Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of Immunotherapy
Background: The wide variability in clinical responses to anti-tumor immunotherapy drives the search for personalized strategies. One of the promising approaches is drug screening using patient-derived models composed of tumor and immune cells. In this regard, the selection of an appropriate in vitr...
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MDPI AG
2025-01-01
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| author | Diana V. Yuzhakova Daria A. Sachkova Anna V. Izosimova Konstantin S. Yashin Gaukhar M. Yusubalieva Vladimir P. Baklaushev Artem M. Mozherov Vladislav I. Shcheslavskiy Marina V. Shirmanova |
| author_facet | Diana V. Yuzhakova Daria A. Sachkova Anna V. Izosimova Konstantin S. Yashin Gaukhar M. Yusubalieva Vladimir P. Baklaushev Artem M. Mozherov Vladislav I. Shcheslavskiy Marina V. Shirmanova |
| author_sort | Diana V. Yuzhakova |
| collection | DOAJ |
| description | Background: The wide variability in clinical responses to anti-tumor immunotherapy drives the search for personalized strategies. One of the promising approaches is drug screening using patient-derived models composed of tumor and immune cells. In this regard, the selection of an appropriate in vitro model and the choice of cellular response assay are critical for reliable predictions. Fluorescence lifetime imaging microscopy (FLIM) is a powerful, non-destructive tool that enables direct monitoring of cellular metabolism on a label-free basis with a potential to resolve metabolic rearrangements in immune cells associated with their reactivity. Objective: The aim of the study was to develop a patient-derived glioma explant model enriched by autologous peripheral lymphocytes and explore FLIM of the redox-cofactor NAD(P)H in living lymphocytes to measure the responses of the model to immune checkpoint inhibitors. Methods: The light microscopy, FLIM of NAD(P)H and flow cytometry were used. Results: The results demonstrate that the responsive models displayed a significant increase in the free NAD(P)H fraction α<sub>1</sub> after treatment, associated with a shift towards glycolysis due to lymphocyte activation. The non-responsive models exhibited no alterations or a decrease in the NAD(P)H α<sub>1</sub> after treatment. The FLIM data correlated well with the standard assays of immunotherapy drug response in vitro, including morphological changes, the T-cells activation marker CD69, and the tumor cell proliferation index Ki67. Conclusions: The proposed platform that includes tumor explants co-cultured with lymphocytes and the NAD(P)H FLIM assay represents a promising solution for the patient-specific immunotherapeutic drug screening. |
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| institution | Kabale University |
| issn | 2073-4409 |
| language | English |
| publishDate | 2025-01-01 |
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| series | Cells |
| spelling | doaj-art-d3706c72e9af44b19644279e62d3e3982025-01-24T13:26:40ZengMDPI AGCells2073-44092025-01-011429710.3390/cells14020097Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of ImmunotherapyDiana V. Yuzhakova0Daria A. Sachkova1Anna V. Izosimova2Konstantin S. Yashin3Gaukhar M. Yusubalieva4Vladimir P. Baklaushev5Artem M. Mozherov6Vladislav I. Shcheslavskiy7Marina V. Shirmanova8Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, RussiaInstitute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, RussiaInstitute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, RussiaDepartment of Neurosurgery, Privolzsky Research Medical University, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, RussiaFederal Research and Clinical Center, Federal Medical and Biological Agency, 28 Orekhovy Blvd., 115682 Moscow, RussiaFederal Research and Clinical Center, Federal Medical and Biological Agency, 28 Orekhovy Blvd., 115682 Moscow, RussiaInstitute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, RussiaInstitute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, RussiaInstitute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, RussiaBackground: The wide variability in clinical responses to anti-tumor immunotherapy drives the search for personalized strategies. One of the promising approaches is drug screening using patient-derived models composed of tumor and immune cells. In this regard, the selection of an appropriate in vitro model and the choice of cellular response assay are critical for reliable predictions. Fluorescence lifetime imaging microscopy (FLIM) is a powerful, non-destructive tool that enables direct monitoring of cellular metabolism on a label-free basis with a potential to resolve metabolic rearrangements in immune cells associated with their reactivity. Objective: The aim of the study was to develop a patient-derived glioma explant model enriched by autologous peripheral lymphocytes and explore FLIM of the redox-cofactor NAD(P)H in living lymphocytes to measure the responses of the model to immune checkpoint inhibitors. Methods: The light microscopy, FLIM of NAD(P)H and flow cytometry were used. Results: The results demonstrate that the responsive models displayed a significant increase in the free NAD(P)H fraction α<sub>1</sub> after treatment, associated with a shift towards glycolysis due to lymphocyte activation. The non-responsive models exhibited no alterations or a decrease in the NAD(P)H α<sub>1</sub> after treatment. The FLIM data correlated well with the standard assays of immunotherapy drug response in vitro, including morphological changes, the T-cells activation marker CD69, and the tumor cell proliferation index Ki67. Conclusions: The proposed platform that includes tumor explants co-cultured with lymphocytes and the NAD(P)H FLIM assay represents a promising solution for the patient-specific immunotherapeutic drug screening.https://www.mdpi.com/2073-4409/14/2/97immunotherapyimmune-checkpoint inhibitor ICIpersonalized therapytumor explantlymphocyteglioma |
| spellingShingle | Diana V. Yuzhakova Daria A. Sachkova Anna V. Izosimova Konstantin S. Yashin Gaukhar M. Yusubalieva Vladimir P. Baklaushev Artem M. Mozherov Vladislav I. Shcheslavskiy Marina V. Shirmanova Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of Immunotherapy Cells immunotherapy immune-checkpoint inhibitor ICI personalized therapy tumor explant lymphocyte glioma |
| title | Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of Immunotherapy |
| title_full | Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of Immunotherapy |
| title_fullStr | Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of Immunotherapy |
| title_full_unstemmed | Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of Immunotherapy |
| title_short | Fluorescence Lifetime Imaging of NAD(P)H in Patients’ Lymphocytes: Evaluation of Efficacy of Immunotherapy |
| title_sort | fluorescence lifetime imaging of nad p h in patients lymphocytes evaluation of efficacy of immunotherapy |
| topic | immunotherapy immune-checkpoint inhibitor ICI personalized therapy tumor explant lymphocyte glioma |
| url | https://www.mdpi.com/2073-4409/14/2/97 |
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