Tumor blood vessels pose obstacles for drug delivery because they are hyper-permeable and non-functional. There is a critical need to identify safe methods to increase chemotherapy delivery to the tumor. PURPOSE: We demonstrated that aerobic exercise improves tumor vasculature function, in multiple disparate tumor models, causing increased chemotherapy delivery and efficacy in mice. Across models, exercise reduced tumor vessel permeability. Because aerobic exercise increases blood flow both in healthy and tumor vessels, we aimed to investigate shear stress responsive mechanisms by which exercise may reduce tumor vessel permeability. METHODS: In vivo approaches including pharmacologic agents, a forced treadmill model of moderate aerobic exercise, and transgenic mouse models were utilized in combination with in vitro modeling of exercise induced shear stress, using a cone and plate viscometer. RESULTS: In tumor endothelium, we found the flow responsive kinase and co-transcriptional activator extracellular signal-regulated kinase 5 (ERK5) regulates tumor vessel permeability, similar to exercise. ERK5 activation in response to exercise was investigated in vivo, using a Krϋppel like factor 2 (KLF2) reporter mouse. KLF2 is a well-defined downstream target of ERK5. KLF2 was upregulated by exercise in the lung and aorta endothelium providing the first evidence for the involvement of ERK5 activation in response to aerobic exercise. Based on this and our previous data demonstrating that exercise induced shear stress upregulates spingosine-1 phosphate receptor 1 (S1PR1) on tumor vessels, we hypothesized that exercise activates ERK5, causing S1PR1 upregulation and decreasing permeability in tumor endothelium. To investigate this, we modeled basal tumor vasculature (low shear stress, 3 dynes/cm²) and exercise-induced flow (high shear stress, 15 dynes/cm²) with a cone and plate viscometer in vitro. We found the ERK5 axis has a similar flow responsive pattern as S1PR1. Further, ERK5 directly regulates S1PR1 in cultured endothelial cells revealing a novel EC pathway, the ERK5-S1PR1 axis. CONCLUSION: In summary, our data identifies the ERK5-S1PR1 axis as a potential exercise responsive pathway in tumor and healthy vasculature. We are currently investigating activation of the ERK5 axis in tumor vasculature.