Single-cell transcriptomics reveals transcriptional reprogramming and microenvironmental remodeling in ectopic...

Ectopic ACTH- and/or CRH-secreting pheochromocytomas (ACTH⁺CRH⁺PCCs) are rare tumors that can mimic Cushing's syndrome and complicate the diagnosis and treatment clinically. However, the tumor-intrinsic and microenvironmental features of ACTH⁺CRH⁺ PCCs remains poorly understood.

Fresh tumor specimens from three ACTH⁺CRH⁺ and three ACTH^- CRH^- PCCs were profiled by single-cell RNA sequencing. Developmental trajectories and transcriptional regulatory networks were analyzed to investigate tumor-intrinsic programs, while CellChat and NicheNet were used to characterize cell-cell communication and infer ligand-mediated microenvironmental remodeling. Findings were also validated using TCGA datasets and immunohistochemical staining.

ACTH⁺ CRH⁺ PCCs showed increased POMC and CRH expression and were enriched for a Schwann cell precursor (SCP)-like population positioned at an early pseudotime state. Regulatory network analysis further identified increased CEBPB activity in ACTH⁺ CRH⁺ tumor cells. Microenvironmental analysis revealed enrichment of SELE⁺ endothelial cells and CD8⁺ T cells in ACTH⁺ CRH⁺ PCCs. Cell-cell communication analysis showed more frequent interactions between ACTH⁺ CRH⁺ tumor cells and SELE⁺ endothelial cells, together with predicted ECM-associated interactions between SELE⁺ endothelial cells and T cells. In contrast, ACTH^- CRH^- PCCs exhibited stronger VEGFA-related angiogenic signaling, greater enrichment of stromal populations and MRC1⁺ M2-like macrophages, and more prominent LGALS9-CD45 signaling between macrophages and T cells.

Our study provides single-cell insights into the tumor-intrinsic and microenvironmental heterogeneity between ACTH⁺CRH⁺ PCCs and ACTH^-CRH^- PCCs, revealing distinct tumor cell states and subtype-specific vascular and immune landscapes.