by sorting different cell subpopulations with combination of surface markers of normal stem cells and further transplantation of them to detect the formation of tumor.Nevertheless, because of the shortage of suitable cultural methods and efficient sorting scheme, there is still no report on the isolation, culture, identification of stem-like subpopulations cells within hepatocellular carcinoma cells. Based on these researches, it was hope to isolate heptoma stem cells by sorting different cell subpopulations with some surface markers of liver stem cells and further test on their abilities to proliferate, differentiate ex vivo and to form tumors in vivo.So, in this project, we plan to sort the hepatoma stem cells with some surface markers of oval cells which was believed to be one kind of stem cells in the liver. Oval cells are a kind of stem cells in liver which have the ability to differentiate both to hepatic cell and biliary ducts cells. A recently serial research support the ideas that hepatocellular carcinoma cells should originate from oval cells, which suggest that surface markers of oval cells could be used in the sorting of hepatoma stem cells. In our earlier study, tumor tissue samples were obtained from 1 patient with HCC after surgery, and then HCC cells were cultured in vitro by primary tissue culture technique. The cell subpopulations were isolated from the tumor cells by immunopanning using oval cell markers CD34, c-kit, Thy-1, CK7, CK14 and CK19. Among all the cell subpopulations, those positive for CD34, c-kit and CK7, respectively, showed a difference ability of proliferation and tumor formation compared with those negative for markers. The CD34~-, c-kit~+ and CK7~+ subpopulations of the cells exhibited stronger capacity of tumor formation even in only 1/4 or 1/10 of their original density, while the ratio of tumor formation decreased compared with the ratios of the cell subpopulations before dilution. The possible reasons for those results were that mixing cells in subpopulations affected the capacity of tumor formation because of the low purity of the cells isolated by immunopanning and hepatoma stem cells could not be completely isolated from hepatocellular cancer cells sorted with single surface marker. The significant difference in the ability to form tumor between single marker-positive positive subpopulation and corresponding negative subpopulation of the surface markers from high to low there are c-kit, CK7, CD34 respectively. So, there is a hope to isolate the hepatoma stem cells from hepatoma carcinoma cells by these three surface markers alone or in combination to sort primary cultured human hepatoma carcinoma cells by flow cytometry. and then the cellular subpopulations were respectively transplanted to BALB/c nude mouse to test the ratio of tumorigenesis and their cell proliferations.ObjectivesThis study aims: 1) To compare the effect of three methods of flow cytometry sorting, magnetic cell sorting and immunopanning isolation in isolating sub-population of hepatoma carcinoma cells so as to decide which might be the possible effective one; 2)To isolate, culture and identify the stem-like subpopulations cells from hepatoma carcinoma cells in vitro, to decide a suitable sorting scheme ; 3)To investigate the basic characteristics of stem-like hepatoma cells.Methods1. Tumor tissue samples were obtained from 1 patient with HCC after surgery, and then HCC cells were cultured in vitro by primary tissue culture technique, c-kit~+ cells in primary cultured human hepatoma carcinoma cells were separated with flow cytometry sorting, magnetic cell sorting and immunopanning isolation respectively. Then, the purities of c-kit~+ cells in each subpopulations were tested by flow cytometry and the recovery rates of three methods were calculated. The viabililties of them was tested by trypanblue exclusion. The Stimulation index (SI) was tested by CCK-8 assay. The cells of each subpopulation were injected into nude mice to measure the ratios of tumorigenesis.2. Comparisons of purities of c-kit~+ hepatoma cells, recovery rates, viabilities, proliferation and ratios of tumorigenesis among subpopulations were performed by one-way ANOVA test. Further multiple comparison between subpopulations were conducted by least significant difference if P＜0.05. The viabilities and SI between cells before-and post-sorted by flow cytometry were compared by Independent t-test.3. c-kit and CK7 were used alone to sort primary cultured human hepatoma carcinoma cells by flow cytometry. The cellular subpopulations were respectively transplanted to BALB/c nude mouse to test the ratio of tumorigenesis.4. If some of the c-kit~- subpopulation and CK7~- subpopulation still have the ability to form implanted tumor, c-kit and CK7 were used in combination to sort primary cultured human hepatoma carcinoma cells by flow cytometry. The cellular subpopulations as positive for both c-kit and CK7 (c-kit~+ck7~+) sub-population and corresponding non-c-kit~+ck7~+ sub-population were respectively transplanted to BALB/c athymic mouse to test the ratio of tumorigenesis.5. If the ratio of tumorigenesis of c-kit~+ck7~+ subpopulations cells and corresponding non-c-kit~+ck7~+ ones were 100 percent and 0 percent, the cells extracted from the implanted tumor of c-kit~+ck7~+ subpopulations were sorted by flow cytometry. Then, the subpopulation cells obtained were also implanted to nude mice to measure the tumor forming ability.6. Basing on the tumor forming ability of subpopulations and their daughter cells, it was determined that which subpopulation was the hepatoma stem cells and how to obtain them.7. The stem-like subpopulations cells and corresponding non- stem-like subpopulations ones were cultured in vitro. The morphologic characteristics of stem-like subpopulations were observed under light microscope and ultra structural characteristics were observed under electronic microscope. Their stimulation index were tested by CCK-8 assay. Their cell cycle and ratios of stem-like cells in stem-like subpopulations after serial subcultivation were both tested by flow cytometry. In turn, these data were analyzed significantly. The above indexes between the stem-like subpopulations cells and corresponding non- stem-like subpopulations ones were analyzed significantly by independent t-test to acquire the basic biologics features of stem-like subpopulations.Results1. Comparison among the effect of three isolating methods of flow cytometry sorting, magnetic cell sorting and immune panning isolation: There was no significant difference among three methods in the viabililty (93.16±4.36%,93.68%±3.20%, 94.46%±3.02% respectively), cell proliferation of cells (SI of 0.92±0.11,0.87±0.13,0.95±0.05 respectively) and the ratios of tumor forming (88.00%±10.95%,88.00%±10.95%,96.00%±8.94% respectively). But there was significant difference among three methods in the purities (99.42%±0.54%,90.64%±4.29%,71.34%±5.22% respectively) and recovery rate of cells (84.40%±3.56%,85.76%±3.72%,63.16%±4.78% respectively). From the higher to the lower, the purities of them are of flow cytometry sorting, magnetic cell sorting and immune panning isolation. The recovery rate of cells sorted by immune panning was lower than flow cytometry sorting and magnetic cell sorting. There was no significant difference in the recovery rates between cells sorted by flow cytometry sorting and magnetic cell sorting. There was no significant difference in the viabililty (94.04±3.27% vs 93.16%±4.36%) and cell proliferation of cells (SI, 1.02%±0.08% vs 0.92%±0.11%) before and after separating by flow cytometry sorting.2. Results of implanted tumor forming of the subpopulation cells isolated by c-kit or CK7 alone: The ratio of tumorigenesis of c-kit~+ subpopulations cells and c-kit~- ones sorted with c-kit were 9/10 and 2/10, respectively. The ratio of tumorigenesis of CK7~+ subpopulations and CK7~- ones sorted with CK7 were 8/10 and 1/10, which means that hepatoma stem cells could not be completely isolated from hepatocellular cancer cells sorted with single surface marker.3. Results of implanted tumor forming of the subpopulation cells isolated by both c-kit and CK7 positively: The ratio of tumorigenesis of c-kit~+ck7~+ subpopulations cells and corresponding non-c-kit~+ck7~+ ones were 10/10 and 0/10. The implanted nude mice tumor cells was extracted and tested by flow cytometry. It was showed that the ratio of c-kit~+ck7~+ subpopulations cells and corresponding non-c-kit~+ck7~+ ones were similar to that of in primary tissues(0.8%,99.2% respectively). The rates of tumors formed after the sorted cells from implanted tumor cells were implanted to nude mice were still similar to those of in primary tissues: the ratio of tumorigenesis of c-kit~+ck7~+ subpopulations cells and corresponding non-c-kit~+ck7~+ ones were 100 percent and 0 percent. Their HE staining and AFP immune staining results are similar.4. The basic biological features of stem-like subpopulations: The c-kit~+ck7~+ subpopulations were in polygon shape when cultivated for 24 hours and in fusiform shape and polygon shape after serial subcultivation, while the corresponding non-c-kit~+ck7~+ ones were in fusiform shape when cultivated for 24 hours and polygon-shaped cells did not emerged. The stimulation index of c-kit~+ck7~+ sub-population was significantly higher than that of non-c-kit~+ck7~+ sub-population(P＜0.05). Along with the serial subcultivation, the ratios of c-kit~+ck7~+ cells declined and were in equal level to those subpopulations before sorting (0.8%). Most of c-kit~+ck7~+ subpopulations were at phase G0/G1 of the cell cycle (60.33%±2.74%), while only significantly lower ratio of corresponding non-c-kit~+ck7~+ ones were at this phase (49.24%±2.79%). The ratio of cells at stage S or S and G2/M and in apoptosis of non-c-kit~+ck7~+ subpopulations was significantly higher than that of c-kit~+ck7~+ subpopulations. The rates of tumors formed after the sorted cells from implanted tumor cells were implanted to nude mice were still similar to those of in primary tissues.Conclusions1. Flow cytometry sorting is an effective and practical method to isolate sub-population in hepatoma carcinoma cells.2. c-kit~+ck7~+ sub-population was possible tumor stem cells within hepatoma carcinoma cells. Sorting with c-kit~+ck7~+ by flow cytometry was a possible effective method to obtain tumor stem cells within hepatocellular carcinoma.3. The c-kit+/ck7+ sub-population cells have the ability to form heterogenic sub-colonies. Most of them were at phase G0/G1 of the cell cycle; They had poorer proliferation ability and showed more homotenous stem cells characteristics. |
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