Fig. 3: Ceralasertib and Olaparib are synergistic in treatment resistant PDAC.

a TKCC10 parent and treatment resistant cell lines were synchronised in G1/S by overnight treatment with hydroxyurea and replicating cells were tracked at 2 hourly intervals following release using BrdU uptake. Cell cycle was assayed by propidium iodide staining b. Progression of replicating cells through S phase was analysed in 3 phases – Early S(S1), Mid S(S2), and Late S(S3); gating strategy is indicated. Percentage of the total population at each cell cycle phase is shown for each timepoint c. TKCC10 parental and resistant cell lines were treated with ceralasertib and olaparib in a 5 × 5 matrix of dose combinations (Ten-fold dilutions, 0–10 μM). Cell viability was assayed by MTS after 8 days’ exposure. Drug synergy was calculated using the interaction potency (ZIP) model across the dose matrix d. TKCC10 parent and resistant cell lines were exposed to 1 μM ceralasertib for 5 hr, at which time treatment containing medium was removed and replaced with growth medium. Rad51 and pRPA2 foci were analysed by confocal microscopy at 16hr, 24hr and 48hr following washout. DAPI was used as a nuclear counterstain e. Clonogenicity analysis of the effect of ceralasertib and olaparib in combination was performed on the TKCC10 cell line using treatment across a 0–0.5 μM dose range. Surviving fraction was calculated relative to the plating efficiency and analysed by 2-way ANOVA. Survival curves were generated using the linear quadratic model f. The response of resistant cell lines to ceralasertib and olaparib combination treatment and ceralasertib monotherapy (1 μM concentrations for each, 24 h exposure) was assayed using Pan-nuclear γH2AX and Pan-nuclear pRPA2 staining as markers for replication catastrophe (Panels g, h). Results were analysed by 2-way ANOVA with Tukey’s test for multiple comparisons (*, p < 0.05; ns, non-significant; results from 2 independent experiments).