Endocrine Abstracts

Adrenocortical carcinoma (ACC) is a rare malignancy with an incidence of 0.7–2.0 per million per year. Prognosis of ACC is generally poor but variable and therapeutic approaches are scarce. While surgical resection presents the best option for definitive cure, mitotane remains the only approved drug for adjuvant therapy in ACC to date. Advancements regarding novel treatment strategies as well as fostered understanding of potential drivers of adrenocortical carcinogenesis have been limited by the lack of tumour models reflecting genetic disease heterogeneity. Currently available cell culture and mouse models for ACC only comprise single patient derived cell lines, corresponding mouse xenograft models and few conventional genetically engineered mouse models mainly targeting components of the Wnt-signaling pathway. To overcome this gap, we developed a workflow for culturing adrenocortical cells from C57BL/6-WT, and C57BL6/J^{6Rosa26Sor-CAGG-SpCas9-IRES-eGFP} mice. After isolation and longitudinal culture of murine adrenal cortices, we performed LC-MS/MS based steroid hormone analyses in supernatant showing sustained secretion of aldosterone (day 7: 1535 ± 697 ng/l), corticosterone (day 7: 1111 ± 123 µg/l), 11-deoxycorticosterone (day 7: 74 ± 14 µg/l) and progesterone (day 7: 9 ± 2 µg/l). Furthermore, qRT-PCR of adrenal targets showed mRNA expression of several steroidogenic enzymes e.g. HSD3B2, HSD11B1, CYP11A1, CYP11B1 and CYP11B2, as well as SF-1, StAR and SCARB1. After confirmation of adrenocortical origin, cultured cells were immortalized via CRISPR/Cas9-mediated targeting of Trp53. This ex vivo setup holds the potential to study adrenal cell homeostasis and biology, and will allow us to replicate oncogenic transformation of adrenocortical cells using CRISPR/Cas9-mediated genome editing. It presents starting point for the development of versatile and clinically relevant isogenic mouse models for adrenocortical tumours.