【作者】 徐永锋；

【导师】 樊嘉； 邱双健；

【作者基本信息】 复旦大学 ， 外科学， 2010， 博士

【摘要】 肝细胞肝癌是最常见的恶性肿瘤之一,在世界范围内肝癌是第六位的常见肿瘤,在肿瘤相关死亡率中排第三位。我国是肝癌的高发国家之一,约占全球肝癌病例一半以上。肝癌的疾病过程进展迅速、具有极高的复发转移倾向。虽然在肝癌的基础与临床研究中已经取得了长足的进展,然而肝癌总体疗效仍然不佳。既往的肝癌研究侧重于研究肝癌本身的生物学行为,忽视了肝癌演进过程中肝癌微环境中其他非肿瘤细胞作用。近年来逐渐重视从微环境与肝癌相互作用的角度进行了研究,以试图全面、深刻认识肝癌发生发展和转移复发的内在机制。本研究的主要内容从两方面入手,一、从影响肿瘤发生发展的外部原因,详细研究了肝癌局部微环境的免疫状态,发现肿瘤局部微环境中免疫细胞表型以抑制性表型为主,并且影响着肝癌复发转移。二、从肿瘤内在疾病基础即基因的改变对复发转移的影响。我们研究了在肝癌发生发展以及治疗中具有重要作用的Raf/MEK/ERK信号通路上游分子PEBP1,发现了PEBP1在肝癌中的异常表达与术后复发转移密切相关。最后我们通过联合分析,发现肿瘤局部免疫状态以及PEBP1的表达共同影响了肝癌进展,并且提示PEBP1除了具有影响肝癌内在的侵袭转移潜能外,可能本身也参与了肿瘤的免疫逃避机制。本研究旨在探索免疫细胞在肝癌患者体内不同的微环境状态下(外周血循环、癌旁肝脏和肝癌病灶局部微环境)的免疫细胞免疫表型与分布的差异。主要侧重分析了在机体抗免疫功能中发挥着重要作用的CD8效应性T细胞,以及肿瘤免疫中占主要地位的调节性T细胞。同时我们也分析了其他相关免疫细胞CD4辅助性T细胞、γδT以及NKT及NK细胞等。首先我们通过酶消化法以及Percoll梯度离心法,建立稳定的获取组织内浸润淋巴细胞分离方法。借助于流式细胞仪,配对比较分析了三处不同来源的免疫的表型与分布差异。结果我们发现,在肝癌外周血循环、癌旁肝脏和肝癌病灶局部的三处不同微环境中,免疫细胞的表型与分布是截然不同的,体现了不同的免疫功能状态。具体为：1.CD8效应性T细胞的分布,肝癌病灶内与外周血循环中的比例均显著低于肝旁肝脏组织。同时我们还发现了相对于健康对照者,肝癌患者CD8效应性T细胞共抑制分子BTLA增加,在成熟活化的记忆效应T细胞阶段时表达呈显著上调,提示CD8细胞在肝癌患者体内功能上是受抑制的。2.肝癌患者外周CD4+CD25+Treg显著高于健康对照者。在肝癌患者体内,肝癌局部微环境中的CD4+CD25+调节性T细胞显著高于外周和癌旁微环境,并且FoxP3的阳性表达率以及表达强度均显著高于外周和癌旁肝脏。另外我们还发现了CD4辅助性T细胞,与CD8T细胞分布情况截然相反,其在癌旁肝脏内分布最低,而外周血和肝癌病灶内均有较高的分布比例。固有免疫78T细胞在肿瘤病灶内比例下调。本实验在离体活细胞水平,发现了在肝癌患者体内,肿瘤局部微环境的免疫状态是免疫抑制表型为主的,包括过继免疫细胞CD8细胞减少,并且在成熟活化阶段明显受到抑制,而免疫抑制性Treg则在肿瘤局部显著增加,同时固有免疫γδT细胞的减少进一步加重了肿瘤局部微环境的免疫状态。在本研究中,我们对大样本的临床病例,通过组织芯片进一步研究肝癌的局部微环境免疫状态,以及对肝癌复发转移的影响,并且通过体外共培养实验初步探讨了Treg对肝癌侵袭能力的影响。首先我们通过免疫组化的方法,在组织芯片上研究了240根治性肝癌切除标本,发现肝癌肿瘤局部CD8+效应性T细胞显著低于癌旁微环境,而FoxP3+Treg在肿瘤局部浸润显著高于癌旁微环境。以CD8/Treg比值代表局部免疫状态,单因素与多因素生存分析表明CD8/Treg比值是肝癌术后复发与总生存的独立危险因素。同时我们发现Treg浸润程度与肝癌的恶性侵袭特征(血管侵犯、分化程度和肿瘤直径)显著相关,提示Treg可能具有促进肝癌侵袭潜能的作用。进一步通过Treg与肝癌细胞株体外共培养实验,证实Treg能在体外促进肝癌细胞的侵袭能力。本实验在免疫组化水平证明了肝癌的局部微环境状态以免疫抑制表型为主,即在肝癌局部CD8效应性T细胞浸润减少而Treg浸润显著增加。肝癌局部的免疫状态(CD8/Treg比值)在肝癌预后的独立危险因素。同时我们也证明了Treg能在体外促进肝癌细胞的侵袭能力。PEBP1是Raf蛋白抑制剂,调节包括Raf/MEK/ERK在内的多种信号通路。PEBP1能抑制肿瘤的侵袭转移,并且在肿瘤免疫监视中发挥重要作用。本研究通过免疫组化和实时定量PCR法,检测了240例原发性肝癌病例中PEBP1的蛋白和基因表达水平。发现PEBP1无论是蛋白水平还是基因水平在肿瘤内表达显著下调,并且下调的PEBP1与肝癌的侵袭性病理特征相关。通过单因素和多因素生存分析,发现低表达的PEBP1(无论是蛋白水平还是基因水平)是肝癌术后不良预后指标。我们的结论在另外一组403例肝癌病例中进一步得到验证。通过与肝癌局部免疫状态的联合分析,我们发现PEBP1与CD8/Treg比值,都是肝癌预后的独立指标,并且肝癌PEBP1的表达差异影响了CD8/Treg预后的价值。通过Western blot实验,我们发现肝癌组织中PEBP1表达下降,与Raf/MEK/ERK信号异常活化显著相关。我们的研究发现PEBP1在肝癌内表达显著下降,是肝癌不良预后的预测指标。肝癌中的PEBP1的低表达不仅与肿瘤的恶性侵袭能力相关,我们的研究也提示PEBP1也参与了肿瘤免疫逃避的机制。肝癌进展的过程受到肿瘤细胞本身的基因异常以及局部微环境状态的共同影响。更多还原

【Abstract】 The immune system can both promote tumor progression or mediated tumor rejection, as a balance between pro-tumor and antitumor immunity. Tumor microenvironment plays an important role in the progression of tumor. Presumably, the local tumor immune microenvironment polarizes host immune response toward a specific phenotype, in favoring or suppressing tumor progression. However, the exact local immune status is unclear in hepatocellular carcinoma.For comprehensive immune characterization, it is also important to investigate T cells from diseased organs/tissues. We compared the immune status of different local microenvironments (peripheral circulation, peritumoral microenvironment and intratumoral microenvironment) in hepatocellular carcinoma. The present study was focused on analyzing the immunophenotypic of tumor infiltrating lymphocytes. We found the number of CD8+ T cells was significantly decreased in intratumoral microenvironment, compared with peripheral circulation and peritumoral microenvironment. We also found CD8+T cell of HCC patients expressed high level of the B and T lymphocyte attenuator (BTLA), a newly discovered coinhibitory molecule, when compared with healthy donors, indicating impaired function of CD8+T cell in HCC. Notably, we also found the enrichment of CD4+CD25+ regulatory T cells (Treg) in intratumoral microenvironment. Moreover, the highest expression level and intensity level of FoxP3 presented in CD4+CD25+ Treg in intratumoral microenvironment. We also found the inherent immunity cell of y8T to be downregulated in intratumoral microenvironment.Our results indicated that the tumor microenvironment of hepatocellular carcinoma is featured by the immunosuppressive status: Tumor immune microenvironment plays an important role in the progression of tumor. We investigated whether immune status of tumor microenvironment correlated with prognosis of hepatocellular carcinoma.Immunohistochemical staining of CD8 and Foxp3 was conducted in a set of tissue microarrays which based on a cohort of 240 hepatocellular carcinoma patients after curative resection. We used the ratio of CD8/Treg as a comprehensive index to represent the immune status of intratumoral microenvironment. Survival analysis was performed by univariate and multivariate analysis.Based on immunohistochemisty, we found that the number of CD8+ infiltrating cells was decreased and FoxP3+ infiltrating cells increased in intratumoral microenvironment, when compared with peritumoral microenvironment. The number of FoxP3+Treg was significantly associated with invasive characteristics (such as vascular invasion, poor differentiation and large size). Multivariate analysis indicated the ratio of CD8/Treg was an independent predictor for time to recurrence and overall survival time. In vitro co-culture study, we found Treg had the ability to promote the invasive potential HCC tumor cells. In conclusion, we found the immunosuppressive status of intratumoral microenvironment was an adverse index for HCC prognosis. Treg had the ability to promote the invasive potential HCC tumor cells which beyond its immunopotency. Phosphatidylethanolamine binding protein 1 (PEBP1) plays a pivotal role in cancer by regulating multiple cellular signaling processes, potentiating immune-mediated apoptosis and suppressing metastasis in animal models. We examined whether RKIP expression in hepatocellular carcinoma (HCC) correlated with the risk of recurrence and survival after resection.A randomly selected cohort of 240 Chinese HCC patients, predominantly hepatitis B-related, formed the basis of the study. PEBP1 expression levels were evaluated by immunohistochemistry and real-time reverse-transcriptase PCR. Immunostaining results of CD8+ cytotoxic to Foxp3+ regulatory T cells (Tregs) ratio was also evaluated as a comprehensive immune index in relation to PEBP1 expression. Survival analysis was performed by univariate and multivariate analysis. The results were further validated in an independent series of 403 patients. The relevance of RKIP to phospho-ERK was determined by Western blot analysis on clinical samples and HCC cell lines.We found that PEBP1 prevalently down-regulated in HCC and was significantly associated with tumor aggressiveness. Both PEBP1 mRNA and protein levels were adverse predictors for time to recurrence and patient overall survival time. The prognostic value of PEBP1 was then confirmed in the validation cohort. In addition, Western blot suggested the loss of PEBP1 led to hyperactivity of MAPK signaling. Subgroup analysis indicated that low level of PEBP1 expression in HCC aggravated the unfavorable prognosis of low intratumoral CD8+/Tregs ratio, while high level of PEBP1 expression reinforced the favorable prognosis of high intratumoral CD8+/Tregs ratio.Our current study indicated that down-regulation of RKIP in HCC was an independent predicator for poor patient outcome, which not only contributed to aggressive tumor behaviors but also may represent a tumor-intrinsic mechanism of active immune escape. The progression of hepatocellular carcinoma is controlled by both the cell-intrinsic mechanism of gene mutation and the cell-extrinsic mechanism of tumor immune microenvironment.更多还原