摘要:癌幹細胞/癌源細胞(Cancer stem/initiating cells (CSIC))由於具有類似幹細胞的特性,包括具有自我更新和分化的能力,因此被認為可能造成腫瘤生成、抗藥性的產生、腫瘤再復發、轉移等,進而影響臨床預後。因此,現今許多證據指出培養癌幹細胞/癌源細胞是急切的; 並且認為研究癌幹細胞/癌源細胞的特性能夠提供診斷與治療癌症的新方法。相較於血液腫瘤而言,實體腫瘤(solid tumor) 的癌幹細胞/癌源細胞分離及培養較不易,尤其是肺癌。在此,本實驗室發展出一個新的體外培養肺癌幹細胞/癌源細胞的方法,可維持肺癌幹細胞/癌源細胞的幹細胞特性。相較於先前文獻大都以細胞表面標誌CD133+、side population (細胞排除Hoechst dye 的能力)、醛類去氫酶(aldehydedehydrogenase)活性等方法以分離癌幹細胞/癌源細胞;在本計畫中,我們利用肺腺癌病人自體來源的腫瘤周遭基質細胞做為培養幹細胞的供給細胞(feeder cell),藉此形成類似癌幹細胞在體內生長的微環境(microenvironment),以此維持肺癌幹細胞/癌源細胞的幹細胞特性。本實驗室初步結果發現利用該方法培養的肺癌幹細胞/癌源細胞,會表現Oct4+/Sox2+/Nanog+/Klf4+,並具有較高比例的side population,較高的醛類去氫酶活性,以及細胞群落形成(colony formation)能力;此外,該細胞也表現出較高的化學藥物抗性,且以50 到100 顆這種肺癌幹細胞/癌源細胞細胞進行orthotopic 或皮下注射的方式即可在免疫缺乏老鼠(NOD/SCID mice)模式中生成腫瘤,並具高度轉移能力;另外,此肺癌幹細胞/癌源細胞也具有分化成脂肪細胞和不同種類肺癌細胞的能力。本計劃中,我們將利用包括基因體學分析、微小核醣核酸表現、和細胞激素蛋白表現微陣列等分析肺癌前驅細胞,找出新穎的肺癌幹細胞/癌源細胞標誌,並將其應用在臨床檢體上,分析其和病人預後的相關性;此外,在此三年計畫中,我們將深入研究肺癌幹細胞/癌源細胞所造成的化學抗藥性和腫瘤細胞分化的相關機制,以及癌幹細胞/癌源細胞與其基質細胞交互作用的可能機制。本計畫的成果將極具應用性,一方面將藉由分析肺癌幹細胞/癌源細胞重要的新穎細胞標誌(CDX2, CDX3, and CDI-1)和訊息傳導路徑(TGF-, EGFR,and BMPs),期許能透過偵測癌幹細胞標誌開發出早期診斷惡性腫瘤的方法,或針對肺癌幹細胞的標靶藥物,應用在臨床肺癌發生的預防與治療上。
Abstract: Cancer stem/initiating cells (CSIC), with stem cell-like properties in self-renewal anddifferentiation, have been suggested to be responsible for carcinogenesis, drug resistance,tumor recurrent, and metastasis, these matters strongly affect the clinical outcome. Evidencesindicate that culture and characterize the CSIC become urgent and may be helpful to developnovel diagnostic and new therapeutic strategies to treat cancers. Compare to hematologiccancers, the solid tumor is relative difficult to isolate and culture the CSIC from clinicalsamples, especially in lung cancer. Here, we developed a new approach to culture andmaintain the stemness of the lung CSIC in vitro. Different from previous isolation methodsusing specific markers (CD133+) or efflux of Hoechst dye (side population) and aldehydedehydrogenase activity; in this project, we use autologous intramural tumor-associated stromacells as the feeder cells to create the microenvironment for culturing and maintaining thetumor sphere-like lung CSIC from patient with adenocarcinoma. Here, we show that thecultured lung CSIC (Oct4+/Sox2+/Nanog+/Klf4+ cells) with relative higher percentages of sidepopulation, ALDH activity, colony formation ability, and more resistance to chemotherapies,could generate adenocarcinoma with highly metastatic ability both in orthotropic andsubcutaneous models in severe combined immunodeficient (NOD/SCID) mice as less as 50 to100 cells. Also, the lung CSIC could be induced into adipogenesis and differentiate intodifferent types of lung cancer cells. Through transcriptomic analysis, microRNAs, andcytokine array approaches, the novel biomarkers of lung CSIC could be identified in suchcultured lung CSIC; and will be validated in clinical samples (isolating tumor CSIC fromtumor tissues and pleural effusion) and correlating to patients’outcome. Furthermore, themechanisms of drug-resistance, cancer stem cell differentiation, and microenvirmentalinteraction between lung CSIC and the feeder cells will also be studied in this three-yearproject. The results of this project should be very helpful to better understand the properties oflung cancer stem cells and through identifying the crucial markers (e.g., CDX2, CDX3, andCDI-1) and signaling pathways (TGF-, EGFR, and BMPs) on these CSIC should beapplicable for clinical applications, earlier detection of malignancies and better prediction oftumor behavior, and ultimately may lead to preventive or therapies for individuals at high riskof developing cancer via targeting on cancer stem cells.