β-catenin信号下调CCL4表达参与食管鳞状细胞癌放疗联合免疫治疗耐受的机制研究

Radiation resistance in esophageal cancer is always an obstacle in clinical. How the tumor intrinsic signaling affects tumor immune microenvironment inducing radiation resistance is known little. Now, we are carrying out the world first clinical trial of radiotherapy combined with anti-PD-1 antibody as the first-line treatment on patients with intolerable concurrent chemoradiotherapy esophageal squamous cancer cell (ESCC). We found part of patients were still resistant to the combination treatment. Little lymphocytes infiltrated in the tumor microenvironment in these patients. Our pre-studies showed that the activation of β-catenin signaling increased and decreased the expression of ATF3 and CCL4, individually. ESCC patients with higher CCL4 expression had improved progress free survival than those with lower CCL4 expression. CCL4 played an important role in recruiting dendritic cells. Radiation activated β-catenin signaling. As a result, our hypothesis is that the activation of β-catenin signaling pathway in ESCC might decrease the expression of CCL4 inducing migration of dendritic cells to the tumor microenvironment, which blocks the T cell anti-tumor response at initial step and devotes to treatment resistance. Based on the findings from our clinical trial and the pre-studies, we will perform the study in detail as following. Firstly, identify the association among the expression of β-catenin, ATF3, CCL4, the tumor immune microenvironment and radiotherapy or radiotherapy combined with immune therapy. Secondly, explored the regulatory mechanisms of β-catenin/ATF3/CCL4 signaling pathway and the influence of radiotherapy in it, and discover novel molecules which interact with ATF3 and involve in adjusting the transcriptional expression of CCL4. Thirdly, confirm the impact of β-catenin/CCL4 signaling pathway in regulating the anti-tumor response in mouse model. Lastly, value the new therapeutic strategies of combination of radiotherapy and immune therapy under the condition of improved tumor immune microenvironment by extrinsic intervention. Our study will provide novel biomarkers for predicting the prognosis for ESCC patients treated by radiotherapy combined immunotherapy and more effective therapeutic strategies for the patients.

食管癌放疗耐受是临床难题，肿瘤内源信号直接调控肿瘤微环境诱导放疗耐受未见报道。我们在国际首个放疗联合PD-1抗体作为一线治疗局部晚期食管癌Ib期临床试验中发现部分患者耐受。耐受者疗前/疗中肿瘤浸润淋巴细胞少，进一步预实验表明食管癌β-catenin信号上调ATF3、下调CCL4表达，高表达CCL4者预后好，CCL4招募DCs，放疗加强β-catenin信号。以上提示食管癌β-catenin/CCL4信号可能通过抑制DCs归巢，使T细胞抗肿瘤免疫阻滞在初始阶段，导致治疗耐受。我们拟研究临床样本β-catenin、ATF3、CCL4与肿瘤免疫微环境及疗效的关系；阐明β-catenin通过ATF3调控CCL4表达的分子机制及放疗对信号通路的影响，寻找ATF3互做分子；体内确定此信号通路对抗肿瘤免疫的抑制作用；以期发现放疗联合免疫治疗的疗效预测新指标，建立改善免疫微环境以提高联合治疗疗效的新策略。

食管癌放疗耐受是临床难题，免疫治疗在晚期食管鳞状细胞癌（ESCC）中已显现良好治疗效果，但其与放疗联合应用作为一线治疗方案应用于局部晚期ESCC的疗效机制、预测标记物研究尚浅。本项目基于两个国际首个放疗/放化疗联合PD-1抗体治疗不可手术局部晚期ESCC的1b期临床研究，探讨了β-catenin、ATF3、CCL4与肿瘤免疫微环境及疗效的关系；β-catenin通过ATF3调控CCL4表达的分子机制及放疗对信号通路的影响；ESCC肿瘤微环境DCs、巨噬细胞亚群空间分布、T细胞免疫组库的特征以及动态变化；放疗联合PD-1抗体治疗过程中外周血系统免疫特征。明确β-catenin通过诱导ATF3降低肿瘤局部CCL4从而抑制DCs浸润是导致ESCC肿瘤免疫受抑的重要原因；ESCC肿瘤微环境DCs、巨噬细胞亚群空间分布为其提呈肿瘤抗原启动抗肿瘤免疫提供必要条件；放疗联合PD-1抗体既有效激活肿瘤局部T细胞，又促进外周血记忆T细胞分化，达到活化、维持T细胞功能的效果。本项目从多个层次阐明影响放疗联合免疫治疗疗效的机制，为进一步提高ESCC治疗疗效、探索联合免疫治疗新方案提供思路。项目资助发表SCI论著11篇，待发表论著1篇。项目投入经费57万元，支出27.3145万元，各项支出基本与预算相符。剩余经费29.6855万元，剩余经费计划用于本项目研究后续支出。