| Background and Objection:Colon cancer is one of the three leading causes of cancer-related death among men and women worldwide, and survival is affected by local recurrence and lymphatic and hematogenous dissemination. This neoplasm frequently metastasizes to the lymph nodes at early stages of the disease. In advanced disease, most patients develop extra lymph node metastases, most often in the livers, lungs, and peritoneum. Knowledge of the factors involved in colon cancer metastasis is largely lacking. Currently, tumor growth and metastatic dissemination are thought to be a result of the deregulation of complex molecular machinery that leads to multiple phenomena, such as the resistance of tumor cells to apoptosis, tumor cell migration, tumor cell invasion, and tumor cell immune escape mechanisms. Recent data have suggested that chemokine receptors may direct lymphatic and hematogenous spread and may additionally influence the sites of metastatic progression of different tumors. The chemokine receptor CXCR4was initially described as a regulator of the homing of lymphocytes in inflammatory tissues. The natural ligand of CXCR4, the stromal cell-derived factor1(SDF-1), is highly expressed in tissues of metastatic growth, such as lung, liver, and lymph node, and attracts lymphocytes to these organs. CXCR4has been experimentally shown to be crucial for cancer cell adhesion and/or migration, suggesting the involvement of CXCR4in tumor invasion and metastasis. Numerous authors have reported on the involvement of the CXCR4/SDF-1axis in promoting a metastatic phenotype in tumors. For example, high CXCR4expression has been shown to be associated with lymph node metastases in breast cancer and oral squamous cell carcinoma.The accumulation of experimental evidence supporting the hypothesis that stem cells have a major role in cancer progression has grown in recent years. These so-called "cancer stem cells" have self-renewing abilities and are thought to drive tumor growth. Thus far, cancer stem cells have been identified in a great number of solid tumors. Initially identified in hematopoietic stem cells, CD133is now recognized as a cancer stem cell marker in multiple types of solid tumors, such as those in the brain, breast, lung, liver, colon, prostate, pancreatic carcinomas, medulloblastoma, and melanoma. Although the function of CD133is unknown, preliminary evidence has proposed that the expression of CD133is associated with the activation of a stemness-related signaling pathway, resistance to apoptosis and bioenergetic stress. Initially identified in hematopoietic stem cells, CD133is now recognized as a cancer stem cell marker in multiple types of solid tumors, such as those in the brain, breast, lung, liver, colon, prostate, pancreatic carcinomas, medulloblastoma, and melanoma. Consequently, the cancer stem cell compartment is increasingly being recognized as a necessary target for the effective treatment of cancers. Supporting data from in vitro and murine tumor models have underlined the key roles of CXCR4and CD133in tumor cell malignancy. However, no data are presently available regarding the co-expression of the two proteins in human colon cancer and the impact of these proteins on disease progression and prognosis. Therefore, we evaluated the expression of CXCR4and CD133in colon cancer specimens and correlated the results with the patients’ clinicopathologic parameters and survival.The present study was aimed to examine the expression of CXCR4and CD133protein in stage Ⅱ or Ⅲ colon cancer and related lymph nodes, and to investigate the clinical and prognostic significance in colon cancer. Immunohistochemistry was performed to examine CXCR4and CD133protein expression in paraffin-embedded stage Ⅱ or Ⅲ primary colon cancer tissues and matched lymph nodes. Furthermore, we also selected four colon cancer cell lines to analysis the expression of CXCR4and CD133, and analysis the cell migrating ability to SDF-1by transwell migration assay in vitro.Methods and materials:One hundred and twenty-five pathologically confirmed specimens were obtained from colon cancer patients with TNM stage Ⅱ or Ⅲ tumors that were subjected to radical resection between January,2001and July,2005in The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China. None of the patients had undergone either chemotherapy or radiotherapy before the collection of the samples. Postoperative therapeutic strategies were applied according to the stage of the disease and the presumed risk of relapse. Immunohistochemistry was performed to examine CXCR4and CD133protein expression in paraffin-embedded stage Ⅱ or Ⅲ primary colon cancer tissues and matched lymph nodes. The specimens were evaluated by pathological doctors who had no knowledge of prognosis and other clinicopathological variables. The correlation between the CXCR4and CD133proteins expression and clinicopathological parameters and the patients’5-year survival was analyzed. We selected four colon cancer cell lines SW480, SW620, HT-29and lovo cell lines, to analysis their expression of CXCR4and CD133preteins. Western blot analysis and flow cytometry were used to analyze the expression of CXCR4and CD133preteins in SW480, SW620, HT-29and lovo cells. The invasion potential of SW480, SW620, HT-29and lovo cells was evaluated by transwell invasion assay in vitro.Statistical analysis:Associations between immunohistochemical scores and clinicopathologic variables of tissue specimens were evaluated by the χ2test. When the subset sample size was small, the corresponding test was performed using Fisher’s exact test. Univariate analysis was performed using the log-rank test. Kaplan-Meier curves were used to estimate the contributions of clinicopathological characteristics to survival. All analyses were performed using the Statistical Package for Social Sciences (SPSS) software, version13.0, for Windows (SPSS Inc., Chicago, IL, USA). Statistical significance in this study was defined as P<0.05. All reported P values are two-sided.Results:Paratumorous normal colon epithelium could be found in102of the125specimens used for this study. Expression of CXCR4in the colon epithelium was found in only11of the102specimens, and expression of CD133in the colon epithelium was found in only8of the102specimens. Seventy-four (59.2%) of the125colon cancer tissues were positive for CXCR4expression. The staining for CXCR4revealed a predominantly cytoplasmic, and in few specimens, an additional weak membranous location of CXCR4. Nuclear staining of CXCR4was not observed. CD133expression was detected in45of the125tumors (36.0%). CD133expression in colon cancer was polar, confined to the apical luminal surface of colon cancer cells with glandular differentiation. Only tumor cells with direct contact with these luminal surfaces were CD133+. Several CD133+tumor cells were usually grouped together, with some glands being completely positive. Among the125stage II or III colon cancer patients, no significant correlation existed between CXCR4or CD133expression and gender, age, location of primary mass, pathological grade, tumor size, AJCC stage, tumor invasion or lymph node status.By combining the expression of CXCR4and CD133, we obtained the following four combinations:CXCR4positive and CD133positive, CXCR4positive and CD133negative, CXCR4negative and CD133positive, and CXCR4negative and CD133negative. Notably, co-expression (both CXCR4and CD133were positve) was detected in29of the125tumors (23.2%), and compared with the other combinations, co-expression of CXCR4and CD133proteins was significantly associated with the AJCC stage (P=0.029) and the lymph node status (P=0.020).Sixty-four stage III colon cancer cases showed metastasis to their related lymph nodes. Among these primary cancer cases, forty-one (64.1%) displayed strong expression of CXCR4. Forty-three (67.2%) of the metastasized tumors in lymph nodes (MTLNs) showed strong expression of CXCR4. Twenty-seven (42.2%) of the64stage III colon cancer cases were positive for CD133expression, but only fifteen (23.4%) of the MTLNs were positive for CD133expression. MTLNs are significantly more likely to show decreased CD133expression. Upon combining the expression of CXCR4and CD133, co-expression of CXCR4and CD133proteins was significantly different between the primary cancer and MTLNs. Notably, the primary cancers revealed strong co-expression of CXCR4and CD133proteins (31.3%), whereas in MTLNs, co-expression was observed in only14.1%of cases (Table4; P=0.020).All125patients were followed-up for survival to assess CXCR4and CD133expression as a prognostic factor. The median follow-up period was6.5years. Of the125patients,36died during the follow-up period, and3cases losted during the follow-up period. Log-rank statistics and multivariate survival analysis showed that AJCC stage, lymph node status, CXCR4expression, CD133expression, and co-expression of CXCR4and CD133proteins were significant prognostic indicators for overall patient survival. Kaplan-Meier survival curves were constructed to assess the prognostic significance of co-expression of CXCR4and CD133proteins, and colon cancer patients with co-expression of CXCR4and CD133proteins tended to have worse outcomes than those with other combinations. Of the patients with co-expression of CXCR4and CD133proteins, the5-year survival rate was only51%; in contrast, the survival rate was77%in patients with tumors that expressed the other combinations (P=0.005).Levels of total cellular CXCR4or CD133protein in SW480, SW620, HT-29and lovo cells was determined by Western blotting. The results demonstrated that the CXCR4protein in SW480, SW620and HT-29cells were expressed in a higher level, and the CXCR4expression level was significantly lower than the above three cells in lovo cells. CD133protein was also expressed in the four colon cancer cell lines. The expression of CD133protein in SW480cells was the strongest one in the four colon cancer cell lines, which were weaker expression in SW620and HT-29cells. And the expression of CD133protein was weakest in lovo cells. The expressions of CXCR4or CD133protein in SW480, SW620, HT-29and lovo cells ware analyzed by flow cytometry. The results shown that in the selected four cell lines, SW480cell has the highest percentage of both CXCR4and CD133positive cells, the proportion of both CXCR4and CD133positive cells in which was39%. The proportion of both CXCR4and CD133positive cells in SW620cell was26%. The proportion of both CXCR4and CD133positive cells in HT-29cell was23%. The proportion of both CXCR4and CD133positive cells in lovo cell was only11%, which was the lowest proportion in the selected four colon cancer cell lines. The invasion potential of SW480, SW620, HT-29and lovo cells was evaluated by transwell invasion assay in vitro. The transwell invasion results shown that the number of cells invaded out the transwell room of SW480cells was the largest in the selected four cell lines, and the average number of cells per high power field was82.4±8.5. The number of cells invaded out the transwell room of SW620and HT-29cells was lower than that of SW480cells, and the average numbers of cells per high power field of SW620and HT-29cell were50.4±8.96and44.4±8.72respectively. The number of cells invaded out the transwell room of lovo cells was the lowest in the selected four cell lines, and the average number of cells per high power field was23.5±6.4.Conclusion:Among the125stage II or III colon cancer patients, no significant correlation existed between CXCR4or CD133expression and clinicopathologic variables. Co-expression of CXCR4and CD133proteins was significantly associated with the AJCC stage and the lymph node status. Log-rank statistics showed that co-expression of CXCR4and CD133proteins were significant prognostic indicators for overall patient survival. Of the patients with co-expression of CXCR4and CD133proteins, the5-year survival rate was significantly decreased. The expressions of CXCR4and CD133proteins were significantly different in the selected four colon cancer cell lines, which affected the migration ability of tumor cells to SDF-1in vitro. The transwell invasion results shown that the migration ability of tumor cells to SDF-1in vitro was concerned with the proportion of both CXCR4and CD133positive cells in tumor cells. The more proportion of both CXCR4and CD133positive cells in tumor cells, the higher migration ability of tumor cells will be.We have shown that co-expression of CXCR4and CD133proteins was a risk factor for worse overall survival in stage Ⅱ or Ⅲ colon cancer patients, and the colon cancer cells which was both CXCR4and CD133positive shows higher migration ability to SDF-1in vitro. These results indicate that co-expression of CXCR4and CD133proteins might contribute to the progression of colon cancer. |
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