2.5. PDX 3D Spheroid Culture

We have successfully generated several patient-derived tumor xenograft (PDX) PC tumors in mice using PC tissue samples procured by biopsy of metastatic sites of patients heavily pre-treated with docetaxel, second generation anti-androgens including abiraterone, and/or enzalutamide. The PDX from these mice were harvested (GUR-017M generation 7, PRJ-88T generation 4, and TMA-027 generation 4), dissociated using MACS human tissue dissociation kit (Order no. 130-095-929) and gentleMACS^TM Octo Dissociated (Order no. 130-095-937) to produce single cells. Cells were plated in Corning Spheroid 96-well Microplate (Ref no. 4520) at a density of 3000–6000 cells per well to form spheroids, incubated at 37 °C and 5% CO₂. Cell culture was maintained using proprietary DMEM-F12 50/50 (Corning ref. 15-090-CV)-based complete spheroid media. Cells were plated in triplicates. On day 5, drug treatment was performed using MT-1 at a concentration between 0.01 µM and 100 µM using 0.1% DMSO as a control. After 72 h of incubation, cell viability assay was performed using CellTiter-Glo^® 3D Cell Viability Assay (cat # G9681, Promega Corporation, Madison, WI, USA) and relative luminescence units (RLU) were obtained and coefficients of variation (CV) and Z-factor (Z’) were determined for quality metrics. A CV of <20% and a Z’ between 0.4 and 1.0 were determined acceptable for assay performance. The relative luminescence was normalized to internal plate controls and plotted against concentration (logarithmic scale) to show a dose response curve. Using GraphPad Prism 8.0, the 50% inhibitory concentration (IC₅₀) with 95% confidence interval (CI) was calculated.