UC Davis

Background: Cancer survivors are at an increased risk of developing cardiovascular diseases such as arrhythmias, left ventricular dysfunction, ischemia, and pericarditis. This has been traced back to the use of anticancer drug treatment that can damage DNA synthesis and repair, create reactive oxygen species, block ion channels, and damage cardiac lipid membranes, among other effects. In this study, we specifically focus on anticancer drug combinations to inhibit ovarian cancer cell growth, as there is a clinical need for more variety in ovarian cancer treatment options. Ovarian cancer does not respond uniformly to treatment, and advanced ovarian cancer patients have increased likelihood of resistance to the clinical standard of care. We therefore aim to bridge these gaps in knowledge and address clinical needs by identifying novel anticancer drug combinations that are highly effective in inhibiting ovarian cancer cell growth (≥ 80% inhibition) while causing limited effects on cardiac intracellular signaling and function.

Objective:We aim to identify highly effective anticancer drug combinations that inhibit ≥ 80% of ovarian cancer cell growth. Subsequently, we intend to use our high-content and physiologically relevant cardiotoxicity screening assay, which investigates the adverse effects of anticancer drug combinations on cardiomyocyte action potential, Ca2+ transient, and contractile signal during both load-free and mechanically loaded (in-gel) conditions. Using mechanically loaded conditions, we mimic high myocardial stresses, and thus, create more physiologically relevant conditions. In addition, we investigate the formation of alternans induced by the anticancer drug combinations in all three signals (AP, Ca2+, contraction). We intend to use drugs of known cardiotoxicity from the Comprehensive In Vitro Proarrhythmia Assay (CiPA) to validate the cardiotoxicity screening assay.

Methods:A MTT (3-(4,5-Dimethylthiazol-2-yl)-25-Diphenyltetrazolium Bromide) Assay is used to obtain the percent inhibition of ovarian cancer cell growth from the anticancer drug combinations. Freshly isolated left ventricular cardiomyocytes are obtained from rabbits and used for the cardiotoxicity screening assay. The optical action potential is measured via a voltage sensitive dye, CRhOMe, created by our collaborator Dr. Evan Miller from UC Berkeley. Intracellular Ca2+ transient measurements use the calcium indicator Fluo-4. Contractile measurements were obtained directly from the cardiomyocyte using IonOptix Software. The cell-in-gel was created using 10% PVA crosslinked with a 10% tetravalent boronate-PEG crosslinker and microbeads to visualize cardiomyocyte displacement and contractile changes. This creates a viscoelastic hydrogel of 3D multi-axial mechanical stresses.

Results:We identified 11 anticancer drug combinations of high efficacy in inhibiting ovarian cancer cell growth: CEL/ACT, CEL/DOX/STR, CEL/DOX, CEL/DOX/MTX, BCNU/CEL/ACT, CEL/DOX/MMC, BCNU/CEL/DOX, DOX/MMC/STR, BCNU/DOX/MMC, DOX/MMC/MTX, and DOX/MMC. Synergism between DOX and STR was investigated based off these results, which was hypothesized to be linked to iron ion usage. Of these 11 combinations, 7 combinations exhibited a high percentage of alternans in load-free conditions (≥ 40%): CEL/ACT, CEL/DOX/STR, CEL/DOX, CEL/DOX/MTX, BCNU/CEL/ACT, CEL/DOX/MMC, and BCNU/CEL/DOX. However, four combinations induced minimal alternans (≤ 33%) in the action potential, Ca2+, and contractile signals in load-free conditions: DOX/MMC/STR, DOX/MMC, DOX/MMC/MTX, and BCNU/DOX/MMC. Specific metrics of the signals were analyzed, with BCNU/DOX/MMC causing a significant increase in contractile fractional shortening (FS%) peak signal during load-free conditions. DOX/MMC was also found to cause a significant decrease in AP peak during load-free conditions. DOX/MMC/MTX and BCNU/DOX/MMC had the lowest percentage of alternans of all combinations. When advanced to in-gel conditions, DOX/MMC/MTX and BCNU/DOX/MMC had increases in alternans formation in comparison to load-free conditions, but still induced minimal alternans formation overall (≤ 33%).

Conclusion:This study identified 11 anticancer drug combinations of high efficacy in inhibiting 80-95% of ovarian cancer cell growth. These anticancer drug combinations were then screened for cardiotoxicity using a high-content screening assay, which investigates the cardiomyocyte action potential, Ca2+, and contractile signals. A cell-in-gel mechanism was also applied in order to induce 3D multi-axial mechanical loads onto the cell, for a more physiologically relevant study. We identified 7 anticancer drug combinations that caused cardiotoxic effects with 4 anticancer drug combinations with minimal effects on the cardiomyocyte, as observed through alternans as well as through an analysis of the AP, Ca2+, and contractile signals. This work highlights the use of a novel high-content and physiologically relevant mechanism for screening of cardiotoxicity, which identifies irregularities in the signals of AP, Ca2+, and contraction on a single-cell cardiomyocyte level under mechanically loaded conditions.