The 3D in vitro Adrenoid cell model recapitulates the complexity of the adrenal gland.

The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell-cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.