Merger of dynamic two-photon and phosphorescence lifetime microscopy reveals dependence of lymphocyte motility on oxygen in solid and hematological tumors

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.

A Window of Opportunity: Targeting Cancer Endothelium to Enhance Immunotherapy

“…Many of these factors, however, contribute to immunosuppression and cancer escape through pro-tumorigenic immune cell polarization (24) or partial leukocyte trafficking blockade. Low hypoxia levels have also been reported to slow down tumor infiltrating lymphocytes (55). Collectively, this data shows how abnormal tumor vasculature contributes to immunosuppression in cancer.…”

Nano versus Molecular: Optical Imaging Approaches to Detect and Monitor Tumor Hypoxia

“…By contrast, the hypoxia at the tumor core was alleviated at 100% O 2 , and as a consequence the T‐lymphocyte motility was significantly improved. [ 170 ]…”

Inhibition of Oxidative Phosphorylation Reverses Bone Marrow Hypoxia Visualized in Imageable Syngeneic B-ALL Mouse Model

“…The observed intracellular hypoxia detected by pimonidazole adduct staining could reflect either inadequate oxygen supply to the BM extracellular spaces, or high rates of intracellular oxygen consumption (or both). To directly quantify the extracellular oxygen tension [pO 2 ] in the BM microenvironment (as opposed to intracellular pimonidazole adduct staining), we utilized an imaging technique that enables high resolution contextual visualization of extracellular molecular oxygen based on probe phosphorescence lifetime along with local microenvironment structure and cellular composition based on multi-reporter fluorescence (36). Using this technique, termed FaST-PLIM (fast-scanning two-photon phosphorescence lifetime imaging microscopy), we performed time course imaging of the calvarial BM oxygenation at various leukemia stages.…”

“…Oxygen imaging was performed by fast scanning two-photon phosphorescence lifetime microscopy (FaST-PLIM) as described (36). Briefly, mice were infused i.v.…”