Abstract
摘要:肺癌與腫瘤相關基質/血管前驅(幹)細胞本子計畫的主旨是探討肺癌(幹)細胞和腫瘤相關基質前驅(幹)細胞之交互作用,本研究將利用多種人類肺癌細胞及基質環境缺陷之基因剔除小鼠為實驗樣本及動物模式,此動物是一內皮唾酸蛋白質(endosialin)基因剔除小鼠,內皮唾酸蛋白質是一種穿膜醣蛋白,又稱為腫瘤內皮細胞標記抗原1 (tumor endothelial marker 1/TEM1)或CD248。本研究將專注於探討肺癌和內皮唾酸蛋白質的關係,計畫之獨特性及重要性有四:第一,在肺癌產生過程中,腫瘤血管新生是重要的議題;第二,內皮唾酸蛋白質被認為是影響血管新生及腫瘤發展的一相當重要蛋白質,且內皮唾酸蛋白質具有高度腫瘤特異性。此論點是根據數個先前重要的研究而來,包括臨床樣本、細胞株及動物模式等;研究發現內皮唾酸蛋白質會大量表達於與腫瘤血管細胞相關之基質前驅(幹)細胞,而非一般組織內皮細胞或人類臍靜脈內皮細胞;且它亦會被專一性地誘導表達在癌症及肉瘤病人之基質內腫瘤相關(或活化的)纖維母細胞;但由於此研究僅包含5個肺癌樣品,樣本數太少,需更進一步探討才能釐清內皮唾酸蛋白質是否亦表達於腫瘤細胞中。第三,先前的細胞實驗結果亦提供一訊息傳遞路徑來解釋內皮唾酸蛋白質如何參與血管形成及血管增生,並證實內皮唾酸蛋白質可透過血小板衍生性生長因子(PDGF)受器之下游訊息傳遞路徑MAP kinase ERK-1/2來調控周細胞增生;而此內皮唾酸蛋白質對細胞具多效影響之機制在肺癌細胞則從未被探討。第四,本計畫將採用活體動物實驗的方式進行研究。本子計畫之研究目的有三:(1) 欲針對各種肺癌亞型、分期、臨床結果與預後來探討並證實內皮唾酸蛋白質之表達與肺癌的關係 (第一年~第二年) 先前的文獻曾指出內皮唾酸蛋白質之表達與肺癌呈強烈的負相關性,是支持此一研究目標的理由,但該項研究的缺點在於缺乏預後相關的研究以及樣本數過少(僅5個樣本)。因此我們預計至少蒐集20至30個樣本以分析每一種亞型,得到具有顯著統計意義的結論。此外,我們亦將確認內皮唾酸蛋白質以及PDGF是否會在原發性肺癌細胞與(/或)基質/血管前驅(幹)細胞表現,及內皮唾酸蛋白質之表達量是否可成為肺癌的預後指標。(2) 欲探討基質/血管前驅(幹)細胞在肺癌轉移機制的角色 (第一年~第三年) 先前文獻曾指出內皮唾酸蛋白質基因剔除小鼠在腫瘤相關的血管新生方面是有缺陷的(Nanda et al., 2006),利用knock-in技術,本計畫共同主持人林淑華教授已建立內皮唾酸蛋白質基因剔除/LacZ嵌入(簡稱為內皮唾酸蛋白質-LacZ嵌入)基因改造小鼠(剛發表於學術期刊Huang et al. 2011),經由與SCID小鼠交配,我們已產製出具免疫缺陷背景的內皮唾酸蛋白質-LacZ嵌入小鼠,這些小鼠將適用於進行人類肺癌細胞之異種移植。在和子計畫2 Dr.林泰元合作下,分離的人類肺癌幹細胞將經靜脈注射送入小鼠體內,而腫瘤細胞將會被攔截於肺臟組織,由於透過血管外滲來進行的癌轉移須仰賴區域性的蛋白酶作用與微血管增生,我們將可進一步剖析基質/血管前驅(幹)細胞在肺癌細胞轉移/血管新生的角色。(3) 欲探討血管新生在肺癌發展中的角色 (第一年~第三年) 本研究將利用小鼠進行實驗。我們將以內皮唾酸蛋白質-LacZ嵌入小鼠和LSL-K-ras 肺癌小鼠(loxp-Stop-loxp K-rasG12D mouse; 購自Jackson Lab)交配以產製LSL-K-ras/內皮唾酸蛋白質-LacZ嵌入小鼠。這個模式將適用於探討肺特異性基質/內皮前驅(幹)細胞在肺癌的原發性、剛發生時、進程發展以及血管新生等各階段的角色。
Abstract: Lung cancer and tumor associated stromal/vessel precursor (stem) cellsThe goal of this subproject is to understand the interaction between lung cancer (stem) cells and tumor associated stromal precursor (stem) cells. Our study will use human samples and knockout mouse models in which the stromal environment is defective. The model contained a targeted deletion of a molecule called endosialin, a transmembrane glycoprotein also known as tumor endothelial marker 1/TEMl or CD248). The rationale and uniqueness of focusing lung cancer on endosialin is the following. First, tumor angiogenesis is an important issue in lung cancer. Secondly, endosialin is proposed to be important for angiogenic neovascularization and tumor development and it is highly tumor specific. The hypothesis is based on clinical samples, cell lines and mouse models. Endosialin is preferentially expressed in stroma precursor (stem) cells associated with tumor vessels but not on HUVECs or endothelial cells of normal tissues. It is specifically induced on the tumor associated (activated) fibroblasts within the stroma of majority of human carcinoma and sarcoma samples, including lung cancer. But because only 5 lung cancer samples were recruited in one study, it needs to be further investigated. Third, a cell-based experiment has provided a signaling pathway to explain how endosialin is involved in tube formation and angiogenesis; endosialin regulates pericyte proliferation through PDGF receptor signaling to the MAP kinase ERK-1/2. This mechanism that endosialin exerts its pleiotropic cellular effects has never been investigated in lung cancer. Forth, we will take in vivo approaches. The specific aims of this subproject are the following.(1) To investigate and confirm endosialin expression in associated with lung cancer, focusing on tumor subtypes, stages, clinical outcomes, and prognostic marker (year 1~2). Several previous reports suggested a positive reverse correlation of endosialin expression with lung cancer which supports the rationale of this aim. The drawback is that there was no correlation of prognosis in these studies. We intend to study 20-30 samples for each subtype to derive statistically significant conclusion. Moreover, we will identify whether endosialin as well as PDGF is expressed in lung cancer cells in-situ and/or stromal/vessel precursor (stem) cells and whether endosialin expression is a prognostic marker.(2) To investigate the role of stroma/vessel stem cells in the metastasis of lung cancer (year 1~3). A previous study has demonstrated endosialin KO mice are defective in generating tumor associated neovascularization (Nanda et al., 2006). An endosialin KO/LacZ knock-in (abbreviated as endosialin-LacZ KI) mouse model has just been published by our Co-PI (Dr. Shu-Wha Lin, Huang et al. 2011). We have crossed the endosialin-LacZ KI mice to SCID mice. These mice are suitable for xenograft of clinical lung cancer samples. In collaboration with Dr. Ling Thai-Yen of subproject 2 to isolate human lung cancer stem cells, we will intravenously inject the cells to trap the cells in the lung. Metastasis of the cells by extravasation will be studied. This will allow us to dissect the role of stromal/vessel precursor (stem) cells in metastasis/angiogeneisis of lung cancer cells.(3) To investigate the role of neovascularization in the development of lung cancer (year 1~3). We will use mouse models in this study. We will breed endosialin-LacZ KI mice to a lung cancer model (loxp-Stop-loxp K-rasG12D or LSL-K-ras, Jackson Lab). The model will be suitable for investigating the lung specific stromal/endothelial precursor (stem) cells in the development of lung cancer in situ; the onset, progression, angiogenesis.
Keyword(s)
肺癌幹細胞
腫瘤相關基質/血管前驅(幹)細胞
內皮唾酸蛋白質基因剔除/LacZ
lung cancer stem cell
tumor associated stromal/vessel precursor (stem) cells