Bystander Effects of Hypoxia-Activated Prodrugs: Agent-Based Modeling Using Three Dimensional Cell Cultures

Intra-tumor heterogeneity represents a major barrier to anti-cancer therapies. One strategy to minimize this limitation relies on bystander effects via diffusion of cytotoxins from targeted cells. Hypoxia-activated prodrugs (HAPs) have the potential to exploit hypoxia in this way, but robust methods for measuring bystander effects are lacking. The objective of this study is to develop experimental models (monolayer, multilayer, and multicellular spheroid co-cultures) comprising ‘activator’ cells with high expression of prodrug-activating reductases and reductase-deficient ‘target’ cells, and to couple these with agent-based models (ABMs) that describe diffusion and reaction of prodrugs and their active metabolites, and killing probability for each cell. HCT116 cells were engineered as activators by overexpressing P450 oxidoreductase (POR) and as targets by knockout of POR, with fluorescent protein and antibiotic resistance markers to enable their quantitation in co-cultures. We investigated two HAPs with very different pharmacology: SN30000 is metabolized to DNA-breaking free radicals under hypoxia, while the dinitrobenzamide PR104A generates DNA-crosslinking nitrogen mustard metabolites. In anoxic spheroid co-cultures, increasing the proportion of activator cells decreased killing of both activators and targets by SN30000. An ABM parameterized by measuring SN30000 cytotoxicity in monolayers and diffusion-reaction in multilayers accurately predicted SN30000 activity in spheroids, demonstrating the lack of bystander effects and that rapid metabolic consumption of SN30000 inhibited prodrug penetration. In contrast, killing of targets by PR104A in anoxic spheroids was markedly increased by activators, demonstrating that a bystander effect more than compensates any penetration limitation. However, the ABM based on the well-studied hydroxylamine and amine metabolites of PR104A did not fit the cell survival data, indicating a need to reassess its cellular pharmacology. Characterization of extracellular metabolites of PR104A in anoxic cultures identified more stable, lipophilic, activated dichloro mustards with greater tissue diffusion distances. Including these metabolites explicitly in the ABM provided a good description of activator and target cell killing by PR104A in spheroids. This study represents the most direct demonstration of a hypoxic bystander effect for PR104A to date, and demonstrates the power of combining mathematical modeling of pharmacokinetics/pharmacodynamics with multicellular culture models to dissect bystander effects of targeted drug carriers.