| BACKGROUND&OBJECTIVEColorectal cancer (Colorectal carcinoma, CRC) is a serious health risk of malignant tumors. In our country, the colorectal cancer is the third nationwide leading cause of digestive tract cancer death. With the improvement of living standard and the change of dietary ingredient, CRC incidence rate still continues to increase. Invasion and metastasis are the major causes of death resulting in colorectal cancer treatment failure. Therefore, it is necessary to understand the underlying mechanisms of cancer occurrence and progression and to improve the medical intervention of diagnosis and treatment for colorectal cancer patients.About50%patients died from colorectal cancer within5years after their have been confirmed the diagnoses even though with surgery, chemotherapy, radiotherapy and biotherapy. Colorectal cancer still accounts for the4-6causes of tumor death according to domestic survey data, which means that treatment will not eradicate to all the tumor cells. Cancer stem cells (cancer stem cell, CSC), which conserve a self-renewal and differentiation potential, are the tumor initiating cells and generate a tumor mass. CSC correlates closely to carcinogenesis, treatment, prognosis, relapse and metastasis. The characteristics of division and differentiation of CSC is very similar with normal stem cells, that the tumor cells population begin with cancer stem cells, progenitor cells, transitting amplification cells and terminated cells.It is wellknown that asymmetric division is responsible to cell growth and differentiation for normal stem cells and CSCs, which give rise to two daughter cells, as one for commiting differentiation and amplification, and the other for self-renewal reservation. However, asymmetrical division is unable to resolve some clinical problems. Firstly, if one tumor stem cells initiate a solid mass with continuously proliferation through asymmetrical division to keep one stem cell for self-renewal, how can one single CSC support a massive cancer population? Secondly, CSCs are completely removed after surgery from primary tumors, why cancer appears again? Where did CSC come from in recurrent tumor? Thirdly, metastases produce independent tumor nodules at distant sites, which means every secondly mass is indispensable to rely on a CSC to maintain proliferation while where these CSC came from? Finally, what is the difference between metastatic and primary tumor stem cells? CSC is still residented at primary site during metastases, where do the metastatic cancer stem cells emerge from? Asymmetrical division is impossible to produce so many CSCs, What’s kind of division can derive a good mount of CSCs?Therefore, we speculate that CSCs have asymmetrical division as well as symmetrical division which can expand the CSC pool to give birth of numerous tumor bulks and provide abundant daughter CSCs for subsequent metastases. If confirmed symmetrical division in CSCs, a possible mechanism will be available to explain tumor growth, recurrence and metastases.In previous studies, we culture SW480for monoclonal cells to acquire multiple CSC clones at same genetic background by limited dilution method, which has been identified as a colorectal CSC cloning with CD133mark and subcutaneously xenografted experiments in nude mice. In this study, we mainly focused on colorectal cancer cell lines SW480cells and colorectal CSCs from SW480(SW480-CSC). To make sure the probability of symmetric division and analyse the mechanism of CRC clinical chemotherapy resistance, we examined the expression of CD133and CK7by immunofluorescence from the daughter cells of single SW480cell and SW480-CSC at first time of division. OBJIECTIVE:1. To explore and optimiz a method of isolating and culturing single cancer stem cell for late research.2. To explore the expression of CD133, CK7in SW480and SW480-CSC and confirm the expression characteristics of SW480-CSC makers after a single SW480-CSC division.3. To analyse the expression of CD133, CK7at the daughter cells of the first division of a single SW480and SW480-CSC and confirm the probability of symmetrical division at SW480-CSC.4. To discusses transformation of the ratio of symmetrical division at SW480-CSC after inducing differentiation and5-Fu chemotherapy, and provide an experimental model for drug resistance of colorectal cancer clinical chemotherapy.METHODS:1. A method for isolating and culturing single SW480-CSC.Methods of isolating single SW480-CSC:a. micromanipulation:a single SW480-CSC was separated on the platform of micromanipulation by outside force; b. a mouth control single cell separation apparatus for separating single cancer stem cells. All of the single SW480-CSCs were cultivated in96-well plates or microdrops.2. To analyse the probability of symmetrical division on colorectal cancer stem cells.Colorectal cancer stem cells and colorectal cells were marked with CD133and CK7by immunofluorescence to make sure that CD133and CK7serve as a marker of colorectal cancer stem cells and analyse the probability of symmetrical division on colorectal cancer stem cells at first division of daughter cells with CD133and CK7.3. The ratio of symmetrical division was evaluated at cancer stem cells after5-Fu induced resistance.CD133and CK7were applied for CSCs markers in determining the transformation of the incidence of symmetrical division of SW480-CSC after experimental chemotherapy with5-Fu. Inhibition rate of SW480-CSC was examined with MTT. Clone forming capacities were tested by soft agar cloning experiments. Western blotting assay was detected the expression of CDK1, CDK4, CDK8, CyclinDl and CyclinBl in CSCs before and after5-Fu treatment.4. Statistical analysis methods:Independent-sample data were analyzed with square x test. ANOVA tests were applied to analyze multiple group data with SPSS13.0software.RESULTS:1. Establishing a method for isolating and culturing single SW480-CSC.Micromanipulating operation was easy to damage cell membranes and cause SW480-CSC death in an attempt to separate single cells. However, mouth control single cell separation apparatus can effectively isolate single SW480-CSC, and the effective survival rate of separated cells was98.99%at the microdrops and95.80%at the96-well plates respectively.No differences were found in cell division between microdrop and96-well plates for culturing single SW480-CSC at the first, second time and thereafter division(x2=1.236,0.750and0.385, P>0.05).2. Symmetrical division assayCD133and CK7were labelled with immunofluorescence staining for characteristic markers of colorectal cancer stem cells. CD133were positively expressed in every cell of SW480-CSC, but only in a few cells from SW480. Oppositely, CK7were negatively staining in SW480-CSC but positively in SW480cells. Stastical analysis revealed significant difference in expression of CD133and CK7in the two group cells (t value were73.451,60.803, P=0.000). CSCs should be CD133+CK7cells.The difference of division pattern between SW480-CSC and SW480cells were statistically signifcant at symmetrical division, asymmetrical division, symmetrical differentiation, single SW480-CSC and single differentiation cell by immunofluorescence.(t value were84.560,38.099,92.392,110.635,95.414, P=0.000) 3. Ratio of symmetrical division in SW480-CSC after5-Fu chemotherapy.1) CD133and CK7were statistically significant difference between SW480-CSC and SW480cell after5-Fu induced,(t value were80.528,106.629respectively, P=0.000). SW480-CSC and SW480cells were also showed statistically significant in division types after5-Fu treatment.(t value were134.918,96.295,29.608,36.083,22.289,23.547respectively, P=0.000). There were differeneces between with or without5-Fu treatment in analysis of SW480-CSC of symmetrical division, asymmetrical division, a single SW480-CSC, a single differentiation cell and apototic cells (t value were11.973,5.541,7.255,3.598,7.585respectively, P<0.005), but there were no difference between symmetrical division and a single SW480-CSC (t value were1.158,0.815respectively, P>0.005), and significant difference both asymmetrical division, symmetrical differentiation, a single SW480-CSC and apoptosis cells as well (t value were6.761,39.539,12.672,41.485respectively, P<0.005). The resistance of5-Fu was closely related to symmetrical division of SW480-CSC, the incidence of symmetrical division was decreased after promoted differentiation and5-Fu treatment.2) A significantly time-dependent inhibitory proliferation was found in four group cells (SW480serum, SW480no serum, SW480-CSC serum, SW480-CSC no serum) by MTT assay in vitro, the inhibition rate increased and then declined gradually with the prolonged time of5-Fu incubation at the group of SW480-CSC serum and SW480-CSC no serum. The control groups showed a continuously increased inhibition and statistically significant both in SW480serum and SW480no serum cells (F=91.739, P=0.000). As expected,5-Fu inhibitory effect was decreased gradually in SW480-CSC with prolonged time of5-Fu incubation.3) The difference of cloning rate was statistically significant at SW480-CSC serum group before and after5-Fu mixed media in soft agar cloning assay (t=9.642, P<0.05) but no difference in SW480-CSC no serum (t=0.346, P>0.05). It indicated that5-Fu was not sensitive to cancer stem cells but could suppress the growth of differentiated cells of SW480-CSC.4) Western-blot assay showed that SW480-CSC cells weakly expressed CDK4 and CyclinDl, but mildly increased CDK4expression with5-Fu treatment. No obvious changes were observed in the expression of CylinD1and CDK8associated with5-Fu incubation. However,5-Fu enhanced higher expression of CDK1and CyclinBl in SW480-CSC than in. After5-Fu incubation, SW480-CSC and SW480were similar to express CDK1and CyclinBl.CONCLUSION:1. Single SW480-CSC can be effectively isolated from mouth control single cell separation device, and cultured in microdrops and96-well plates as well.2. CD133+CK7-served as markers of SW480-CSC.3. SW480-CSC divided symmetrically to expand CSC pool.4. Symmetrical division of SW480-CSC was closely related to resistance of5-Fu, the incidence of symmetrical division was significantly reduced after promoted differentiation and5-Fu chemotherapy. |
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