Experiment on Cultivation and Identification of Colorectal Cancer ...

[Abstract]

Background and Objective:Recent studies have shown that a rare population of undifferentiated cells are responsible for tumour formation and maintenance, they express definite cell surface markers and possess some differentiation potential. In vivo xenograft transplantation assay,they show high tumorigenic potential, so they are defined as cancer stem cells. Further study show that the features of self-renewal and differentiation potential they possess are preserved and regulated by microenvironment such as tumor stroma cells and vascular endothelial cells. They also express drug resistance protein, more efficient at repairing of damaged DNA, so they can resistant to drug and radiation. Thus cancer stem cells are the root of cancer,and responsible for cancer metastasis,recurrence and drug resistance. Similar studies have shown that subpopulations of tumor cells from blood, breast, brain, pancreas and colon.Cancer cell line is seen as classic model for studying cancer. There are certain progress in the study of cancer stem cells about cancer cell line since Toru Kondo has been found that cancer stem cells persist in many cancer cell lines, but only limited to glioma and breast cancer cell line. Zhu found that spheres could isolated from glima cell line under serum-free culture condition containing growth factor, those spheres were enrich in cancer stem cells, so he called them brain tumor spheres. As so far, the report about cancer stem cells in colorectal cancer cell line is rare. Therefore we choose colorectal cancer cell line Colo205 as research objective and aim to study how Colo205 generate colorectal cancer sphere under special culture condition, and identify its biological characteristics. Colo205 cell line was established in 1975 by Toni U. Semple, from ascitic fluid of a 70-year-old Caucasian male with carcinoma of the colon. It growth property is mixed with adherent and suspension.Methods:1. Culture colorectal cancer sphere Serum-free medium(SFM) contains B27, epidermal growth factor(EGF), basic fibroblast growth factor(bFGF), Leukemia inhibitory factor(LIF) and glutamine.Colo205 cells were cultured in SFM,while routine Colo205 cells cultured in 1640 medium containing 10% fetal bovine serum were as control. Observe the process of Colo205 cells generating colorectal cancer spheres and detect expressions of intestal stem cells marker Musashi-1 by immunocytochemical.2. Differentiation assay Colorectal cancer spheres were cultivated without EGF and bFGF in the presence of 10% serum to induce to differentiation. Cells morphology were observed by optical microscope. Then we collected cells from colorectal cancer spheres, post-differentiated sphere cells and routine Colo205 cells, and labeled anti-CD44-FITC and anti-CD133-PE antibody respectively, and analysed expressions of stem cell surface markers CD133 and CD44 among them by flow cytometry.3. Cell cycle analyse We collected colorectal cancer spheres and routine Colo205 cells, and labeled PI after adjusting to the same concentration, then we compared cell cycle between two groups by flow cytometry.4. Spectral karyotype analyse We collected colorectal cancer spheres and routine Colo205 cells, spectral karyotype was performed on tumor metaphase cells after adjusting to the same concentration, and then made statistical analysis on the number of Chromosomes.5. Three-dimensional culture When typical Colorectal cancer spheres were generated, we mechanically and enzymatically dissociated into a single-cell suspension, and changed the medium into medium containing serum, and adjusted to the concentration of 60000/ml, while the same concentration of routine Colo205 cells were as control. Three-dimensional culture was conducted using Matrigel matrix, then we observed how spheres differentiate into crypt-like structures.6. Detect sternness genes by RT-PCR We extracted RNA of colorectal cancer spheres and routine Colo205 respectively, and then we synthesised cDNA and second strand DNA consecutively. Semi-quantitative PCR detected the expression of Bmi-1 and Notch-1.7 . Xenograft transplantation assay After having set up multiple concentrations, we compared tumorigenic capacity between the two groups at the same concentration. Then transmission electron microscope observation, Haematoxylin-eosin analysis and Immunohistochemical analysis of xenograft were conducted.Results:1. Colo205 could survived in serum-free medium containing growth factor, when cultured at 7 day, most of adherent cells were apoptosis, a rare population of cells could survived , proliferated and formed the suspended colorectal cancer spheres. The spheres can generate serially passaged spheres, demonstrating their self-renewal feature. When changed the serum-free medium into the medium containing serum, we observed that the cells in sphere migrate out of it and formed single-cell layer after 10 days. We also found that colorectal cancer spheres were high expression of intestinal stem cells marker Musashi-1, while routine Colo205 cells did not express Musashi-1.2. The proportions of CD133~+, CD44~+, CD44~+CD133~+ cells in routine Colo205 cells were (13.27?5.62)% ,(62.92?8.38)% and (10.77?4.96%) respectively,while the three indicators in colorectal cancer spheres were (52.71?16.97)%, (79.06?12.10)% and (46.89?19.17%) respectively, three indicators for post-differentiated cells were (16.47?2.45)% ,(47.80?2.57)% and (12.41?2.27%) respectively. There were statistical difference between the groups, and F values were 17.517,10.752,12.294 respectively, P values were 0.001,0.003,0.002 respectively, the proportion of CD133~+, CD44~+, CD44~+CD133~+ cells in colorectal cancer spheres were significantly higher than post-differentiated cells and routine Colo205 cells (P 0.05):3. The proportion of routine Colo205 cells in G1 phase was (73.8?2.9)%, and (26.2?2.9%) for S/G2 phase, while the proportion of colorectal cancer spheres in G1 phase was (51.9?1.6)%, and (48.1?1.6%) for S/G2 phase. The proportion of cells in S/G2 phase was significantly higher in colorectal cancer spheres than in routine Colo205 cells (P = 0.000), indicating that colorectal cancer spheres were in a high-proliferation state.4. We could not find any noticeable difference in the number of chromatosomes between the colorectal cancer spheres and the routine Colo205 cells(P=0.070), and the two groups were all of aneuploidy.5. The typical crypt-like structures were emerged in 18 days, a cell-ring interconnection structure and overlap upward growth trend was formed in colorectal cancer spheres group, while the control group did not formed any crypt-like structure, with cells stopping proliferation. Those demonstrated that colorectal cancer spheres had a strong proliferation and differentiation potential, while routine Colo205 cells only had limited cell proliferation potential and without any differentiation potential. When at day of 18, the numbers of crypt-like structures in routine Colo205 cells group was 0 at one level, while the number in tumor spheres group were (8.6?1.1). 95% confidence interval was [7.184, 10.016], and it did not contain 0, then we can see that the difference was significantly.6. Semi-quantitative RT-PCR analysis found that expressions of Bmi-1 and Notch-1 in tumor spheres were significantly higher than the control group (P value were 0.002 and 0.031 respectively ), indicating that colorectal cancer spheres had a strong self-renewal capacity.7. Xenograft transplantation assay showed that 104 colorectal cancer sphere cells were tumorigenicity, while the same number of routine Colo205 cells did not. The volume of tumor generated by 105 colorectal cancer sphere cells was significantly larger than the control group, indicating that colorectal cancer spheres were high tumorigenicity. HE analyse found similar differentiation structure between the two groups, and intestinal stem cells Musashi-1 -positive cells were similar between two groups of xenograft, and similar malignant phenotypes were found by transition electron microscopy in both of two xenografts. This suggested that in spite of the number of cancer stem cells that implantated in mice between two groups are vary greatly, once they initiated the foundation of tumor growth in mice, and in the active of basement membrane elements, same cytokines and vascular endothelial cell which induce to differentiation, the final of tissue structure, degree of differentiation and degree of malignancy are similar, showing that colorectal cancer stem cells possess the self-renewal and differentiation capacity in vivo.Conclusion:1. There were a small population of cancer stem cells persist in Colo205 cells. Colorectal cancer spheres in which enrich cancer stem cells can be generated under serum-free culture condition with EGF and bFGF.2. Colorectal cancer spheres are high expression of cancer stem cell marker CD133, CD44, when they were induced to differentiation by adding serum, the expression of both markers were markedly decrease. We first discovered and identified CD44 as a useful colorectal cancer stem cell marker at home and abroad.3. Colorectal cancer stem cells possess infinite self-renewal and certain differentiation potential in vitro,and high expression of sternness gene Bmi-1 and Notch-1. In three-dimensional culture they can be differentiated into ?crypt-like? structure.4. Colorectal cancer stem cells are more tumorigenic than routine Colo205 cells.

Title: Experiment on Cultivation and Identification of Colorectal Cancer Stem Cell-derived Spheres in Vivo and in Vitro

Category: Tumor Biology

Filename: Experiment on Cultivation and Identification of Colorectal Cancer Stem Cell-derived Spheres in Vivo and in Vitro.pdf

Pages: 158

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