Pituitary Neuroendocrine Tumor Organoids Generated from Induced Pluripotent Stem Cells Reveal the Dysregulation of Cell Lineage Differentiation in Cushing’s Disease

Abstract

Background: Cushing’s Disease (CD) is an endocrine disease typically caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET). Corticotroph subtype PitNETs subsequently stimulate the adrenal glands to overproduce causing a wide range of detrimental effects on health, including increased stroke rates, diabetes, obesity, depression, anxiety, and a threefold increase in the risk of death from cardiovascular disease. In some instances, CD is a manifestation of genetic mutation syndromes that include MEN1, FIPA, CDH23 and Carney complex. In other cases, somatic mutations underlie the development of CD including mutations in the deubiquitinase gene USP8, identified in 35-62 % of sporadic corticotroph PitNETs and USP48 as a driver mutation in USP8 wild-type tumors. There are currently no in vitro models to study the impact of genetic mutations on the pathways leading to PitNET development in humans. Objective: To define the cellular complexity of corticotroph subtype PitNETs at a spatial single-cell resolution. Methodology: Organoids were generated from a CD patient expressing a CDH23 mutation (hPitNETO iPSCDH23) as reported by our group (Mallick et al., 2023, Translational Research). CRISPR-Cas9 gene editing of iPSCs was also used to model the development of corticotroph PitNETs expressing USP48 and USP8 mutations. Human PitNET organoids (PitNETOs) were generated from tumor tissue harvested during transsphenoidal surgery. Cell lineage trajectory analysis was performed on scRNA-seq data collected during the iPSC differentiation program. ScRNA-seq was used to compare the transcriptomic profile and cell composition of CD patient PitNETOs to the patient’s own tissue using the NanoString CosMxTM spatial transcriptomic analysis. Results: Organoids generated from a CD patient expressing a CDH23 mutation (hPitNETO iPSCDH23) exhibited tumor behavior consistent with the expected pathology and function of sparsely granulated corticotroph PitNETs. Cell lineage trajectories of hPitNETO iPCCtrl control cultures revealed multiple branching paths from the stem-like (SOX2/PAX7+) base cluster leading to clusters enriched for markers of pituitary cell lineages (lactotroph, somatotroph, thyrotroph, gonadotroph and corticotroph). Trajectory analysis for hPitNETO iPSCDH23 showed dysregulation in the normal pituitary cell lineage differentiation leading to the development of poorly differentiated corticotrophs (PAX7+/TPit+) and PAX7+/stem cells. CosMxTM spatial gene mapping revealed a cell population co-expressing PAX7 and stem cell markers SOX2 and CD44 that was abundant in Crooke cell and sparsely granulated TPit+ PitNET subtypes. An increase in the PAX7/SOX2/CD44 PitNET cell population correlated with a higher Knosp grade, tumor size and invasiveness, when compared to densely granulated PitNETs. Conclusions: Defining the complexity of the PitNET microenvironment will provide insights into the pathogenesis of CD and potentially lead to the stratification of high-risk patients for disease recurrence. 1R01DK133325. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.