白血病小鼠動物模式的腫瘤發生機轉和治療研發的研究

Abstract

白血病是發生在白血球的癌化現象，有別於其他的腫瘤，血癌細胞隨著血流遍佈全身不易成為治療標的，而且容易造成癌細胞轉移（metastasis）到其他組織器官，使得臨床上治療較為困難。 RL ♂1 是一株T 細胞淋巴癌（T cell leukemia），會表現CD4、CD25、IL-10及TGF-β，篩選自放射線誘發癌症的BALB/c小鼠，我們發現 RL ♂1 會表現Foxp3，並具有許多特性和調節性T細胞（regulatory T cell, Treg cells）類似， 本論文利用 RL ♂1 細胞株建立白血病的小鼠動物模式，來研究探討白血病生成的可能機轉，並試圖從已知的致病機轉中找出可行的輔助治療方式。 第一部份的研究中，以RL ♂1 的細胞碎片予樹突細胞（dendritic cells，DCs）吞食後，再利用腺病毒載體（adenoviral vector），將介白質12（interleukin 12，IL-12）送入樹突細胞中，來改善以樹突細胞為基礎的腫瘤免疫療法。 將同時呈現腫瘤抗原和表現介白質12的樹突細胞， 打入白血病小鼠體內發現，第40天以後的存活率仍有75%，優於只用腫瘤細胞碎片或是介白質12改造的樹突細胞治療的對照組，存活率分別為33%及50%。體外細胞實驗（in vitro）證實， 腫瘤抗原和介白質12改造過的樹突細胞治療的腫瘤鼠所誘發的毒殺型T細胞的活性高於對照組，利用抗體將CD8+ T 細胞去除之後，並看不到此樹突細胞所誘發前述的保護效果，實驗結果說明了， 同時呈現腫瘤抗原和表現介白質12的樹突細胞能誘發較佳的抗腫瘤免疫反應，同時也優於傳統的以樹突細胞為基礎的免疫療法。許多實驗證實，樹突細胞會誘發自體免疫反應，我們的實驗結果也發現，以樹突細胞誘發出抗腫瘤的效應，同時也會產生高量的抗雙股螺旋去氧核糖核酸抗體（anti-ds DNA antibody）。流式細胞儀分析中發現，抗雙股螺旋去氧核糖核酸單株抗體（monoclonal antibody），能夠辨認腫瘤細胞的表面分子，體外實驗也發現，此抗體有能力透過補體反應將腫瘤細胞摧毀。直接用此單株抗體打入腫瘤小鼠體內，也可觀察到腫瘤的生長速率有意義地變慢。 本實驗結果認為，自體抗體可達到抑制腫瘤的效果，而自體抗體結合腫瘤細胞上的哪個分子，以及是否會引發其他的反應，則須進一步的研究。 本論文的第二部份在探討淋巴癌細胞在體內生成的可能原因，以及從可能的生成機制中尋找適當的治療方式 。實驗中發現， 表現Foxp3的RL ♂1 T細胞淋巴癌在體外培養幾代之後，失去了表現Foxp3的能力，也無法在正常的老鼠皮下生成腫瘤。體外實驗發現，失去表現Foxp3能力的腫瘤細胞，同時也失去了抑制T細胞分裂的能力，有趣的是，這些細胞在經過老鼠腹腔培養後（in vivo passage），皮下生成腫瘤和體外抑制T細胞分裂的能力都恢復了，這些實驗結果認為， 腫瘤細胞的生成能力，Foxp3可能扮演極為重要的角色。利用Lentivirus 轉殖系統（transduction system），將Foxp3的微干擾核糖核酸（siRNA）送入腫瘤細胞後， Foxp3的表現被抑制了，同時種植在老鼠皮下的腫瘤生長速度明顯降低，而且對於用ConA刺激後的CD4+CD25-T細胞分裂能力的抑制效果也較未轉殖Fxop3微干擾核糖核酸的腫瘤細胞，低三倍之多，當直接將Lentivirus Foxp3的微干擾核糖核酸注入老鼠的皮下腫瘤位置，腫瘤的生長速度也明顯地被抑制了。實驗數據證實了，利用微干擾核塘核酸抑制T細胞淋巴癌的Foxp3基因表現，也有效地抑制了癌細胞的生長。 本論文實驗中，利用腫瘤細胞碎片和IL-12改造的樹突細胞誘發出有效的抗腫瘤免疫反應，也發現樹突細胞所誘發的自體免疫反應可以抑制腫瘤生長。同時實驗結果顯示，Foxp3參與腫瘤的生成過程，我們利用Lentivirus Foxp3的微干擾核糖核酸減低腫瘤細胞抑制免疫系統的能力，來避免免疫系統被腫瘤細胞抑制並攻擊腫瘤的生成， 雖然尚有許多的機制有待釐清，但已初步獲得正面的治療效果， 希望這些實驗結果對於未來在白血病的臨床治療上能有所助益。

Leukemia is a cancer of white blood cells that are disseminated by blood circulation. Because their features differ from solid tumors, leukemia cells metastasize to other tissues and organs more easily through the blood or lymphatic systems. RL ♂1 cell, which expressed CD4, CD25, IL-10, TGF-b and also expressed Foxp3, is a regulatory T cell (Treg)-like leukemia cell line. In this study, we established a murine model of leukemia with RL ♂1 to study the pathogenic mechanism of leukemia and intend to look for applicable approaches for cancer therapy. In the part I of the studies, we tried to improve conventional dendritic cells (DCs) based immunotherapy by administering engineered DCs that transduced with adenoviral vector expressing IL-12 (AdIL-12) pulsed with tumor cell lysate (TCL). Tumor mice treated with engineered DCs had a longer survival rate of 75% at day forty significantly improved the survival rate, 33% and 50%, of tumor mice treated with DCs pulsed with TCL alone or transduced with AdIL-12 respectively. In addition, IL-12 transduced and TCL pulsed DCs treated mice had higher CTL response compared to that of control group. Depletion of CD8+ T cells with specific antibodies abrogated the protective effects in tumor mice with DCs treatment. The results suggested that IL-12 gene modified DCs pulsed with TCL can stimulate immune response against tumor better than conventional DCs based tumor immunotherapy in animal model of leukemia. However, many studies have shown that DC-based tumor vaccines in animal tumor models can inhibit tumor growth and induce autoantibodies transiently. In this study, anti-ds DNA monoclonal antibodies recognized RL ♂1 cells but not normal cells by FACs analysis. The autoantibodies were demonstrated to lyse tumor cells via complement-mediated reaction in vitro and also exhibit the antitumor effects when the antibody was injected into tumor implanted mice. The data suggested that autoantibodies exert anti-tumor activity in vivo. In the future, it would be important to clarify the surface molecule of tumor recognized by autoantibody induced by DCs. The second part of the thesis discussed the mechanism of leukemia development in vivo and tried to explore adequate solution for cancer therapy. The data suggested that RL ♂1 cells lost Foxp3 expression and the ability of tumor growth simultaneously when subcutaneously transplanted into mice after in vitro culture for several generations. Interestingly, the phenomenon could be rescued or reversed after in vivo passage in the peritoneal cavity of mice. In addition, we transfer Foxp3 siRNA into tumor cells using lentiviral transduction system to inhibit Foxp3 expression. With infection of Lenti-Foxp3-siRNA in RL ♂ 1 cells, Foxp3 gene expression was abrogated and decreased the suppressive function to CD4+CD25- effector cells stimulated with ConA. Furthermore, lentiviral-mediated Foxp3 RNAi transduced into RL ♂ 1 cell or intratumoral injection of Lenti-Foxp3 siRNA showed suppressive effects of tumor growth and prolonged the survial time of tumor-transplanted mice. These results suggested that inhibition of Foxp3 gene expression by shRNAs effectively decreased tumor growth of regulatory T cell-like leukemia. In the study, engineered DCs activate specific immune response and produce autoantibodies inhibit tumor growth. In addition, Lenti-Foxp3-siRNA abrogates the chance of tumor to escape from immunosurveillance system. The studies in the thesis might provide a novel strategy for future clinical immunotherapy of leukemia