Activation of
prodrugs in
tumors (e.g., by bioreduction in hypoxic zones) has the potential to generate active metabolites that can diffuse within the tumor microenvironment. Such "bystander effects" may offset spatial heterogeneity in
prodrug activation but the relative importance of this effect is not understood. Here, we quantify the contribution of bystander effects to antitumor activity for the first time, by developing a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model for
PR-104, a
phosphate ester pre-
prodrug that is converted systemically to the
hypoxia-activated
prodrug PR-104A. Using
Green's function methods we calculated concentrations of
oxygen,
PR-104A and its active metabolites, and resultant cell killing, at each point of a mapped three-dimensional
tumor microregion. Model parameters were determined in vitro, using single cell
suspensions to determine relationships between
PR-104A metabolism and clonogenic cell killing, and multicellular layer (MCL) cultures to measure tissue diffusion coefficients. LC-MS/MS detection of active metabolites in the extracellular medium following exposure of anoxic single cell
suspensions and MCLs to
PR-104A confirmed that metabolites can diffuse out of cells and through a tissue-like environment. The SR-PK/PD model estimated that bystander effects contribute 30 and 50% of
PR-104 activity in SiHa and HCT116
tumors, respectively. Testing the model by modulating PR-104A-activating
reductases and
hypoxia in
tumor xenografts showed overall clonogenic killing broadly consistent with model predictions. Overall, our data suggest that bystander effects are important in
PR-104 antitumor activity, although their reach may be limited by macroregional heterogeneity in
hypoxia and
reductase expression in
tumors. The reported computational and experimental techniques are broadly applicable to all targeted anticancer
prodrugs and could be used to identify strategies for rational
prodrug optimization.