High-content monitoring of drug effects in a 3D spheroid model upon IDX and cis treatments.
(A) Spheroids were pre-formed for 3 days with 20,000 cells/well in the presence of 0.1 uM CellEvent and 0.2 uM Lysotracker and then treated with indicated concentrations of IDX and cis. Representative images were acquired at day 3 using the InCell 6000. The intensity of C17 spheroid staining with CellEvent was proportional to IDX and cis concentrations. When LysoTracker was added to the spheroids, the staining pattern was complementary to that of CellEvent, with the LysoTracker constantly accumulating in the outer and presumably metabolically active layers of the spheroids. (B) CellEvent was used to determine the IC50 values for IDX and cis. (C) Schematic representation of the protocol used for the coculture of C17/NPC43+ve spheroids and PBMCs from healthy donors and subsequent imaging and flow cytometric analysis. (D) Representative images were acquired at the indicated time points using the InCell 6000 where PKH26-stained and CellEvent-stained spheroids were cocultured. (E) Numbers of infiltrated cells were calculated by Fuji ImageJ software. (F) Apoptotic tumor cells were measured according to CellEvent intensity. (G) Pre-formed tumor spheroids were pre-stained with CellEvent followed by coculture with PKH26-stained PBMCs in the presence or absence of anti-CXCL10 (10 µg/mL) neutralizing antibody everyday throughout 3 days of coculture. Images were obtained at 10 µm increments on the GE InCell6000 instrument using a 10x objective. Representative images of 10 stacked planes are shown. Representative images of 3D pictures were generated using “Clear Volume”. (H) Quantification of immune cells infiltrating the spheroids upon IDX2 treatment alone or in combination of Cis1.4 was done. * p≤0.05 when compared to IDX treatment alone.