肝癌患者CD4~+CD25~+CD127~(low/-)CD49d~-调节性T细胞与肝癌复发转移关系的研究
摘要
肝细胞肝癌(hepatocellular carcinoma, HCC)是全球最常见的恶性肿瘤之一,在恶性肿瘤相关死亡率中位居第三位。我国是肝癌高发地区,约占全球肝癌病例一半以上。肝癌严重影响我国人民的健康事业,并带来了巨大的社会负担。由于肝癌早期诊断较难,患者就诊时往往已属晚期。虽然近来手术切除、肝移植、射频消融、肝动脉栓塞化疗和分子靶向治疗等手段对肝癌的治疗取得了很大的进步,但总体上术后高复发率和高死亡率近来未有明显改善。免疫治疗作为肿瘤最有希望的治疗手段越来越受到重视。同时肝脏作为免疫特惠器官又是免疫治疗理想的靶器官。既往对肝癌的研究集中在肝癌细胞本身的生物学行为上,忽略了肝癌演进过程中肝癌细胞与肿瘤微环境(tumor microenvironment)中各种免疫细胞之间的相互作用,而后者被认为在肿瘤的发展、侵袭中发挥重要的作用。具有免疫抑制性作用的调节性T细胞(regulatory T cell, Treg)在肿瘤细胞逃避局部微环境的免疫监视从而发生免疫逃逸的过程中,扮演着重要的角色。
本研究从肿瘤微环境中调节性T细胞的特异性表面标志入手,证实了在免疫微环境中CD4+CD25+CD127low/-CD49d-调节性T细胞的浸润和表达,表现为更强的免疫抑制表型,并与肝癌的临床病理特征相关。进而我们通过全基因芯片筛选出较重要的调控肿瘤浸润调节性T细胞的免疫抑制功能相关的功能基因,分析肿瘤浸润调节性T细胞与肿瘤细胞相互作用的免疫信号通路。基于凝集素芯片检测HCC患者外周血调节性T细胞膜特征性的聚糖改变。最后通过对调节性T细胞与galectin-1在HCC中表达的相关性分析,为HCC预后的预测和免疫治疗提供新的选择。
第一部分HCC患者CD4+CD25+CD127low/-CD49d-调节性T细胞的表达和表型研究
本部分研究旨在探索CD4+CD25+CD127loW/-CD49d-调节性T细胞在HCC患者体内不同部位(外周血、癌旁肝脏和癌灶)的分布和表达。
我们通过梯度离心结合酶消化法,建立了稳定的获取外周血、组织内浸润淋巴细胞分离方法。借助于流式细胞仪,首次采用四色荧光分选淋巴细胞,配对比较分析了三处不同来源的CD4+CD25+CD127low/-CD49d-调节性T细胞的分布表达和表型。发现在HCC患者外周血、癌旁肝组织和癌灶中,CD4+CD25+CD127low/-CD49d-调节性T细胞的分布和免疫表型截然不同。首先,HCC患者肿瘤微环境中存在高表达FoxP3的CD4+CD25+CD127low/-CD49d-调节性T细胞。其次,HCC患者外周血调节性T细胞占整个CD4+T细胞比例显著高于健康对照者。HCC患者肿瘤浸润调节性T细胞占整个CD4+T细胞比例显著高于配对的癌旁肝组织和外周血。最后,HCC患者癌灶浸润调节性T细胞表达较高的免疫抑制表型,如FoxP3、CTLA-4和GITR。混合淋巴细胞实验显示CD4+CD25+CD127low/-CD49d-调节性T细胞对活化分子CD154的抑制率较对照明显增强,表明具有较强的免疫抑制能力。结合体外扩增实验结果,这群淋巴细胞分泌高水平的免疫抑制因子IL-10和TGF-B1,扩增后的细胞仍保持较高的抑制表型。我们分选出的肿瘤浸润的这群具有抑制能力的淋巴细胞较癌旁肝组织和外周血来源的显示出更强的免疫抑制能力。
我们首次采用四色荧光分选出的CD4+CD25+CD127low/-CD49d-T淋巴细胞高表达调节性T细胞的特异性标志FoxP3,较其他相关研究文献可获得更高纯度的调节性T细胞,并且这群高表达FoxP3的T淋巴细胞保持活性状态,可用于下游的功能实验。CD4+CD25+CD127low/-CD49d-T细胞高表达于肝癌肿瘤微环境中,表现出更强的免疫抑制表型,体现了肿瘤免疫微环境中调节性T细胞的免疫抑制功能,参与HCC的肿瘤生物学进程。
第二部分基于全基因芯片的CD4+CD25+CD127low/-CD49d-调节性T细胞基因表达谱分析
结合前部分研究结果和其他相关文献报道,我们可以认为癌灶浸润性调节性T细胞之所以表达增强的抑制表型,必然是受到肿瘤微环境中肿瘤细胞和其他免疫细胞的影响,导致其与免疫抑制功能相关的基因表达上调和信号通路激活。但具体参与的调控基因和信号通路尚未有系统的报道。在本部分研究中,我们利用Agilent全基因芯片对9例HCC患者配对的癌灶和癌旁肝组织中的调节性T细胞进行全基因表达谱的对比分析,以期找出与免疫抑制功能相关的关键差异基因及相应的信号通路。
我们利用酶消化法和梯度离心法分离出癌灶和癌旁肝组织浸润淋巴细胞,再利用流式细胞仪分选CD4+CD25+CD127low/-CD49d-调节性T细胞。细胞RNA抽提后与Agilent全基因芯片杂交,GenePix4000B芯片扫描仪扫描分析结果。结果显示Hierarchical聚类分析样本划分不同组别明显。将标准设为Fold Change≥2,P<0.05后,癌灶浸润调节性T细胞相对于癌旁肝组织浸润调节性T细胞共有1032个基因表达上调,1357个基因表达下调。PATHWAY结果显示两组间有7条上调通路和9条下调通路差异。GO分析显示癌灶浸润调节性T细胞相对于癌旁肝组织调节性T细胞有431条GO条目上调,237条GO条目下调。我们选取与肿瘤免疫和T细胞免疫相关的GO条目并按照Fold Change大小,从前15条中选取基因CD46、BCL6、TNFRSF11A、MDM4、P2RX7、CD276和IL6R进行分析,并对选取的基因进行RT-PCR验证。
其中高表达RANK (TNFRSF11A基因编码)的调节性T细胞与高表达RANKL的HCC肿瘤细胞之间的RANK-RANKL信号通路,高表达IL-6R的调节性T细胞与高表达IL-6的HCC肿瘤细胞之间的IL6-IL-6R信号通路在HCC肿瘤免疫逃逸中可能起到很重要的作用。
第三部分凝集素芯片检测CD4+CD25+CD127low/-CD49d-调节性T细胞和肝癌细胞株HCCLM3、MHCC97H、MHCC97L和Hep3B特征性糖基化改变
肿瘤特征性糖基化改变影响着肿瘤细胞的恶性生物学行为。同样,异常的糖基化改变也影响着调节性T细胞的功能。目前,糖生物方兴未艾,是研究肿瘤发生发展的一个新的领域。肿瘤细胞异常的糖基化改变已证实与肿瘤细胞的恶性生物学行为相关。糖基转移酶调控糖基化改变。现已证实糖基化改变在淋巴细胞的成熟,阴性阳性分化选择,归巢,凋亡等过程中起着非常重要的作用。调节性T细胞的功能也必然伴随着糖基化的改变而改变。凝集素是一种可以与特定糖链结合的糖蛋白,利用此特性可以检测出特定糖链的表达和改变。国内目前尚未广泛开展基于凝集素芯片的肝癌相关研究。
我们已于前期成功构建检测糖基化改变的凝集素芯片,并检测出不同来源的甲胎蛋白之间细微的聚糖改变,为我们这部分的实验奠定了基础。本部分实验首先在前期的基础上扩大凝集素芯片上凝集素的种类至32种,以便能够更准确地捕捉糖链的改变。我们构建的对细胞膜表面糖链可进行即时、高通量检测的凝集素芯片,优点在于将凝集素与芯片技术相结合,利用芯片高通量、高特异性、高灵敏度等优点,对提取的膜蛋白糖链进行自动捕获,成功地实现对细胞膜表面糖链进行高通量的检测分析,获得细胞膜表面糖链全面而综合的信息。甘露糖抑制实验和胎球蛋白实验充分显示我们构建的凝集素芯片具有糖链结合的特异性、准确性和稳定性。最后我们将分选的HCC患者与健康志愿者的外周血CD4+CD25+CD127low/-CD49d-调节性T细胞提取细胞膜蛋白后与芯片杂交后发现HCC患者外周血调节性T细胞较健康对照的外周血调节性T细胞膜表面α1-6岩藻糖、α1-6甘露糖、GalNac、β型半乳糖链、复杂型N-聚糖增加。另外,我们选取转移潜能梯度降低的人肝癌细胞株HCCLM3、MHCC97H、MHCC97L和几乎不转移的Hep3B,经细胞膜蛋白提取和Trizol提取RNA后,分别与凝集素芯片和糖基转移酶PCR芯片杂交。结果提示,HCCLM3相对于Hep3B,代表唾液酸糖链的MAL-Ⅰ、MAL-Ⅱ、WGA明显增加；代表甘露糖链的ConA和GNA明显增加；代表岩藻糖链的LTL、LCA、PSA和UEA-1明显增加；代表半乳糖链的BPL、EEL、jacalin, GSL-1增加；代表GlcNAc的PHA-L增加；代表GalNAc的VVA、DBA、SBA明显增加。糖基转移酶PCR芯片结果提示HCCLM3较Hep3B,ST3Ga11、FUT8和MGAT5等转移酶增高。其中MAL-Ⅰ,MAL-Ⅱ和WGA变化相对于ST3Gall, PSA、LCA相对于FUT8,PHA-L相对于MGAT5。
因此我们可以认为调节性T细胞特征性糖基化改变可能伴随着免疫功能的变化。HCC细胞的特征性糖基化改变及相应的糖基转移酶改变为肿瘤的治疗从糖生物学的角度提供了新的方向。
第四部分Galectin-1对HCC复发转移的影响及与调节性T细胞相关性的研究
异常的糖基化改变影响着肿瘤的恶性生物学行为。Galectin-1是半乳凝素家族重要成员之一,通过与肿瘤细胞表面聚糖(glycan)形成galectin-glycan结构参与调控肿瘤的分化、迁移、凋亡和信号转导等生物学过程。在许多肿瘤中都发现galectin-1表达的增高与肿瘤的浸润、转移密切相关。许多高表达galectin-1的肿瘤都提示不良预后。近来研究表明调节性T细胞参与介导肿瘤细胞免疫逃逸的过程中受到肿瘤细胞来源的galectin-1的信号调控。同时,我们前面的实验结果也显示HCC患者外周血Treg膜表面表达较高的半乳糖链。目前在HCC中尚未有关galectin-1和Treg的表达及相关性的报道。
在本部分研究中我们通过免疫组化的方法,对包含386例HCC患者的组织芯片检测FoxP3+Treg和galectin-1的表达,并通过生存分析观察galectin-1与预后的关系。同时通过流式细胞仪和ELISA分别检测31例HCC患者的外周血中的CD4+CD25+FoxP3+Treg和galectin-1的表达,以期发现两者在组织和外周血之间的相关性。最后我们利用ELISA法验证了147例HCC患者血清中galectin-1的表达水平。研究结果发现galectin-1在HCC肿瘤组织中高表达,是独立的预后指标。高表达galectin-1的HCC患者预后较差。Galectin-1与HCC的临床病理特征相关,如肿瘤大小、包膜是否完整和血管侵犯等等。在BCLC早期的HCC患者中,galectin-1也显示出预测预后的价值。肝癌组织中Galectin-1的表达与FoxP3+Treg数量两者呈正相关,但是在外周血中两者的表达无相关性。
我们的结果提示肿瘤微环境中的galectin-1可能促进Treg在HCC中扩增,进而影响肿瘤的发展和预后,为肿瘤的免疫治疗提供了新的靶点。
结论
1.CD4+CD25+CD127low/-CD49d-T淋巴细胞高表达调节性T细胞的特异性标志FoxP3。
2.肿瘤浸润性调节性T细胞可能通过IL6WIL6、RANK/RANKL信号通路参与HCC肿瘤细胞的免疫逃逸。
3.HCC患者外周血调节性T细胞较健康对照的外周血调节性T细胞膜表面α1-6岩藻糖、α1-6甘露糖、GalNac、p型半乳糖链、复杂型N-聚糖增加。调节性T细胞特征性糖基化的改变可能伴随着细胞免疫抑制功能的变化。
4.肝癌细胞具有α2-3,α2-6唾液酸、岩藻糖、平分型GlcNAc等糖链表达上调,可能受糖基转移酶ST6GALNAC1、ST3Ga11、FUT8和MGAT5等调控。
5. Galectin-1在HCC中的高表达伴随着调节性T细胞数量的增加,联合调节性T细胞和galectin-1可增加预测HCC患者预后的准确性。可作为HCC免疫治疗的靶点。
创新点
1.首次在HCC患者中分选出具有活性的可供功能实验所需的CD4+CD25+CD127low/-CD49d-调节性T细胞。
2.首次提出IL6WIL6、RANK/RANKL信号通路可能参与HCC肿瘤浸润性调节性T细胞介导的肿瘤免疫逃逸。
3.HCC患者外周血调节性T细胞特征性糖基化的改变可能伴随着细胞免疫抑制功能的变化。
4.首次报道galectin-1在HCC患者预后中的作用。
研究展望
1.进一步研究HCC肿瘤浸润性调节性T细胞的免疫相关差异表达基因和信号通路的作用机制,为HCC的治疗提供更为准确的治疗靶点。
2.进一步研究调节性T细胞和肿瘤细胞特征性糖基化改变及相关性,从新的角度研究肿瘤的侵袭、转移机制。
Part I Increased prevalence of CD4+CD25+CD127low/-CD49d FoxP3+regulatory T cells in HCC tumor microenvironment
Objective Expansion of regulatory T cells (Tregs) in tumor microenvironment was one of the mechanisms by which cancer cells escaped host defense. The purpose of this study was to investigate the increasing prevalence of Tregs in HCC cancer microenvironment. Methods Levels of CD4+CD25+CD127low/-CD49d-FoxP3+Tregs in peripheral blood mononuclear cells (PBMCs) from HCC patients (n=58) and healthy donors (n=12), tumor infiltrating lymphocytes (TILs) extracted from HCC (n=20), matched peri-tumor and PBMCs were measured by flow cytometry. Their suppression were determined by using activation marker expression (CD154). Superana levels of interleukin (IL)-10and transforming growth factor (TGF)-β1were measured by enzyme linked immunosorbent assay. Results The prevalence of Tregs in PBMCs from HCC patients was significantly higher than that from healthy donors and expressed high levels of IL-10and TGF-β1Similarly, the frequency of Tregs in tumor tissues was increased compared with that in matched peri-tumor and PBMCs. TIL Tregs were more suppressible by approximately30%than their peri-tumor and PBMCs counterparts. CTLA-4, GITR and MFI of FoxP3at higher levels on tumour-infiltrating Tregs compared with matched peri-tumor and periphery. Conclusions This study identified an increased frequency of CD4+CD25+CD127low/-CD49d-FoxP3+Tregs in patients with HCC. Further effort is needed to establish new immunotherapeutic strategies to modulate Tregs to promote a competent antitumor response.
Part Ⅱ Comparative gene expression profile of CD4+CD25+CD127low/-CD49d-regulatory T cells in human hepatocellular carcinoma
Objective The aim of this study was to compare gene expression profile of regulatory T cell (Treg) in HCC tumor sits with that of peri-tumor by Agilent whole genome oligo microarray. Methods Levels of CD4+CD25+CD127low/-CD49d-Tregs in HCC tumor (n=9), matched peri-tumor and PBMCs were measured by flow cytometry. Tregs from tumor and peri-tumor were hybridized on an Agilent whole genome microarray. Functional analysis of the microarray data was performed using KEGG PATHWAY and Gene Ontology (GO) analyses. Results The prevalence of Tregs in HCC tumor was significantly higher than that in peri-tumor. There were1032genes up-regualtion and1357genes down-regulation in tumor Tregs compared to peri-tumor. KEGG PATHWAY showed that7pathway up-regulation and9down-regulation of intratumoral Tregs compared to peri-tumor. GO showed that431terms up-regulation and237terms down-regulation. Microarray gene expression analysis showed that CD46, BCL6, TNFRSF11A, MDM4, P2RX7, CD276, TNFRSF18and IL6R were among the most upregulated genes in tumor Tregs compared with peri-tumor Tregs. The selected genes were validated by RT-PCR. Functional analysis showed that the complement and p53pathways were significantly upregulated in tumor Tregs, and that the GO terms related to cell differentiation, proliferation, apoptosis and migration were significantly affected. Conclusions This study identified IL6/IL6R, RANK/RANKL signalling might play an impotant role in intratumoral Tregs between HCC tumor cells.
Part III Lectin Microarray based glycan profiling change of periphery regulatory T cell in patients with HCC
Objective The aims of this section was to assess the applicability of application of lectin microarray to screen the glycosylation changes in complex biological systems. Based on that, glycosylation profilings were screened to search for the characteristic glycan structures associated with periphery regulatory T cells in HCC patients. Methods Firstly, we selected one pair of model protein:FET and its sialic acid defective strains ASF, of which characteristic glycan structures on the cell surface were well defined. Integrated protein was labeled with Cy3, then frozen in liquid nitrogen to terminatate the labeling. Next, membrane protein of Tregs was extracted by natural membrane protein extraction kit.10μg of protein was added to each chip and incubated for2hours, after washing, scanning and statistical analysis, lectin affinity profiling was obtained from the chip. Different batches of samples were loaded to detect the reproducibility of lectin microarray. HCC cell lines HCCLM3, MHCC97H, MHCC97L and Hep3B were used to hydratied with lectinarray and glycotranferase PCR microarray. Results The results showed that the characteristic glycosylation difference between ASF and FET were dtermined by defined lectin microarray. Comparing with ASF, FET showed increased affinity and decreased signals for SNA, MAL-Ⅰ and MAL-Ⅱ. HCC patients periphery Tregs presented elevated signals for LCA, UEA-Ⅰ, VVA, SBA, ACA, LEL and decreased signals for RCA-I with contrast to healthy controls. MAL-Ⅰ, MAL-Ⅱ, WGA, PHA-L were correlated with ST6GALNAC1、ST3Ga11、FUT8and MGAT5. Conclusions Our results proved that lectin microarray is an applicable glcan analysis tool, and could be used in clinical to screen HCC related Tregs glycan profiling, in search of better glycol-biomarkers for tumor immuno-escape. Besides, our study also presented reference and technology platform for the evaluation of the relationship between complex biological systems and glycan alteration.
Part IV Overexpression of Galectin-1Associates with Poor Prognosis in Human Hepatocellular Carcinoma Following Resection
Objective High expression of the galectin-1predicted poor patient outcome in several tumors. The aim of this study was to investigate its prognostic value in patients with hepatocellular carcinoma (HCC) after resection. Methods Galectin-1and tumor-infiltrating FoxP3+regulatory T cells (Tregs) were validated by tissue microarrays from HCC patients (n=386) and statistically assessed for correlations with the clinical profiles and the prognosis of the patients. Serum galectin-1levels and frequency of FoxP3+Tregs in circulation was measured by ELISA and flowcyte, respectively. And serum levels of galectin-1was validated by ELISA in147HCC patients. Results We found that galectin-1, prevalently up-regulated in HCC, was significantly associated with tumor invasive characteristics (such as vascular invasion, incomplete encapsulation, poor differentiation, multiple number and large tumor size). Patients with high galectin-1expression had a significantly poorer tumor recurrence (p=0.025) and overall survival (p=0.021) than those with low galectin-1expression. Even in early-stage disease, high galectin-1expression also independently associated with shortened survival (p<0.001) and increased tumor recurrence (p=0.005). Multivariate Cox proportional hazards analysis showed that galectin-1was an independent marker for predicting poor prognosis of HCC. Galectin-llevel was positively related to number of tumor-infiltrating FoxP3+Tregs (r=0.416,p<0.001) and their combination served as a better prognosticator. The postoperative tumor recurrence and survival of HCC patients with galectin-1high and FoxP3high were significantly poorer than the other groups (both p<0.001). Conclusions Galectin-1may be a new prognostic factor for HCC after resection and potentially be a high-priority therapeutic target.
本研究从肿瘤微环境中调节性T细胞的特异性表面标志入手,证实了在免疫微环境中CD4+CD25+CD127low/-CD49d-调节性T细胞的浸润和表达,表现为更强的免疫抑制表型,并与肝癌的临床病理特征相关。进而我们通过全基因芯片筛选出较重要的调控肿瘤浸润调节性T细胞的免疫抑制功能相关的功能基因,分析肿瘤浸润调节性T细胞与肿瘤细胞相互作用的免疫信号通路。基于凝集素芯片检测HCC患者外周血调节性T细胞膜特征性的聚糖改变。最后通过对调节性T细胞与galectin-1在HCC中表达的相关性分析,为HCC预后的预测和免疫治疗提供新的选择。
第一部分HCC患者CD4+CD25+CD127low/-CD49d-调节性T细胞的表达和表型研究
本部分研究旨在探索CD4+CD25+CD127loW/-CD49d-调节性T细胞在HCC患者体内不同部位(外周血、癌旁肝脏和癌灶)的分布和表达。
我们通过梯度离心结合酶消化法,建立了稳定的获取外周血、组织内浸润淋巴细胞分离方法。借助于流式细胞仪,首次采用四色荧光分选淋巴细胞,配对比较分析了三处不同来源的CD4+CD25+CD127low/-CD49d-调节性T细胞的分布表达和表型。发现在HCC患者外周血、癌旁肝组织和癌灶中,CD4+CD25+CD127low/-CD49d-调节性T细胞的分布和免疫表型截然不同。首先,HCC患者肿瘤微环境中存在高表达FoxP3的CD4+CD25+CD127low/-CD49d-调节性T细胞。其次,HCC患者外周血调节性T细胞占整个CD4+T细胞比例显著高于健康对照者。HCC患者肿瘤浸润调节性T细胞占整个CD4+T细胞比例显著高于配对的癌旁肝组织和外周血。最后,HCC患者癌灶浸润调节性T细胞表达较高的免疫抑制表型,如FoxP3、CTLA-4和GITR。混合淋巴细胞实验显示CD4+CD25+CD127low/-CD49d-调节性T细胞对活化分子CD154的抑制率较对照明显增强,表明具有较强的免疫抑制能力。结合体外扩增实验结果,这群淋巴细胞分泌高水平的免疫抑制因子IL-10和TGF-B1,扩增后的细胞仍保持较高的抑制表型。我们分选出的肿瘤浸润的这群具有抑制能力的淋巴细胞较癌旁肝组织和外周血来源的显示出更强的免疫抑制能力。
我们首次采用四色荧光分选出的CD4+CD25+CD127low/-CD49d-T淋巴细胞高表达调节性T细胞的特异性标志FoxP3,较其他相关研究文献可获得更高纯度的调节性T细胞,并且这群高表达FoxP3的T淋巴细胞保持活性状态,可用于下游的功能实验。CD4+CD25+CD127low/-CD49d-T细胞高表达于肝癌肿瘤微环境中,表现出更强的免疫抑制表型,体现了肿瘤免疫微环境中调节性T细胞的免疫抑制功能,参与HCC的肿瘤生物学进程。
第二部分基于全基因芯片的CD4+CD25+CD127low/-CD49d-调节性T细胞基因表达谱分析
结合前部分研究结果和其他相关文献报道,我们可以认为癌灶浸润性调节性T细胞之所以表达增强的抑制表型,必然是受到肿瘤微环境中肿瘤细胞和其他免疫细胞的影响,导致其与免疫抑制功能相关的基因表达上调和信号通路激活。但具体参与的调控基因和信号通路尚未有系统的报道。在本部分研究中,我们利用Agilent全基因芯片对9例HCC患者配对的癌灶和癌旁肝组织中的调节性T细胞进行全基因表达谱的对比分析,以期找出与免疫抑制功能相关的关键差异基因及相应的信号通路。
我们利用酶消化法和梯度离心法分离出癌灶和癌旁肝组织浸润淋巴细胞,再利用流式细胞仪分选CD4+CD25+CD127low/-CD49d-调节性T细胞。细胞RNA抽提后与Agilent全基因芯片杂交,GenePix4000B芯片扫描仪扫描分析结果。结果显示Hierarchical聚类分析样本划分不同组别明显。将标准设为Fold Change≥2,P<0.05后,癌灶浸润调节性T细胞相对于癌旁肝组织浸润调节性T细胞共有1032个基因表达上调,1357个基因表达下调。PATHWAY结果显示两组间有7条上调通路和9条下调通路差异。GO分析显示癌灶浸润调节性T细胞相对于癌旁肝组织调节性T细胞有431条GO条目上调,237条GO条目下调。我们选取与肿瘤免疫和T细胞免疫相关的GO条目并按照Fold Change大小,从前15条中选取基因CD46、BCL6、TNFRSF11A、MDM4、P2RX7、CD276和IL6R进行分析,并对选取的基因进行RT-PCR验证。
其中高表达RANK (TNFRSF11A基因编码)的调节性T细胞与高表达RANKL的HCC肿瘤细胞之间的RANK-RANKL信号通路,高表达IL-6R的调节性T细胞与高表达IL-6的HCC肿瘤细胞之间的IL6-IL-6R信号通路在HCC肿瘤免疫逃逸中可能起到很重要的作用。
第三部分凝集素芯片检测CD4+CD25+CD127low/-CD49d-调节性T细胞和肝癌细胞株HCCLM3、MHCC97H、MHCC97L和Hep3B特征性糖基化改变
肿瘤特征性糖基化改变影响着肿瘤细胞的恶性生物学行为。同样,异常的糖基化改变也影响着调节性T细胞的功能。目前,糖生物方兴未艾,是研究肿瘤发生发展的一个新的领域。肿瘤细胞异常的糖基化改变已证实与肿瘤细胞的恶性生物学行为相关。糖基转移酶调控糖基化改变。现已证实糖基化改变在淋巴细胞的成熟,阴性阳性分化选择,归巢,凋亡等过程中起着非常重要的作用。调节性T细胞的功能也必然伴随着糖基化的改变而改变。凝集素是一种可以与特定糖链结合的糖蛋白,利用此特性可以检测出特定糖链的表达和改变。国内目前尚未广泛开展基于凝集素芯片的肝癌相关研究。
我们已于前期成功构建检测糖基化改变的凝集素芯片,并检测出不同来源的甲胎蛋白之间细微的聚糖改变,为我们这部分的实验奠定了基础。本部分实验首先在前期的基础上扩大凝集素芯片上凝集素的种类至32种,以便能够更准确地捕捉糖链的改变。我们构建的对细胞膜表面糖链可进行即时、高通量检测的凝集素芯片,优点在于将凝集素与芯片技术相结合,利用芯片高通量、高特异性、高灵敏度等优点,对提取的膜蛋白糖链进行自动捕获,成功地实现对细胞膜表面糖链进行高通量的检测分析,获得细胞膜表面糖链全面而综合的信息。甘露糖抑制实验和胎球蛋白实验充分显示我们构建的凝集素芯片具有糖链结合的特异性、准确性和稳定性。最后我们将分选的HCC患者与健康志愿者的外周血CD4+CD25+CD127low/-CD49d-调节性T细胞提取细胞膜蛋白后与芯片杂交后发现HCC患者外周血调节性T细胞较健康对照的外周血调节性T细胞膜表面α1-6岩藻糖、α1-6甘露糖、GalNac、β型半乳糖链、复杂型N-聚糖增加。另外,我们选取转移潜能梯度降低的人肝癌细胞株HCCLM3、MHCC97H、MHCC97L和几乎不转移的Hep3B,经细胞膜蛋白提取和Trizol提取RNA后,分别与凝集素芯片和糖基转移酶PCR芯片杂交。结果提示,HCCLM3相对于Hep3B,代表唾液酸糖链的MAL-Ⅰ、MAL-Ⅱ、WGA明显增加；代表甘露糖链的ConA和GNA明显增加；代表岩藻糖链的LTL、LCA、PSA和UEA-1明显增加；代表半乳糖链的BPL、EEL、jacalin, GSL-1增加；代表GlcNAc的PHA-L增加；代表GalNAc的VVA、DBA、SBA明显增加。糖基转移酶PCR芯片结果提示HCCLM3较Hep3B,ST3Ga11、FUT8和MGAT5等转移酶增高。其中MAL-Ⅰ,MAL-Ⅱ和WGA变化相对于ST3Gall, PSA、LCA相对于FUT8,PHA-L相对于MGAT5。
因此我们可以认为调节性T细胞特征性糖基化改变可能伴随着免疫功能的变化。HCC细胞的特征性糖基化改变及相应的糖基转移酶改变为肿瘤的治疗从糖生物学的角度提供了新的方向。
第四部分Galectin-1对HCC复发转移的影响及与调节性T细胞相关性的研究
异常的糖基化改变影响着肿瘤的恶性生物学行为。Galectin-1是半乳凝素家族重要成员之一,通过与肿瘤细胞表面聚糖(glycan)形成galectin-glycan结构参与调控肿瘤的分化、迁移、凋亡和信号转导等生物学过程。在许多肿瘤中都发现galectin-1表达的增高与肿瘤的浸润、转移密切相关。许多高表达galectin-1的肿瘤都提示不良预后。近来研究表明调节性T细胞参与介导肿瘤细胞免疫逃逸的过程中受到肿瘤细胞来源的galectin-1的信号调控。同时,我们前面的实验结果也显示HCC患者外周血Treg膜表面表达较高的半乳糖链。目前在HCC中尚未有关galectin-1和Treg的表达及相关性的报道。
在本部分研究中我们通过免疫组化的方法,对包含386例HCC患者的组织芯片检测FoxP3+Treg和galectin-1的表达,并通过生存分析观察galectin-1与预后的关系。同时通过流式细胞仪和ELISA分别检测31例HCC患者的外周血中的CD4+CD25+FoxP3+Treg和galectin-1的表达,以期发现两者在组织和外周血之间的相关性。最后我们利用ELISA法验证了147例HCC患者血清中galectin-1的表达水平。研究结果发现galectin-1在HCC肿瘤组织中高表达,是独立的预后指标。高表达galectin-1的HCC患者预后较差。Galectin-1与HCC的临床病理特征相关,如肿瘤大小、包膜是否完整和血管侵犯等等。在BCLC早期的HCC患者中,galectin-1也显示出预测预后的价值。肝癌组织中Galectin-1的表达与FoxP3+Treg数量两者呈正相关,但是在外周血中两者的表达无相关性。
我们的结果提示肿瘤微环境中的galectin-1可能促进Treg在HCC中扩增,进而影响肿瘤的发展和预后,为肿瘤的免疫治疗提供了新的靶点。
结论
1.CD4+CD25+CD127low/-CD49d-T淋巴细胞高表达调节性T细胞的特异性标志FoxP3。
2.肿瘤浸润性调节性T细胞可能通过IL6WIL6、RANK/RANKL信号通路参与HCC肿瘤细胞的免疫逃逸。
3.HCC患者外周血调节性T细胞较健康对照的外周血调节性T细胞膜表面α1-6岩藻糖、α1-6甘露糖、GalNac、p型半乳糖链、复杂型N-聚糖增加。调节性T细胞特征性糖基化的改变可能伴随着细胞免疫抑制功能的变化。
4.肝癌细胞具有α2-3,α2-6唾液酸、岩藻糖、平分型GlcNAc等糖链表达上调,可能受糖基转移酶ST6GALNAC1、ST3Ga11、FUT8和MGAT5等调控。
5. Galectin-1在HCC中的高表达伴随着调节性T细胞数量的增加,联合调节性T细胞和galectin-1可增加预测HCC患者预后的准确性。可作为HCC免疫治疗的靶点。
创新点
1.首次在HCC患者中分选出具有活性的可供功能实验所需的CD4+CD25+CD127low/-CD49d-调节性T细胞。
2.首次提出IL6WIL6、RANK/RANKL信号通路可能参与HCC肿瘤浸润性调节性T细胞介导的肿瘤免疫逃逸。
3.HCC患者外周血调节性T细胞特征性糖基化的改变可能伴随着细胞免疫抑制功能的变化。
4.首次报道galectin-1在HCC患者预后中的作用。
研究展望
1.进一步研究HCC肿瘤浸润性调节性T细胞的免疫相关差异表达基因和信号通路的作用机制,为HCC的治疗提供更为准确的治疗靶点。
2.进一步研究调节性T细胞和肿瘤细胞特征性糖基化改变及相关性,从新的角度研究肿瘤的侵袭、转移机制。
Part I Increased prevalence of CD4+CD25+CD127low/-CD49d FoxP3+regulatory T cells in HCC tumor microenvironment
Objective Expansion of regulatory T cells (Tregs) in tumor microenvironment was one of the mechanisms by which cancer cells escaped host defense. The purpose of this study was to investigate the increasing prevalence of Tregs in HCC cancer microenvironment. Methods Levels of CD4+CD25+CD127low/-CD49d-FoxP3+Tregs in peripheral blood mononuclear cells (PBMCs) from HCC patients (n=58) and healthy donors (n=12), tumor infiltrating lymphocytes (TILs) extracted from HCC (n=20), matched peri-tumor and PBMCs were measured by flow cytometry. Their suppression were determined by using activation marker expression (CD154). Superana levels of interleukin (IL)-10and transforming growth factor (TGF)-β1were measured by enzyme linked immunosorbent assay. Results The prevalence of Tregs in PBMCs from HCC patients was significantly higher than that from healthy donors and expressed high levels of IL-10and TGF-β1Similarly, the frequency of Tregs in tumor tissues was increased compared with that in matched peri-tumor and PBMCs. TIL Tregs were more suppressible by approximately30%than their peri-tumor and PBMCs counterparts. CTLA-4, GITR and MFI of FoxP3at higher levels on tumour-infiltrating Tregs compared with matched peri-tumor and periphery. Conclusions This study identified an increased frequency of CD4+CD25+CD127low/-CD49d-FoxP3+Tregs in patients with HCC. Further effort is needed to establish new immunotherapeutic strategies to modulate Tregs to promote a competent antitumor response.
Part Ⅱ Comparative gene expression profile of CD4+CD25+CD127low/-CD49d-regulatory T cells in human hepatocellular carcinoma
Objective The aim of this study was to compare gene expression profile of regulatory T cell (Treg) in HCC tumor sits with that of peri-tumor by Agilent whole genome oligo microarray. Methods Levels of CD4+CD25+CD127low/-CD49d-Tregs in HCC tumor (n=9), matched peri-tumor and PBMCs were measured by flow cytometry. Tregs from tumor and peri-tumor were hybridized on an Agilent whole genome microarray. Functional analysis of the microarray data was performed using KEGG PATHWAY and Gene Ontology (GO) analyses. Results The prevalence of Tregs in HCC tumor was significantly higher than that in peri-tumor. There were1032genes up-regualtion and1357genes down-regulation in tumor Tregs compared to peri-tumor. KEGG PATHWAY showed that7pathway up-regulation and9down-regulation of intratumoral Tregs compared to peri-tumor. GO showed that431terms up-regulation and237terms down-regulation. Microarray gene expression analysis showed that CD46, BCL6, TNFRSF11A, MDM4, P2RX7, CD276, TNFRSF18and IL6R were among the most upregulated genes in tumor Tregs compared with peri-tumor Tregs. The selected genes were validated by RT-PCR. Functional analysis showed that the complement and p53pathways were significantly upregulated in tumor Tregs, and that the GO terms related to cell differentiation, proliferation, apoptosis and migration were significantly affected. Conclusions This study identified IL6/IL6R, RANK/RANKL signalling might play an impotant role in intratumoral Tregs between HCC tumor cells.
Part III Lectin Microarray based glycan profiling change of periphery regulatory T cell in patients with HCC
Objective The aims of this section was to assess the applicability of application of lectin microarray to screen the glycosylation changes in complex biological systems. Based on that, glycosylation profilings were screened to search for the characteristic glycan structures associated with periphery regulatory T cells in HCC patients. Methods Firstly, we selected one pair of model protein:FET and its sialic acid defective strains ASF, of which characteristic glycan structures on the cell surface were well defined. Integrated protein was labeled with Cy3, then frozen in liquid nitrogen to terminatate the labeling. Next, membrane protein of Tregs was extracted by natural membrane protein extraction kit.10μg of protein was added to each chip and incubated for2hours, after washing, scanning and statistical analysis, lectin affinity profiling was obtained from the chip. Different batches of samples were loaded to detect the reproducibility of lectin microarray. HCC cell lines HCCLM3, MHCC97H, MHCC97L and Hep3B were used to hydratied with lectinarray and glycotranferase PCR microarray. Results The results showed that the characteristic glycosylation difference between ASF and FET were dtermined by defined lectin microarray. Comparing with ASF, FET showed increased affinity and decreased signals for SNA, MAL-Ⅰ and MAL-Ⅱ. HCC patients periphery Tregs presented elevated signals for LCA, UEA-Ⅰ, VVA, SBA, ACA, LEL and decreased signals for RCA-I with contrast to healthy controls. MAL-Ⅰ, MAL-Ⅱ, WGA, PHA-L were correlated with ST6GALNAC1、ST3Ga11、FUT8and MGAT5. Conclusions Our results proved that lectin microarray is an applicable glcan analysis tool, and could be used in clinical to screen HCC related Tregs glycan profiling, in search of better glycol-biomarkers for tumor immuno-escape. Besides, our study also presented reference and technology platform for the evaluation of the relationship between complex biological systems and glycan alteration.
Part IV Overexpression of Galectin-1Associates with Poor Prognosis in Human Hepatocellular Carcinoma Following Resection
Objective High expression of the galectin-1predicted poor patient outcome in several tumors. The aim of this study was to investigate its prognostic value in patients with hepatocellular carcinoma (HCC) after resection. Methods Galectin-1and tumor-infiltrating FoxP3+regulatory T cells (Tregs) were validated by tissue microarrays from HCC patients (n=386) and statistically assessed for correlations with the clinical profiles and the prognosis of the patients. Serum galectin-1levels and frequency of FoxP3+Tregs in circulation was measured by ELISA and flowcyte, respectively. And serum levels of galectin-1was validated by ELISA in147HCC patients. Results We found that galectin-1, prevalently up-regulated in HCC, was significantly associated with tumor invasive characteristics (such as vascular invasion, incomplete encapsulation, poor differentiation, multiple number and large tumor size). Patients with high galectin-1expression had a significantly poorer tumor recurrence (p=0.025) and overall survival (p=0.021) than those with low galectin-1expression. Even in early-stage disease, high galectin-1expression also independently associated with shortened survival (p<0.001) and increased tumor recurrence (p=0.005). Multivariate Cox proportional hazards analysis showed that galectin-1was an independent marker for predicting poor prognosis of HCC. Galectin-llevel was positively related to number of tumor-infiltrating FoxP3+Tregs (r=0.416,p<0.001) and their combination served as a better prognosticator. The postoperative tumor recurrence and survival of HCC patients with galectin-1high and FoxP3high were significantly poorer than the other groups (both p<0.001). Conclusions Galectin-1may be a new prognostic factor for HCC after resection and potentially be a high-priority therapeutic target.
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