【作者】 王琳；

【导师】 刁宏燕；

【作者基本信息】 浙江大学 ， 内科学（传染病）， 2018， 博士

【摘要】 研究背景与实验目的:结直肠癌(Colorectal cancer,CRC)在世界范围内具有较高的死亡率,严重威胁人类健康。外科手术通常作为早期结直肠癌的首选治疗,但在首次手术后患者复发率高达30-40%。此外,据报道约60%的CRC患者后期会发生肿瘤转移。研究表明CRC的高发病率,复发和转移均与CRC患者免疫细胞功能低下有关。文献报道显示低活性的自然杀伤(Natural killer,NK)细胞增加了癌症风险的发生。NK细胞体外活化后回输治疗可以作为转移性CRC患者的有效免疫疗法。也有研究表明,与健康成年人相比,转移性CRC患者的CD8+T细胞的数量显著减少。西妥昔单抗(Cetuximab,CET)是一种人-小鼠嵌合型表皮生长因子受体(Epidermal growth factor receptor,EGFR)的单克隆抗体。虽然有一些关于体外西妥昔单抗对肿瘤细胞作用的报道,但是在CRC治疗期间,西妥昔单抗的作用机制由于缺乏体内研究而尚未完全阐明。因此,我们分析了 CRC患者治疗过程中外周血的免疫学特性。我们发现在西妥昔单抗治疗中,NK细胞和CD8+T细胞发挥了重要作用。与体内、外实验相结合研究西妥昔单抗对患者免疫细胞数量和功能上的影响,旨在找到CRC治疗过程中西妥昔单抗发挥作用的免疫机制及标志西妥昔单抗治疗效果的早期指标。研究方法:通过收集,整理和详细分析63例CRC患者和健康成年人的临床资料,收集患者外周抗凝血样本,分离出样本中的单个核细胞(peripheral blood monouclear cells,PBMC),检测健康成年人和患者治疗前后体内淋巴细胞亚群的的比例和分布情况。进一步通过体外实验检测其功能。利用流式细胞仪和RTCA检测肿瘤患者和健康成年人的外周血单个核细胞对肿瘤细胞系的杀伤作用。同时应用酶联免疫实验(Enzyme-l inked immunosorbent assay,ELISA)和液相芯片检测 CRC 相关细胞因子IFN-y,IL-10和IL-6的表达水平。进一步收集整理和分析结肠癌患者的临床资料和影像学诊断将其分为西妥昔单抗治疗有效组和治疗无效组,按照治疗有效组、无效组再次进行数据统计分析,检测大肠癌患者体内淋巴细胞的数目比例以及分布情况,比较治疗有效组和治疗无效组的免疫细胞活化的差异。检测患者外周血中IL-33的表达水平,了解其变化趋势。分离患者PBMC,检测IL-33对骨桥蛋白(osteopontin,OPN)、IL-4、IL-10以及TNF-α等细胞因子产生的影响。观察OPN对肿瘤细胞侵袭作用的影响。研究结果:第一部分:研究结果提示与健康对照相比,CRC患者治疗前外周血的CD3+T细胞、CD8+T细胞和NK细胞数量都显著下降;而CD4+T细胞与健康成年人组相比无显著差异,恒定自然杀伤性T(invariant natural killer T,iNKT)细胞与CD3+CD56+ NKT细胞的数量较之健康组无明显差异。另外,CRC患者树突状(Dendritic cells,DC)细胞表面的BDCA1共刺激分子并没有显著变化。而治疗后的CRC患者与治疗前患者相比,CD3+T细胞、CD8+T细胞和NK细胞数量都显著上升;调节性T(regulatory T,Treg)细胞治疗后逐渐降低,免疫抑制作用得到了缓解;CD4+T细胞、CD3+CD56+NKT、iNKT细胞和BDCA1共刺激分子在治疗后的患者与治疗前相比无明显差异。表明NK细胞和CD8+T细胞可能在西妥昔单抗治疗过程中发挥了重要作用。CD107a和CD137分子与脱颗粒和杀伤靶细胞功能相关,CRC患者治疗前CD107a和CD137分子在NK、CD8+T表面的表达水平与健康成人相比明显减低,而CRC患者治疗后与治疗前患者相比,CD107a和CD137分子在NK、CD8+T细胞表面的表达水平显著升高。研究还发现与健康组相比CRC患者治疗前外周血中,IL-6、IL-10细胞因子水平均升高,而治疗后4周表达水平逐渐下降;CRC患者治疗前与健康成年人相比IFN-y细胞因子水平降低,治疗后4周表达水平明显上升。第二部分:我们根据患者治疗后的临床症状和影像学诊断将CRC患者分为西妥昔单抗治疗有效组和无效组,发现在西妥昔单抗治疗有效组外周血中IL-33水平在治疗2周后明显上升,OPN水平显著降低;而无效组患者治疗前后外周血中IL-33、OPN的水平无明显变化;另外治疗有效组患者与治疗前相比血浆中IL-10水平显著降低。而治疗无效组IL-10水平无显著差异。IL-4、TNF-α水平治疗前后两组中均没有变化;治疗有效组患者外周血单个核细胞培养中添加IL-33后,IL-10、OPN水平明显降低。我们推测,IL-33在大肠癌早期可能调控IL-10与OPN的表达水平发挥抗肿瘤作用。结论:在西妥昔单抗治疗CRC过程中,CD8+T、NK细胞在西妥昔单抗治疗CRC过程中发挥重要作用;西妥昔单抗的治疗可以增加CD107a和CD137分子在NK、CD8+T细胞表面的表达水平。与此同时,在体外实验中,也表明西妥昔单抗可以增加NK、CD8+T细胞对肿瘤细胞的细胞毒作用,IL-33炎性因子与西妥昔单抗治疗大肠癌的疗效密切相关,IL-33可能通过调节OPN以及IL-10的水平发挥抗肿瘤作用。更多还原

【Abstract】 Backgrouds:Colorectal cancer(CRC)carries a high mortality worldwide.Surgery is first-line treatment for CRC but up to 30-40%of patients relapse within the first few years after initial surgery.Also,it has been reported that ≈60%CRC patients will go on to suffer from tumor metastasis.Studies have indicated a high prevalence of mortality,recurrence and metastasis of CRC to be associated with immunocompromised patients.One epidemiologic survey showed low activity of natural killer(NK)cells to be associated with an increased risk of cancer,and that infiltration of CD8+T cells and NK cells are closely related to the clinical outcome of CRC.One study demonstrated that NK cells can kill human tumor cells isolated immediately from colon carcinomas.Moreover,NK cells can serve as effective immunotherapy for patients with metastatic CRC.For example,the adoptive transfer of NK cells to patients is an emerging method to treat the metastasis of CRC.Cetuximab is a chimeric human-mouse anti-epidermal growth factor receptor(EGFR)immunoglobulin(Ig)G1monoclonal antibody.It has been used to inhibit the proliferation,invasion,and metastasis of tumor cells.Cetuximab was approved recently by the US Food and Drug Administration for first-line treatment of advanced CRC.By crosslinking CD16a(FcyRⅢa),cetuximab mediates the crosstalk between NK cells and dendritic cells(DCs),which is important for the induction of antitumor cellular immunity.Cetuximab-induced activation of NK cells can upregulate expression of the costimulatory receptor CD137(4-1BB),which is closely related to the efficiency of NK cells,and subsequently promotes T cell-based immunity in vitro.Importantly,if DCs and NK cells are removed from peripheral blood,the antitumor activity of cetuximab is attenuated significantly.It has been shown that the number of CD8+T cells is reduced significantly in patients with metastatic CRC compared with healthy individuals.Some in vitro and mouse-model experiments have investigated the role of cetuximab in CRC treatment,but few studies have tried to determine the mechanism of action(MoA)of cetuximab during CRC treatment by analyzing the lymphocyte subsets of CRC patients.Mice-model experiments have suggested that cetuximab may target tumor cells by antibody-dependent cell-mediated cytotoxicity(ADCC),and that cetuximab can intensify the ADCC antitumor activity of adoptive NK cells towards CRC with increased expression of the EGFR.Several studies have provided some information on how cetuximab acts on CRC in vitro,but the MoA of cetuximab during CRC treatment is not known due to a lack of in vivo studies.Cetuximab is used widely for the treatment of metastatic CRC,but its MoA is not known.Thus,we analyzed the immune characteristics of the peripheral blood of CRC patients.We found that NK cells and CD8+T cells have important roles in cetuximab treatment.These findings provide further understanding of the MoA of cetuximab during CRC treatment.Methods:Collect and analyze the clinical data of 63 CRC patients and healthy control.Isolate peripheral blood mononuclear cells for studying the proportion and function by in vivo and vitro experiments.Then test the number and the proportion of NK and CD8+T cells and their ability to kill tumor cells through flow cytometry,Detect the levels of CRC-related cytokines through enzyme-linked immune response(ELISA)and enzyme immunoassay.Results:Part1 The results showed that compared with healthy controls,the number of CD3+ T,CD8+ T,and natural killer(NK)cells was increased significantly and T-regulatory cells reduced gradually after cetuximab treatment.There was no significant difference in CD4+T cells compared with healthy controls.The number of CD3+CD56+ NKT(natural killer)like cells was not significantly different from the healthy group.Whereas,Tregs(Treg cells)has immunosuppressive effects.It was found that Tregs in CRC patients had a significantly lower inhibitory effect than healthy controls;In addition,there was no significant change in the BDCA1 co-stimulatory molecules on DC surface of CRC patients;There was also no significantly different in iNKT(invariant natural killer T)cells between CRC patients and healthy group.invariant NKT.It is further proved that NK cells and CD8+T are important immune cells,they are associated with CD 107a and CD 137 molecules with degranulation and cytotoxic functions.The level of CD 107α and CD 137 expression on NK and CD8+T cells was increased significantly after 4 weeks of cetuximab therapy.In vitro cetuximab treatment markedly increased expression of CD 137 and CD 107a on NK and CD8+ T cells.Cetuximab treatment promoted the cytotoxic activity of NK and CD8+ T cells against tumor cells.It is also found that plasma levels of cytokines IL-6 and IL-10 in peripheral blood of CRC patients decreased;IFN-ylevels increased.Part2 We divided CRC patients into effective and inactive groups according to the clinical symptoms and imaging diagnosis after treatment.We found that IL-33 levels in the peripheral blood of Cetuximab-effective group were increased for 2 weeks.The level of OPN was significantly decreased;The levels of IL-33 and OPN in the peripheral blood of the patients in the ineffective group had no significant changes before and after treatment;In addition,the IL-10 levels in the plasma of the patients in the effective treatment group were significantly lower than no treatment.There was no significant difference in IL-10 levels in the inactive group.There was no change in the levels of IL-4 and TNF-a before and after treatment.The levels of IL-33,IL-10 and OPN were significantly lower in the culture supernatant of peripheral blood mononuclear cells in patients with effective treatment.We speculate that IL-33 may play an anti-tumor role in regulating the expression of IL-10 and OPN in peripheral blood in the early stages of colorectal cancer.Conclusions:Cetuximab treatment promotes activation of the immune response but alleviates immunosuppression:this might be the underlying anti-CRC effect of cetuximab.In the process of treatment of CRC,CD8+ T and NK cells play an important role in the mechanism is closely related with cetuximab;At the same time,in vitro,CD8+T cells and NK cells play an important role in anti-tumor activity.IL-33 inflammatory factors are closely related to the early onset of colorectal cancer,and may regulate tumor-related cytokines through the two key factors of OPN and IL-10.更多还原