The Cell Cycle Of Cancer Stem Cells And Its Pathological Significance In Colorectal Carcinoma

PurposeTo investigate the relationship between the expressions of CD44, PCNA (S-phase-marker), P27 and their clinic-pathologic significance in primary colorectal carcinoma, and to explore the state of proliferation and cell cycle of CD44+ colorectal cancer stem cells, furtherly to examine the quantity, distribution, morphological characteristics of CD44+/PCNA-, CD44+/P27high colorectal cancer stem cells (Co-CSCs), and to provide more evidence to colorectal cancer stem cells; To investigate the expressions of CD44, PCNA and P27 in human well-differentiated colorectal carcinoma cell line (SW480), and to analyse the cell cycle and the morphologic features of SW480; To compare the difference between the expression of CD44 and hMSH2 and to explore the genesis of CD44+ colorectal cancer stem cells.Methods61 cases of human colorectal carcinoma,10 cases of normal mucosa and 18 cases of adenoma with atypical hyperplasia were collected and made into three tissue microarrays each containing 42 dots. Expressions of CD44, PCNA, P27 proteins were detected by SP (streptavidin HRP) immunohistochemical staining to these tissue microarrays. The location, quantity and distribution of CD44+/PCNA-, CD44+/P27high and CD44+/hMSH2+ cells were detected by double immunohistochemical staining. The expressions of CD44, PCNA and P27 proteins of human well-differentiated colorectal carcinoma cell line (SW480) were detected by SP immunohistochemical staining to analyze the cell cycle and its morphologic features of SW480.Results1. In colorectal carcinoma, the rate of CD44+ tumor cells was about 0.1～55% (30.9±19.29). There were two kinds of morphology of CD44+ tumor cells:one was bigger vacuolus nucleus, chromatin margination, clear nuclear membrane and nucleolus. Another one was smaller nucleus, chromatin dense. The expressions of CD44 in colorectal carcinoma were significantly higher than in normal mucosa and adenoma with atypical hyperplasia. Significant relationship was not found among the expression of CD44 and lymphatica metastasis, pathological grade and Dukes stage (P >0.05).2. In colorectal carcinoma, the rate of PCNA+ tumor cells was about 85-98% (93±4.21). The expressions of PCNA in colorectal carcinoma were significantly higher than in normal mucosa and adenoma with atypical hyperplasia. There was no significant relationship among the expression of CD44 and lymphatical metastasis, pathological grade and Dukes stage (P>0.05).3. In colorectal carcinoma, the amount of PCNA+ tumor cells was remarkably more than CD44+. More than 90% of CD44+ tumor cells were PCNA+, but just 0.1～6%(3.38±2.08) showed PCNA- and the CD44+/PCNA- was very few and with smaller nucleus and dense chromatin. CD44+/PCNA- was as spot or focus in all kinds of colorectal carcinoma4. In colorectal carcinoma, the rate of P27+ tumor cells was accounted for 0.1～35%(20.9±12.21) in all tumor cells. The expressions of P27 in colorectal carcinoma were significantly lower than in normal mucosa and adenoma with atypical hyperplasia. Significant relationship was not found among the expression of P27 and lymphatical metastasis, pathological grade and Dukes stage (P>0.05). The expression of P27 was not uniformity in a colorectal carcinoma tissue and the strong staining cells (P27high) showed diffuse distribution or focus which accounted about 0.1～15% (9.79±4.68) of the total cancer cells.5. In colorectal carcinoma, the amount of CD44+/P27high cells of CD44+ was about 0.1～5%(3.12±1.18) and showed spot or focus distribution in all kinds of colorectal carcinoma.6. There were two kinds of morphology of SW480 tumor cells:one was large-polygon with abundant cytoplasm and bigger irregular nucleus, rough chromatin; another one was small-round with few cytoplasm and maller nucleus, chromatin dense. The rates of CD44, P27 and PCNA in colorectal carcinoma were about 65.80±5.26%, 31.28±5.14% and 55±6.57%. Few of CD44+/P27+ and CD44+/PCNA- cells in G0/G1 phase were observed in SW480 tumor cells. The rate of CD44+/P27+ and CD44+/ PCNA- were about 8±3.51% and 11±5.36% respectively. 7. In colorectal carcinoma, CD44 was showed diffused or focus expression and the human mismatch repair gene (hMSH2) was reported as a marker of stem cells and expressed widespread. Significant relationship was not found among the expression of CD44, hMSH2 and lymphatica metastasis, pathological grade and Dukes stage (P >0.05), and all of CD44 positive cells showed positive hMSH2, but part of hMSH2 positive cells showed positive for CD44.Conclusion1. In colorectal carcinoma, most of CD44+ tumor cells are expressed PCNA and in S-phase, only few of CD44+ tumor cells are negative for PCNA and in G0/G1 phase.2. In colorectal carcinoma, just few of CD44+ tumor cells are strongly expressed P27 (P27high), it may be staying in G0/G1 phase.3. In colorectal carcinoma, CD44+/PCNA- and CD44+/P27high tumor cells show the same morphology features and stay in the same phase of cell cycle, both CD44+/PCNA- and CD44+/P27high may be stay in G0/G1 phase and can be regarded as cancer stem cell and are effective markers of cancer stem cells.4. In the human well-differentiated colorectal carcinoma cell line (SW480), the expressions of CD44, P27 and PCNA are presented in different degree. The quantity of CD44+/PCNA- and CD44+/P27high cells staying in G0/G1 phase are obviously more than solid tumor, and have a similar morphology with solid tumor.5. In colorectal carcinoma, CD44+cancer stem cells have the same phenotype with normal stem cells. It suggests that the CSCs are originated from normal stem cells possibly.