{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T07:01:55Z","timestamp":1791874915728,"version":"4.3.4"},"reference-count":30,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,4,18]],"date-time":"2026-04-18T00:00:00Z","timestamp":1776470400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Ectopic ACTH-secreting pheochromocytomas are rare and life-threatening endocrine tumors responsible for hypertension, paroxysmal symptoms, and Cushing\u2019s syndrome. The cellular origin of ACTH and the tumor\u2019s molecular characteristics remain poorly understood. Single-cell RNA sequencing was performed on tumor specimens and adjacent adrenal tissues from three patients with ectopic ACTH-secreting pheochromocytomas. Integrated bioinformatic analyses, including differential expression, functional enrichment, cell\u2013cell communication, and pseudotemporal trajectory inference, were conducted. Key findings were supported by immunofluorescence and immunohistochemical staining. Our study integrated single-cell transcriptomic profiling with detailed clinical characterization of three cases of ectopic ACTH-secreting pheochromocytomas. All patients presented classic Cushing\u2019s features and variable catecholamine secretory patterns. Hormone levels improved after surgical resection. Single-cell analysis revealed a complex tumor microenvironment comprising 11 distinct cell populations. Chromaffin cells expressing the ACTH precursor gene POMC were identified within the tumor cell population, suggesting that these cells may represent the source of ectopic ACTH production. This finding was further supported by immunofluorescence and immunohistochemical staining demonstrating ACTH expression in CHGA-positive chromaffin tumor cells and absence of staining for the adrenocortical marker \u03b1-inhibin. These tumor cells exhibited metabolic reprogramming characterized by upregulation of oxidative phosphorylation pathways and downregulation of adaptive immune responses. Cell\u2013cell communication analysis suggested interactions between POMC-expressing chromaffin cells and cytotoxic immune cells. Pseudotemporal trajectory analysis further suggested that these chromaffin cells did not transition toward a steroidogenic fate. This study provided a single-cell atlas of ectopic ACTH-secreting pheochromocytomas. Our integrated analysis suggested POMC-expressing chromaffin cells may represent the cellular source of ectopic ACTH production and revealed a transcriptional signature involving metabolic activation and immune modulation that might contribute to tumor progression. These findings offered new insights into the pathophysiology of this rare disease and provided a framework for future investigations into the molecular mechanisms underlying ectopic ACTH production.<\/jats:p>","DOI":"10.3390\/ijms27083625","type":"journal-article","created":{"date-parts":[[2026,4,20]],"date-time":"2026-04-20T08:24:23Z","timestamp":1776673463000},"page":"3625","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Single-Cell Transcriptomic Profiling of Ectopic ACTH-Secreting Pheochromocytoma Reveals the Chromaffin Cell Origin of Ectopic Hormone Production"],"prefix":"10.3390","volume":"27","author":[{"given":"Xu","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Penghu","family":"Lian","sequence":"additional","affiliation":[{"name":"Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guoyang","family":"Zheng","sequence":"additional","affiliation":[{"name":"Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenda","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8058-9702","authenticated-orcid":false,"given":"Yushi","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"249","DOI":"10.4158\/EP.6.3.249","article-title":"Pheochromocytoma: Clinical observations from a Brooklyn tertiary hospital","volume":"6","author":"Ariton","year":"2000","journal-title":"Endocr. Pract."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1530\/EC-17-0321","article-title":"Initial clinical presentation and spectrum of pheochromocytoma: A study of 94 cases from a single center","volume":"7","author":"Falhammar","year":"2018","journal-title":"Endocr. Connect."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2331","DOI":"10.1097\/01.hjh.0000251887.01885.54","article-title":"Pheochromocytoma: Presentation, diagnosis and treatment","volume":"24","author":"Reisch","year":"2006","journal-title":"J. Hypertens."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1590\/s1677-5538.ibju.2023.0038","article-title":"The Pheochromocytoma\/Paraganglioma syndrome: An overview on mechanisms, diagnosis and management","volume":"49","author":"Lima","year":"2023","journal-title":"Int. Braz. J. Urol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Negro, A., Verzicco, I., Tedeschi, S., Campanini, N., Zanelli, M., Negri, E., Farnetti, E., Nicoli, D., Palladini, B., and Santi, R. (2021). Case Report: Irreversible Watery Diarrhea, Severe Metabolic Acidosis, Hypokalemia and Achloridria Syndrome Related to Vasoactive Intestinal Peptide Secreting Malignant Pheochromocytoma. Front. Endocrinol., 12.","DOI":"10.3389\/fendo.2021.652045"},{"key":"ref_6","first-page":"158","article-title":"Vasoactive Intestinal Peptide-Secreting Pheochromocytoma: A Case Report and Review of Literature","volume":"8","author":"Yee","year":"2022","journal-title":"AACE Clin. Case Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1210\/clinem\/dgaa488","article-title":"Ectopic ACTH- and\/or CRH-Producing Pheochromocytomas","volume":"106","author":"Elliott","year":"2021","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Huang, J., Lin, C., Fang, L., Gu, L., Cai, X., Zeng, H., Chen, K., Mu, Y., and Liu, J. (2025). Ectopic ACTH Syndrome Caused by Pheochromocytoma: A Case Report and Systematic Review. Endocr. Metab. Immune Disord. Drug Targets.","DOI":"10.2174\/0118715303372676250628161600"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1507\/endocrj.EJ24-0459","article-title":"Pheochromocytoma associated with ectopic ACTH-producing tumor and hyper-interleukin-6emia: A case report with review of literature","volume":"72","author":"Nakahira","year":"2025","journal-title":"Endocr. J."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1016\/j.tem.2023.12.003","article-title":"A clinical perspective on ectopic Cushing\u2019s syndrome","volume":"35","author":"Ragnarsson","year":"2024","journal-title":"Trends Endocrinol. Metab."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"4955","DOI":"10.1210\/jc.2004-2527","article-title":"Cushing\u2019s syndrome due to ectopic corticotropin secretion: Twenty years\u2019 experience at the National Institutes of Health","volume":"90","author":"Ilias","year":"2005","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1210\/jc.2005-1542","article-title":"The ectopic adrenocorticotropin syndrome: Clinical features, diagnosis, management, and long-term follow-up","volume":"91","author":"Isidori","year":"2006","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e240029","DOI":"10.1530\/ERC-24-0029","article-title":"Cushing syndrome from an ACTH-producing pheochromocytoma or paraganglioma: Structured review of 94 cases","volume":"31","author":"Kishlyansky","year":"2024","journal-title":"Endocr. Relat. Cancer"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"RP87586","DOI":"10.7554\/eLife.87586","article-title":"Molecular classification and tumor microenvironment characteristics in pheochromocytomas","volume":"12","author":"Qin","year":"2024","journal-title":"Elife"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e2413493","DOI":"10.1002\/advs.202413493","article-title":"Single-Cell Atlas Reveals Tumorigenic Profiles and Immune Dynamics of Adrenal Incidentalomas","volume":"12","author":"Wang","year":"2025","journal-title":"Adv. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2547","DOI":"10.1038\/s41388-025-03391-3","article-title":"Single-cell chromosome and bulk transcriptome analysis as a diagnostic tool to differentiate between localized and metastatic pheochromocytoma and sympathetic paraganglioma","volume":"44","author":"Berends","year":"2025","journal-title":"Oncogene"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e68436","DOI":"10.7554\/eLife.68436","article-title":"Single-cell transcriptome analysis identifies a unique tumor cell type producing multiple hormones in ectopic ACTH and CRH secreting pheochromocytoma","volume":"10","author":"Zhang","year":"2021","journal-title":"Elife"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1007\/s12022-022-09708-2","article-title":"Overview of the 2022 WHO Classification of Neuroendocrine Neoplasms","volume":"33","author":"Rindi","year":"2022","journal-title":"Endocr. Pathol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/s12022-022-09710-8","article-title":"Overview of the 2022 WHO Classification of Adrenal Cortical Tumors","volume":"33","author":"Mete","year":"2022","journal-title":"Endocr. Pathol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1007\/s12022-022-09704-6","article-title":"Overview of the 2022 WHO Classification of Paragangliomas and Pheochromocytomas","volume":"33","author":"Mete","year":"2022","journal-title":"Endocr. Pathol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1029","DOI":"10.1126\/science.1160809","article-title":"Understanding the Warburg effect: The metabolic requirements of cell proliferation","volume":"324","author":"Cantley","year":"2009","journal-title":"Science"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1016\/j.cmet.2015.12.006","article-title":"The Emerging Hallmarks of Cancer Metabolism","volume":"23","author":"Pavlova","year":"2016","journal-title":"Cell Metab."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"5001","DOI":"10.1158\/1078-0432.CCR-16-0606","article-title":"Pheochromocytoma: The First Metabolic Endocrine Cancer","volume":"22","author":"Jochmanova","year":"2016","journal-title":"Clin. Cancer Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"3787","DOI":"10.1158\/1078-0432.CCR-12-3922","article-title":"Genotype-specific abnormalities in mitochondrial function associate with distinct profiles of energy metabolism and catecholamine content in pheochromocytoma and paraganglioma","volume":"19","author":"Rao","year":"2013","journal-title":"Clin. Cancer Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2211","DOI":"10.1098\/rstb.2005.1762","article-title":"Glucose-sensing mechanisms in pancreatic beta-cells","volume":"360","author":"MacDonald","year":"2005","journal-title":"Philos. Trans. R. Soc. B Biol. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1122","DOI":"10.1038\/s41467-023-36769-6","article-title":"Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma","volume":"14","author":"Calsina","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1229","DOI":"10.1016\/j.cell.2015.08.016","article-title":"Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression","volume":"162","author":"Chang","year":"2015","journal-title":"Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1038\/s41596-024-01045-4","article-title":"CellChat for systematic analysis of cell-cell communication from single-cell transcriptomics","volume":"20","author":"Jin","year":"2025","journal-title":"Nat. Protoc."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1016\/j.ccell.2017.01.001","article-title":"Comprehensive Molecular Characterization of Pheochromocytoma and Paraganglioma","volume":"31","author":"Fishbein","year":"2017","journal-title":"Cancer Cell"},{"key":"ref_30","unstructured":"(2026, February 01). Transparency in the Reporting of Artificial INtelligence\u2014The TITAN Guideline\u2014Premier Science. Available online: https:\/\/premierscience.com\/pjs-25-950\/."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/8\/3625\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T04:14:06Z","timestamp":1776744846000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/8\/3625"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,18]]},"references-count":30,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["ijms27083625"],"URL":"https:\/\/doi.org\/10.3390\/ijms27083625","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,18]]}}}