Development of a patient-derived tumoroid culture system to explore novel medical treatments for refractory prolactinomas

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ABSTRACT Prolactinomas (PRL-omas), the most common secreting pituitary tumors, frequently cause hypogonadism and subfertility. Although dopamine agonist therapy often works well initially and 1/3 of patients experience long-term remission after 2 years of therapy, 1/3 of patients require long-term D2 agonist therapy to maintain normal PRL levels and a further 1/3 of patients are either refractory to DA therapy or intolerant of their side effects. Although surgical resection can be offered in some, remission rates in large locally invasive tumors is <50%. Translational research has been hampered by the lack of any human PRL-oma cell models. Using current methodologies, human PRL-oma cultures only survive for 7-10 days in vitro. There is an urgent unmet need for establishment of a human PRL-secreting tumor model that retains its highly differentiated phenotype and allows sufficient long-term expandability of cells for use in preclinical translational research and development of novel treatment options. We compared the global transcriptome landscape in consecutive passages from two human prolactin-secreting pituitary tumor primary cultures using bulk RNA-seq to understand the molecular events leading to loss of hormone secretion during in vitro human prolactinoma culture. As pituitary tumor cells lost hormone secretion, we observed a reduction in angiogenesis, survival signals and immune responses in parallel with increased collagen catabolism, cell adhesion and extracellular matrix organization. Guided by these findings, we developed a unique 3-dimensional (3D) PRL-oma culture system and for the first time, we have generated long-term (> 6 months) expandible PRL-secreting 3D human pituitary tumor cultures (>6 months). In the first of two aims, we will use our unique patient-derived 3D PRL-oma culture model in a high throughput screen to identify small molecule inhibitors of PRL secretion and proliferation. We have already demonstrated the feasability of this approach and identified a compound of interest in a pilot screen and now wish to conduct an expanded HTS to identify novel therapies for patients with refractory PRL-omas. Our primary screen will include pharmacological validation and repurposing-, targeted-, lead-like- and diverse- libraries. Initial hits will be selected using robust z-score statistics and transitioned to aim 2 for further development. Aim 2 will employ a cascade of follow-up assays to validate potential hit compounds. These will include dose-response curves in both rat and human PRL-oma cells, assessment and exclusion of non-specific overly toxic compounds. And tests of specificity on hormone secretion in various murine and human neuroendocrine tumors. Additionally, in-silica target prediction will be combined with RNA-seq transcriptome profiling to segregate drug target pathways and deconvolute the MOA of resultant hits. Finally, potential actions of hits on identified pathway targets will be corroborated by pathway disruption using short hairpin RNA (shRNA) and CRISPR libraries in both rat and human PRL-omas to define a group of first-in-class hits.