Photodynamic treatment of glioblastoma plus endothelial cell spheroid models: increased proliferative and migratory aggressiveness of surviving tumor cells due to iNOS/NO upregulation

This study developed three-dimensional glioblastoma spheroid models to investigate tumor-endothelial interactions, mechanical properties, photodynamic therapy response, and the role of inducible nitric oxide synthase in the behavior of surviving tumor cells. U87-MG and LN229 human glioblastoma cells were cultured as spheroids and, for the broader tumor-microenvironment model, combined with HMEC-1 endothelial cells at defined ratios. U87-containing spheroids grew more rapidly and were larger and mechanically softer than LN229 spheroids. Mechanical testing showed a mean Young’s modulus of 4.2 ± 0.3 kPa for LN229 spheroids compared with 2.4 ± 0.4 kPa for U87 spheroids, indicating substantially greater deformability of the U87 model. The softer U87 phenotype was also associated with deeper HMEC-1 endothelial infiltration, with mean infiltration depths of 340 ± 46 µm in U87 spheroids versus 145 ± 20 µm in LN229 spheroids. The authors then evaluated 5-aminolevulinic-acid-mediated photodynamic therapy, showing that protoporphyrin IX initially accumulated at spheroid surfaces before becoming more uniformly distributed. Increasing light exposure produced progressively greater cell killing, although substantial numbers of tumor cells survived. Surviving cells showed increased proliferation and migration after photodynamic stress, accompanied by substantial iNOS upregulation. The selective iNOS inhibitor 1400W reduced post-treatment spheroid regrowth, supporting a role for stress-induced iNOS signaling in the aggressive behavior of surviving glioblastoma cells. The authors conclude that the mixed 3D spheroid system captures important features of the glioblastoma microenvironment and that combining photodynamic therapy with iNOS inhibition may improve treatment effectiveness.