Functional single-cell omics in gynecological cancers immunotherapy: from heterogeneity dissection to clinical...

Ovarian, endometrial, and cervical cancers represent major threats to women's health worldwide. The profound heterogeneity of their immune microenvironments has rendered immune checkpoint blockade (ICB) therapies, particularly PD-1/PD-L1 inhibitors, far less effective than anticipated in gynecological malignancies. Conventional research approaches, constrained by coarse analytical resolution, are insufficient to unravel the underlying mechanisms of this therapeutic shortfall. Functional single-cell omics technologies, including single-cell RNA sequencing, single-cell chromatin accessibility profiling, spatial transcriptomics, and multi-omics integration, have provided a new framework for systematically studying the tumor immune microenvironment across three progressive dimensions: cellular identity, functional state, and spatial localization. This review systematically synthesizes the applications of this technological toolkit in the three major gynecological cancers. It reveals distinct bottlenecks across the three dimensions. In ovarian cancer, the core obstacle lies in the spatial dimension, where cancer-associated fibroblast-mediated physical barriers and pro-angiogenic macrophage subsets together construct a "spatial exclusion" microenvironment. In cervical cancer, the principal bottleneck resides in the functional dimension, where HPV status and histological subtype drive a "functional disability" phenotype, in which T cells infiltrate but fail to achieve effective activation, with therapy resistance-associated immune evasion clones further exacerbating functional impairment. In endometrial cancer, however, the immune fate is governed by the identity dimension, where molecular subtypes dictate the baseline nature of immune infiltration, and autophagy-related cellular subpopulations may offer new insights into intratumoral heterogeneity. Although single-cell omics has identified immunologically actionable targets with translational potential, including TREM2⁺ macrophages and the LAG-3/TIM-3 dual-target axis, clinical translation remains constrained by technical and biological bottlenecks, including stringent requirements for tissue sample quality, high assay costs, lack of standardized analytical pipelines, and the discordance between transcriptomic profiles and protein-level functionality.