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

ABSTRACT: BACKGROUND:Low 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. METHODS:Here, 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. RESULTS:Both 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. CONCLUSIONS:This 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.

SUBMITTER: Rytelewski M

PROVIDER: S-EPMC6423744 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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

Rytelewski Mateusz M Haryutyunan Karine K Nwajei Felix F Shanmugasundaram Meenakshi M Wspanialy Patrick P Zal M Anna MA Chen Chao-Hsien CH El Khatib Mirna M Plunkett Shane S Vinogradov Sergei A SA Konopleva Marina M Zal Tomasz T

Journal for immunotherapy of cancer 20190318 1

<h4>Background</h4>Low 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 indi ...[more]

PMID: 30885258

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Project description:Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids' (RtOgs') long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.

Dataset Information

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

Publications

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

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