Phosphorescence-optimized 2-photon lifetime and kinetic imaging reveals reanimation of tumor immune surveillance by hyper-oxygenation

Abstract

Lymphocytes encounter varying oxygen levels as they traverse through healthy and diseased tissue. Hypoxia is a hallmark of the tumor microenvironment and can affect the anti-tumor immune response. However, despite the importance of T cell adaptation to differing oxygen concentrations in the tumor niche, there has been no method to study T cell spatiotemporal dynamics in the context of oxygen in vivo. To this end, we developed phosphorescence-optimized 2-photon lifetime and kinetic (2pOLAK) microscopy which enables co-imaging of phosphorescence lifetimes and cellular dynamics in highly fluorescent biological reporter systems. In conjunction with the PtP-C343 oxygen probe, 2pOLAK microscopy revealed the tissue oxygen landscapes and individual oxygen “experiences” of T cells as they moved through tissues in syngeneic models of metastatic lung cancer and acute leukemia. We found that T cells experienced hypoxia in leukemic bone marrow, and that the motility of these cells was significantly decreased relative to non-hypoxic T-cells in healthy bone marrow. Inhibition of oxidative phosphorylation slowed non-hypoxic T cell motility to a level comparable with that of hypoxic T cells. T cell motility was also decreased in hypoxic lung tumor cores, and it was significantly lower than in the tumor margin, where T cells experienced higher oxygen. Supplemental oxygenation increased the oxygen experienced by T cells in the tumor core and reanimated T cell motility. These studies describe a novel method for co-imaging tissue oxygen and cellular behavior, shed light on the role that oxygen availability plays in T cell dynamics in vivo, and suggest that counteracting hypoxia can improve tumor immune surveillance by restarting T cell motility.