肺鳞状细胞癌（LUSC）的高度异质性和复杂的肿瘤微环境导致许多患者对免疫治疗无反应。因此，表征LUSC患者肿瘤微环境的异质性，进一步探讨导致免疫抵抗的免疫特征和分子机制，将有助于提高该类患者免疫治疗的疗效。在此，我们回顾性分析了 513 个 LUSC 样本的 RNA 测序 (RNA-seq) 数据以及其他多组学和单细胞 RNA-seq 数据，并使用多重免疫组织化学验证了关键特征。我们根据RNA-seq数据将这些样本分为6个亚型（CS1-CS6），发现CS3以高水平淋巴细胞浸润激活免疫反应，聚集大量晚期疾病患者，但表达量增加耗尽标记细胞毒性 T 淋巴细胞相关蛋白 4、淋巴细胞激活基因 3 和程序性死亡 1。对免疫治疗反应的预测显示CS3对免疫检查点阻断治疗具有潜在抵抗性，多组学数据分析显示CS3特异性表达免疫抑制相关蛋白B细胞淋巴瘤2、GRB2相关结合蛋白和双特异性磷酸酶4，驱动基因ATP结合盒亚家族A成员13的突变率较高。此外，单细胞RNA-seq验证了CS3亚型的淋巴细胞浸润，揭示了LAMC2-CD44的表达与免疫抵抗之间的正相关关系。 LAMC2和CD44是调节肿瘤增殖的上皮间质转化相关基因，多色免疫荧光验证了LAMC2-CD44的表达与免疫浸润之间的负相关关系。因此，我们在 LUSC 患者中发现了一种淋巴细胞浸润亚型（CS3），该亚型对免疫检查点阻断疗法表现出抵抗力，并且 LAMC2-CD44 的共高表达有助于免疫抵抗，这可能通过靶向该分子对来提高免疫功效与免疫疗法相结合。© 2024 作者。由爱思唯尔有限公司出版

The high heterogeneity of lung squamous cell carcinomas (LUSC) and the complex tumor microenvironment lead to non-response to immunotherapy in many patients. Therefore, characterizing the heterogeneity of the tumor microenvironment in patients with LUSC and further exploring the immune features and molecular mechanisms that lead to immune resistance will help improve the efficacy of immunotherapy in such patients. Herein, we retrospectively analyzed the RNA sequencing (RNA-seq) data of 513 LUSC samples with other multiomics and single-cell RNA-seq data and validated key features using multiplex immunohistochemistry. We divided these samples into six subtypes (CS1-CS6) based on the RNA-seq data and found that CS3 activates the immune response with a high level of lymphocyte infiltration and gathers a large number of patients with advanced-stage disease but increases the expression of exhausted markers cytotoxic T-lymphocyte associated protein 4, lymphocyte-activation gene 3, and programmed death-1. The prediction of the response to immunotherapy showed that CS3 is potentially resistant to immune checkpoint blockade therapy, and multi-omic data analysis revealed that CS3 specifically expresses immunosuppression-related proteins B cell lymphoma 2, GRB2-associated binding protein, and dual-specificity phosphatase 4 and has a high mutation ratio of the driver gene ATP binding cassette subfamily A member 13. Furthermore, single-cell RNA-seq verified lymphocyte infiltration in the CS3 subtype and revealed a positive relationship between the expression of LAMC2-CD44 and immune resistance. LAMC2 and CD44 are epithelial-mesenchymal transition-associated genes that modulate tumor proliferation, and multicolor immunofluorescence validated the negative relationship between the expression of LAMC2-CD44 and immune infiltration. Thus, we identified a lymphocyte-infiltrated subtype (CS3) in patients with LUSC that exhibited resistance to immune checkpoint blockade therapy, and the co-hyperexpression of LAMC2-CD44 contributed to immune resistance, which could potentially improve immunological efficacy by targeting this molecule pair in combination with immunotherapy.© 2024 The Authors. Published by Elsevier Ltd.