肿瘤干细胞（CSCs）是恶性肿瘤的“种子”细胞，但迄今对其免疫逃逸所知甚少。我们前期经临床样本组学技术鉴定了结直肠癌（CRC）新抗原（Neoantigen）；并发现CRC微环境重要炎症信号轴IL6/STAT3不仅正调控CRC干细胞（CRC-CSCs）干性从而促进恶性进展；而且初步发现其还可能负调控CRC-CSCs新抗原表达从而促进免疫逃逸。本项目拟经IL6/STAT3诱导和分选CRC-CSCs和CRC-nonCSCs，分析两类细胞的特异和共同新抗原；确定IL6/STAT3对CRC-CSCs新抗原和抗原加工呈递分子表达以及免疫原性的负调控作用，阐明IL6/STAT3、CRC-CSCs干性、及T细胞免疫逃逸三者之间的调控关系和通路机制；结合动物模型和临床样本分析，验证靶向CRC-CSCs新抗原及炎症微环境对其表达负调控机制的联合干预效应，为清除CRC-CSCs的新精准免疫治疗策略提供实验依据。

Cancer stem cells (CSCs) are believed to be “seed cells” for tumor development and progression as they are capable of self-renewal and can resist therapeutic killings and differentiate into cancer cells. However, it is still unclear how the small amount of CSCs can escape the strong killing effects of our host immune system. We have previously identified the profiles and characteristics of neoantigens in clinical colorectal cancer (CRC) tissues by whole exome sequencing and transcriptome sequencing, HLA I/II epitope prediction, and in vitro and in vivo tests for immunogenicity. We not only found that IL6/STAT3, one of the most important inflammatory signal axes in CRC microenvironment, positively regulated the stemness of CRC-CSCs promoting malignant progression, but also showed with preliminary data that it negatively regulated the neoantigen expression of CRC-CSCs leading to immune evasion. .In the present proposed study, CD133+/CD44+ CRC-CSCs and CD133-/CD44- CRC-non-CSCs will be separated by fluorescence activated cell sorting (FACS) after IL6/STAT3 induction, and the specific and shared neoantigens for the CRC-CSCs and CRC-non-CSCs will be identified. Then, the expression levels of neoantigens and antigen processing/presenting machinery molecules, the immunogenicity of the neoantigens, as well as the malignent phenotypes of the CRC-CSCs and CRC-non-CSCs following IL6/STAT3 stimulation will be characterized. Furthermore, the molecular mechanisms of the possible crass-talk regulatory networks between IL6/STAT3 inflammatory axis and CSCs plasticity and / or other signaling pathways in negatively regulating the neoantigen expression of CRC-CSCs leading to T cell immune evasion will be determined and clarified through cell-based studies and validated in NSG mouse xenografts and clinical CRC specimens. Finally, the potential intervention effects on eliminating CRC-CSCs by combined treatment with CRC-CSCs specific/shared neoantigens-targeted health donors-derived CD8+ T cells, blockades of IL6/STAT3 signaling, and inhibitors of CSCs plasticity and / or other pathways will be investigated in zebrafish and NSG mouse xenograft models. The study on the novel therapeutic strategy integratively targeting CRC-CSCs and the inflammatory microenvironment network will provide a basis for clinical trials to eradicate CRC in the future.