||Tumors contain a small subpopulation of cancer-initiating cells, known as cancer stem cells (CSCs), which exhibit a self-renewing capacity and are responsible for tumor generation. CSCs are reputed not to be typical cancer cells, and they may persist in tumors as a distinct population, causing relapse and metastasis by giving rise to new tumors. The first evidence for CSCs was reported in 1997 in a study that an isolated subpopulation of leukemic cells that expressed a specific surface marker, CD34, but lacked the CD38 marker; established that the CD34+/CD38- subpopulation was capable of initiating tumors in non-obese diabetic/severe combined immunodeficiency (SCID) mice and that these tumors were histologically similar to the primary leukemic tumors.|
CSCs can form tumors and it has been suggested that CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. The hypothesis that stem cells drive tumorigenesis in colon cancer raises the question of whether current anticancer therapies can efficiently target the tumorigenic cell population that is responsible for tumor growth and metastasis since current therapies mostly fail to eradicate CSC clones and instead favor expansion of the CSC pool and/or select for drug-resistant CSC clones. The isolation and characterization of tumorigenic colon CSCs should enable the development of novel diagnostic and therapeutic procedures.
Specific surface markers for colon CSCs have been reported, and CD133 is the most studied surface marker for colon CSCs. Using CD133 to identify and confirm expansion of human colon CSCs has been reported. CD133+ colon cancer cells injected subcutaneously readily generated a tumor in SCID mice, whereas CD133- cells did not form tumors but with controversial results.
Using a knock-in lacZ reporter mouse (CD133lacz/+) in which the expression of lacZ was driven by the endogenous CD133 promoters, CD133 expression in the colon was found not to be restricted to stem cells alone; CD133 was ubiquitously expressed on differentiated colonic epithelia. An examination of CD133 expression did not reveal the entire population of CSCs in human metastatic colon cancer; both CD133+ and CD133- metastatic tumor subpopulations were capable of long-term tumorigenesis in a non-obese diabetic/SCID xenotransplantation model.
Other colon CSC markers have been proposed including epithelial specific antigen (EpCAM, BerEp4; cell adhesion molecule), CD44 (CDW44; cell adhesion molecule, hyaluronic acid receptor), CD166 (ALCAM; cell adhesion molecule), Msi-1 (Musashi-1; RNA-binding protein), CD29 (integrin β1; cell adhesion molecule), CD24 (HSA; cell adhesion molecule), Lgr5 (GPR49; Wnt targeting gene), and ALDH-1 (ALDC; enzyme). Exact and reliable surface markers of colon CSCs, however, have not yet been identified. Presently, the only reliable method for identifying and quantifying CSCs is to observe tumor formation in a serial xenotransplantation model.
It is generally accepted that CSCs express active transmembrane ATP-binding cassette (ABC) transporter family members, such as the multidrug-resistant transporter 1 and ABC sub-family G member 2 (ABCG2),7 which render them drug resistant. Drug-resistant cells from human colorectal adenocarcinoma tumors produced two orders higher than normal levels of carcinoembryonic antigen (CEA) per cell. Only 1% of cells treated with acetylsalicylic acid (aspirin) in their culture medium survived, compared with cells grown in the normal expansion medium. This raised questions about whether the drug-resistant colorectal cells, which are increased by adding anticancer drugs into the culture medium, might be CSCs; if so, this method might be the simplest isolation method for CSCs. It will also be important to determine which anticancer drugs or chemotherapy treatments can efficiently deplete CSCs when colon cancer cells are subcutaneously xenotransplanted into mice after the cells have been treated with anticancer drugs.
In this paper, the higher levels of CEA secreted by the LoVo colon carcinoma cell line, which was cultured in serum-free and serum-containing media containing anticancer drugs will be evaluated. Drug-resistant LoVo cells were analyzed to determine whether those cells had CSC characteristics, e.g., small size of the cells/colonosphere and strong expression of CSC surface markers, as indicated by flow cytometry and immunohistochemistry analysis. Finally, in vivo tumorigenesis was examined by subcutaneously xenotrans- planting the isolated drug-resistant LoVo cells into mice. Drug-resistant cells isolated in this study were CSCs will be evaluated.
In conclusion the production of CEA by LoVo cells can be stimulated by the addition of anticancer drugs as well as aspirin in both serum-free and serum-containing media. CSCs are believed to be drug resistant cells; however, although the drug-resistant subpopulation of LoVo colon cancer cells, which were isolated by the addition of anticancer drugs to the culture medium, could stimulate the production of CEA in both serum-free and serum-containing media, these cells did not act as CSCs in in vivo tumor generation experiments. In the clinic, a CEA surge or flare has been observed as an early biochemical phenomenon in metastatic colorectal cancer during chemotherapy in approximately 10% of the patients who experience a clinical benefit. In light of this clinical evidence, we speculate that drug-resistant can?cer cells are not CSCs because patients with CEA surges experience a clinical benefit, which is inconsistent with the CSC theory that predicts that these patients would have a worse prognosis.
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