Hybrid Tumor Spheroid Formation

Hybrid tumor/Ma spheroids were formed as previously described.^{12 (link)} Prior to spheroid formation, both nanoparticle-loaded and empty Ma were incubated for 1 hour in 20 µg/mL mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) to inhibit cell division and their subsequent contribution to spheroid growth. Hybrid spheroids using either 5 × 10³ ACBT cells and 1 × 10³ or 2 × 10³ loaded Ma (designated Ma^NS or Ma^NR) were generated. The cell combinations were alloquated into the wells of ultra-low attachment 96-well round-bottom plates (Corning, Corning, NY, USA) in 200 µL culture medium. The plates were centrifuged at 1,000 ×g for 10 min. The plates were incubated for 48 h to allow the spheroids to stabilize. Previous experiments employing two-photon microscopy have shown an even distribution of the Ma throughout the spheroids.^{12 (link)}