新型新辅助免疫疗法联合化疗（neoICT）改善了食管鳞状细胞癌（ESCC）患者的预后，但低反应率和治疗耐药性仍然存在挑战。对于 ESCC 肿瘤微环境 (TME) 的肿瘤内异质性知之甚少，这种异质性是对新辅助治疗的不同反应的基础。我们应用单细胞 RNA 测序 (scRNA-seq) 分析和多重免疫荧光染色来彻底解读来自新辅助抗 PD1 联合治疗临床试验的 ESCC 标本中的 TME。新辅助治疗后，癌症相关成纤维细胞（CAF）群体的丰度发生了显着变化。具体来说，IL6  CCL2  免疫调节CAF和新型CD248  机械响应CAF子集表现出浸润增加。从机制上讲，CD248 机械反应性 CAF 接近并排列在肿瘤巢内，以物理方式阻断 CD8  T 细胞的浸润和药物输送，而 IL6 CCL2  免疫调节性 CAF 则通过不同的 IL-6 表达诱导治疗抵抗。在接受 neoICT 治疗的患者中，我们观察到显着的 CAF-T 细胞相互作用。特别是，NECTIN2-TIGIT 配体-受体对在处理的样品中富集，TIGIT 被确定为 T 细胞的主要抑制检查点。我们的研究结果表明，TME 成分对新辅助免疫治疗的反应发生了明显改变，并确定了与患者不利治疗反应相关的 CAF 功能表型。这提供了增强 ESCC 新辅助治疗反应的潜在目标。© 2024。作者。

Novel neoadjuvant immunotherapy combined with chemotherapy (neoICT) has improved outcomes for patients with esophageal squamous-cell carcinoma (ESCC), but challenges persist in low response rates and therapy resistance. Little is known about the intra-tumoral heterogeneity in the ESCC tumor microenvironment (TME) that underlies differential responses to neoadjuvant therapy. We applied single-cell RNA sequencing (scRNA-seq) profiling and multiplexed immunofluorescence staining to thoroughly decipher the TME in ESCC specimens from a neoadjuvant anti-PD1 combination therapy clinical trial. The cancer-associated fibroblasts (CAFs) population showed the significant alteration in abundance following neoadjuvant therapy. Specifically, IL6 + CCL2 + immunomodulatory CAFs and a novel CD248 + mechanoresponsive CAFs subset exhibited increasing infiltration. Mechanistically, CD248 + mechanoresponsive CAFs approached and lined the tumor nest to physically block the infiltration of CD8 + T cells and drug delivery, while IL6 + CCL2 + immunomodulatory CAFs induced therapeutic resistance with distinct IL-6 expression. Among patients treated with neoICT, we observed prominent CAF-T cell interactions. In particular, the NECTIN2-TIGIT ligand-receptor pair was enriched in treated samples, and TIGIT was identified as the major inhibitory checkpoint of T cells. Our findings demonstrate distinct alterations in TME constituent responses to neoadjuvant immunotherapy and identify functional phenotypes of CAFs associated with unfavorable therapeutic responses in patients. This provides potential targets to enhance responses to neoadjuvant therapy in ESCC.© 2024. The Author(s).