MON-466 Transcriptome Dynamics of in vitro Human Pituitary Tumor Primary Culture

Abstract

Lack of an immortalized or long-term hormone-secreting human pituitary tumor in vitro cell line has significantly hampered studies of etiopathogenesis and development of targeted therapies for pituitary tumors. Various media, vessels, substrates and additives have been explored to optimize primary cultures. Some methods have prolonged cell survival time but maintaining hormone secretion is still a major challenge. We have recently developed methodology to maintain robust hormone secretion in human prolactinoma, somatotroph and corticotroph tumors in vitro for up to 9 months. To gain insight into the changes in gene expression profiles that occurred prior to/coincident with loss of hormone secretion during in vitro human pituitary tumor cultures, we compared the global transcriptiome landscape in serial passages from two human prolactinoma primary cultures. We obtained 27.1 million quality filtered reads per sample of which ~90% were exonic reads detecting an average of 16,000 genes. 3D principal component analysis identified 3 distinct clusters, with close similarity between cell passages 1-2, those beyond passage 4-5 and a stand-alone passage 3. Analysis of differential gene expression (DEG) over time using DAVID knowledgebase and hierarchical clustering demonstrated that most changes in gene expression occurred in the first 1-2 weeks in in vitro culture. During the first week of in vitro culture, although the cells maintained robust PRL secretion, reduced activation of angiogenesis and growth signal pathways (PI-3K, MAPK pathways, cell adhesion, GTPase activity, cell proliferation and cell-cell junction assembly) was observed. In the following 1-2 weeks, the cell population maintained PRL secretion but exhibited continued loss of survival signals, with increased expression of extracellular matrix (ECM) and surface adhesion molecules consistent with development of a fibroblastic phenotype that dominated later stage cultures. Based on our findings, we propose that the molecular events underpinning loss of hormone production in primary pituitary tumor cultures represent transition from endocrine cells to pituitary specific fibroblastic folliculo-stellate cell likely selected by the in vitro culture conditions. Our data further reveal that underexpression of several GPCRs and resultant downregulation of the PKA pathway may lead to loss of hormone production in parallel with loss of cell survival signals. In summary, our study defines a temporal progressive transcriptome transition signature to explain loss of hormone secretion and morphological alterations observed during human pituitary tumor in vitro culture and may provide molecular targets to further optimize our culture methodology to attain “immortalized” hormone-producing human pituitary tumor cell-lines.