Multicellular tumor spheroids and cell viability and proliferation assays.

3D cell cultures were essentially prepared as described by Ho et al. (64). Briefly, spheroids were produced by seeding U-bottom wells previously coated with 1% agarose with 2 × 10⁴ cells suspended in 200 μL DMEM/F-12 medium containing 10% FBS. With this setup, cells do not adhere to the plastic matrix and are thus allowed to associate, forming a multicellular 3D structure. The size of the aggregates was monitored every 24 hours in the inverted microscope for a total of 72 hours. The sulforhodamine B (SRB) cell viability assay and BrdU proliferation assay were performed as recently described (17).