Cancer stem cells and metastatic colon cancer: Identification, characterization and phenotypic examination of tumor cells

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Colon cancer is a very common cancer, second only to lung cancer. Distant metastases are one of the worst prognostic signs as they place the patient in the most advanced staging category. Colon cancers, like most of the cancers, generally spread through the lymphatics or through the portal venous system to the liver. The liver is the most frequent visceral site of metastatic dissemination and is the initial site of distant spread in one-third of recurring colon cancers, with two-thirds of patients having liver involvement at the time of death. The median survival after the detection of distant metastases ranges from 6 to 9 months (with heavy liver involvement) to 24 to 30 months (with initially small liver nodules). It is believed that the successful metastatic cancer cell must leave the primary tumor, enter the lymphatic and blood circulation, survive within the circulation, overcome host defenses, extravasate and grow as a vascularized metastatic colony. We know that tumors shed large numbers of tumor cells, but less than 0.01% of these cells develop into metastasis. A metastatic tumor may be more efficient at producing metastasis because it has a higher proportion of the rare metastatic cells or because the average tumor cell in the colony has stronger metastatic fitness. The model of cancer stem cells may bring some answer to these long-standing questions about the metastatic process and its origin. Methods: We isolated tumor cells from metastatic colon cancer specimens obtained from patients. We optimized an in vitro culture assay to grow and generate tumor cell colonies. Morphologic and phenotypic analyses of the generated tumor cell populations were examined using four-color flow cytometry and immunohistochemistry. Different tumor cell populations were identified and isolated by cell sorting, and clonogenic cultures were successfully implemented. Results: We expanded in vitro tumor cells from isolated metastatic cancer cells and showed that these colonies differentiated into organoid bodies. Tumor cells are constituted by different cell morphologies indicating heterogeneous cell populations. Side population analysis and clonal expansion of tumor cells were carried out and confirmed the heterogeneity of the expanded tumor cell population. Transplantation of the tumor cell populations in immunodeficient animal models has been initiated. Conclusion: This preliminary study on metastatic colon cancer indicates phenotypic heterogeneity among tumor cells derived from metastatic colon cancer. We hypothesized that cancer initiating cells or cancer stem cells are responsible for this heterogeneity, which is at the origin of the metastatic process and that a hierarchy model can be developed.