(A) Experimental schedule for studying BPD uptake and distribution in homotypic and heterotypic spheroids using PICs. Cells were seeded in CellCarrier Spheroid ULA 96-well Microplates followed by incubation with Visudyne® for 90 min. The spheroids were then washed, fixed in PFA, counterstained with Hoechst and visualized under a confocal fluorescence microscope. (B) Representative confocal images of the central optical section of the 3D spheroids. Mia PaCa-2 cells and PCAFs are pseudo-colored as cyan and green. Nuclei are stained with Hoechst (pseudo-colored as blue) and BPD is shown in red. Profile plots were generated using Image J to monitor BPD distribution across the central plane. As suggested by the profile-plots, the distribution of BPD was homogeneous for spheroids generated with 0%, 10% and 25% PCAFs. However, as the PCAF percentage increased beyond 50% the distribution of BPD appeared to be heterogeneous with higher signal on the periphery and lower signal in the spheroid core (scale bar = 500 μm).