口腔鳞癌免疫原性细胞死亡调控MDSC功能转化的机制研究

How to effectively improve the anti-tumor immune response is a hot and important topic in cancer research. This project try to answer the scientific question that how to improve the response of oral squamous cells carcinoma (OSCC) to immune checkpoint blockade based on our previous and pilot study. We hypothesize that induced immunogenicity cell death (ICD) could regulate functional transformation of myeloid-derived suppressor cells (MDSCs) , which may improve the response of OSCC to the immune checkpoint blockade. Taking advantage of immunocompetent OSCC mouse model, we will perform mechanistic studies on the following aspects: Firstly, we will apply the Opal multiplex immunohistochemistry and multi-color flow cytometry to identify the correlation of ICD with MDSCs as well as antigen presenting cells (APCs) in human and mouse OSCC. Secondly, radiotherapy, photothermal therapy as well as chemotherapy will be utilized to induce OSCC ICD, and then the effect of ICD on the function and proportion of MDSCs and APCs will be explored. Thirdly, we try to figure out how ICD regulates the differentiation of MDSCs to APCs by co-culture system, and identify the dominant transcriptional factors using next generation sequencing. Finally, utilizing the transgenic mouse model, flow cytometry sorting technology and magnetic-activated cell sorting, we intend to investigate the role and mechanism of ICD to PD-L1 expression on myeloid cells in vitro and in vivo, as well as enhance the potential to response of PD-1/PD-L1 blockade in OSCC. This study is expected to provide new adjuvant therapeutic strategies for the immunotherapy of OSCC.

本研究围绕“如何提高口腔鳞癌对免疫检查点阻断治疗的响应性”这一科学问题，结合前期研究，提出“诱导口腔鳞癌免疫原性细胞（ICD）能够调控髓源性抑制细胞（MDSCs）发生功能转化，从而提高口腔鳞癌对免疫检查点阻断治疗响应性”的科学假说。利用特有的免疫健全小鼠口腔鳞癌模型，拟从以下方面展开机制研究：①应用多标记染色技术、多色流式等技术明确人及小鼠口腔鳞癌 ICD 与 MDSCs 及抗原递呈细胞（APCs）的相关性；②通过放疗、光热治疗及化疗等方法诱导口腔鳞癌 ICD，明确ICD 对MDSCs、APCs比例及功能的改变；③利用体外共培养及二代测序技术，明确ICD 如何调控MDSCs分化及其相关转录因子；④利用转基因小鼠模型、流式及磁珠分选等方法，体内、体外探究ICD 对髓系细胞 PD-L1 表达的作用机制，及能否增强PD-1/PD-L1阻断治疗的响应性。有望为口腔鳞癌免疫治疗提供新辅助策略。

肿瘤细胞受到外界刺激发生死亡的同时，由非免疫原性转变为免疫原性从而刺激机体产生抗肿瘤免疫应答的过程称为免疫原性细胞死亡（ICD）。肿瘤细胞发生ICD的同时，会产生一系列的信号分子，启动一系列的细胞学反应，激活宿主的抗肿瘤免疫反应，然而口腔鳞癌ICD产生的分子机制及诱导方法尚不清楚，ICD对肿瘤微环境中髓源性抑制细胞（MDSCs）分化功能影响尚不明确。在本项目中，我们利用口腔鳞癌人类标本及转基因小鼠模型探索了MDSCs在头颈鳞癌中的表达水平及其与临床病理参数的相关性；明确了放疗、化疗、光热光动力治疗对口腔鳞癌ICD的诱导作用，探索了肿瘤细胞免疫原性死亡对MDSCs分化的影响及分子机制。进一步通过学科交叉应用新型生物材料诱导肿瘤精准高效的产生ICD，调控MDSCs的功能，进而增强头颈鳞癌免疫治疗的响应率。特别关注了焦亡这种特殊形式的ICD提高抗肿瘤免疫的作用并提出了阻断MDSCs增强抗肿瘤免疫的六条策略。通过完成本项目，我们可以得出以下结论：①可以通过Calreticulin，HMGB1，ATP，Annexin A1的表达及转位并利用体内rechallenge实验表征ICD；②放疗、化疗、光热光动力治疗及溶瘤病毒治疗均能引起强烈的ICD及细胞焦亡，提高抗肿瘤免疫；③浆细胞样树突细胞、TIGIT/CD155通路及NLRP3参与口腔鳞癌MDSCs的免疫抑制作用，Tadalafil可以有效的降低肿瘤中MDSCs；④应用ROS/pH双应答纳米前药联合融瘤病毒能够有效诱导肿瘤细胞ICD并有效减少肿瘤微环境中MDSC的数量，增强PD-1免疫治疗的疗效。