Investigating the relationship between hypoxia, hypoxia-inducible factor 1, and the optical redox ratio in response to radiation therapy.

Significance: Radiation 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.

Aim: We 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.

Approach: Two-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.

Results: Histogram 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.

Conclusions: A 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.