A Novel Human Pituitary Adenoma Organoid Model to Accelerate Targeted Treatment of Cushing's Disease

Objective: Cushing's disease (CD) is a serious endocrine disorder by which tumors secrete excessive and unregulated amounts of adrenocorticotropic hormone (ACTH), that stimulates the adrenal glands to overproduce cortisol. Chronic exposure to excess cortisol has detrimental effects on health, including increased stroke rates, diabetes, obesity, cognitive impairment, anxiety, depression, and death. The first-line treatment for CD is pituitary surgery, and despite multiple treatments, biochemical control is not achieved in approximately 30% of patients, suggesting that in routine clinical practice, initial and long-term remission is not achieved in a substantial number of CD patients. The lack of specificity of current standard of care treatments with low efficacy and tolerability makes CD a medical therapeutic challenge for endocrinologists. One major limitation to developing new medical therapies is the lack of model systems. While pituitary cell lines predominantly represent hormonal lineages, these cultures do not reproduce the primary pituitary tissue. The development of pituitary spheroids from mouse tissue and induced pluripotent stem cells is documented in the literature, but there are no reports of true organoid cultures derived from patient pituitary adenomas. In this study, we developed a carefully cultured organoid model derived from pituitary adenomas that replicated much of the cellular complexity and functionality of the patient's tumor.

Major Findings: Human pituitary adenoma tissue was harvested fresh during transsphenoidal pituitary surgery from 12 patients to generate organoids. Bright-field microscopy images of hPITOs that were generated from corticotroph, lactotroph, gonadotroph, and somatotroph adenomas revealed morphological diversity among the organoid lines between individual patients and among subtypes ([Fig. 1A]). Unique cell populations were detected via pseudocolor smooth density plots of t-SNE maps generated from each organoid line ([Fig. 1B]). Proliferation was measured within the cultures using 5-ethynyl-2′-deoxyuridine (EdU) uptake and showed that the percentage of EdU+ve cells/total Hoechst+ve nuclei directly correlated with the pathology MIB-1 (Ki67) score (red, R ² = 0.9256; [Fig. 1C, D]). To validate the similarity in cell lineages identified between the organoidline and the patient's tumor, we compared the immunohistochemistry from the neuropathology report, to the expression pattern of pituitary adenoma specific markers ([Fig. 2A–F]). The location of cells that are found in each cluster based on the highly expressed antigens are shown in the representative heatmap t-SNE maps (red cell populations, [Fig. 2A–E]). For example, while organoids derived from corticotroph adenomas highly expressed ACTH and T-Pit, gonadotroph adenoma-derived hPITOs expressed SF-1 and alpha-subunit ([Fig. 2A–E]). Data were summarized in a heatmap based on quantified cell abundance (percent of total cells) using spectral flow cytometry ([Fig. 2F]). ACTH concentration that was measured by ELISA using organoid conditioned culture media collected from each hPITO line, showed the highest expression in the corticotroph adenoma organoids ([Fig. 2G]). A high-throughput drug screen, including drugs commonly used for the treatment of pituitary adenomas, demonstrated patient-specific drug responses of hPITOs among each adenoma subtype ([Fig. 3]).

Conclusion: To the best of our knowledge, our study is the first report that documents the development of patient adenoma-derived organoids to accelerate targeted treatment of pituitary diseases.

Fig. 1 Characterization of patient pituitary adenoma-derived organoids (hPITOs).

Fig. 2 Qualification of cell types within patient pituitary adenoma-derived organoids (hPITOs) by Cytek Aurora spectral flow cytometry.

Fig. 3 Drug responses in patient pituitary adenoma-derived organoids (hPITOs).

Publikationsverlauf

Artikel online veröffentlicht:
15. Februar 2022