ScRNA-seq profiles to identify HES1 associated with ACTH-secreting tumors and the distinct microenvironment between ACTH+/CRH+ and ACTH-/CRH- pheochromocytomas.

e16604 Background: Ectopic ACTH and/or CRH-secreting pheochromocytomas are rare catecholamine-secreting tumors originating from adrenal medulla, which could also secrete ectopic adrenocorticotropic hormone (ACTH) and corticotrophin-releasing hormone (CRH), mimicking Cushing syndrome and complicating the diagnosis clinically. The extremely low incidence limits the comprehensive understanding and management for specific individuals accompanied with this kind of tumor. Furthermore, mechanisms of tumorigenesis and ectopic ACTH secretion in pheochromocytomas are unknown. Studying this rare type of tumor will help tailor therapies to the specifically molecular and metabolic characteristics of pheochromocytomas. Methods: Fresh tumor and peritumoral specimens were collected from three ACTH+&CRH+ pheochromocytoma patients and three ACTH-&CRH- pheochromocytoma patients, and then were digested and sent to construct single-cell library as well as sequencing. Subsequent analysis and immunohistochemical staining were conducted to explore the difference of ACTH+&CRH+ pheochromocytomas from the ACTH-&CRH- ones and normal tissues. Results: We constructed the linear trajectory of 2 types of pheochromocytomas. The cluster of intermediate cells that differentiated into mature ACTH+&CRH+ pheochromocytomas showing high expression of HES1, and the immunohistochemical staining revealed that HES1 was higher in ACTH+&CRH+ ones. Besides, Higher expression of POMC and PNMT was observed in ACTH+&CRH+ pheochromocytomas compared with ACTH-&CRH- ones, being in accordance with the clinical information that three ACTH+&CRH+ pheochromocytoma patients had relatively higher catecholamine levels. T cells in ACTH+&CRH+ pheochromocytomas were activated by signals, like HLA-B, promoting their cytotoxic functions, while T cells in ACTH-&CRH- ones were subject to inhibitory signals, especially from M2 macrophages. What's more, a population of endothelial cells marked by SELE was identified in ACTH+&CRH+ pheochromocytomas, showing strong interaction with CD8+ T cells and facilitating lymphocyte infiltration, particularly the GZMK⁺IL7R⁺ T cells. While ACTH-&CRH- pheochromocytomas were enriched in glycolysis and HIF signaling pathways, and characterized by lacking T cell infiltration, with macrophages being the dominant immune cells. Conclusions: HES1 plays a significant role in the fate transition of pheochromocytomas and may drive ACTH secretion. In addition, ACTH+&CRH+ pheochromocytomas are considerated as relatively immunologically“hot”tumors but poor in angiogenesis. In contrast, ACTH-&CRH- pheochromocytomas lack immune infiltration while exhibiting extensive angiogenesis. Immunotherapies may be effective for the former, and the latter may benefit from anti-angiogenic therapies.