| 摘要: | 腫瘤細胞裡包含了一小群亞型的腫瘤起始細胞被稱為癌症幹細胞，這群細胞可以自我更新以及具備增生的能力。癌症幹細胞被廣泛認為不是一種典型的癌症細胞，會以不同的族群持續的存在腫瘤裡，導致復發以及轉移而生成新的腫瘤。在1997年的一個研究中癌症幹細胞首次被證實出來，有一亞型的白血球細胞會特別表現一個細胞表面標記CD34但不會表現CD38的標記，因此而建立了CD34+/CD38-的亞群，且這一亞群癌細胞可在免疫缺陷的老鼠裡引發腫瘤。其腫瘤細胞在組織學上亦與最原先的白血球細胞非常相似。
曾有研究提過癌症幹細胞在大腸癌裡可驅使腫瘤的生成，這一假說質疑了現有的抗癌藥物療效因為現有的抗癌藥物只針對癌細胞毒殺，並且容易使細胞產生抗藥性，而這些抗藥性的細胞通常都被認為是癌症幹細胞。如能分離出這些癌症幹細胞以及去瞭解其特性就可開發新穎的診斷及治療的步驟。
CD133已被證實為大腸癌幹細胞特有的細胞表面標記，因此有研究利用CD133分離出大腸癌裡的癌症幹細胞。當細胞表面標記CD133+的癌幹細胞注入免疫缺乏老鼠的皮下時，這一群的細胞很容易就生成新的腫瘤，而細胞表面標記CD133-的細胞則否。但這發現是有爭議性的。
為了探討CD133的表態，利用建立的lacZ基因崁入小鼠(CD133lacZ/+)驅使lacZ受內生性的CD133 啓動子表現，發現CD133的表態不只是僅限於大腸癌裡的癌幹細胞，CD133也表現於已分化的大腸上皮細胞。如此只針對CD133表態分離出大腸癌裡所有的幹細胞是有爭議的，因為不是所有的癌幹細胞都有CD133表態。不管是CD133+或是CD133-的細胞注入免疫缺乏的老鼠裡皆可產生新的腫瘤。
除了CD133，另外有被發現的大腸癌幹細胞表面標記有上皮特定抗原(EpCAM, BerEp4; 細胞粘附分子), CD44 (CDW44;細胞粘附分子;玻尿酸接受體), CD166 (ALCAM;細胞粘附分子), Msi-1 (Musashi-1; RNA結合蛋白), CD29 (組合蛋白beta1;細胞粘合分子), CD24 (HAS;細胞粘合分子), Lgr5 (GPR49;Wnt 標靶基因)以及ALDH-1 (ALDC;酵素)。無論如何，確定的大腸癌幹細胞的表面標記並未完全被認定。近期的實驗技術裡，如要有效地分離出癌症幹細胞仍需靠動物模式觀察新的腫瘤的生成。
癌症幹細胞會表態ABC運輸蛋白家族是普遍的被接受，譬如ABCG2蛋白(multidrug-resistant transporter 1 與 ABC sub-family G member 2)，而這些蛋白賦予腫瘤細胞抗藥的能力。在人類的大腸癌腫瘤中有抗藥性的細胞亦可以產生遠高於正常的癌症胚胎抗原(CEA)。我們發現當加了阿斯匹靈的培養基與沒有加的培養基相比時，含阿斯匹靈培養基中只有1% 的細胞會存活。指出了，培養基加入抗癌藥物後產生抗藥的大腸癌細胞有可能是大腸癌幹細胞， 或許是一種分離大腸癌幹細胞的方法。
在這文章中，我們將會探討培養在無血清或有血清的培養基中的大腸癌LoVo細胞其所分泌的CEA是否會被加入的抗癌藥物所影響。利用流式細胞儀以及細胞免疫染色分離，檢驗這些抗藥性的大腸癌LoVo細胞是否具有癌幹細胞的特徵如細胞較小，癌症次球體(colonosphere)的產生以及表現大腸癌幹細胞特有的表面標記)。最後，在體內實驗裡，我們會將這些有抗藥的大腸癌LoVo細胞注入免疫缺乏小鼠的皮下後再觀察新腫瘤的生成。
不管是在沒有血清或有血清的培養基裡，大腸癌LoVo細胞所產出的CEA量可被抗癌藥物以及阿斯匹靈刺激分泌。癌症幹細胞已被相信擁有抗藥的特性，然而在我們小鼠體內的實驗裡這些相信擁有抗藥的特性癌幹細胞並沒有生成的新腫瘤。在臨床上， 有10％的早期大腸癌轉移的病人在接受化癌藥物治療時，CEA 升高(CEA surge或CEA flare)的產生被認為是對治療是有效的。在臨床上我們一佐證了抗癌藥物的癌細胞並不是癌症幹細胞，不是大眾所認為因為癌幹細胞的存在會產生更負面的治療效果。
;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. |
