A human ACTH-secreting corticotroph tumoroid model

Abstract

Background Cushing disease (CD), although rare, is a life-threatening disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary adenoma, which leads to excess adrenal-derived cortisol. Efficacious and safe medical therapies that control both hormonal hypersecretion and pituitary corticotroph tumor growth remain an unmet need in the management of CD. Translational research in pituitary tumors has been significantly hampered by limited quantities of surgically resected tissue for ex vivo studies, and unavailability of human pituitary tumor cell models. Methods To characterize human corticotroph tumors at the cellular level, we employed single cell RNA-sequencing (scRNA-seq) to study 4 surgically resected tumors. We also used microarrays to compare individualized paired consecutive culture passages to understand transcriptional shifts as in vitro cultures lost ACTH secretion. Based on these findings, we then modified our in vitro culture methods to develop sustained ACTH-secreting human corticotroph tumoroid cultures. Findings scRNA-seq identified 4 major cell populations, namely corticotroph tumor (73.6%), stromal (11.2%), progenitor (8.3%), and immune cells (6.8%). Microarray analysis revealed striking changes in extracellular matrix, cell adhesion and motility-related genes concordant with loss of ACTH secretion during conventional 2D culture. Based on these findings, we subsequently defined a series of crucial culture nutrients and scaffold modifications that provided a more favorable trophic and structural environment that could maintain ACTH secretion in in vitro human corticotroph tumor cultures for up to 4 months. Interpretation Our human corticotroph tumoroid model is a significant advance in the field of pituitary tumors and will further enable translational research studies to identify critically needed therapies for CD. Funding This work was partly funded by NCI P50-CA211015 and the Warley Trust Foundation.

Organoid models of the pituitary gland in health and disease

“…Zhang and colleagues strived to develop organoids from corticotrope tumors ( 63 ). Cells were embedded in a Matrigel drop on top of a thick Matrigel coating and cultured in an optimized medium containing 10% FBS and typical (pituitary) organoid growth factors, such as EGF, insulin like growth factor-1 (IGF-1), FGF8, triiodothyronine (T3), TRH, and bovine hypothalamus extract.…”

“…ACTH secretion was rapidly lost in the 2D culture system, whereas hormone secretion was sustained up to 18 weeks in the 3D tumoroid approach. The 3D cultures were described to be expandable, although methodological details are missing ( 63 ) ( Table 1 ).…”

A novel human tumoroid 3D model of sustained ACTH-secreting cell cultures to study critically needed therapies for Cushing's disease

“…In this article of EBioMedicine, Zhang et al. [7] developed a new sustained ACTH-secreting human corticotroph tumoroid cell culture model from surgically resected human tumours based on the identification of four major cell subpopulations derived from human corticotroph tumours [namely corticotroph tumour (73.6%), stromal (11.2%), progenitor (8.3%), and immune (6.8%) cells] as well as on the transcriptional shifts experienced by the cells after several passages. To do that, the authors characterized the transcriptional patterns of these cellular subpopulations at a single cell level (high-throughput scRNA-seq) and compared individualized paired consecutive culture passages using a microarray expression method.…”

“…Finally, the authors developed an in vitro 3D structure that maintains corticotroph tumour cell-cell and cell-extracellular matrix interactions mimicking the tumour microenvironment assembly with a defined medium using matrigel. All these analyses and conditions allowed the development of a 3D culture methodology for research use, able to maintain human corticotroph tumour ACTH secretion and propagate sufficient human corticotroph tumour cells and proliferation of the progenitor cell population over time [7] .…”

“…From a clinical point of view, this elegant model provides a novel tool to gain a better understanding of the molecular pathogenesis of corticotroph tumours and to identify critically needed therapies for Cushing's disease, with more reproducible results to be applied in humans [eg, wide drug sensitivity screening, detailed mechanism(s) of action of therapeutic agents at the cellular and subcellular level] [7] . Hence, this model solves two challenging aspects to study this tumour type.…”