The successes with immune checkpoint blockade (ICB) and chimeric antigen receptor (CAR)-T-cell therapy in treating multiple cancer types have established immunotherapy as a powerful curative option for patients with advanced cancers. Unfortunately, many patients do not derive benefit or long-term responses, highlighting a pressing need to perform complete investigation of the underlying mechanisms and the immunotherapy-induced tumor regression or rejection. In recent years, a large number of single-cell technologies have leveraged advances in characterizing immune system, profiling tumor microenvironment, and identifying cellular heterogeneity, which establish the foundations for lifting the veil on the comprehensive crosstalk between cancer and immune system during immunotherapies. In this review, we introduce the applications of the most widely used single-cell technologies in furthering our understanding of immunotherapies in terms of underlying mechanisms and their association with therapeutic outcomes. We also discuss how single-cell analyses help to deliver new insights into biomarker discovery to predict patient response rate, monitor acquired resistance, and support prophylactic strategy development for toxicity management. Finally, we provide an overview of applying cutting-edge single-cell spatial-omics to point out the heterogeneity of tumor–immune interactions at higher level that can ultimately guide to the rational design of next-generation immunotherapies.

免疫检查点阻断(ICB) 和嵌合抗原受体 (CAR)-T 细胞疗法在治疗多种癌症类型方面取得的成功，使免疫疗法成为晚期癌症患者的强大治疗选择。不幸的是，许多患者没有获得益处或长期反应，这凸显了迫切需要对潜在机制和免疫治疗诱导的肿瘤消退或排斥进行全面研究。近年来，大量单细胞技术在表征免疫系统、分析肿瘤微环境和识别细胞异质性方面取得了进展，为揭开免疫治疗过程中癌症与免疫系统之间全面串扰的面纱奠定了基础。在这篇综述中，我们介绍了最广泛使用的单细胞技术的应用，以加深我们对免疫疗法的基本机制及其与治疗结果的关联的理解。我们还讨论了单细胞分析如何帮助为生物标志物发现提供新的见解，以预测患者的反应率、监测获得性耐药性并支持毒性管理的预防策略开发。最后，我们概述了应用尖端单细胞空间组学来指出更高水平的肿瘤免疫相互作用的异质性，这最终可以指导下一代免疫疗法的合理设计。