Endocrine Abstracts

The adrenal gland displays an important role in integrating neuronal, immune, vascular, metabolic and endocrine signals under a common organ capsule. The adrenal is the central organ of the stress response system, responsible for responses to acute and chronic stress stimuli and, thereby, playing a major role in numerous stress-related disorders. While for other diseases translational scientists developed programs to generate healthy cells to regenerate damaged tissues in patients, such approaches have been widely overlooked in other fields of endocrinology, except for type-I-diabetes. Moreover, tumor formation is very common in the adrenal gland and these tumors are furthermore highly heterogeneous. However, high throughput applications reflecting this heterogeneity and furthermore relevant 3D-structures in vitro are still widely lacking. Recently, we initiated the development of standardizable multidimensional models of adrenal organoids and tumor spheroids. For this purpose we utilized microwell plates providing stem cell quality clustering without adhesion, enormous scalability and medium change possibility. In a first step, we assessed for the human adrenocortical cell lines NCI-H295R and MUC-1 the impact of different cell numbers, incubation periods and methods of spheroid preparation. Based on these experiments we were able to successfully establish and dimensionally preserve stable tumor spheroids for both tumor models. Subsequent histological and immunohistochemical investigations revealed viable spheroids with characteristic cellular abundance and localizations for important tumor and steroidogenic marker such as Ki-67, SF-1 and 3betaHSD. Real time PCR analyses demonstrate no significant differences in SF-1 and Ki67 expression compared with monolayers. Our experiments indicate furthermore potential applicability for drug screenings. Treatment with 75 µM mitotane over 72 h revealed significantly decreased Ki67 Indices compared with untreated controls for both models (NCI-H295R: P <0.001; MUC-1; P <0.01) while adavosertib affected primarily MUC-1 spheroids (P <0.01). Moreover, we aimed at the development of bovine derived adrenal organoids and we are now also able to establish viable (H&E), as for organoids expected low/not proliferating (Ki67) and steroidogenic active (SF-1 and 3betaHSD) adrenal organoids. Preliminary data indicate furthermore mixed cell populations originating from both, adrenal cortex and medulla. Moreover, cell culture supernatants have been analyzed in a pilot experiment by LC–MS/MS and confirm specific secretion of hormones such as aldosterone, cortisol, and progesterone among others. In next steps, these models will be extended to human primary tumors of adrenal origin and assessed regarding further applications and read outs. Thereby, we hope to provide new avenues for adrenal tissue replacement and mechanistic insights into adrenal (tumor) biology.