Supplementary Data for "Canine adrenomedullary and pheochromocytoma organoids: a novel in vitro model"

Methods

Normal adrenal medullas from healthy dogs and tumor tissue from client-owned dogs with PCC were used to develop organoids. Primary cell suspensions were cultured in a 3D matrix, and organoids were established under optimized conditions. Organoids were characterized using histology, immunohistochemistry, immunofluorescence, qPCR, and metanephrine analysis by LC-MS/MS.

Results Five adrenomedullary organoid lines were successfully established, demonstrating sustained growth. Organoid cultures were also derived from nine PCCs, although expansion was limited after passages 1-2. Both adrenomedullary and PCC organoids expressed differentiation markers (chromogranin A, synaptophysin, phenylethanolamine N-methyltransferase) and stem/progenitor markers (nestin, SOX10).

Organoids retained key functional traits, as indicated by metanephrine levels in culture supernatants, which initially mirrored primary tumor patterns. A decline in both differentiation marker expression and metanephrine levels was observed over time, possibly due to organoid dedifferentiation or selective loss of differentiated chromaffin cells. Conclusions This study demonstrates the establishment of the first adrenomedullary and PCC organoid lines.

While further optimization is needed, these organoids offer valuable potential as an in vitro model to investigate PCC pathophysiology and explore novel treatment strategies for this therapeutically challenging tumor.