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

Photodynamic therapy (PDT) is a unique oxidative stress-based anti-tumor modality that has proven highly effective for various solid malignancies. Intrinsic and acquired resistance is a significant challenge for all cancer treatments, including PDT. We showed previously that several human cancer cell lines in 2D cultures can exploit nitric oxide (NO) from stress-upregulated inducible nitric oxide synthase (iNOS) to (i) resist photokilling sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin IX, and (ii) promote growth and mobility aggressiveness of surviving tumor cells. We describe here a mixed-spheroid model consisting of glioblastoma (LN229 or U87) cells and normal human (HMEC-1) epithelial cells. To systematically investigate the interactions between tumor and endothelial cells in a 3D microenvironment, we developed a stepwise workflow: first, generating mixed spheroids with defined cell ratios; second, characterizing their growth, mechanical properties, and cellular organization using fluorescent labeling and confocal microscopy; third, assessing ALA-induced PpIX distribution and PDT efficacy; and finally, evaluating post-PDT responses including iNOS upregulation, proliferation, migration, and the impact of iNOS inhibition. Using high resolution confocal microscopy, we visualized the process of development of heterospheroid models. We analyzed the distribution of ALA-induced protoporphyrin IX within preformed spheroids. PpIX formed initially in the periphery of spheroid reaches even distribution within the spheroid in ~ 12 h of equilibration time. The survival of spheroid cells subjected to photodynamic action was determined. In general, higher doses of LED light were needed to achieve the same killing ratio for spheroids, as compared to 2D cultures. The effects of ALA/light treatment on the expression of iNOS, and proliferative potential of surviving tumor cells are reported. The impact of iNOS inhibitor (1400W) on the process of spheroid re-growth after induced photodynamic action was also analyzed. Photodynamic stress of glioma cells spheroids increases their intrinsic iNOS expression and NO-dependent proliferation. This increase is inversely proportional to the initial/constitutive expression of iNOS: less aggressive LN229 cells show greater stress-induced increase of iNOS and proliferation, than constitutively more aggressive U87 cells. The results presented suggest, that introduction of an iNOS inhibitor could significantly increase the effectiveness of photodynamic therapy.