SignificanceRadiation resistance is a major contributor to cancer treatment failure and is likely driven by multiple pathways. Multivariate visualization that preserves the spatial co-localization of factors could aid in understanding mechanisms of resistance and identifying biomarkers of response.AimWe aim to investigate the spatial and temporal relationship between hypoxia, hypoxia-inducible factor 1 (HIF-1α), and metabolism in response to radiation therapy in two cell lines of known radiation resistance and sensitivity.ApproachTwo-photon excited fluorescence and fluorescence lifetime imaging microscopy were used to quantify the optical redox ratio (ORR) and NAD(P)H fluorescent lifetime and bound fraction in frozen tumor sections and co-registered with immunohistochemical stain-based imaging of hypoxic fraction and HIF-1α.ResultsHistogram analysis of hypoxia, HIF-1α, and ORR revealed an increase in the ORR in regions of low hypoxia and high HIF-1α, indicating that the stabilization of HIF-1α is likely due to an increase in reactive oxygen species following radiation therapy. In addition, the bound NAD(P)H fraction was higher in regions with a low ORR in resistant tumors following radiation, suggesting an increase in fatty acid synthesis.ConclusionsA multivariate histogram approach can reveal hidden trends not observed in bulk analysis of tumor images and may be useful in understanding biomarkers and mechanisms of radiation resistance.
Multi-photon microscopy was used to determine the differential response to radiation at multiple time points between radiation-resistant and -susceptible human head and neck cancer tumor xenografts grown in a cohort of 60 athymic mice. Mice were randomly divided into groups for cell line, treatment or control, and time points relative to treatment — baseline, 1 hour-, 24 hours-, and 48 hours-post-treatment. We quantified the optical redox ratio, lifetime of NAD(P)H, and heterogeneity in ORR and lifetime endpoints. We saw a significant effect of cell line, treatment, and the interaction of all three factors — line, treatment, and time point.
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