Digital dispenser

All SPR measurements were run on a BIAcore S200 (GE Healthcare) using running buffer; 10 mM HEPES, 150 mM NaCl, 0.05% Tween 20, 5 mM MgCl₂, pH 7.4. Full-length VCP (LD Biopharma) was immobilized on EDC/NHS-activated NID500L chip (Xantec) utilizing the 6xHis-tag for pre-concentration of protein on the chip to a final level of 5500 ± 500 RU before addition of 1 M ethanolamine. Concentration series of compounds (n = 3) were dispensed using a Digital dispenser (Tecan), normalized to 0.5% DMSO, and injected at 30 μL/min for 1 min. Binding levels were fitted to a Langmuir 1:1 interaction model to extract steady-state affinity (K_d).

Reporter cell lines were seeded in triplicate at a density of 1 million cells/ml in 100 μl per well in a 96 well plate and measured once. Ligand was added by Digital Dispenser (Tecan), and incubated at 37°C for the appropriate time before reading by flow cytometry. Cells were analyzed for GFP vs mCherry expression. GFP expression was normalized to mCherry signal and ligand treatments were compared to DMSO controls.

Organoids were dissociated into single cells using a diluted solution of TrypLE Express (Thermo Fisher Scientific). Equal numbers of single cells (1,500 viable cells per well) were plated in a 20 μL suspension of 10% BME in ultra-low attachment 384-well plates (Corning). Twenty-four hours after plating, reformation of organoids was visually verified, and therapeutic compounds were applied using a digital dispenser (Tecan). Chemotherapies were tested in triplicate 10-point dose-response curves. Gemcitabine, paclitaxel, SN-38 (active metabolite of irinotecan), and trametinib ranged from 0.5 nM to 5μM; oxaliplatin and 5-fluorouracil ranged from 50 nM to 100 μM. Compounds were dissolved in DMSO, and all treatments were normalized to 0.5% DMSO content. Mouse organoids underwent treatment for 3 days, while human organoids underwent treatment for 5 days. After treatment, cell viability was assessed using Cell Titer Glo (Promega) per manufacturer’s instructions on a Tecan Spark Cyto plate reader. A 4-parameter log-logistic (LL4) function with upper limit equal to the mean of the lowest dose values was fit to the data (viability versus dose) with Graphpad Prism 9. The IC₅₀ values, AUC, and Sy.x for each dose-response curve were calculated using Graphpad Prism 9. Each pharmacotyping experiment was carried out at least 2 times.