A. At varying concentrations, ARI-4175 does not alter the growth of MC38-CEA cells. Vehicle-treated and untreated cells served as controls; medium and compound were replenished daily. The total number of viable cells was determined at various time points by trypan blue exclusion. Data represent mean ± SEM from 2 replicate wells. B. MC38-CEA cells undergo immunogenic modulation following exposure to ARI-4175. Cells were treated daily with 10 μM ARI-4175 for 72 h, harvested, and analyzed by flow cytometry for cell-surface expression of Fas, ICAM-1, CEA, MHC I (H2Kb/H2Db), and calreticulin. Vehicle-treated and untreated cells served as controls. Numbers indicate percentage of positive cells (MFI in parentheses). Bold type indicates marked up-regulation (> 10% increase in percent of cells or MFI) relative to controls. C. Exposure of MC38-CEA cells to ARI-4175 increases sensitivity to antigen-specific CTL killing. Tumor cells treated with 10 μM ARI-4175 for 72 h were used as targets in an 18-h CTL lysis assay. CEA- or gp70-specific CD8+ T cells were used as effector cells at an effector:target ratio of 30:1 and 50:1, respectively. Data represent mean ± SEM for 3 replicate wells. Asterisks denote significance (P < 0.05) relative to untreated wells. Data are representative of one experiment. Experiments were repeated 2–4 times with similar results.