Long-term in vitro 2D-culture of SDHB and SDHD-related human paragangliomas and pheochromocytomas

Jean Pierre Bayley*, Heggert G. Rebel, Kimberly Scheurwater, Dominique Duesman, Juan Zhang, Francesca Schiavi, Esther Korpershoek, Jeroen C. Jansen, Abbey Schepers, Peter Devilee

*Corresponding author for this work

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Abstract

The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDHrelated PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to the classical 2D-culture of SDH-related human paragangliomas and pheochromocytomas, with the ultimate goal of identifying a viable tumour-derived cell line. PPGL tumour tissue/cells (chromaffin cells) were cultured in a variety of media formulations and supplements. Tumour explants and dissociated primary tumour cells were cultured and stained with a range of antibodies to identify markers suitable for use in human PPGL culture. We cultured 62 PPGLs, including tumours with confirmed SDHB, SDHC and SDHD variants, as well as several metastatic tumours. Testing a wide range of basic cell culture media and supplements, we noted a marked decline in chromaffin cell numbers over a 4-8 week period but the persistence of small numbers of synaptophysin/ tyrosine hydroxylase-positive chromaffin cells for up to 99 weeks. In cell culture, immunohistochemical staining for chromogranin A and neuron-specific enolase was generally negative in chromaffin cells, while staining for synaptophysin and tyrosine hydroxylase was generally positive. GFAP showed the most consistent staining of type II sustentacular cells. Of the media tested, low serum or serum-free media best sustained relative chromaffin cell numbers, while lactate enhanced the survival of synaptophysin-positive cells. Synaptophysin- positive PPGL tumour cells persist in culture for long periods but show little evidence of proliferation. Synaptophysin was the most consistent cell marker for chromaffin cells and GFAP the best marker for sustentacular cells in human PPGL cultures.

Original languageEnglish
Article numbere0274478
JournalPLoS ONE
Volume17
Issue number9 September
DOIs
Publication statusPublished - 30 Sep 2022

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© 2022 Bayley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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