Characterization of glioblastoma spheroid models for drug screening and phototherapy assays

Glioblastoma (GBM) spheroids present as a promising alternative testing platform for in vivo models of brain cancer, aiming at the 3Rs principle of animal use in pre-clinical trials. However, the poor characterization and irreproducibility of various GBM-based spheroids have hampered the achievement of statistically relevant data for drug screening and photodynamic therapy (PDT) assays. In a previous study (Calori et al., 2022), we defined the conditions for inducing tight compaction of U87MG, T98G, UW473, A172, and U251 GBM cell lines into reproducible 3D spheroids using type I collagen as an extracellular matrix. Herein, GBM spheroids were characterized in their growth profile, proliferation rate, cell viability, necrotic nucleus, and cell death mechanisms and applied in PDT assays. Driven by proliferation, U87MG and A172 spheroids grew with time, at least up to day 12, whereas T98G and U251 demonstrated little change. The order of cell viability of spheroids over time was U87MG > A172 > U251 > T98G. A necrotic nucleus was formed over time for all cell lines, as shown by histological assays and the inverse size-cell viability relationship. When subjected to PDT, all spheroids showed a dose-dependent response, with an excellent dose-light-cell viability correlation. The higher light dose required for PDT of GBM 3D spheroids compared with the simplest 2D monolayers and its proximity to in vivo dose response in the literature is proof of its applicability in pre-clinical tests to reduce the use of animals in research.