a Hit compounds from the primary screen were tested on T-cell/spheroid co-cultures where both cell types were primed with the same antigen (CEF Peptide Pool), cell types were primed with different antigens (CEF Scramble) or spheroid monocultures with no T-cells (No T-cells). Cytotoxic compounds that targeted key cellular machinery exhibited anti-tumor properties independent of T-cell presence (MTi β-tubulin inhibitor, HDACi histone deacetylase inhibitor, MAPKi mitogen-activated protein kinase inhibitor). ECM-targeting compounds could be distinguished by their unique, though non-toxic, phenotypes (MMPi matrix metalloproteinase inhibitor). Spheroid size is quantitated in bar graphs on right, relative to DMSO controls (ns = not significant and error bars indicate standard deviation of three biologically independent samples). b Viability readout of spheroids from a using HCT116-Luc reporter spheroids in the presence of T-cells and hit compounds with matching viral antigens (CEF Peptide Pool). HDAC inhibitors and β-tubulin inhibitors demonstrated sigmoidal dose–response curves, ERK inhibitors demonstrated high potencies and MMP inhibitors did not significantly affect tumor spheroid cell viability.