【摘要】 背景与目的 免疫治疗反应率低，可部分归因于异质性肿瘤微环境导致的肿瘤免疫逃逸机制，本研究旨在通过组织学层面确定炎症型非小细胞肺癌（non-small cell lung cancer, NSCLC）对新辅助免疫联合化疗疗效的影响，并探究特定CD8~+T及CD4~+T细胞的数量和空间接近性在疗效预测中的价值。方法 回顾性纳入2021年1月至2023年6月在山东省肿瘤医院接受新辅助免疫联合化疗的43例NSCLC患者，收集术前活检标本并进行多重免疫荧光染色[CD8/程序性细胞死亡受体1(programmed cell death protein 1, PD-1)/T细胞免疫球蛋白及黏蛋白结构域蛋白3(T-cell immunoglobulin and mucin-domain containing protein 3, TIM-3)/CD4/叉头框蛋白3(forkhead box protein 3,FoxP3)/细胞角蛋白（cytokeratin, CK）/4’,6-二脒基-2-苯基吲哚（4’,6-diamidino-2-phenylindole, DAPI）]。使用InForm软件行组织分割（上皮区和间质区），并量化肿瘤细胞、CD8~+T细胞及分群（细胞毒性、预耗竭和耗竭）、CD4~+T细胞及分群（常规和调节性）的密度及其空间接近性。基于CD8~+T细胞在上皮及间质区中的相对浸润程度，将NSCLC分为炎症型（上皮区及间质区均>10/1000）、排除型（上皮区<10/1000，间质区>10/1000）和荒漠型（上皮区及间质区均<10/1000）。使用Kolmogorov-Smirnov检验、Fisher??s精确检验、Mann-Whitney U检验及Logistic回归确定与主要病理缓解（major pathological response, MPR）有关的因素。结果 炎症型、排除型及荒漠型NSCLC分别占65.1%、27.9%和7.0%。与非炎症型NSCLC患者相比，炎症型MPR率更高（71.4%vs 33.3%, P=0.016）。单因素以及多因素Logistic回归分析均证实炎症型是NSCLC患者获得MPR的独立保护因素（OR=0.20,95%CI:0.05-0.74,P=0.020；校正后OR=0.17,95%CI:0.03-0.80,P=0.030）。在炎症型NSCLC上皮区内分析CD8~+T与CD4~+T细胞空间距离时发现，MPR组上皮区内调节性CD4~+T细胞30μm半径内细胞毒性CD8~+T细胞的有效密度低于non-MPR组（0.00 vs 0.33,P=0.037）。结论 炎症型NSCLC患者接受新辅助免疫联合化疗的疗效更佳，这可能与调节性CD4~+T细胞与细胞毒性CD8~+T细胞接近性减弱有关。更多还原

【Abstract】 Background and objective The low response rate to immunotherapy can be partially attributed to tumor immune escape mechanisms arising from the heterogeneous tumor microenvironment. This study aims to determine the impact of inflammatory non-small cell lung cancer(NSCLC) on the efficacy of neoadjuvant immunotherapy combined with chemotherapy at the histological level, and to investigate the predictive value of specific CD8~+ and CD4~+ T cell numbers, as well as spatial interactions, in treatment response. Methods A retrospective study included 43 patients with NSCLC who underwent neoadjuvant immunotherapy combined with chemotherapy at Shandong Cancer Hospital from January 2021 to June 2023. Preoperative biopsy specimens were collected and subjected to multiplex immunofluorescence staining [CD8/programmed cell death protein 1(PD-1)/T cell immunoglobulin and mucin-domain containing protein 3(TIM-3)/CD4/forkhead box protein 3(FoxP3)/cytokeratin(CK)/4’,6-diamidino-2-phenylindole(DAPI)]. InForm software was used to perform tissue segmentation(epithelial and stromal regions) and quantify the density and spatial proximity of tumor cells, CD8~+ T cells and their subsets(cytotoxic, pre-exhausted and exhausted), as well as CD4~+ T cells and their subsets(conventional and regulatory). NSCLC was classified into three subtypes based on the relative infiltration levels of CD8~+ T cells in both the epithelial and stromal compartments: inflamed(both compartments>10/1000), excluded(epithelial compartment<10/1000 and stromal compartment>10/1000), and desert(both compartments<10/1000). The Kolmogorov-Smirnov test, Fisher??s exact test, Mann-Whitney U test and Logistic regression were used to identify factors associated with major pathological response(MPR). Results Inflamed, excluded, and desert NSCLC accounted for 65.1%, 27.9% and 7.0%, respectively. Compared with patients with non-inflamed NSCLC, those with inflamed NSCLC exhibit a higher MPR rate(71.4% vs 33.3%, P=0.016). Both univariate and multivariate Logistic regression analyses confirmed that the inflamed subtype is an independent protective factor against the acquisition of MPR in NSCLC patients(OR=0.20, 95%CI: 0.05-0.74, P=0.020; adjusted OR=0.17, 95%CI: 0.03-0.80, P=0.030). Analysis of the spatial distance between CD8~+ and CD4~+ T cells within the epithelial regions of inflamed NSCLC revealed that the effective density of cytotoxic CD8~+ T cells within a 30 μm radius of regulatory CD4~+ T cells was lower in the MPR group than in the non-MPR group(0.00 vs 0.33, P=0.037). Conclusion Patients with inflamed NSCLC demonstrate superior efficacy when receiving neoadjuvant immunotherapy combined with chemotherapy. This may be due to reduced proximity between regulatory CD4~+ T cells and cytotoxic CD8~+ T cells.更多还原