No abstract

BackgroundLow availability of oxygen in tumors contributes to the hostility of the tumor microenvironment toward the immune system. However, the dynamic relationship between local oxygen levels and the immune surveillance of tumors by tumor infiltrating T-lymphocytes (TIL) remains unclear. This situation reflects a methodological difficulty in visualizing oxygen gradients in living tissue in a manner that is suitable for spatiotemporal quantification and contextual correlation with individual cell dynamics tracked by typical fluorescence reporter systems.MethodsHere, we devise a regimen for intravital oxygen and cell dynamics co-imaging, termed ‘Fast’ Scanning Two-photon Phosphorescence Lifetime Imaging Microscopy (FaST-PLIM). Using FaST-PLIM, we image the cellular motility of T-lymphocytes in relation to the microscopic distribution of oxygen in mouse models of hematological and solid tumors, namely in bone marrow with or without B-cell acute lymphocytic leukemia (ALL), and in lungs with sarcoma tumors.ResultsBoth in bone marrow leukemia and solid tumor models, TILs encountered regions of varying oxygen concentrations, including regions of hypoxia (defined as pO2 below 5 mmHg), especially in advanced-stage ALL and within solid tumor cores. T cell motility was sustained and weakly correlated with local pO2 above 5 mmHg but it was very slow in pO2 below this level. In solid tumors, this relationship was reflected in slow migration of TIL in tumor cores compared to that in tumor margins. Remarkably, breathing 100% oxygen alleviated tumor core hypoxia and rapidly invigorated the motility of otherwise stalled tumor core TILs.ConclusionsThis study demonstrates a versatile and highly contextual FaST-PLIM method for phosphorescence lifetime-based oxygen imaging in living animal tumor immunology models. The initial results of this method application to ALL and solid lung tumor models highlight the importance of oxygen supply for the maintenance of intratumoral T cell migration, define a 5 mmHg local oxygen concentration threshold for TIL motility, and demonstrate efficacy of supplementary oxygen breathing in TIL motility enhancement coincident with reduction of tumor hypoxia.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0543-y) contains supplementary material, which is available to authorized users.

No abstract

No 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.