Pulsed electromagnetic field stimulation subtly modulates doxorubicin sensitivity in a spheroid co-culture model of osteosarcoma

Osteosarcoma is a rare but aggressive bone cancer primarily affecting children and adolescents. Current treatment options are limited by toxicity and reduced efficacy in advanced stages. Pulsed electromagnetic field (PEMF) stimulation has shown promise as a non-invasive anticancer strategy, though inconsistent experimental models and exposure protocols limit translational progress. In the present study, a co-culture spheroid model of osteosarcoma was optimized using human osteosarcoma U2OS cells and bone marrow-derived mesenchymal stem cells (hBMSCs) and used to investigate whether PEMF exposure (1.5 mT, 75 Hz, sinusoidal) enhances spheroid sensitivity to doxorubicin (DOX). The model was characterized by structural integrity, mechanical properties, and functionality: while U2OS monoculture spheroids lacked structural stability, 1:3 MSC-U2OS co-cultures produced compact, stiff spheroids with migratory potential. PEMF stimulation for 3 days at 4 h per day reduced cell metabolic activity and spheroid stiffness and caused the downregulation of several genes associated with proliferation, survival, and invasion. These changes were most evident when combined with low-dose DOX, suggesting altered sensitivity; however, the effect was not uniformly detectable across all assessment methods. Thus, PEMF stimulation induces biological changes consistent with increased chemosensitivity in a subset of conditions, though the effect is subtle and assay-dependent. The developed co-culture model offers a relevant platform for further investigation of PEMF as a modulator of anticancer treatment response.