Spatial and single-cell transcriptomics reveal cellular heterogeneity and a novel cancer-promoting Treg cell subset in human clear-cell renal cell carcinoma

Background: Clear cell renal cell carcinoma (ccRCC) is the most common histologic type of RCC. However, the spatial and functional heterogeneity of immunosuppressive cells and the mechanisms by which their interactions promote immunosuppression in the ccRCC have not been thoroughly investigated.

Methods: To further investigate the cellular and regional heterogeneity of ccRCC, we analyzed single-cell and spatial transcriptome RNA sequencing data from four patients, which were obtained from samples from multiple regions, including the tumor core, tumor-normal interface, and distal normal tissue. On the basis, the findings were investigated in vitro using tissue and blood samples from 15 patients with ccRCC and validated in the broader samples on tissue microarrays.

Results: In this study, we revealed previously unreported subsets of both stromal and immune cells, as well as mapped their spatial location at finer resolution. In addition, we validated the clusters of tumor cells after removing batch effects according to six characterized gene sets, including epithelial-mesenchymal transition^high clusters, metastatic clusters and proximal tubule^high clusters. Importantly, we identified a special regulatory T (Treg) cell subpopulation that has the molecular characteristics of terminal effector Treg cells but expresses multiple cytokines, such as interleukin (IL)-1β and IL-18. This group of Treg cells has stronger immunosuppressive function and was associated with a worse prognosis in ccRCC cohorts. They were colocalized with MRC1 ⁺ FOLR2 ⁺ tumor-associated macrophages (TAMs) at the tumor-normal interface to form a positive feedback loop, maintaining a synergistic procarcinogenic effect. In addition, we traced the origin of IL-1β⁺ Treg cells and revealed that IL-18 can induce the expression of IL-1β in Treg cells via the ERK/NF-κB pathway.

Conclusions: We demonstrated a novel cancer-promoting Treg cell subset and its interactions with MRC1 ⁺ FOLR2 ⁺TAMs, which provides new insight into Treg cell heterogeneity and potential therapeutic targets for ccRCC.