臺灣博碩士論文加值系統

肺癌在世界上是最常發生的惡性腫瘤，平均五年存活率低於14%，在台灣，每年有八千餘人死於肺癌，為惡性腫瘤造成死亡原因的首位。臨床上，肺癌因為早期診斷困難，化學藥物治療容易出現抗藥性，造成癌症復發和轉移的現象。肺臟細胞型態多元，而誘發肺癌發生的危險因子眾多，包括吸菸、氡氣暴露、石棉接觸等等，其中肺臟幹源細胞(stem/progenitor cells, PSCs)被認為是肺臟發生癌化的起源，我們認為肺臟幹源細胞是探討肺臟癌化過程重要的研究平台。腫瘤誘發細胞(cancer initiating cells, CICs)又稱作癌症幹細胞，負責腫瘤組織的生長並於化學治療時展現抗藥性，與癌症的惡性行為具有高度相關，因此針對腫瘤誘發細胞發展標靶策略是重要的治療方針，標的腫瘤誘發細胞以表面抗原辨識為主，例如肺癌的腫瘤誘發細胞利用CD133、CD117或ALDH活性進行辨識分離，目前腫瘤誘發細胞的研究平台是癌症細胞株和臨床腫瘤組織，然而腫瘤誘發細胞在這些細胞或組織中比例低不易分離，且分離後其細胞特性難以維持，因此對於腫瘤誘發細胞的研究仍有許多爭議。過去研究指出，Oct-4異常表現與腫瘤誘發細胞的行為特性有關，臨床上，Oct-4高度表現的肺癌病患常對傳統化學治療出現抗藥性，且病患平均存活時間較短。目前實驗室以coxsackievirus/adenovirus receptor (CAR)作為陽性標記成功分離出小鼠肺臟幹源細胞(CAR+ mouse pulmonary stem/progenitor cells, CAR+/mPSCs)，本研究利用逆轉病毒轉植，使Oct-4高度表達於CAR+/mPSCs，發現其誘導形成細胞群落，並建立出CAR+/mPSCsOct-4_hi細胞株，包括C1，E9，和C7; CAR+/mPSCsOct-4_hi細胞週期分析發現，S期和G2/M期的細胞分佈比例明顯上升，並且CAR+/mPSCsOct-4_hi細胞展現繼代培養能力及端粒酶(telomerase)活性; 進一步利用體內異種移植腫瘤試驗(in vivo xenograft tumor assay)證明，Oct-4高度表達誘導CAR+/mPSCs走向癌化，並展現高度肺癌腫瘤誘發細胞的行為，包括腫瘤再生和轉移的能力; 分析肺癌腫瘤誘發細胞特性，發現CAR+/mPSCsOct-4_hi細胞具CD133表現和ALDH活性，並且展現化療藥物(Cisplatin和Palitaxel)處理的耐受性; 利用次世代定序技術分析基因表現，發現血管新生調控機制在CAR+/mPSCsOct-4_hi細胞中高度活化，其中CAR+/mPSCsOct-4_hi細胞展現豐富的促血管新生因子表達並誘導內皮細胞(SVEC4-10)建立管狀結構(tube formation)，同時在雞胚尿囊膜試驗(chorioallantonicmembrane inoculation，CAM assay)中也發現CAR+/mPSCsOct-4_hi細胞促進血管形成，而在CAR+/mPSCsOct-4_hi腫瘤組織中，利用組織免疫染色，發現內皮細胞(CD31+)所佔比例較高; 利用共軛焦顯微鏡觀察CAR+/mPSCsOct-4_hi細胞與內皮細胞的交互作用，發現CAR+/mPSCsOct-4_hi細胞會吸引內皮細胞匯集並建立出血管脈絡，以綠色螢光蛋白(GFP)標記CAR+/mPSCsOct-4_hi C1細胞株，利用體內異種移植腫瘤試驗形成的腫瘤組織，發現內皮細胞(CD31+)中有18%為GFP陽性表現; 進一步分析蛋白質表現，CAR+/mPSCsOct-4_hi細胞中Angiopoietins/Tie2訊息高度活化，而Tie2抑制劑處理有效降低CAR+/mPSCsOct-4_hi細胞誘導血管形成的行為，並抑制CAR+/mPSCsOct-4_hi細胞形成腫瘤。綜合以上結論，異常Oct-4高度表現誘導小鼠肺臟幹細胞走向癌化，並伴隨高度癌症誘發細胞特徵表現，此外，Oct-4高度表達的CAR+/mPSCsOct-4_hi細胞具有誘發血管新生的能力，並且會直接參與血管形成，其中機制可能與Angiopoietins/Tie2訊息活化有關。腫瘤誘發細胞的標靶策略是治療癌症的重要方針，本研究提供一個新的腫瘤誘發細胞平台，將有助於肺癌治療策略的發展，此外，本研究發現腫瘤誘發細胞可能經由不同於內皮細胞的訊息傳遞，直接參與腫瘤血管新生行為，期待這項研究成果有助於未來肺癌疾病的診斷與治療。

Lung cancer is the leading cause of cancer-related mortality worldwide. The overall 5-year survival rate is less than 14% and most cases are diagnosed in the late stages of the disease, which tend to exhibit chemotherapy resistance, tumor recurrence, and metastasis, resulting in poor prognoses. A number of factors may increase the risk of lung cancer, such as smoking, radon exposure, and asbestos inhalation. The lung is a highly complex organ comprised of a diversity of cell types. Recent studies suggest that stem/progenitor cells are susceptible to tumorigenic force. Thus, lung stem/progenitor cells are an important model for exploring the tumorigenesis process. Cancer initiating cells (CICs), also called cancer stem cells (CSCs), are responsible for chemotherapy resistance, tumor recurrence, and metastasis. Currently, the identification of CICs is highly dependent on surface markers. For example, lung cancer CICs are identified by CD133, CD117, or ALDH activity. However, through surface marker identification, studies have yielded conflicting results on CICs in different types of cancer. Clues to more effective treatments targeting tumorigenic cells could be developed with an accelerated understanding of tumor biology aided by characterization of CICs. Oct-4 is one of the most important transcription factors for the pluripotency of stem cells. Meanwhile, an increasing number of studies have demonstrated that aberrant expression of Oct-4 can contribute to CICs-like behavior in various somatic cancers. In previous studies, we have reported on the enrichment of mouse pulmonary stem/progenitor cells (mPSCs) by serum-free primary selection culture followed by FACS isolation using the coxsackievirus/adenovirus receptor (CAR) as the positive selection marker. In the current study, we demonstrated that overexpression of the pluripotent transcription factor Oct-4 is sufficient to induce CAR+/mPSCs transformation, the result of which we name CAR+/mPSCsOct-4_hi (C1, E9, and C7 clones). CAR+/mPSCsOct-4_hi clones exhibit a progressive cell cycle, proliferation potential, and telomerase activity. With a xenograft tumor formation assay, CAR+/mPSCsOct-4_hi clones showed cancer initiating and metastasis capacity. In further validating their CICs-like abilities, CAR+/mPSCsOct-4_hi clones express CD133, ALDH activity, as well as chemoresistance potential. Next generation sequencing (NGS) analysis of CAR+/mPSCsOct-4_hi clones revealed that angiogenesis-associated mechanisms were highly activated. CAR+/mPSCsOct-4_hi clones show remarkable expression of certain proangiogenic factors, including angiopoietins (ANGs) and VEGF, and enhanced angiogenic potential. Moreover, CAR+/mPSCsOct-4_hi actively participated in tumor blood vessel formation and triggered a novel angiogenic mechanism, the angiopoietins/Tie2 signaling pathway. The present study indicates that by overexpressing Oct-4 at high levels in a subset of pulmonary stem/progenitor cells, CAR+/mPSCs, can transform the cells such that they exhibit tumorigenic potential and CICs-like properties. The transformed cells not only showed enhanced angiogenic potential, but also actively participated in tumor blood vessel formation and activated the ANGs/Tie2 signaling pathway. This study provides critical evidence supporting a possible origin of CICs and helps elucidate the pathways responsible for CICs-mediated blood vessel formation.

目錄
口試委員會審定書------------------------------------------I
謝誌---------------------------------------------------II
中文摘要------------------------------------------------IV
ABSTRACT-----------------------------------------------VI

1. INTRODUCTION-----------------------------------------1
2. MATERIALS AND METHODS--------------------------------9
3. RESULTS---------------------------------------------21
4. DISCUSSION------------------------------------------29
5. CONCLUSION------------------------------------------38
6. FURTHER WORK----------------------------------------39
7. REFERENCES------------------------------------------45
8. FIGURES---------------------------------------------52
9. TABLES----------------------------------------------82
10. PUBLICATIONS---------------------------------------87

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