Single-cell transcriptomic reveals transcriptional reprogramming and microenvironmental remodeling in ectopic ACTH/CRH-secreting pheochromocytomas

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.