Extended Data Fig. 9: CYP3A activity mediates irinotecan tolerance in CYP3A-positive PDOs.
From: Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC
a) Cell proliferation assay showing doubling time for resistant (n = 3) and susceptible (n = 3) PDOs. The results represent n = 3 independent biological experiments. b) Bar plot showing doubling time between resistant (n = 3) and susceptible (n = 3) PDOs. Mann-Whitney (two-sided) P-value is shown on the plot. P-value was not adjusted for multiple testing. Bar plots represent mean values ± s.d. c) Bar plot of Ki67 protein expression in selected resistant (n = 5) and susceptible (n = 6) PDOs. Mann-Whitney (two-sided) P-value is shown on the plots. P-value was not adjusted for multiple testing. Bar plots represent mean values ± s.d. d) CYP3A enzyme activity in selected PDOs as determined by luminescence assay. CYP3A enzyme activity was normalized to total cell number. PDOs were treated with increasing concentrations of the CYP3A inhibitors ketoconazole and cobicistat to determine the optimum concentrations for combination treatments. Dunnett’s multiple comparison test (two-sided) was performed to identify concentrations sufficient to significantly inhibit CYP3A activity. P-values adjusted by multiple correction are shown on the plot. Bar plots show mean values of n = 3 independent biological experiments ± s.d. e) Cell viability assays showing PDO responses to increasing concentrations of the CYP3A inhibitors ketoconazole and cobicistat. Dunnett’s multiple comparison test (two-sided) was performed to identify concentrations sufficient to significantly reduce cell viability. P-values adjusted by multiple correction are shown on the plot. Bar plots show mean values of n = 3 independent biological replicates ± s.d. f) Compound analysis by UPLC-MS/MS of relative resistant and relative susceptible PDOs showing relative irinotecan to SN-38 conversion. Bar plots show mean values of n = 3 independent biological experiments ± s.d. g) Compound analysis by UPLC-MS/MS of relative resistant and relative susceptible PDOs showing relative SN-38 in the supernatant. Bar plots show mean values of n = 3 independent biological experiments ± s.d. h) Treatment of selected PDOs with irinotecan, SN-38, or paclitaxel in combination with either ketoconazole or cobicistat as indicated. Combination treatment with ketoconazole increases drug sensitivity to irinotecan and paclitaxel but not SN-38. IC50 values are provided for the indicated treatments. The results represent n = 3 independent biological experiments. Data are presented as mean values +/− s.e.m.
