血管内皮抑素协同肿瘤特异性DC-T细胞的抗肿瘤效应及其机制探讨
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摘要
近年来,恶性肿瘤已成为全球主要的公共卫生问题,对于大多数肿瘤患者,手术、化疗和放疗等传统治疗未能有效控制病情。作为一种新兴的治疗模式,细胞免疫治疗能够有效控制某些化放疗失败的肿瘤,但总体而言其临床有效率仍然偏低。目前肿瘤生物治疗研究的热点之一就是寻找具有较强靶向性的、安全有效的肿瘤联合治疗新模式,以提高细胞免疫治疗恶性肿瘤的有效性。
血管新生是肿瘤的基本生物学特征,靶向肿瘤的血管新生是一种有良好前景的控制肿瘤生长的治疗策略。研究证实单独使用抗血管新生药物不能带来长期的生存获益,联合细胞免疫治疗可能获得持续性的肿瘤消退,这提示抗血管新生药物与细胞免疫治疗有协同效应。血管内皮抑素(Endostatin)被认为是一种没有毒副作用且不易耐药的、有潜力的抗血管新生药物。但迄今为止,Endostatin对细胞免疫治疗抗肿瘤效应的影响尚不清楚,上述的协同效应能否延伸到Endostatin还需明确。Endostatin与细胞免疫治疗联合应用的研究将为抗肿瘤效应强大而持久的发挥提供新的视角。
炎性微环境是肿瘤的主要构成特征,对于细胞免疫治疗的成败起决定性作用,其通过MDSC等多种抑制性因素使回输的杀伤性T细胞转换为抑制性调节T细胞(Treg)。此外,肿瘤微环境内的骨髓来源抑制细胞(MDSC)、肿瘤相关巨噬细胞(TAM)、未成熟树突状细胞因子(imDC)等免疫细胞及促炎细胞因子(IL-6、IL-17、 TNF-α及趋化因子等)能通过刺激血管新生和组织重构促进肿瘤发展,并形成肿瘤微环境内抑制性的免疫状态。新近研究证实抗血管新生药物Sunitinib可减少肿瘤微环境中的免疫抑制性细胞(MDSC、Treg)的数量、下调抑制性细胞因子[IL-10、转化生长因子(TGF)-β]和程序性死亡受体-1(PD-1)的表达,缓解与肿瘤相关的免疫抑制,促进肿瘤特异性TIL细胞在肿瘤组织内的浸润。但至今Endostatin对肿瘤微环境内免疫状态及对过继免疫细胞抗肿瘤效应的影响及机制尚未明确。本课题通过Lewis肺癌荷瘤鼠模型,研究Endostatin在发挥抗肿瘤效应过程中对肿瘤微环境内免疫网络的影响,进一步明确Endostatin抗肿瘤效应的具体机制,从而为开展抗血管新生的分子靶向治疗与细胞免疫治疗的联合寻找理论依据。
精确评估抗血管新生治疗后肿瘤微环境的改变对于明确其具体的抗肿瘤效应、优化治疗策略是十分重要的。肿瘤微环境中的炎性介质与促血管新生因子之间存在密切关系。其中,肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)是肿瘤微环境内重要的促血管新生炎性细胞因子,在调节免疫细胞、促进血管新生中发挥重要作用。TNF-α驱动的CXC趋化因子(CXCLs)与其受体(CXCR2)轴通路在肺损伤所致的血管新生中发挥关键作用。研究发现非小细胞肺癌(NSCLC)患者血清中TNF-α的水平较健康人群显著升高。IL-6主要通过转录信号传导子与激活子3(STAT3)信号通路促进VEGF的转录、内皮细胞的增殖和转移,从而在多种实体瘤的肿瘤微环境中发挥促血管新生的作用。目前国际上已有关于TNF-α和IL-6基因多态性与疾病的相关性研究。在本课题的工作中,我们探讨了肿瘤微环境中的TNF-α和IL-6基因型与肺癌发生的相关性,以进一步论证肿瘤微环境促血管新生的炎性因子与肺癌发生的关系,并为深入研究Endostatin抗肿瘤效应的主要作用靶点及机制提供新的线索。
为了刺激血管新生,肿瘤上调了一系列与血管新生相关的因子,这些发现为抗血管新生治疗的发展提供了理论基础。近年来,国际上抗血管新生的机制研究主要集中于VEGF及其受体通路,VEGF-A和其受体VEGFR-2是抗血管新生药物的关键靶点。而有关Endostatin抗血管新生的作用靶点及其机制的研究少有。一项对人类90%基因组的分析结果揭示Endostatin下调了微血管内皮细胞的调节血管生成因子的信号通路,包括VEGF、乏氧诱导因子-1α (HIF-1α)、肿瘤坏死因子受体(TNFR)等。这一研究提示Endostatin是一种多靶点的抗血管新生药物。鉴于肿瘤微环境内的免疫细胞、细胞因子及趋化因子在肿瘤血管新生中发挥的重要作用,使其成为抗血管新生治疗的理想靶点。本课题以肿瘤微环境中的TNF-α、IL-6或其受体、信号通路下游因子等作为主要靶目标,采用蛋白芯片技术筛选Endostatin抗肿瘤效应的主要作用靶点,进一步明确Endostatin与免疫系统间的相互作用,有望为合理设计抗血管新生的靶向药物Endostatin与细胞免疫治疗联合的最佳模式(应用顺序、时间及剂量)提供理论和实验基础。
总之,本课题组前期成功获得Lewis肺癌肿瘤抗原特异性的DC-T细胞,并经流式细胞术分析其细胞亚群主要为细胞毒性的CD8+T细胞。在此基础上,本课题研究了Endostatin对肿瘤特异性DC-T细胞抗肿瘤效应的影响,进而探求其对肿瘤微环境内的免疫网络的作用,以明确Endostatin与细胞免疫治疗的联合抗肿瘤的效应机制；从遗传多态性的角度,探讨肿瘤微环境的重要促血管新生炎性因子TNF-α和IL-6的基因型与中国北方汉族人群肺癌发生的相关性；进而以TNF-α和IL-6及其受体、信号通路下游因子为主要靶目标,利用蛋白芯片筛选Endostatin抗肿瘤效应的主要作用靶点,为合理设计抗血管新生的Endostatin与细胞免疫治疗联合的最佳模式提供了理论和实验基础。
第一部分血管内皮抑素协同肿瘤特异性DC-T细胞的抗肿瘤效应
目的探讨血管内皮抑素(Endostatin)对肿瘤特异性DC-T细胞抗肿瘤效应的影响。
方法
1.建立Lewis肺癌C57BL/6鼠移植瘤模型,设DC-T组、DC-T+Endostatin组及PBS对照组,每组均设C57BL/6鼠7只,小鼠种瘤后第7天开始给予治疗干预,第14天给药后24小时处死小鼠；隔日观察肿瘤生长情况、测量瘤径,并绘制肿瘤生长曲线；
2. DC-T+Endostatin组中,每只C57BL/6鼠给予重组人血管内皮抑素(rhEndostatin, Simcere公司)15mg/Kg/d,经鼠尾静脉注射,共14天；
3.取健康C57BL/6小鼠抗凝静脉血,采用Ficoll梯度离心法分离外周血单个核细胞(PBMC),尼龙毛柱法分离得到T淋巴细胞,将培养5天的DC细胞与Lewis肺癌细胞株反复冻融所获得的肿瘤抗原共同孵育3天,转入培养第9天的T淋巴细胞培养瓶内,再共同培养24小时,所获得的细胞即为肿瘤特异性DC-T细胞,经流式细胞分析验证其主要的细胞亚群；
4.DC-T组及DC-T+Endostatin组中,每只C57BL/6鼠于种瘤后第7天,给予肿瘤特异性DC-T细胞5×106个,经鼠尾静脉输注,共1次；
5.分离小鼠体内的肿瘤组织,观察肿瘤组织内的坏死情况,检测各组肿瘤组织微血管密度(MVD)评价血管新生情况；
6.Western blot、免疫组化分别检测各组肿瘤组织内VEGF和HIF-1α蛋白表达；
7.制备肿瘤组织单细胞悬液,流式细胞术检测各组肿瘤组织细胞悬液内CD8+T、mDC、TAM (M1/M2)和MDSC等细胞的比例；
8.酶联免疫吸附试验(ELISA)检测肿瘤组织细胞悬液中IL-6、IL-10、IL-17、TGF-β及干扰素-γ(INF-γ)等细胞因子表达。
结果
1.相较于对照组,肿瘤特异性DC-T细胞组有效抑制肿瘤生长(P=0.021)；肿瘤特异性DC-T细胞联合Endostatin治疗较对照组显著抑制了肿瘤的生长(P=0.009)。免疫组化检测显示,肿瘤特异性DC-T细胞治疗组的肿瘤组织内微血管密度(MVD)少于对照组(P=0.027)；而肿瘤特异性DC-T细胞联合Endostatin治疗组肿瘤组织的MVD显著减少(P=0.002),肿瘤组织坏死明显增加；
2.Western blot检测显示,相较于对照组,肿瘤特异性DC-T细胞治疗使肿瘤组织内的VEGF蛋白表达明显下降(P=0.002),且HIF-1α蛋白表达增加(P=0.000)；而肿瘤特异性DC-T细胞联合Endostatin治疗组VEGF表达显著下降(P=0.000),HIF-1α蛋白表达明显增加(P=0.000)；免疫组化染色结果支持了上述变化。ELISA检测显示,相较于对照组,肿瘤特异性DC-T细胞治疗使肿瘤组织内的促血管新生因子IL-6、IL-17表达明显减少(P值分别为0.026,0.044),而抑制血管新生的IFN-γ表达增加(P=0.026)；肿瘤特异性DC-T细胞联合Endostatin治疗使IL-6、IL-17显著减少(P值分别为0.008,0.01),而IFN-γ表达明显增加(P=0.009)；
3.流式细胞分析显示,相较于对照组,DC-T细胞治疗使肿瘤组织内免疫抑制性细胞MDSC(P=0.03)比例下降,TAM细胞的免疫增强M1型升高(P=0.045)而免疫抑制性的M2型下降(P=0.038)；肿瘤特异性DC-T细胞联合Endostatin治疗更显著地减少肿瘤组织内MDSC(P=0.009)和M2型TAM明显下降(P=0.008),M1型TAM细胞显著增加(P=0.005)。DC-T细胞治疗单独较对照组增加了肿瘤组织内mDC(P=0.038)和CD8+T细胞的浸润(P=0.019)；肿瘤特异性DC-T细胞联合Endostatin治疗显著上调mDC比例(P=0.005),明显增加肿瘤组织内CD8+T细胞的浸润(P=0.008)；
4.ELISA检测进一步证实了流式细胞分析的结果,相较于对照组,DC-T细胞治疗单独应用下调了肿瘤组织内的免疫抑制性细胞因子IL-6、IL-10、TGF-β和IL-17(P值分别为0.026,0.032,0.029,0.044),增加了参与溶瘤作用的IFN-γ表达(P=0.026)；肿瘤特异性DC-T细胞联合Endostatin治疗较PBS对照更显著地下调肿瘤组织内的IL-6、IL-10、TGF-β和IL-17表达(P值分别为0.008,0.008,0.002,0.01),IFN-γ表达明显增加(P=0.009)。
结论
1.肿瘤特异性DC-T细胞联合Endostatin治疗显著地减少了肿瘤组织内的血管结构,强力抑制肿瘤血管新生,明显减缓肿瘤生长,并促进了肿瘤坏死；
2.两种治疗的联合显著抑制了多种促血管新生因子表达,并上调抑制血管新生因子的表达,同时使HIF-1α蛋白表达显著增加,加剧了肿瘤组织内的低氧状态；
3.肿瘤特异性DC-T细胞联合Endostatin治疗显著地抑制了肿瘤组织内的免疫抑制性的MDSC和M2型TAM细胞比例,上调了免疫增强的M1型TAM比例,并使肿瘤组织内成熟DC和CD8+T细胞浸润明显增加,逆转了肿瘤微环境的免疫抑制；
4.两种治疗联合显著地下调了肿瘤组织内免疫抑制性的IL-6、IL-10、TGF-β和IL-17表达,增加了参与溶瘤作用的IFN-y表达,进一步支持了Endostatin协同肿瘤特异性DC-T细胞治疗在强力抑制血管新生和肿瘤生长的同时,有效逆转肿瘤微环境的免疫抑制,使肿瘤细胞更易被抗肿瘤免疫反应损伤。
第二部分血管内皮抑素有效纠正肺癌炎性肿瘤微环境的免疫抑制
目的探讨Endostatin对肺癌肿瘤微环境内的免疫抑制细胞及相关细胞因子的影响。
方法
1.建立Lewis肺癌C57BL/6鼠移植瘤模型,分为对照组、低剂量Endostatin、高剂量Endostatin三组,每组均设C57BL/6鼠7只。对照组给予PBS0.2ml/只/天,低剂量Endostatin组给予rhEndostatin7.5mg/Kg/d,高剂量Endostatin组给予rhEndostatin15mg/Kg/d,三组均于小鼠种瘤第7日开始给予治疗干预,均为经鼠尾静脉注射药物,治疗共14天。第14天给药后24小时处死荷瘤鼠。隔日观察肿瘤生长情况、测量瘤径,绘制肿瘤生长曲线；
2.分离小鼠体内的肿瘤组织,观察肿瘤组织内的坏死情况,检测肿瘤组织MVD,评价肿瘤血管新生；
3.Western blot、免疫组化分别检测各组肿瘤组织内VEGF和HIF-la蛋白表达；
4.制备肿瘤组织单细胞悬液,流式细胞术检测各组肿瘤组织细胞悬液内CD8+T、mDC、TAM (M1/M2)和MDSC等细胞的比例；
5.ELISA检测各组肿瘤组织细胞悬液中IL-6、IL-10、IL-17、TGF-β和IFN-γ等细胞因子的表达。
结果
1.相对于对照组,Endostatin使肿瘤生长明显受抑(P=0.013),且低剂量与高剂量Endostatin组间无明显差别(P>0.05)。免疫组化检测显示,CD31作为血管内皮细胞的标记,内皮细胞胞浆被染为黄棕色。低剂量Endostatin使肿瘤组织内微血管密度(MVD)较对照组明显减少(P=0.014)；高剂量Endostatin组肿瘤组织的MVD显著减少(P=0.002),肿瘤坏死明显增加；且低剂量与高剂量Endostatin组间存在明显差别(P<0.05)；
2.Western blot检测显示,相较于对照组,低剂量Endosattin治疗使肿瘤组织内的VEGF蛋白表达明显下降(P=0.001),且HIF-1α蛋白表达增加(P=0.000)；而高剂量Endostatin治疗组VEGF表达显著下降(P=0.000),HIF-1α蛋白表达明显增加(P=0.001)；免疫组化染色结果支持了上述变化。ELISA检测显示,相较于对照组,低剂量Endosattin治疗使肿瘤组织内的促血管新生因子IL-6、IL-17表达明显减少(P值分别为0.022,0.038),而抑制血管新生的IFN-y表达增加(P=0.044)；而高剂量Endostatin治疗组IL-6、IL-17显著减少(P值分别为0.012,0.029),而IFN-γ表达明显增加(P=0.036)。而低剂量与高剂量Endostatin组之间的上述变化无统计学差异(P>0.05)；
3.流式细胞分析显示,相较于对照组,低剂量Endosattin治疗使肿瘤组织内免疫抑制性细胞MDSC(P=0.035)和M2型TAM(P=0.042)比例下降,免疫增强的M1型TAM升高(P=0.031)；高剂量Endostatin治疗更显著地减少了肿瘤组织内MDSC(P=0.019)和M2型TAM(P=0.013),而M1型TAM显著增加(P=0.017)。低剂量Endosattin治疗较对照增加了肿瘤组织内mDC(P=0.034)和CD8+T细胞的浸润(P=0.038)；高剂量Endostatin治疗显著上调mDC比例(P=0.018),明显增加肿瘤组织内CD8+T细胞的浸润(P＝0.017)。而低剂量与高剂量Endostatin组间的上述变化无显著差异(P>0.05)；
4ELISA检测进一步证实了流式细胞分析的结果,相较于PBS对照,低剂量Endostatin治疗后荷瘤鼠肿瘤组织内的免疫抑制性细胞因子IL-6、IL-10、IL-17及TGF-p表达明显下降(P值分别为0.022,0.020,0.038,0.018),增加了参与溶瘤作用的IFN-γ,表达(P=0.044)；高剂量Endostatin治疗更显著地下调肿瘤组织内的IL-6、IL-10、TGF-p和IL-17表达(P值分别为0.012,0.018,0.014,0.029),IFN-γ表达明显增加(P=0.036)。而低剂量与高剂量Endostatin组间的上述变化无统计学差异(P>0.05)。
结论
1. EEndostatin以剂量依赖模式显著地抑制肿瘤血管新生,促进肿瘤坏死,从而强力抑制肿瘤的生长；
2.Endostatin通过抑制肿瘤组织内多种促血管新生因子的表达,上调抑制血管新生因子的表达,并使HIFla蛋白表达明显增加,加剧了肿瘤组织内的低氧状态；
3.Endostatin通过下调肿瘤微环境内免疫抑制性细胞MDSC和M2型TAM的比例,上调了免疫增强的M1型TAM细胞比例,并使肿瘤组织内成熟DC和CD8+T细胞浸润明显增加,有效纠正肿瘤微环境的免疫抑制；
4.Endostatin使肿瘤组织内的多种免疫抑制性细胞因子表达减少,同时增加了参与溶瘤作用的IFN-γ表达,进一步支持Endostatin可有效改善肿瘤微环境的免疫抑制,从而发挥协同DC-T细胞治疗的抗肿瘤效应。
第三部分促炎细胞因子TNF-α和IL-6的基因多态性与肺癌的发生相关
目的研究促炎细胞因子TNF-α和IL-6的基因启动子区多态性与肺癌发生的相关性。
方法
1.提取中国北方地区汉族人群中138例肺癌患者(肺癌组)和138位健康人(对照组)外周血基因组总DNA,两组的年龄和性别分布无统计学差异(P>0.05)；
2.用PCR方法分别扩增TNF-α基因启动子区-238G位点和IL-6基因启动子区-572C位点的片段；
3.PCR扩增TNF-α基因位点的产物片段约290个碱基对,采用Bgl Ⅱ酶进行酶切以明确-238G位点是否发生了G-to-A单碱基转换；
4.PCR扩增IL-6基因位点的产物片段约400碱基对,采用BsrBI酶进行酶切以明确-572C位点是否发生了C-to-G单碱基转换；
5.以χ2-Test验证基因型频率是否符合与Hardy-Weinberg平衡定律,采用非条件Logistic回归校正混杂因素,行多态性与肺癌风险关联性的统计学分析。
结果
1TNF-a基因-238G位点的单核苷酸多态性(SNP)检测
两组276例含290碱基对片段的PCR扩增产物,经Bgl II酶切产生了三种条带型,分别为80、210和290碱基对条带。由于酶Bgl II识别AGATCT,-238G位点发生单碱基G-to-A转换产生80和210碱基对条带,生成一个Bgl Ⅱ酶切位点。IRFLP结果提示TNF-a基因-238位点有三种SNP基因型,这三种多态性包括无转换、两个等位基因-238G位点都有一个单碱基G-to-A转换、等位基因中的一个基因-238G位点有一个单碱基G-to-A转换,即分别为野生型(wt),G-A/G-A和G-A基因型。
2IL-6基因-572C位点的SNP检测
两组276例含400碱基片段的PCR扩增产物,经BsrBI酶切产生了三种条带型分别为150、250和400碱基对条带。由于酶BsrBI识别CCGCTC,-572C位点发生了单碱基C-to-G转换产生150和250碱基对条带,生成一个BsrBI酶切位点。RFLP结果提示IL-6基因-572C位点有三种SNP基因型,这三种多态性包括无转换、两个等位基因-572C位点都有一个单碱基C-to-G转换、等位基因中的一个基因-572C位点有一个单碱基C-to-G转换,即分别为野生型(wt),G-A/G-A和G-A基因型。
3TNF-a基因-238G位点单碱基G-to-A转换的高频率分布与肺癌的发生相关
对TNF-a基因-238G位点的SNP进行统计学分析,证实两个等位基因-238GSNP的基因频率分布符合Hardy-Weinberg平衡定律(P>0.05)。大多数的病例具有-238G位点的野生型(wt) SNP(对照组113例,肺癌组99例)；对照组和肺癌组有相同数量的G-A基因型,对照组有2例G-A/G-A基因型,而肺癌组有14例G-A/G-A基因型。肺癌组G-A SNP数量的增加提示两个等位基因上G-A SNP与肺癌的发生相关(P=0.005,OR=1.74395%C10.232-0.682)。
4IL-6基因-572C位点单碱基C-to-G转换的高频率分布与肺癌的发生相关
对IL-6基因-572C位点的SNP进行统计学分析,证实两个等位基因-572C位点SNPs的基因频率分布符合Hardy-Weinberg平衡定律(P>0.05)。在IL-6基因-572C位点的SNP,对照组和肺癌组有相同数量的野生型(wt)或C-G SNP,而对照组有3例C-G/C-G基因型,肺癌组有9例C-G/C-G基因型。肺癌组C-G/C-GSNP数量的增加提示两个等位基因上纯合型C-G/C-G SNP与肺癌的发生相关(P=0.029,OR=I.23495%CI1.412-2.727)。
结论
1.肿瘤微环境中重要的促血管新生炎性因子TNF-a和IL-6的基因多态性是中国北方汉族人群肺癌遗传易感性的关键因素；
2. TNF-a基因启动子区-238G位点单碱基G-to-A转换和IL-6基因启动子区-572C位点单碱基C-to-G转换的高频率分布与肺癌的发生相关。
第四部分趋化因子CXCL1-2/CXCR2轴通路参与血管内皮抑素的抗肿瘤效应
目的采用蛋白芯片技术筛选Endostatin抗肿瘤效应的主要作用靶点。
方法
1.建立Lewis肺癌C57BL/6鼠移植瘤模型,设对照组和实验组,每组均设C57BL/6小鼠7只,于小鼠种瘤后第7天开始给予治疗干预,对照组给予PBS0.2m1/只/天,实验组给予rhEndostatin15mg/Kg/d,均经鼠尾静脉注射给药,治疗共14天。第14天末次治疗后24小时处死小鼠；
2.分离小鼠体内的肿瘤组织,取各组肿瘤组织块行蛋白芯片检测。检测项目包括CCL11、CCL2、CCL3、GM-CSF、IFN-γ、IL-12p70、IL-2、IL-4、IL-6、 IL-9、CXCL1、CXCL2、MCP5、SCF、sTNFR1、TIMP1和VEGF-A等19种促／抗血管新生因子。统计学分析采用Significance Analysis of Microarrays(SAM)基因芯片分析软件；
3.将对照组和实验组的样本量均扩大一倍,并重复上述实验,经处理后留取肿瘤组织行蛋白芯片检测。并根据第一次筛选的结果调整检测项目。统计学分析采用Significance Analysis of Microarrays (SAM)基因芯片分析软件,并将肿瘤样品的蛋白芯片检测结果进行聚类分析。
结果
1.初次实验中蛋白芯片杂交图和SAM分析结果显示,肿瘤组织内趋化因子CXCL1和CXCL2、CCL3的水平显著升高(分别为4.1倍,2.4倍,2.3倍),而VEGF-A、GM-CSF、sTNFR1/2、TIMP1等重要的促／抗血管新生因子的水平未发生明显变化；
2.根据第一次筛选的结果调整检测项目,包括GM-CSF、CCL27、CXCL1、 CXCL2、IL-2、MCP5、sTNFR1、TIMP1和VEGF-A等9种促／抗血管新生因子。重复实验中蛋白芯片杂交图和SAM分析结果显示,肿瘤组织内的趋化因子CXCL2水平仍呈显著升高,同时VEGFA、GM-CSF亦明显升高(分别为1.964倍,1.505倍,2.18倍),聚类分析结果验证了上述因子的变化趋势。
结论
1.肿瘤微环境内的多个促血管新生因子在Endostatin治疗后发生显著变化,证实了Endostatin具有多靶点抗血管新生的特征；
2.除了VEGF信号通路,肿瘤微环境内的趋化因子CXCL1-2/CXCR2轴信号通路也参与了Endostatin的抗肿瘤效应。
In recent years, malignant tumors have been a worldwide issue of public health. To most patients with tumors, conventional treatment strategies such as surgery, chemotherapy and radiotherapy couldn't effectively control diseases. As an emerging antitumor therapeutic method, cellular immunotherapy has remarkable curative effect on tumors, even those refractory to chemotherapy and radiotherapy. However, its clinical benefits in cancer therapy are very modest. At present, how to improve antitumor effect of cellular immunotherapy by combination with different safty and targeted therapies is always the key point in cancer research of antitumor biotherapy.
Angiogenesis is one of the basic hallmarks of cancer. Antiangiogenic therapy is a new and good prospective treatment strategy. Studies showed that monotherapy with antiangiogenic agents is failing to produce longlasting clinical responses in most patients, however, antiangiogenic molecules may contribute to persistent tumor's remission in combination with immunotherapy. And it suggested that antiangiogenic agents synergized with cellular immunotherapy. Endostatin is regarded as one of the promising antiangiogenesis agents with less adverse side effects and seldom susceptible to the resistance to antigiogenesis. However, it's rare to study on the role and mechanism of Endostatin playing in cellular therapeutic potency. To explore the relationship between Endostatin and cellular immunotherapy on antitumor treatment and provide a theoretical basis for raising the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy.
The inflammatory tumor microenvironment forms a major part of tumors and plays a vital role in the antitumor effect of cellular therapy, and adoptive cytotoxic T cells would be changed into immunosuppressive regular T cells (Treg) through many immunosuppressive factors such as MDSC in the tumor microenvironment. The immune inhibitory cells (MDSC, TAM, and imDC) and proinflammatory cytokines (IL-6,Il-17, TNF-α and chemokines) promote angiogenesis and tissue remodeling and contribute to tumor development and innumosuppression in the tunor micro-environment. Recent studies demonstrated that antiangiogenic agent Sunitinib decreased immunosuppressive cells such as MDSC and Treg, immunosuppressive cytokines IL-10and TGF-β, as well as T cell inhibitory factor PD-1and reversed immunosuppression in the tumor microenvironment. However, it's rare to study on the role and mechanism of Endostatin playing in the immune cells and related cytokines in the tumor microenvironment. In a Lewis lung cancer tumor-bearing mice model, we investigated the mechanism of Endostatin antitumor potency and provided a theoretical basis for designing the optimal strategy of Endostatin combined with cellular immunotherapy in cancer treatment.
Accurate evaluation of the changes in the tumor microenvironment after antiangiogenic therapy is very important to definite the mechanism of antitumor effect and design the optimal strategy in cancer treatment. Inflammatory cells and mediators are tightly related to proangiogenesis factors in the tumor microenvironment. Both TNF-α and IL-6are the vital proinflammatory cytokines in the tumor microenvironment. TNF-α promotes CXC chemokines and their receptor CXCR2axis signaling and plays the crucial role in neuvascularization of lung tissue in pulmonary inflammation and ischemia injury. The level of TNF-α is significantly higher in the serums of patients with non-small cell lung cancer (NSCLC) than that of healthy individuals. IL-6induces VEGF transduction, proliferation of endothial cells, and tumor metastasis via STAT3signaling in many solid tumors. Studies have demonstrated that genic polymorphisms of TNF-α and IL-6are associated with increased occurring risk of some benign or malignant diseases. In the present study, we studied SNP markers in the promoter-regulating regions of TNF-α and IL-6genes in the ethnic group Han of North China, to explore the SNP polymorphisms of the TNF-α and IL-6genes relevant to occurring of lung cancer. It will further investigate the relationship between pro-angiogenic inflammatory cytokines in the tumor microenvironment and occurring of lung cancer. Moreover, we may provide the theoretical basis for the main targets and mechanism of Endostatin antitumor effect.
Induction of angiogenesis requires a shift/switch towards activation/upregulation of inducers of angiogenesis over suppression of angiogenic inhibitors in the tumor microenvironment. Preclinical studies and clinical trials mainly focused on VEGF and its receptors signaling, and demonstrated that VEGF-A and its receptor VEGFR-2are key targets of antiangiogenic agents. However, it's rare to study on role and the molecular mechanism of Endostatin antitumor effect. In the human Genome study, the data demonstrated that Endostatin down-regulated proangiogenic signaling relevant to the endothelial cells such as VEGF, HIF-lα, and TNF-a receptor (sTNFR) and so on.The data suggested that Endostatin is a multi-target antiangiogenic agent. The cells and mediators of inflammation play the crucial role in tumor angiogenesis, so they are the promising therapeutic standpoints in antiangiogenic therapy.To screen the proflammatory cytokines TNF-α, IL-6and their receptors as well as down-stream factors of their signaling pathways by Propeome Profiling microarray and definite the main targets and mechanism of Endostatin antitumor effect, so as to provide a new perspective and theoretical basis for the optimal strategy of Endostatin combined with cellular immunotherapy.
Based on the previous study that we successfully cultured and achieved tumor antigen special DC-T cells of Lewis lung cancer, further demonstrated cytotoxic CD8+T cells are the main subpopulation of the DC-T cells detected by Flow cytometry (FCM). In this project, we explored the relationship between Endostatin and cellular immunotherapy on antitumor treatment, the role and molecular mechanisms of Endostatin playing its antitumor effect in the tumor microenvironment, then provided a theoretical basis for the main targets and mechanism of Endostatin antitumor effect and promoting the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy. Moreover, we invetigated the SNP polymorphisms of the TNF-a and IL-6genes relevant to occurring of lung cancer in the ethnic group Han of North China.Furthermore, we screened the main targets of Endostatin antitumor effect by Propeome Profiling microarray, we may provide a theoretical basis for the optimal strategy of raising the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy.
Part I Endostatin Synergized with Tumor Special DC-T Cellular Therapy on Antitumor Effect
OBJECTIVE To investigate the role of Endostatin playing in tumor special DC-T cellular therapy on antitumor effect.
METHODS
1. The transplanted Lewis lung cancer models of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells (LLC). The tumor-bearing mice were randomly divided into three groups, including DC-T cells combined with Endostatin group, DC-T cells group, and PBS control group.There were seven mice in each group. Since day7, the tumor-bearing mice had been administered by Endostatin or DC-T cells or PBS.The body weight and tumor diameter were measured every other day, respectively; and the tumor growth curve was drawn.
2. In DC-T cells combined with Endostatin group, each tumor-bearing mouse had been treated by rhEndostatin15mg/Kg/d by tail vein injection since day7after transplanted with LLCs, for14days.
3. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy C57BL/6mice by Ficoll density gradient centrifugation, T lyphoytes were enriched from PBMCs by Nylon wool column method. On day5, DCs and tumor antigen from LLC by repetitive freeze thaw method were cocultured for3days, then transferred into the culture flasks containing T cells, and cocultured for24hours, tumor antigen special DC-T cells were harvested. And, the T cell subpopulation was detected by Flow cytometry (FCM).
4. In DC-T cells group and DC-T cells combined with Endostatin group, each tumor-bearing mouse was treated by DC-T cells5×106by tail vein injection on day7after transplanted with LLCs.
5. All the mice were sacrificed24hours later after the last time of administration. Separated the tumor tissue of tumor-bearing mice and observed the tumor necrosis. The microvessel density (MVD) in the tumor tissue of tumor-bearing mice was detected by immunohistochemistry.
6. The VEGF and HIF-1α expressions were determined by Western blot and immunohistochemical staining, respectively.
7. The proportions of CD8+T, mDC, TAM (M1/M2), and MDSC in suspended tumor cells of tumor tissue were detected by FCM.
8. The expressions of IL-6, IL-10, IL-17, TGF-β and IFN-γ in suspended tumor cells of tumor tissue were detected by ELISA.
RESULTS:
1. Compared with control group, tumor special DC-T cells inhibited the tumors growth (P=0.021); tumor special DC-T cells combined with Endostatin remarkably regressed the tumors better than PBS control (P=0.009). The data of immunohistochemical staining are shown that CD31was emplored as the marker of vascular endothelial cell and its cytoplasm was dyed yellow-brown. The Microenvironment Density in DC-T cells group was slightly lower than that in the control group (P=0.027); the MVD of tumor special DC-T cells combined with Endostatin was deeply lowest in comparison with control group (P=0.002), concurrently tumor necrosis obviously increased in comparison with control group.
2. The expressions of VEGF, IL-6, IL-17in the tumors were decreased, concurrently those of IFN-γ and HIF-la were increased administered with DC-T cells compared with PBS control (P<0.05, respectively). Tumor special DC-T cells combined with Endostatin alleviated VEGF, IL-6, and IL-17, concurrently elevated IFN-y and HIF-la remarkably better than PBS control (P<0.01, respectively).
3. Flow cytometric data are shown that the proportions of MDSC and TAM (M2type) were significantly lower, those of mDC and TAM (M1type) were up-regulated, and CD8+T cells were recruited to infiltrated the tumors by DC-T cells therapy, compared with PBS control(P<0.05, respectively). The changes in tumor special DC-T cells combined with Endostatin were strongly more than PBS control (P<0.01, respectively).
4. Tumor special DC-T cells combined with Endostatin potently reduced the expressions of IL6, IL-10, TGF-β and IL-17in the tumor tissue, enhanced that of IFN-γ, concurrently recruited CD8+T cells into the tumor tissue in comparison with control group (P<0.01, respectively); The changes in DC-T cells group were significantly more than control group(P<0.05, respectively).
CONCLUSIONS:
1. Tumor special DC-T cells combined with Endostatin potently reduced tumor vasculature and tumor growth, and augmented tumor necrosis, implying the better antitumor effects than monotherapy.
2. Tumor special DC-T cells combined with Endostatin strongly inhibited tumor angiogenesis via regulating the pro/anti-angiogenic factors, and intensified the hypoxia in the tumors.
3. Tumor special DC-T cells combined with Endostatin remarkably inhibited immunosuppressive MDSC and TAM (M2type), enhanced mature DC and TAM (M1type), moreover, increased the number of CD8+T cells infiltrating the tumors, efficiently reversed the immunosuppression of the tumor microvironment.
4. Tumor special DC-T cells combined with Endostatin effectively provided the advantage of reducing the levels of immunosuppressive cytokines in the tumor regions, further exhibited synergistic and much better antitumor effects than monotherapy strategy, making them more vulnerable to the immune reactions.
Part Ⅱ Endostatin Effectively Relieved Immunosuppression of the Inflammatory Tumor Microenvironment in Lung Caner
OBJECTIVE To investigate the role of Endostatin playing in immunosuppressive cells and related cytokines in the inflammatory tumor microenvironment in lung caner.
METHODS
1. The transplanted Lewis lung cancer model of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells(LLC). The tumor-bearing mice were randomly divided into three groups, including high-dose Endostatin group (rhEndostatin15mg/Kg/d), low-dose Endostatin (rhEndostatin7.5mg/Kg/d) group, and control group(PBS0.2ml/d), all the mice were administrated by tail vein injection since day7after transplanted with LLCs, for14days.There were seven mice in each group. All the mice were sacrificed24hours later after the last time of administration. The body weight and tumor diameter were measured every other day, respectively; and the tumor growth curve was drawn.
2. Separated the tumor tissue of tumor-bearing mice and observed the tumor necrosis. The microvessel density (MVD) in the tumor tissue of tumor-bearing mice was detected by immunohistochemistry.
3. The VEGF and HIF-1α expressions were determined by Western blot and immunohistochemical staining, respectively.
4. The proportions of CD8+T, mDC, TAM(M1/M2), and MDSC in suspended tumor cells of tumor tissue were detected by Flow cytometry (FCM).
5. The expressions of IL-6, IL-10, IL-17, TGF-β and IFN-γ in suspended tumor cells of tumor tissue were detected by ELISA.
RESULTS
1. Compared with PBS control group, Endostatin inhibited the tumors growth (P<0.05), and there were no significant difference between high-dose and low-dose Endostatin (P>0.05); the Microenvironment Density in low-dose Endostatin group was slightly lower than that in the control group (P<0.05), that strongly reduced in high-dose Endostatin (P<0.01). There were significant difference between high-dose and low-dose Endostatin (P<0.05).
2. Endostatin greatly decreased the expressions of VEGF, IL-6and IL-17, enhanced the expressions of IFN-y and HIF-la compared with control group (P<0.05, respectively) by Western blot, immunohistochemistry or ELISA. There were significant difference between high-dose and low-dose Endostatin (P<0.05).
3. Flow cytometric data are shown that the proportions of MDSC and TAM (M2type) were significantly lower, those of mDC and TAM (M1type) were up-regulated, and CD8+T cells were recruited to infiltrated the tumors by Endostatin therapy, compared with PBS control(P<0.05, respectively). And there were no significant difference between high-dose and low-dose Endostatin (P>0.05).
4. Endostatin significantly reduced the expressions of IL6, IL-10, TGF-β and IL-17in the tumor tissue, and concurrently elevated that of IFN-γ in comparison with control group (P<0.05, respectively); There were no significant difference between high-dose and low-dose Endostatin (P>0.05).
CONCLUSIONS
1. Endostatin strongly reduced tumor vasculature and tumor growth, and augmented tumor necrosis.
2. Endostatin inhibited tumor angiogenesis via regulating the pro/anti-angiogenic factors in dose-dependent manner, and intensified the hypoxia in the tumors.
3. Endostatin remarkably inhibited immunosuppressive MDSC and TAM, remarkably inhibited immunosuppressive MDSC and TAM (M2type), enhanced mature DC and TAM (M1type), moreover, increased the number of CD8+T cells infiltrating the tumors, efficiently reversed the immunosuppression of the tumor microvironment.
4. Endostatin effectively provided the advantage of reducing the levels of immune inhibitory cytokines in the tumor regions, further demonstrated that Endostatin efficantly reversed immunosuppression of the inflammatory tumor microenvironment in lung caner, promoted mature DC and CD8+T cells to infiltrate the tumors, so as to synergize with tumor special DC-T cellular therapy on antitumor effect.
Part Ⅲ Pro-inflammatory cytokines TNF-α and IL-6gene polymorphisms were associated with occurring of lung cancer
OBJECTIVE To investigate the relationship between SNP polymorphisms of pro-inflammatory cytokines TNF-α and IL-6genes and occurring of lung cancer.
METHODS
1.138consecutive patients with primary lung cancer were recruited in this study. The control group consisted of138healthy individuals. All of them were from the ethnic group Han of North China. The ages, sex distribution, and behaviors (such as smoking or not) between lung cancer group and healthy control group had no significant difference statistically (P>0.05). Total DNAs were isolated from Peripheral blood mononuclear cells of138lung cancer patients and138healthy individuals.
2. PCR were performed and amplified-238G locus of TNF-α gene and-572C locus of IL-6gene, respectively. Further, the gene polymorphisms of two cytokines were revealed by restriction fragment length polymorphism (RFLP).
3. The PCR produced fragments of-238G locus of TNF-α gene about290bp as expected. The PCR products were then subjected to BglⅡ digestion to explore if a G-to-A single base alteration occurring at-238locus.
4. The PCR produced fragments of-572C locus of IL-6gene about400bp as expected. The PCR products were digested with BsrBI to explore if a C-to-G single base alteration occurring at-572C locus.
5. The correlation between the polymorphisms and risks of lung cancer was indicated by odds ratio (OR) and their95%confidence interval (CI). OR values and their95%CI were calculated using a non-conditional logistic regression model, which were adjusted by ages, gender, smoking and other factors.
RESULTS
1Detection of SNP polymorphisms at-238G locus of the TNF-a gene
The PCR produced fragments about290bp as expected, digestion of the290bp fragments from the276individuals in both groups with BglⅡ generated three kinds of band patterns on agarose gels:(ⅰ) a band of about290bp,(ⅱ) two bands with sizes of about80and210bp, respectively, or (ⅲ) three bands with sizes of80,210, and290bp, respectively.
Since BglⅡ recognizes AGATCT, generation of the80and210bp bands may be explained by a single-base G-to-A alteration occurred at-238G locus, which would generate a BglⅡ locus. Three kinds of SNP patterns were found at-238G locus of the TNF-a gene, including no alteration, a single-base G-to-A alteration at-238G locus of both alleles, a single-base G-to-A alteration at-238G locus of one of both alleles, were named as wt, G-A/G-A, and G-A, respectively.
2Detection of SNP polymorphisms at-572C locus of the IL-6gene
The PCR produced fragments about400bp as expected, digestion of the400bp fragments from the276individuals in both groups with BsrBI generated3kinds of band patterns on agarose gels:(ⅰ) a band of about400bp,(ⅱ) two bands with sizes of about150and250bp, respectively, or (ⅲ) three bands with sizes of150,250, and400bp, respectively.
Since BsrBI recognizes CCGCTC, generation of the150and250bp bands may be explained by a single-base C-to-G alteration at-572C locus, which would generate a BsrBI locus.These three polymorphisms, including no alteration, a single-base C-to-G alteration at-572C locus of both alleles, a single-base C-to-G alteration at-572C locus of one of both alleles, were named as wt, C-G/C-G, and C-G, respectively.
3High rates of single-base G-to-A alteration at-238G locus of the TNF-a gene
correlated with occurring of lung cancer
It was founded that the frequency distribution of-238G SNPs at both alleles fits the Hardy-Weinberg equilibrium (.P>0.05). Most cases possessed a wt SNP at-238G locus (113and99for healthy control group and lung cancer group, respectively). The control group and lung cancer group had similar number of G-A cases, respectively. The control group had2G-A/G-A cases, while lung cancer group had14G-A/G-A cases. It was suggested that correlations between G-A/G-A cases of-238G SNPs and occurring of lung cancer (P=0.005, OR=1.74395%CI0.232-0.682)
4High rates of single-base C-to-G alteration at-572C locus of the IL-6gene correlated with tumorgenesis of lung cancer.
Similar situations were also found for the SNPs on-572C locus of the IL-6gene. The healthy control group and lung cancer group had similar numbers of wt or C-G SNPs. However, the control group had three C-G/C-G cases, while lung cancer group had nine C-G/C-G cases. The increased numbers of C-G/C-G SNPs in lung cancer group suggested that the homozygous C-G/C-G SNPs on both alleles may be related to occurring of lung cancer (P=0.029, OR=1.23495%CI1.412-2.727).
CONCLUSIONS
1. The TNF-α and IL-6gene polymorphisms may be a critical risk for the genie susceptibility to lung cancer in the ethnic group Han of North China.
2. The high rates of singlebase G-to-A alteration at-238G locus of both alleles of the TNF-α gene and high rates of single-base C-to-G alteration at-572G locus of both alleles of the IL-6gene correlated with tumorgenesis of lung cancer.
Part Ⅳ Chemokines CXCL1-2/CXCR2Axis Signaling were Involved in the Antitumor Effect of Endostatin
OBJECTIVE To screen the main targets of the antitumor effect of Endostatin using Proteome Profiler arrays.
METHODS
1. The transplanted Lewis lung cancer model of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells (LLC). The tumor-bearing mice were randomly divided into two groups, including Endostatin group (rhEndostatin15mg/Kg/d) and control group (PBS0.2ml/d). There were seven mice in each group. All the mice were administrated by tail vein injection since day7after transplanted with LLCs, for14days. All the mice were sacrificed24hours later after the last time of administration.
2. The tumor specimens were separated from tumor-bearing mice. Proteome Profiling array of tumor specimens was performed, which included19pro/anti-angigenic factors such as CCL11, CCL2, CCL3, GM-CSF, IFN-γ, IL-12p70, IL-2, IL-4, IL-6, IL-9, CXCL1, CXCL2, MCP5, SCF, sTNFRl, TIMP1and VEGF-A so on. Significance Analysis of Microarrays (SAM) soft was used for statistical analysis.
3. The sample sizes of Endostatin group and control group both were expanded doubly, and the experiment was repeated for further validation. Moreover, the molecular profiling was adjusted based on the data of the first experiment. Significance Analysis of Microarrays (SAM) soft and clustering analysis were used for statistical analysis.
RESULTS
1. The levels of CXCL1, CXCL2, and CCL3in the tumors were remarkably enhanced (4.1fold,2.4fold, and2.3fold, respectively), however, the critical pro-angiogenic factors including VEGF-A, GM-CSF, sTNFR1/2and TIMP1had no significant change.
2. Based on the above data, the Proteome profiling was adjusted to include the key pro/anti-angiogenic factors such as GM-CSF、CCL27、CXCL1、CXCL2、IL-2、 MCP5、sTNFR1、TIMP1and VEGF-A. In the repeated experiment, according to SAM soft and clustering analysis, CXCL2was still strongly elevated, concurrently VEGF-A and GM-CSF were significantly enhanced (1.964fold, 1.505fold, and2.18fold, respectively).
CONCLUSIONS
1. The data demonstrated that multiple proangiogenic factors had remarkable changes after receiving Endostatin therapy, and strongly supported that Endostatin have the characteristic of multi-targeted antiangiogenic efficancy.
2. Besides VEGF signaling pathway, chemokines CXCL1-2/CXCR2axis signaling in the tumor microvironment participated in the antitumor effect of Endostatin.
血管新生是肿瘤的基本生物学特征,靶向肿瘤的血管新生是一种有良好前景的控制肿瘤生长的治疗策略。研究证实单独使用抗血管新生药物不能带来长期的生存获益,联合细胞免疫治疗可能获得持续性的肿瘤消退,这提示抗血管新生药物与细胞免疫治疗有协同效应。血管内皮抑素(Endostatin)被认为是一种没有毒副作用且不易耐药的、有潜力的抗血管新生药物。但迄今为止,Endostatin对细胞免疫治疗抗肿瘤效应的影响尚不清楚,上述的协同效应能否延伸到Endostatin还需明确。Endostatin与细胞免疫治疗联合应用的研究将为抗肿瘤效应强大而持久的发挥提供新的视角。
炎性微环境是肿瘤的主要构成特征,对于细胞免疫治疗的成败起决定性作用,其通过MDSC等多种抑制性因素使回输的杀伤性T细胞转换为抑制性调节T细胞(Treg)。此外,肿瘤微环境内的骨髓来源抑制细胞(MDSC)、肿瘤相关巨噬细胞(TAM)、未成熟树突状细胞因子(imDC)等免疫细胞及促炎细胞因子(IL-6、IL-17、 TNF-α及趋化因子等)能通过刺激血管新生和组织重构促进肿瘤发展,并形成肿瘤微环境内抑制性的免疫状态。新近研究证实抗血管新生药物Sunitinib可减少肿瘤微环境中的免疫抑制性细胞(MDSC、Treg)的数量、下调抑制性细胞因子[IL-10、转化生长因子(TGF)-β]和程序性死亡受体-1(PD-1)的表达,缓解与肿瘤相关的免疫抑制,促进肿瘤特异性TIL细胞在肿瘤组织内的浸润。但至今Endostatin对肿瘤微环境内免疫状态及对过继免疫细胞抗肿瘤效应的影响及机制尚未明确。本课题通过Lewis肺癌荷瘤鼠模型,研究Endostatin在发挥抗肿瘤效应过程中对肿瘤微环境内免疫网络的影响,进一步明确Endostatin抗肿瘤效应的具体机制,从而为开展抗血管新生的分子靶向治疗与细胞免疫治疗的联合寻找理论依据。
精确评估抗血管新生治疗后肿瘤微环境的改变对于明确其具体的抗肿瘤效应、优化治疗策略是十分重要的。肿瘤微环境中的炎性介质与促血管新生因子之间存在密切关系。其中,肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)是肿瘤微环境内重要的促血管新生炎性细胞因子,在调节免疫细胞、促进血管新生中发挥重要作用。TNF-α驱动的CXC趋化因子(CXCLs)与其受体(CXCR2)轴通路在肺损伤所致的血管新生中发挥关键作用。研究发现非小细胞肺癌(NSCLC)患者血清中TNF-α的水平较健康人群显著升高。IL-6主要通过转录信号传导子与激活子3(STAT3)信号通路促进VEGF的转录、内皮细胞的增殖和转移,从而在多种实体瘤的肿瘤微环境中发挥促血管新生的作用。目前国际上已有关于TNF-α和IL-6基因多态性与疾病的相关性研究。在本课题的工作中,我们探讨了肿瘤微环境中的TNF-α和IL-6基因型与肺癌发生的相关性,以进一步论证肿瘤微环境促血管新生的炎性因子与肺癌发生的关系,并为深入研究Endostatin抗肿瘤效应的主要作用靶点及机制提供新的线索。
为了刺激血管新生,肿瘤上调了一系列与血管新生相关的因子,这些发现为抗血管新生治疗的发展提供了理论基础。近年来,国际上抗血管新生的机制研究主要集中于VEGF及其受体通路,VEGF-A和其受体VEGFR-2是抗血管新生药物的关键靶点。而有关Endostatin抗血管新生的作用靶点及其机制的研究少有。一项对人类90%基因组的分析结果揭示Endostatin下调了微血管内皮细胞的调节血管生成因子的信号通路,包括VEGF、乏氧诱导因子-1α (HIF-1α)、肿瘤坏死因子受体(TNFR)等。这一研究提示Endostatin是一种多靶点的抗血管新生药物。鉴于肿瘤微环境内的免疫细胞、细胞因子及趋化因子在肿瘤血管新生中发挥的重要作用,使其成为抗血管新生治疗的理想靶点。本课题以肿瘤微环境中的TNF-α、IL-6或其受体、信号通路下游因子等作为主要靶目标,采用蛋白芯片技术筛选Endostatin抗肿瘤效应的主要作用靶点,进一步明确Endostatin与免疫系统间的相互作用,有望为合理设计抗血管新生的靶向药物Endostatin与细胞免疫治疗联合的最佳模式(应用顺序、时间及剂量)提供理论和实验基础。
总之,本课题组前期成功获得Lewis肺癌肿瘤抗原特异性的DC-T细胞,并经流式细胞术分析其细胞亚群主要为细胞毒性的CD8+T细胞。在此基础上,本课题研究了Endostatin对肿瘤特异性DC-T细胞抗肿瘤效应的影响,进而探求其对肿瘤微环境内的免疫网络的作用,以明确Endostatin与细胞免疫治疗的联合抗肿瘤的效应机制；从遗传多态性的角度,探讨肿瘤微环境的重要促血管新生炎性因子TNF-α和IL-6的基因型与中国北方汉族人群肺癌发生的相关性；进而以TNF-α和IL-6及其受体、信号通路下游因子为主要靶目标,利用蛋白芯片筛选Endostatin抗肿瘤效应的主要作用靶点,为合理设计抗血管新生的Endostatin与细胞免疫治疗联合的最佳模式提供了理论和实验基础。
第一部分血管内皮抑素协同肿瘤特异性DC-T细胞的抗肿瘤效应
目的探讨血管内皮抑素(Endostatin)对肿瘤特异性DC-T细胞抗肿瘤效应的影响。
方法
1.建立Lewis肺癌C57BL/6鼠移植瘤模型,设DC-T组、DC-T+Endostatin组及PBS对照组,每组均设C57BL/6鼠7只,小鼠种瘤后第7天开始给予治疗干预,第14天给药后24小时处死小鼠；隔日观察肿瘤生长情况、测量瘤径,并绘制肿瘤生长曲线；
2. DC-T+Endostatin组中,每只C57BL/6鼠给予重组人血管内皮抑素(rhEndostatin, Simcere公司)15mg/Kg/d,经鼠尾静脉注射,共14天；
3.取健康C57BL/6小鼠抗凝静脉血,采用Ficoll梯度离心法分离外周血单个核细胞(PBMC),尼龙毛柱法分离得到T淋巴细胞,将培养5天的DC细胞与Lewis肺癌细胞株反复冻融所获得的肿瘤抗原共同孵育3天,转入培养第9天的T淋巴细胞培养瓶内,再共同培养24小时,所获得的细胞即为肿瘤特异性DC-T细胞,经流式细胞分析验证其主要的细胞亚群；
4.DC-T组及DC-T+Endostatin组中,每只C57BL/6鼠于种瘤后第7天,给予肿瘤特异性DC-T细胞5×106个,经鼠尾静脉输注,共1次；
5.分离小鼠体内的肿瘤组织,观察肿瘤组织内的坏死情况,检测各组肿瘤组织微血管密度(MVD)评价血管新生情况；
6.Western blot、免疫组化分别检测各组肿瘤组织内VEGF和HIF-1α蛋白表达；
7.制备肿瘤组织单细胞悬液,流式细胞术检测各组肿瘤组织细胞悬液内CD8+T、mDC、TAM (M1/M2)和MDSC等细胞的比例；
8.酶联免疫吸附试验(ELISA)检测肿瘤组织细胞悬液中IL-6、IL-10、IL-17、TGF-β及干扰素-γ(INF-γ)等细胞因子表达。
结果
1.相较于对照组,肿瘤特异性DC-T细胞组有效抑制肿瘤生长(P=0.021)；肿瘤特异性DC-T细胞联合Endostatin治疗较对照组显著抑制了肿瘤的生长(P=0.009)。免疫组化检测显示,肿瘤特异性DC-T细胞治疗组的肿瘤组织内微血管密度(MVD)少于对照组(P=0.027)；而肿瘤特异性DC-T细胞联合Endostatin治疗组肿瘤组织的MVD显著减少(P=0.002),肿瘤组织坏死明显增加；
2.Western blot检测显示,相较于对照组,肿瘤特异性DC-T细胞治疗使肿瘤组织内的VEGF蛋白表达明显下降(P=0.002),且HIF-1α蛋白表达增加(P=0.000)；而肿瘤特异性DC-T细胞联合Endostatin治疗组VEGF表达显著下降(P=0.000),HIF-1α蛋白表达明显增加(P=0.000)；免疫组化染色结果支持了上述变化。ELISA检测显示,相较于对照组,肿瘤特异性DC-T细胞治疗使肿瘤组织内的促血管新生因子IL-6、IL-17表达明显减少(P值分别为0.026,0.044),而抑制血管新生的IFN-γ表达增加(P=0.026)；肿瘤特异性DC-T细胞联合Endostatin治疗使IL-6、IL-17显著减少(P值分别为0.008,0.01),而IFN-γ表达明显增加(P=0.009)；
3.流式细胞分析显示,相较于对照组,DC-T细胞治疗使肿瘤组织内免疫抑制性细胞MDSC(P=0.03)比例下降,TAM细胞的免疫增强M1型升高(P=0.045)而免疫抑制性的M2型下降(P=0.038)；肿瘤特异性DC-T细胞联合Endostatin治疗更显著地减少肿瘤组织内MDSC(P=0.009)和M2型TAM明显下降(P=0.008),M1型TAM细胞显著增加(P=0.005)。DC-T细胞治疗单独较对照组增加了肿瘤组织内mDC(P=0.038)和CD8+T细胞的浸润(P=0.019)；肿瘤特异性DC-T细胞联合Endostatin治疗显著上调mDC比例(P=0.005),明显增加肿瘤组织内CD8+T细胞的浸润(P=0.008)；
4.ELISA检测进一步证实了流式细胞分析的结果,相较于对照组,DC-T细胞治疗单独应用下调了肿瘤组织内的免疫抑制性细胞因子IL-6、IL-10、TGF-β和IL-17(P值分别为0.026,0.032,0.029,0.044),增加了参与溶瘤作用的IFN-γ表达(P=0.026)；肿瘤特异性DC-T细胞联合Endostatin治疗较PBS对照更显著地下调肿瘤组织内的IL-6、IL-10、TGF-β和IL-17表达(P值分别为0.008,0.008,0.002,0.01),IFN-γ表达明显增加(P=0.009)。
结论
1.肿瘤特异性DC-T细胞联合Endostatin治疗显著地减少了肿瘤组织内的血管结构,强力抑制肿瘤血管新生,明显减缓肿瘤生长,并促进了肿瘤坏死；
2.两种治疗的联合显著抑制了多种促血管新生因子表达,并上调抑制血管新生因子的表达,同时使HIF-1α蛋白表达显著增加,加剧了肿瘤组织内的低氧状态；
3.肿瘤特异性DC-T细胞联合Endostatin治疗显著地抑制了肿瘤组织内的免疫抑制性的MDSC和M2型TAM细胞比例,上调了免疫增强的M1型TAM比例,并使肿瘤组织内成熟DC和CD8+T细胞浸润明显增加,逆转了肿瘤微环境的免疫抑制；
4.两种治疗联合显著地下调了肿瘤组织内免疫抑制性的IL-6、IL-10、TGF-β和IL-17表达,增加了参与溶瘤作用的IFN-y表达,进一步支持了Endostatin协同肿瘤特异性DC-T细胞治疗在强力抑制血管新生和肿瘤生长的同时,有效逆转肿瘤微环境的免疫抑制,使肿瘤细胞更易被抗肿瘤免疫反应损伤。
第二部分血管内皮抑素有效纠正肺癌炎性肿瘤微环境的免疫抑制
目的探讨Endostatin对肺癌肿瘤微环境内的免疫抑制细胞及相关细胞因子的影响。
方法
1.建立Lewis肺癌C57BL/6鼠移植瘤模型,分为对照组、低剂量Endostatin、高剂量Endostatin三组,每组均设C57BL/6鼠7只。对照组给予PBS0.2ml/只/天,低剂量Endostatin组给予rhEndostatin7.5mg/Kg/d,高剂量Endostatin组给予rhEndostatin15mg/Kg/d,三组均于小鼠种瘤第7日开始给予治疗干预,均为经鼠尾静脉注射药物,治疗共14天。第14天给药后24小时处死荷瘤鼠。隔日观察肿瘤生长情况、测量瘤径,绘制肿瘤生长曲线；
2.分离小鼠体内的肿瘤组织,观察肿瘤组织内的坏死情况,检测肿瘤组织MVD,评价肿瘤血管新生；
3.Western blot、免疫组化分别检测各组肿瘤组织内VEGF和HIF-la蛋白表达；
4.制备肿瘤组织单细胞悬液,流式细胞术检测各组肿瘤组织细胞悬液内CD8+T、mDC、TAM (M1/M2)和MDSC等细胞的比例；
5.ELISA检测各组肿瘤组织细胞悬液中IL-6、IL-10、IL-17、TGF-β和IFN-γ等细胞因子的表达。
结果
1.相对于对照组,Endostatin使肿瘤生长明显受抑(P=0.013),且低剂量与高剂量Endostatin组间无明显差别(P>0.05)。免疫组化检测显示,CD31作为血管内皮细胞的标记,内皮细胞胞浆被染为黄棕色。低剂量Endostatin使肿瘤组织内微血管密度(MVD)较对照组明显减少(P=0.014)；高剂量Endostatin组肿瘤组织的MVD显著减少(P=0.002),肿瘤坏死明显增加；且低剂量与高剂量Endostatin组间存在明显差别(P<0.05)；
2.Western blot检测显示,相较于对照组,低剂量Endosattin治疗使肿瘤组织内的VEGF蛋白表达明显下降(P=0.001),且HIF-1α蛋白表达增加(P=0.000)；而高剂量Endostatin治疗组VEGF表达显著下降(P=0.000),HIF-1α蛋白表达明显增加(P=0.001)；免疫组化染色结果支持了上述变化。ELISA检测显示,相较于对照组,低剂量Endosattin治疗使肿瘤组织内的促血管新生因子IL-6、IL-17表达明显减少(P值分别为0.022,0.038),而抑制血管新生的IFN-y表达增加(P=0.044)；而高剂量Endostatin治疗组IL-6、IL-17显著减少(P值分别为0.012,0.029),而IFN-γ表达明显增加(P=0.036)。而低剂量与高剂量Endostatin组之间的上述变化无统计学差异(P>0.05)；
3.流式细胞分析显示,相较于对照组,低剂量Endosattin治疗使肿瘤组织内免疫抑制性细胞MDSC(P=0.035)和M2型TAM(P=0.042)比例下降,免疫增强的M1型TAM升高(P=0.031)；高剂量Endostatin治疗更显著地减少了肿瘤组织内MDSC(P=0.019)和M2型TAM(P=0.013),而M1型TAM显著增加(P=0.017)。低剂量Endosattin治疗较对照增加了肿瘤组织内mDC(P=0.034)和CD8+T细胞的浸润(P=0.038)；高剂量Endostatin治疗显著上调mDC比例(P=0.018),明显增加肿瘤组织内CD8+T细胞的浸润(P＝0.017)。而低剂量与高剂量Endostatin组间的上述变化无显著差异(P>0.05)；
4ELISA检测进一步证实了流式细胞分析的结果,相较于PBS对照,低剂量Endostatin治疗后荷瘤鼠肿瘤组织内的免疫抑制性细胞因子IL-6、IL-10、IL-17及TGF-p表达明显下降(P值分别为0.022,0.020,0.038,0.018),增加了参与溶瘤作用的IFN-γ,表达(P=0.044)；高剂量Endostatin治疗更显著地下调肿瘤组织内的IL-6、IL-10、TGF-p和IL-17表达(P值分别为0.012,0.018,0.014,0.029),IFN-γ表达明显增加(P=0.036)。而低剂量与高剂量Endostatin组间的上述变化无统计学差异(P>0.05)。
结论
1. EEndostatin以剂量依赖模式显著地抑制肿瘤血管新生,促进肿瘤坏死,从而强力抑制肿瘤的生长；
2.Endostatin通过抑制肿瘤组织内多种促血管新生因子的表达,上调抑制血管新生因子的表达,并使HIFla蛋白表达明显增加,加剧了肿瘤组织内的低氧状态；
3.Endostatin通过下调肿瘤微环境内免疫抑制性细胞MDSC和M2型TAM的比例,上调了免疫增强的M1型TAM细胞比例,并使肿瘤组织内成熟DC和CD8+T细胞浸润明显增加,有效纠正肿瘤微环境的免疫抑制；
4.Endostatin使肿瘤组织内的多种免疫抑制性细胞因子表达减少,同时增加了参与溶瘤作用的IFN-γ表达,进一步支持Endostatin可有效改善肿瘤微环境的免疫抑制,从而发挥协同DC-T细胞治疗的抗肿瘤效应。
第三部分促炎细胞因子TNF-α和IL-6的基因多态性与肺癌的发生相关
目的研究促炎细胞因子TNF-α和IL-6的基因启动子区多态性与肺癌发生的相关性。
方法
1.提取中国北方地区汉族人群中138例肺癌患者(肺癌组)和138位健康人(对照组)外周血基因组总DNA,两组的年龄和性别分布无统计学差异(P>0.05)；
2.用PCR方法分别扩增TNF-α基因启动子区-238G位点和IL-6基因启动子区-572C位点的片段；
3.PCR扩增TNF-α基因位点的产物片段约290个碱基对,采用Bgl Ⅱ酶进行酶切以明确-238G位点是否发生了G-to-A单碱基转换；
4.PCR扩增IL-6基因位点的产物片段约400碱基对,采用BsrBI酶进行酶切以明确-572C位点是否发生了C-to-G单碱基转换；
5.以χ2-Test验证基因型频率是否符合与Hardy-Weinberg平衡定律,采用非条件Logistic回归校正混杂因素,行多态性与肺癌风险关联性的统计学分析。
结果
1TNF-a基因-238G位点的单核苷酸多态性(SNP)检测
两组276例含290碱基对片段的PCR扩增产物,经Bgl II酶切产生了三种条带型,分别为80、210和290碱基对条带。由于酶Bgl II识别AGATCT,-238G位点发生单碱基G-to-A转换产生80和210碱基对条带,生成一个Bgl Ⅱ酶切位点。IRFLP结果提示TNF-a基因-238位点有三种SNP基因型,这三种多态性包括无转换、两个等位基因-238G位点都有一个单碱基G-to-A转换、等位基因中的一个基因-238G位点有一个单碱基G-to-A转换,即分别为野生型(wt),G-A/G-A和G-A基因型。
2IL-6基因-572C位点的SNP检测
两组276例含400碱基片段的PCR扩增产物,经BsrBI酶切产生了三种条带型分别为150、250和400碱基对条带。由于酶BsrBI识别CCGCTC,-572C位点发生了单碱基C-to-G转换产生150和250碱基对条带,生成一个BsrBI酶切位点。RFLP结果提示IL-6基因-572C位点有三种SNP基因型,这三种多态性包括无转换、两个等位基因-572C位点都有一个单碱基C-to-G转换、等位基因中的一个基因-572C位点有一个单碱基C-to-G转换,即分别为野生型(wt),G-A/G-A和G-A基因型。
3TNF-a基因-238G位点单碱基G-to-A转换的高频率分布与肺癌的发生相关
对TNF-a基因-238G位点的SNP进行统计学分析,证实两个等位基因-238GSNP的基因频率分布符合Hardy-Weinberg平衡定律(P>0.05)。大多数的病例具有-238G位点的野生型(wt) SNP(对照组113例,肺癌组99例)；对照组和肺癌组有相同数量的G-A基因型,对照组有2例G-A/G-A基因型,而肺癌组有14例G-A/G-A基因型。肺癌组G-A SNP数量的增加提示两个等位基因上G-A SNP与肺癌的发生相关(P=0.005,OR=1.74395%C10.232-0.682)。
4IL-6基因-572C位点单碱基C-to-G转换的高频率分布与肺癌的发生相关
对IL-6基因-572C位点的SNP进行统计学分析,证实两个等位基因-572C位点SNPs的基因频率分布符合Hardy-Weinberg平衡定律(P>0.05)。在IL-6基因-572C位点的SNP,对照组和肺癌组有相同数量的野生型(wt)或C-G SNP,而对照组有3例C-G/C-G基因型,肺癌组有9例C-G/C-G基因型。肺癌组C-G/C-GSNP数量的增加提示两个等位基因上纯合型C-G/C-G SNP与肺癌的发生相关(P=0.029,OR=I.23495%CI1.412-2.727)。
结论
1.肿瘤微环境中重要的促血管新生炎性因子TNF-a和IL-6的基因多态性是中国北方汉族人群肺癌遗传易感性的关键因素；
2. TNF-a基因启动子区-238G位点单碱基G-to-A转换和IL-6基因启动子区-572C位点单碱基C-to-G转换的高频率分布与肺癌的发生相关。
第四部分趋化因子CXCL1-2/CXCR2轴通路参与血管内皮抑素的抗肿瘤效应
目的采用蛋白芯片技术筛选Endostatin抗肿瘤效应的主要作用靶点。
方法
1.建立Lewis肺癌C57BL/6鼠移植瘤模型,设对照组和实验组,每组均设C57BL/6小鼠7只,于小鼠种瘤后第7天开始给予治疗干预,对照组给予PBS0.2m1/只/天,实验组给予rhEndostatin15mg/Kg/d,均经鼠尾静脉注射给药,治疗共14天。第14天末次治疗后24小时处死小鼠；
2.分离小鼠体内的肿瘤组织,取各组肿瘤组织块行蛋白芯片检测。检测项目包括CCL11、CCL2、CCL3、GM-CSF、IFN-γ、IL-12p70、IL-2、IL-4、IL-6、 IL-9、CXCL1、CXCL2、MCP5、SCF、sTNFR1、TIMP1和VEGF-A等19种促／抗血管新生因子。统计学分析采用Significance Analysis of Microarrays(SAM)基因芯片分析软件；
3.将对照组和实验组的样本量均扩大一倍,并重复上述实验,经处理后留取肿瘤组织行蛋白芯片检测。并根据第一次筛选的结果调整检测项目。统计学分析采用Significance Analysis of Microarrays (SAM)基因芯片分析软件,并将肿瘤样品的蛋白芯片检测结果进行聚类分析。
结果
1.初次实验中蛋白芯片杂交图和SAM分析结果显示,肿瘤组织内趋化因子CXCL1和CXCL2、CCL3的水平显著升高(分别为4.1倍,2.4倍,2.3倍),而VEGF-A、GM-CSF、sTNFR1/2、TIMP1等重要的促／抗血管新生因子的水平未发生明显变化；
2.根据第一次筛选的结果调整检测项目,包括GM-CSF、CCL27、CXCL1、 CXCL2、IL-2、MCP5、sTNFR1、TIMP1和VEGF-A等9种促／抗血管新生因子。重复实验中蛋白芯片杂交图和SAM分析结果显示,肿瘤组织内的趋化因子CXCL2水平仍呈显著升高,同时VEGFA、GM-CSF亦明显升高(分别为1.964倍,1.505倍,2.18倍),聚类分析结果验证了上述因子的变化趋势。
结论
1.肿瘤微环境内的多个促血管新生因子在Endostatin治疗后发生显著变化,证实了Endostatin具有多靶点抗血管新生的特征；
2.除了VEGF信号通路,肿瘤微环境内的趋化因子CXCL1-2/CXCR2轴信号通路也参与了Endostatin的抗肿瘤效应。
In recent years, malignant tumors have been a worldwide issue of public health. To most patients with tumors, conventional treatment strategies such as surgery, chemotherapy and radiotherapy couldn't effectively control diseases. As an emerging antitumor therapeutic method, cellular immunotherapy has remarkable curative effect on tumors, even those refractory to chemotherapy and radiotherapy. However, its clinical benefits in cancer therapy are very modest. At present, how to improve antitumor effect of cellular immunotherapy by combination with different safty and targeted therapies is always the key point in cancer research of antitumor biotherapy.
Angiogenesis is one of the basic hallmarks of cancer. Antiangiogenic therapy is a new and good prospective treatment strategy. Studies showed that monotherapy with antiangiogenic agents is failing to produce longlasting clinical responses in most patients, however, antiangiogenic molecules may contribute to persistent tumor's remission in combination with immunotherapy. And it suggested that antiangiogenic agents synergized with cellular immunotherapy. Endostatin is regarded as one of the promising antiangiogenesis agents with less adverse side effects and seldom susceptible to the resistance to antigiogenesis. However, it's rare to study on the role and mechanism of Endostatin playing in cellular therapeutic potency. To explore the relationship between Endostatin and cellular immunotherapy on antitumor treatment and provide a theoretical basis for raising the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy.
The inflammatory tumor microenvironment forms a major part of tumors and plays a vital role in the antitumor effect of cellular therapy, and adoptive cytotoxic T cells would be changed into immunosuppressive regular T cells (Treg) through many immunosuppressive factors such as MDSC in the tumor microenvironment. The immune inhibitory cells (MDSC, TAM, and imDC) and proinflammatory cytokines (IL-6,Il-17, TNF-α and chemokines) promote angiogenesis and tissue remodeling and contribute to tumor development and innumosuppression in the tunor micro-environment. Recent studies demonstrated that antiangiogenic agent Sunitinib decreased immunosuppressive cells such as MDSC and Treg, immunosuppressive cytokines IL-10and TGF-β, as well as T cell inhibitory factor PD-1and reversed immunosuppression in the tumor microenvironment. However, it's rare to study on the role and mechanism of Endostatin playing in the immune cells and related cytokines in the tumor microenvironment. In a Lewis lung cancer tumor-bearing mice model, we investigated the mechanism of Endostatin antitumor potency and provided a theoretical basis for designing the optimal strategy of Endostatin combined with cellular immunotherapy in cancer treatment.
Accurate evaluation of the changes in the tumor microenvironment after antiangiogenic therapy is very important to definite the mechanism of antitumor effect and design the optimal strategy in cancer treatment. Inflammatory cells and mediators are tightly related to proangiogenesis factors in the tumor microenvironment. Both TNF-α and IL-6are the vital proinflammatory cytokines in the tumor microenvironment. TNF-α promotes CXC chemokines and their receptor CXCR2axis signaling and plays the crucial role in neuvascularization of lung tissue in pulmonary inflammation and ischemia injury. The level of TNF-α is significantly higher in the serums of patients with non-small cell lung cancer (NSCLC) than that of healthy individuals. IL-6induces VEGF transduction, proliferation of endothial cells, and tumor metastasis via STAT3signaling in many solid tumors. Studies have demonstrated that genic polymorphisms of TNF-α and IL-6are associated with increased occurring risk of some benign or malignant diseases. In the present study, we studied SNP markers in the promoter-regulating regions of TNF-α and IL-6genes in the ethnic group Han of North China, to explore the SNP polymorphisms of the TNF-α and IL-6genes relevant to occurring of lung cancer. It will further investigate the relationship between pro-angiogenic inflammatory cytokines in the tumor microenvironment and occurring of lung cancer. Moreover, we may provide the theoretical basis for the main targets and mechanism of Endostatin antitumor effect.
Induction of angiogenesis requires a shift/switch towards activation/upregulation of inducers of angiogenesis over suppression of angiogenic inhibitors in the tumor microenvironment. Preclinical studies and clinical trials mainly focused on VEGF and its receptors signaling, and demonstrated that VEGF-A and its receptor VEGFR-2are key targets of antiangiogenic agents. However, it's rare to study on role and the molecular mechanism of Endostatin antitumor effect. In the human Genome study, the data demonstrated that Endostatin down-regulated proangiogenic signaling relevant to the endothelial cells such as VEGF, HIF-lα, and TNF-a receptor (sTNFR) and so on.The data suggested that Endostatin is a multi-target antiangiogenic agent. The cells and mediators of inflammation play the crucial role in tumor angiogenesis, so they are the promising therapeutic standpoints in antiangiogenic therapy.To screen the proflammatory cytokines TNF-α, IL-6and their receptors as well as down-stream factors of their signaling pathways by Propeome Profiling microarray and definite the main targets and mechanism of Endostatin antitumor effect, so as to provide a new perspective and theoretical basis for the optimal strategy of Endostatin combined with cellular immunotherapy.
Based on the previous study that we successfully cultured and achieved tumor antigen special DC-T cells of Lewis lung cancer, further demonstrated cytotoxic CD8+T cells are the main subpopulation of the DC-T cells detected by Flow cytometry (FCM). In this project, we explored the relationship between Endostatin and cellular immunotherapy on antitumor treatment, the role and molecular mechanisms of Endostatin playing its antitumor effect in the tumor microenvironment, then provided a theoretical basis for the main targets and mechanism of Endostatin antitumor effect and promoting the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy. Moreover, we invetigated the SNP polymorphisms of the TNF-a and IL-6genes relevant to occurring of lung cancer in the ethnic group Han of North China.Furthermore, we screened the main targets of Endostatin antitumor effect by Propeome Profiling microarray, we may provide a theoretical basis for the optimal strategy of raising the longlasting and potent therapeutic efficacy of Endostatin combined with cellular immunotherapy.
Part I Endostatin Synergized with Tumor Special DC-T Cellular Therapy on Antitumor Effect
OBJECTIVE To investigate the role of Endostatin playing in tumor special DC-T cellular therapy on antitumor effect.
METHODS
1. The transplanted Lewis lung cancer models of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells (LLC). The tumor-bearing mice were randomly divided into three groups, including DC-T cells combined with Endostatin group, DC-T cells group, and PBS control group.There were seven mice in each group. Since day7, the tumor-bearing mice had been administered by Endostatin or DC-T cells or PBS.The body weight and tumor diameter were measured every other day, respectively; and the tumor growth curve was drawn.
2. In DC-T cells combined with Endostatin group, each tumor-bearing mouse had been treated by rhEndostatin15mg/Kg/d by tail vein injection since day7after transplanted with LLCs, for14days.
3. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy C57BL/6mice by Ficoll density gradient centrifugation, T lyphoytes were enriched from PBMCs by Nylon wool column method. On day5, DCs and tumor antigen from LLC by repetitive freeze thaw method were cocultured for3days, then transferred into the culture flasks containing T cells, and cocultured for24hours, tumor antigen special DC-T cells were harvested. And, the T cell subpopulation was detected by Flow cytometry (FCM).
4. In DC-T cells group and DC-T cells combined with Endostatin group, each tumor-bearing mouse was treated by DC-T cells5×106by tail vein injection on day7after transplanted with LLCs.
5. All the mice were sacrificed24hours later after the last time of administration. Separated the tumor tissue of tumor-bearing mice and observed the tumor necrosis. The microvessel density (MVD) in the tumor tissue of tumor-bearing mice was detected by immunohistochemistry.
6. The VEGF and HIF-1α expressions were determined by Western blot and immunohistochemical staining, respectively.
7. The proportions of CD8+T, mDC, TAM (M1/M2), and MDSC in suspended tumor cells of tumor tissue were detected by FCM.
8. The expressions of IL-6, IL-10, IL-17, TGF-β and IFN-γ in suspended tumor cells of tumor tissue were detected by ELISA.
RESULTS:
1. Compared with control group, tumor special DC-T cells inhibited the tumors growth (P=0.021); tumor special DC-T cells combined with Endostatin remarkably regressed the tumors better than PBS control (P=0.009). The data of immunohistochemical staining are shown that CD31was emplored as the marker of vascular endothelial cell and its cytoplasm was dyed yellow-brown. The Microenvironment Density in DC-T cells group was slightly lower than that in the control group (P=0.027); the MVD of tumor special DC-T cells combined with Endostatin was deeply lowest in comparison with control group (P=0.002), concurrently tumor necrosis obviously increased in comparison with control group.
2. The expressions of VEGF, IL-6, IL-17in the tumors were decreased, concurrently those of IFN-γ and HIF-la were increased administered with DC-T cells compared with PBS control (P<0.05, respectively). Tumor special DC-T cells combined with Endostatin alleviated VEGF, IL-6, and IL-17, concurrently elevated IFN-y and HIF-la remarkably better than PBS control (P<0.01, respectively).
3. Flow cytometric data are shown that the proportions of MDSC and TAM (M2type) were significantly lower, those of mDC and TAM (M1type) were up-regulated, and CD8+T cells were recruited to infiltrated the tumors by DC-T cells therapy, compared with PBS control(P<0.05, respectively). The changes in tumor special DC-T cells combined with Endostatin were strongly more than PBS control (P<0.01, respectively).
4. Tumor special DC-T cells combined with Endostatin potently reduced the expressions of IL6, IL-10, TGF-β and IL-17in the tumor tissue, enhanced that of IFN-γ, concurrently recruited CD8+T cells into the tumor tissue in comparison with control group (P<0.01, respectively); The changes in DC-T cells group were significantly more than control group(P<0.05, respectively).
CONCLUSIONS:
1. Tumor special DC-T cells combined with Endostatin potently reduced tumor vasculature and tumor growth, and augmented tumor necrosis, implying the better antitumor effects than monotherapy.
2. Tumor special DC-T cells combined with Endostatin strongly inhibited tumor angiogenesis via regulating the pro/anti-angiogenic factors, and intensified the hypoxia in the tumors.
3. Tumor special DC-T cells combined with Endostatin remarkably inhibited immunosuppressive MDSC and TAM (M2type), enhanced mature DC and TAM (M1type), moreover, increased the number of CD8+T cells infiltrating the tumors, efficiently reversed the immunosuppression of the tumor microvironment.
4. Tumor special DC-T cells combined with Endostatin effectively provided the advantage of reducing the levels of immunosuppressive cytokines in the tumor regions, further exhibited synergistic and much better antitumor effects than monotherapy strategy, making them more vulnerable to the immune reactions.
Part Ⅱ Endostatin Effectively Relieved Immunosuppression of the Inflammatory Tumor Microenvironment in Lung Caner
OBJECTIVE To investigate the role of Endostatin playing in immunosuppressive cells and related cytokines in the inflammatory tumor microenvironment in lung caner.
METHODS
1. The transplanted Lewis lung cancer model of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells(LLC). The tumor-bearing mice were randomly divided into three groups, including high-dose Endostatin group (rhEndostatin15mg/Kg/d), low-dose Endostatin (rhEndostatin7.5mg/Kg/d) group, and control group(PBS0.2ml/d), all the mice were administrated by tail vein injection since day7after transplanted with LLCs, for14days.There were seven mice in each group. All the mice were sacrificed24hours later after the last time of administration. The body weight and tumor diameter were measured every other day, respectively; and the tumor growth curve was drawn.
2. Separated the tumor tissue of tumor-bearing mice and observed the tumor necrosis. The microvessel density (MVD) in the tumor tissue of tumor-bearing mice was detected by immunohistochemistry.
3. The VEGF and HIF-1α expressions were determined by Western blot and immunohistochemical staining, respectively.
4. The proportions of CD8+T, mDC, TAM(M1/M2), and MDSC in suspended tumor cells of tumor tissue were detected by Flow cytometry (FCM).
5. The expressions of IL-6, IL-10, IL-17, TGF-β and IFN-γ in suspended tumor cells of tumor tissue were detected by ELISA.
RESULTS
1. Compared with PBS control group, Endostatin inhibited the tumors growth (P<0.05), and there were no significant difference between high-dose and low-dose Endostatin (P>0.05); the Microenvironment Density in low-dose Endostatin group was slightly lower than that in the control group (P<0.05), that strongly reduced in high-dose Endostatin (P<0.01). There were significant difference between high-dose and low-dose Endostatin (P<0.05).
2. Endostatin greatly decreased the expressions of VEGF, IL-6and IL-17, enhanced the expressions of IFN-y and HIF-la compared with control group (P<0.05, respectively) by Western blot, immunohistochemistry or ELISA. There were significant difference between high-dose and low-dose Endostatin (P<0.05).
3. Flow cytometric data are shown that the proportions of MDSC and TAM (M2type) were significantly lower, those of mDC and TAM (M1type) were up-regulated, and CD8+T cells were recruited to infiltrated the tumors by Endostatin therapy, compared with PBS control(P<0.05, respectively). And there were no significant difference between high-dose and low-dose Endostatin (P>0.05).
4. Endostatin significantly reduced the expressions of IL6, IL-10, TGF-β and IL-17in the tumor tissue, and concurrently elevated that of IFN-γ in comparison with control group (P<0.05, respectively); There were no significant difference between high-dose and low-dose Endostatin (P>0.05).
CONCLUSIONS
1. Endostatin strongly reduced tumor vasculature and tumor growth, and augmented tumor necrosis.
2. Endostatin inhibited tumor angiogenesis via regulating the pro/anti-angiogenic factors in dose-dependent manner, and intensified the hypoxia in the tumors.
3. Endostatin remarkably inhibited immunosuppressive MDSC and TAM, remarkably inhibited immunosuppressive MDSC and TAM (M2type), enhanced mature DC and TAM (M1type), moreover, increased the number of CD8+T cells infiltrating the tumors, efficiently reversed the immunosuppression of the tumor microvironment.
4. Endostatin effectively provided the advantage of reducing the levels of immune inhibitory cytokines in the tumor regions, further demonstrated that Endostatin efficantly reversed immunosuppression of the inflammatory tumor microenvironment in lung caner, promoted mature DC and CD8+T cells to infiltrate the tumors, so as to synergize with tumor special DC-T cellular therapy on antitumor effect.
Part Ⅲ Pro-inflammatory cytokines TNF-α and IL-6gene polymorphisms were associated with occurring of lung cancer
OBJECTIVE To investigate the relationship between SNP polymorphisms of pro-inflammatory cytokines TNF-α and IL-6genes and occurring of lung cancer.
METHODS
1.138consecutive patients with primary lung cancer were recruited in this study. The control group consisted of138healthy individuals. All of them were from the ethnic group Han of North China. The ages, sex distribution, and behaviors (such as smoking or not) between lung cancer group and healthy control group had no significant difference statistically (P>0.05). Total DNAs were isolated from Peripheral blood mononuclear cells of138lung cancer patients and138healthy individuals.
2. PCR were performed and amplified-238G locus of TNF-α gene and-572C locus of IL-6gene, respectively. Further, the gene polymorphisms of two cytokines were revealed by restriction fragment length polymorphism (RFLP).
3. The PCR produced fragments of-238G locus of TNF-α gene about290bp as expected. The PCR products were then subjected to BglⅡ digestion to explore if a G-to-A single base alteration occurring at-238locus.
4. The PCR produced fragments of-572C locus of IL-6gene about400bp as expected. The PCR products were digested with BsrBI to explore if a C-to-G single base alteration occurring at-572C locus.
5. The correlation between the polymorphisms and risks of lung cancer was indicated by odds ratio (OR) and their95%confidence interval (CI). OR values and their95%CI were calculated using a non-conditional logistic regression model, which were adjusted by ages, gender, smoking and other factors.
RESULTS
1Detection of SNP polymorphisms at-238G locus of the TNF-a gene
The PCR produced fragments about290bp as expected, digestion of the290bp fragments from the276individuals in both groups with BglⅡ generated three kinds of band patterns on agarose gels:(ⅰ) a band of about290bp,(ⅱ) two bands with sizes of about80and210bp, respectively, or (ⅲ) three bands with sizes of80,210, and290bp, respectively.
Since BglⅡ recognizes AGATCT, generation of the80and210bp bands may be explained by a single-base G-to-A alteration occurred at-238G locus, which would generate a BglⅡ locus. Three kinds of SNP patterns were found at-238G locus of the TNF-a gene, including no alteration, a single-base G-to-A alteration at-238G locus of both alleles, a single-base G-to-A alteration at-238G locus of one of both alleles, were named as wt, G-A/G-A, and G-A, respectively.
2Detection of SNP polymorphisms at-572C locus of the IL-6gene
The PCR produced fragments about400bp as expected, digestion of the400bp fragments from the276individuals in both groups with BsrBI generated3kinds of band patterns on agarose gels:(ⅰ) a band of about400bp,(ⅱ) two bands with sizes of about150and250bp, respectively, or (ⅲ) three bands with sizes of150,250, and400bp, respectively.
Since BsrBI recognizes CCGCTC, generation of the150and250bp bands may be explained by a single-base C-to-G alteration at-572C locus, which would generate a BsrBI locus.These three polymorphisms, including no alteration, a single-base C-to-G alteration at-572C locus of both alleles, a single-base C-to-G alteration at-572C locus of one of both alleles, were named as wt, C-G/C-G, and C-G, respectively.
3High rates of single-base G-to-A alteration at-238G locus of the TNF-a gene
correlated with occurring of lung cancer
It was founded that the frequency distribution of-238G SNPs at both alleles fits the Hardy-Weinberg equilibrium (.P>0.05). Most cases possessed a wt SNP at-238G locus (113and99for healthy control group and lung cancer group, respectively). The control group and lung cancer group had similar number of G-A cases, respectively. The control group had2G-A/G-A cases, while lung cancer group had14G-A/G-A cases. It was suggested that correlations between G-A/G-A cases of-238G SNPs and occurring of lung cancer (P=0.005, OR=1.74395%CI0.232-0.682)
4High rates of single-base C-to-G alteration at-572C locus of the IL-6gene correlated with tumorgenesis of lung cancer.
Similar situations were also found for the SNPs on-572C locus of the IL-6gene. The healthy control group and lung cancer group had similar numbers of wt or C-G SNPs. However, the control group had three C-G/C-G cases, while lung cancer group had nine C-G/C-G cases. The increased numbers of C-G/C-G SNPs in lung cancer group suggested that the homozygous C-G/C-G SNPs on both alleles may be related to occurring of lung cancer (P=0.029, OR=1.23495%CI1.412-2.727).
CONCLUSIONS
1. The TNF-α and IL-6gene polymorphisms may be a critical risk for the genie susceptibility to lung cancer in the ethnic group Han of North China.
2. The high rates of singlebase G-to-A alteration at-238G locus of both alleles of the TNF-α gene and high rates of single-base C-to-G alteration at-572G locus of both alleles of the IL-6gene correlated with tumorgenesis of lung cancer.
Part Ⅳ Chemokines CXCL1-2/CXCR2Axis Signaling were Involved in the Antitumor Effect of Endostatin
OBJECTIVE To screen the main targets of the antitumor effect of Endostatin using Proteome Profiler arrays.
METHODS
1. The transplanted Lewis lung cancer model of C57BL/6mice were established by left extremity axillary subcutaneous injection of Lewis lung cancer cells (LLC). The tumor-bearing mice were randomly divided into two groups, including Endostatin group (rhEndostatin15mg/Kg/d) and control group (PBS0.2ml/d). There were seven mice in each group. All the mice were administrated by tail vein injection since day7after transplanted with LLCs, for14days. All the mice were sacrificed24hours later after the last time of administration.
2. The tumor specimens were separated from tumor-bearing mice. Proteome Profiling array of tumor specimens was performed, which included19pro/anti-angigenic factors such as CCL11, CCL2, CCL3, GM-CSF, IFN-γ, IL-12p70, IL-2, IL-4, IL-6, IL-9, CXCL1, CXCL2, MCP5, SCF, sTNFRl, TIMP1and VEGF-A so on. Significance Analysis of Microarrays (SAM) soft was used for statistical analysis.
3. The sample sizes of Endostatin group and control group both were expanded doubly, and the experiment was repeated for further validation. Moreover, the molecular profiling was adjusted based on the data of the first experiment. Significance Analysis of Microarrays (SAM) soft and clustering analysis were used for statistical analysis.
RESULTS
1. The levels of CXCL1, CXCL2, and CCL3in the tumors were remarkably enhanced (4.1fold,2.4fold, and2.3fold, respectively), however, the critical pro-angiogenic factors including VEGF-A, GM-CSF, sTNFR1/2and TIMP1had no significant change.
2. Based on the above data, the Proteome profiling was adjusted to include the key pro/anti-angiogenic factors such as GM-CSF、CCL27、CXCL1、CXCL2、IL-2、 MCP5、sTNFR1、TIMP1and VEGF-A. In the repeated experiment, according to SAM soft and clustering analysis, CXCL2was still strongly elevated, concurrently VEGF-A and GM-CSF were significantly enhanced (1.964fold, 1.505fold, and2.18fold, respectively).
CONCLUSIONS
1. The data demonstrated that multiple proangiogenic factors had remarkable changes after receiving Endostatin therapy, and strongly supported that Endostatin have the characteristic of multi-targeted antiangiogenic efficancy.
2. Besides VEGF signaling pathway, chemokines CXCL1-2/CXCR2axis signaling in the tumor microvironment participated in the antitumor effect of Endostatin.
引文
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