D300e digital drug dispenser

Drugs were administered by a Tecan D300e digital drug dispenser. Wells were randomized during drug administration and re-organized during data analysis to avoid systematic spatial bias on the plates. Drugs were administered as single agents, pairwise, triplicate, and quadruplicate combinations in concentration ratios determined by their C_sustained values. C_sustained refers to a clinically relevant concentration based on measurements in patients’ serum up to 6 hours after administration^{12 (link)}; these are: 4-HC, 15 μM^{9 (link),39 (link)}; doxorubicin, 150 nM^{9 (link)}; prednisolone, 5 μM^{40 (link),41 (link)}; vincristine 5 nM^{9 (link)}. Dose response measurements spanned concentrations from 0% to 500% C_sustained in log-spaced steps. Cells were incubated with drugs for 72 hours, which spans the in vivo elimination half-lives of these drugs⁴² .

The optimal seeding density for each cell line was determined to ensure exponential growth for the entire duration of the assay. Cell suspensions were automatically seeded in 2D in black 384-well plates using the OT-2 pipetting robot (Opentrons) and allowed to settle overnight. Afterward, cells were treated with AF (0–6.5 μM), olaparib (0–30 μM), and their combination using the D300e digital drug dispenser (Tecan, Männedorf, Switzerland) with synergy wizard to treat cells with a 7-point drug titration matrix for each compound. Cell survival and cell death were assessed using an endpoint assay after 72 h. Therefore, cells were stained with 1000 nM Hoechst 33342 (blue) to determine the number of live cells and with 50 nM green-fluorescent marker Cytotox Green to measure the level of cell death in each well. The Tecan Spark Cyto live-cell imaging system was used to acquire images at 72 h to monitor drug response. The number of blue and green cells was counted using Tecan’s Image Analyzer Software. Potential synergism between AF and olaparib was determined using Synergy Finder (version 3.0) [34 (link)]. Synergy scores above 10 indicated synergism, scores between −10 and 10 implied an additive response, and those below −10 indicated an antagonistic effect.