CombiCTx: Screening diffusion gradients of anti-cancer drug combinations

Abstract

The reduced effectiveness of chemotherapy in many patients highlights the need for novel drug combinations and optimal ratios that target multiple survival mechanisms, which tumors may engage to confer drug resistance. Dynamic conditions within the tumor microenvironment shape cell behavior and influence the response to anti-cancer drugs, varying by cell type and local context. Accordingly, assays that identify effective concentrations and drug interactions (additive, synergistic, or antagonistic) in a relevant tumor tissue model are required to discover new combination treatments. To address this need for combinatorial chemotherapeutic (CTx) screening assays, we reconfigured CombiANT, a device for testing antibiotic interactions, and present a new assay called CombiCTx. The assay uses a device with three reservoirs containing gels loaded with chemotherapeutics or other anti-cancer drugs. The drug-loaded device is inverted and placed in a standard culture dish containing cancer cells, and both are then enclosed in gel. As drugs diffuse from the reservoirs, cancer cells are exposed to overlapping dynamic gradients of anti-cancer drugs, which can interact in various ways. We imaged doxorubicin diffusion in the assay using timelapse microscopy and employed the apoptotic agent staurosporine as a model drug, and established an imaging protocol for quantifying MDA-MB-231 breast cancer cell apoptosis along drug diffusion gradients. Finally, we tested navitoclax and gemcitabine to demonstrate the capacity of CombiCTx to evaluate combined cytotoxic effects while accounting for drug diffusion. Evaluating drug combinations with an assay that accounts for drug properties that influence diffusion in a complex tumor matrix may provide clinically relevant information.