MUC4介导NK细胞分离失能及其促胰腺癌转移、吉西他滨耐药的研究

Natural killer (NK) cells can be involved in the selection and differentiation of stem cells by split anergy(SA) of NK with losing the ability of cytotoxicity but gaining the ability to secrete cytokines. In tumor microenvironment, split anergy of NK cells (SA of NK) cannot effectively kill cancer stem cells (CSC), but promote the differentiation and proliferation of CSC, so this is a new mechanism for tumor metastasis and drug resistance. Whether the occurrence of SA of NK is involved in the malignant progression of pancreatic cancer is an unconfirmed but significant problem because proving the occurrence of SA of NK which causes CSC survival and accumulation and revealing the mechanisms is of great importance for finding good ways to inhibit metastasis and gemcitabine resistance of pancreatic cancer. Results from our preliminary studies showed that NK cell dysfunction occurs in the patients with pancreatic cancer and this dysfunction is associated with indicators of malignant progression (such as histological grade, depth of invasion, lymph node metastasis). Our results also showed that MUC4 plays pivotal role in the malignant progression of human pancreatic cancer and the level of MUC4 membrane expression of pancreatic cancer cell strongly positively correlated with SA of NK. Based on these significant findings, we proposed our hypothesis: MUC4-mediated split anergy of NK cell can be one of mechanisms of the metastasis and gemcitabine resistance of pancreatic cancer. To confirm the hypothesis, we will conduct a series of studies. Firstly, we will detect the expression of MUC4, the occurrence of SA of NK and the proliferation of CSC in clinical samples of the patients with pancreatic cancer and analysis the correlation. Secondly, we will verify that pancreatic cancer cells using the function of MUC4 induce the occurrence of SA of NK with the proliferation of CSC in vitro co-culture experiments. Then, we will clarify the molecular mechanisms of MUC4-mediated SA of NK by block experiment. Thirdly, we will investigate the role of MUC4-mediated SA of NK, a series of reverse solutions of SA of NK and combination therapy of gemcitabine and reversing SA of NK in vivo experiments. Lately, we will examine the effects of autogeneic NK, reversing SA of autogeneic NK or allogeneic NK from healthy volunteers on the tumor cells from pancreatic cancer tissue. Therefore, the aims of our project are to find valuable new indicator monitoring malignant progression and prognosis of pancreatic cancer, demonstrate the role of MUC4-mediated SA of NK on metastasis and gemcitabine resistance of pancreatic cancer, disclose the best reverse solution of SA of NK and the effect of combination therapy of gemcitabine and reversing SA of NK, and to provide theoretical basis and experimental basis for designing strategies of the immunotherapy of pancreatic cancer using NK cells combined with chemotherapeutics.

NK细胞通过发生“分离失能”，不仅失去原有的细胞毒功能，还获得分泌细胞因子的能力，参与干细胞的选择和分化过程。在肿瘤微环境中，分离失能的NK细胞既不能有效杀伤肿瘤干细胞（CSC），还促进CSC的分化和增殖，成为肿瘤转移和耐药的新机制。申请者前期发现胰腺癌患者体内环境中NK细胞为失能表型，与疾病恶性进展密切相关，但是否发生分离失能尚未证实。因此，确认胰腺癌中存在NK细胞分离失能造成CSC的存活和积累及具体机制，对于抑制胰腺癌转移和解决吉西他滨耐药意义重大。本课题在证明胰腺癌枢纽分子MUC4与NK细胞分离失能相关的基础上，拟通过临床样本的检测与分析、体内外实验，深入探讨MUC4介导NK细胞分离失能、促胰腺癌转移、吉西他滨耐药的作用及分子机制，寻找有价值的监测胰腺癌恶性进展和预后的新指标及逆转NK细胞分离失能的最佳方案，为设计基于NK细胞的胰腺癌免疫治疗与化疗联用的策略提供实验依据。

本课题组在前期研究中发现胰腺癌患者体内环境中NK细胞为失能表型，与疾病恶性进展密切相关，但是否存在分离失能和分子机制尚未证实。按照原标书计划执行实验中，我们发现人外周血中 “分离失能”表型的NK群的占比极低，在胰腺癌病例与对照组之间没有数量差异。实验中我们也发现缺乏稳定的研究“分离失能”机制的NK细胞研究模型。MUC4在胰腺癌特异性表达，我们证明与总结了MUC4介导促胰腺癌转移的作用及分子机制的相关工作。在建立吉西他滨耐药株的基础上，我们发现胰腺癌细胞可能通过自噬和内吞作用增强了对代谢物的再循环利用，所发生代谢重编程的作用过程可能增强了胰腺癌细胞对吉西他滨的抵抗性，使耐药株获得刺激Treg细胞发育的能力，使耐药株逃逸免疫的能力进一步增强。我们发现与证明一批能指示胰腺癌恶性进展和预后的新指标，如SOX2OT、PEG10、 PIK3R3、HK1、GAPDH、PKM2和LDHA。我们首次建立具有PD-L1/PD1负信号逆变器和IL-15/IL-15Ra复合物共同修饰的嵌合抗原受体(CAR)-NK92细胞系(简称SP)，证明SP的稳定增殖可不依赖添加外源IL-2；SP细胞能将PD-L1 / PD1的免疫抑制信号转换为激活信号，对PD-L1阳性的胰腺癌细胞具有很强的杀伤活性，并能抵抗PD-L1和IFN-γ诱导的负面效应；证明SP发挥更强杀伤效应的途径与机制是CAR结构可增强SP细胞对靶细胞的黏附力、反应力、脱颗粒能力、TRAIL的表达。首次建立除了检测CD107a指标以外，能适时、定量检测NK类效应细胞对靶细胞的反应力、脱颗粒能力和杀伤能力的新方法。为找到改善胰腺癌患者预后的过继免疫治疗的新策略奠定基础。我们建立胰腺癌自发成瘤的Pdx1-Cre; K-RasG12D/+（简称KC）基因工程鼠模型，模拟人体胰腺癌发生发展过程，应用全景单细胞测序技术，构建人类胰腺癌原发灶和配对肝转移灶的高分辨率单细胞图谱，旨在解析胰腺癌发生发展特定的时间和组织空间中NK的表型功能与机制变化，因为时间与经费有限，进一步的探索还在进展中。