| Background and Objective:Colon cancer is one of the maligancies to human health, the morbidity and mortality showed an increasing trend in recent years. Studies suggest that tumor blood vessels has an important role in tumor growth and metastasis and anti-angiogenesis therapy has become one of the most important anti-tumor strategies. However, some reporters indicate that anti-angiogenesis therapy could not inhibit tumor growth and could even promote tumor progression in some patients.There exist a small portion of cells with self-renewal and differentiation potential in the tumor tissue, although the number of cells is very small, they are closely related to the invasive growth, recurrence and drug resistance of tumor, this small part of the tumor cells are named stem cell-like tumor cells. Studies have shown that some endothelial cells derive from the stem cell-like tumor cells in glioblastoma, suggesting that in the process of tumor formation, stem cell-like tumor cells have the role of tumor angiogenesis cells. These stem cell-like tumor cells can be directly involved in tumor microcirculation. Vasculogenic mimicry (vasculogenic mimicry, VM) is a special blood supply pattern through endothelial-independent cells. Our previous studies have found that VM exists in colon cancer and it may be closely related to the presence of tumor cells "stem-like". Therefore, we placed colon cancer cells in a specific micro-environment, analyzed the relationship between the morphology changes and differentiation of colon cancer cells and the relationship between the morphology changes and the characteristics of "stem-like". Therefore we select stem cell-like tumor cells, and they are induced to differentiate. Thus we suggest that stem cell-like tumor cells can simulate endothelial cell function to become VM, furtherly they can differentiate into endothelial cells with endothelial cell-specific molecular markers under certain circumstances. This hypothesis can rich the theory of colon cancer angiogenesis, and have the significance to solve the clinical anti-angiogenesis drugs resistance phenomenon.Methods:1) We choosed three different levels of differentiation of colon cancer cells HCT116, SW480and HT29, cultured in37℃, a humidified5%CO2incubator.2) We take three growing well colon cancer cells, cultured in conditioned medium containing endothelial cells inducible factor, and morphology changes of cells were observed daily.3) Western bloting was used to analyze the expression level of endothelial cell-specific markers in the three colon cancer cells after induced15days.4) Western bloting and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression level of endothelial cell-specific markers in colon cancer HCT116cells after induced with increasing number of days.5) Immunofluorescence staining was used to detect the situ expression level of endothelial cell-specific markers between common HCT116cells and HCT116cells after induced.6) We cultured HCT116cells with stronger "stem-like" properties in serum-free medium to select stem cell-like tumor cells, immunofluorescence staining was used to detect the expression level of CD133and Lgr5in HCT116stem cells spheres.7) We cultured HCT116stem cells spheres in conditioned medium containing endothelial cells inducible factor, Western bloting and immunofluorescence staining were used to detect the expression level of endothelial cell-specific markers in HCT116stem cells spheres between in conditioned medium containing endothelial cells inducible factor and in common medium.8) The ability of VM formation of HCT116stem cells spheres after endothelial-induced was detected in three-dimensional culture. Results1) The three colon cancer cells cultured in endothelial induction medium, with the extension of the induction time, poorly-differentiated HCT116cells became adherent and expressed polygonal then gradually formed tubular structure,15days later, the cells showed tubular structure. Moderately-differentiated SW480cells became from short fusiform to long fusiform and cells gradually have formed the trend of tubular structure, but not obvious. Well-differentiated HT29cells after induced for two weeks, basically unchanged. 2) Western bloting showed that three colon cancer cells cultured in endothelial induction medium after15days, CD31and CD34expression in endothelial-inducing medium HCT116cells (poorly differentiated) were higher than in the normal medium, while the CD31and CD34expression in SW480cells (moderately differentiated) and HT29cells (well differentiated) in the two cultural mediums were not notably changed.3) According to the results of Western bloting, RT-PCR and immunofluorescence staining, colon cancer HCT116cells after induced endothelial cell culture medium in a few days, with the extension of the induction time, the expression of specific molecular markers of endothelial cells gradually increased.4) According to the results of cell immunofluorescence, the selected HCT116stem cell spheres can express stem cell-related molecules CD133and Lgr5.5) According to the results of Western bloting and immunofluorescence staining, the selected HCT116stem cell spheres after induced endothelial stem cell culture medium in a few days, the expression of specific molecular markers of endothelial cells gradually increased.6) According to the results of Matrigel three-dimensional culture, the analysis of HCT116stem cell spheres in endothelial induction medium and the general culture medium showed that:under the three-dimensional culture conditions, the environment of endothelial induction medium can promote HCT116stem cell spheres to form tubular structure, and shorten the time of tubular structure formed.Conelusions:1) Colon cancer cells have the ability of differentiation towards vascular endothelial cells in endothelial cell culture medium in vitro.2) Colon cancer cells after cultured in endothelial cell culture medium a few days, the cell morphology changes and the differentiation express negative correlation. Poorly-differentiated colon cancer cells after induced gradually formed a tubular structure, well-differentiated colon cancer cells basically unchanged.3) Colon cancer cells HCT116after induced endothelial cell culture medium in a few days, the expression of specific molecular markers of endothelial cells increased, indicate that colon cancer cells have the ability of differentiation towards vascular endothelial cells.4) Endothelial cell culture medium can promote colon cancer cells with stronger "stem-like" properties differentiation towards vascular endothelial cells. 5) Colon cancer cells with stronger stem cell-like properties have a strong capacity of tubular structure formation, stem cell-like tumor cells may simulate the function of endothelial cell to form tubular structure, further differentiation become endothelial cells with endothelial cell-specific molecular markers under certain circumstances. |
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