The challenges of performing ring test studies to evaluate the efficacy of complex drug combinations in vitro

<p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" class="first" dir="auto" id="d6315192e306">Within the REMEDi4ALL project reproducibility received special attention as a key barrier to successful drug repurposing (DR) in different fields from their original application. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d6315192e308">Three different partners of the REMEDi4ALL community (Germany, Sweden and Italy) performed, in the frame of Demonstrator2, a ring test study to analyze the critical steps necessary to achieve robust and reproducible preclinical outcomes. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d6315192e310">The DR hypothesis of Demonstrator-2 is based on the original results obtained in the Italian center demonstrating the synergistic effect of the histone deacetylase inhibitor (HDACi), valproic acid (VPA), combined with the lowering-cholesterol drug, simvastatin (SIM), in potentiating the antitumoral activity of the first-line gemcitabine/nab-paclitaxel chemotherapy in pancreatic ductal adenocarcinoma (PDAC). <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d6315192e312">To ensure reproducibility, an experimental workflow was developed following three main steps: material sharing (compounds, cells, legal documentation); alignment of experimental conditions (visiting scientists, protocols); synergy screening, data sharing and analysis. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d6315192e314">A first drug screening was performed across three PDAC cell lines (BxPC3, MiaPaca-2, PanC-1) testing the combinations of HDACis (vorinostat, entinostat, panobinostat, VPA) plus statins (lovastatin, atorvastatin, SIM). A second drug screening was performed to assess the ability of VPA and SIM to enhance the cytotoxic effects of chemotherapy in the same cell lines. Across the tested compound concentrations, we obtained the MSA score by Synergy Finder tool, corresponding to the highest synergistic area. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d6315192e316">Despite the initial development of a common workflow, technical and experimental challenges occurred, highlighting the need to realign the experimental conditions. Through the full alignment of the three institutes, we achieved consistent reproducibility of Demonstrator-2 data. <p xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" dir="auto" id="d6315192e318">This success enabled us to develop standard operative procedures (SOPs) as a valuable resource for the REMEDi4ALL.