BACKGROUND: Pheochromocytomas and paragangliomas (PPGLs) are rare catecholamine-producing tumors arising from chromaffin tissue. In a PPGL subgroup, dysregulation of hypoxia signaling pathways, in particular mediated through stabilization of hypoxia-inducible factor 2 alpha (HIF2α), have been suggested to drive tumorigenesis through altering downstream transcriptional activity. OBJECTIVE: This study evaluated the use of mCherry-transgenic mouse pheochromocytoma (MPC^mCherry) spheroids as in vitro models for investigating consequences of HIF2α expression on aggregation behavior, morphology, growth, glucose consumption, amino acid uptake, and somatostatin type 2 receptors under stable hypoxic conditions. METHODS: MPC^mCherry spheroids were monitored using confocal laser scanning microscopy. Hypoxic regions were detected using pimonidazole. Radiotracer incubation was performed using 2-[¹⁸F]fluoro-2-deoxyglucose ([¹⁸F]FDG), O-3-(2[¹⁸F]fluoroethoxy)-4-hydroxyphenylalanine ([¹⁸F]OFED), and [⁶⁸Ga]Ga-(Tyr³)octreotate ([⁶⁸Ga]Ga-DOTA-TATE). RESULTS: Both HIF2α-expressing and empty vector (EV) control spheroids showed regions of stable cellular hypoxia. Expression of HIF2α in MPC^mCherry spheroids was associated with less symmetric morphology, faster growth, and decreased uptake of [⁶⁸Ga]Ga-DOTA-TATE (somatostatin type 2 receptors) compared to controls, whereas, uptake of [¹⁸F]FDG (glucose transporter 1 and hexokinases) and [¹⁸F]OFED (system L amino acid transporter 1) remained unaffected. CONCLUSIONS: The recent study proved MPC^mCherry spheroids to be complex three-dimensional tumor cell models for investigating morphologic and metabolic consequences of dysregulated hypoxia pathways under hypoxic conditions.