Analysis of intratumoral immune heterogeneity reveals spatial organisation of immunosuppression in breast cancer

Abstract

Recent advances in single-cell transcriptomics have illuminated intratumoral heterogeneity (ITH) in breast cancer, yet the spatial organization and functional states of immune populations within luminal tumors remain poorly understood. Here, we generated a spatially and clonally resolved single-cell immune atlas of early-stage luminal breast cancer by integrating paired single-cell RNA/TCR sequencing with multiplex immunohistochemistry across three spatially distinct tumor regions: central (core-proximal), intermediate and peripheral (invasive margin-proximal), in five patients. Peripheral regions are enriched in naive CD4 T cells, monocytes, dendritic cells, and memory CD8 T cells with bystander properties, alongside TCF1+ exhausted progenitors (Tpex), and displayed elevated antigen presentation and tertiary lymphoid structures (TLS) signatures. In contrast, central regions harbor macrophages, clonally expanded regulatory T cells (Tregs), and two subsets of terminally exhausted CD8 T cells (Tex), one tissue-resident and one circulating, both expressing tumor-reactive features. Clonal overlap was observed between peripheral Tpex and central, tissue-resident Tex cells, revealing a spatially confined differentiation trajectory toward exhaustion within the tumor microenvironment. Central T cells also upregulate TGF-{beta} signaling and metabolic pathways including fatty acid oxidation and oxidative phosphorylation, consistent with adaptation to an immunosuppressive microenvironment. Together, these findings reveal a spatially structured immune landscape in luminal breast cancer, marked by progressive immunosuppression from the tumor margin to the core, providing a framework for future region-specific immunotherapeutic strategies.