Development of a 3D Tumor Spheroid Model from the Patient's Glioblastoma Cells and Its Study by Metabolic Fluorescence Lifetime Imaging.

Autor:	Yuzhakova DV; Researcher, Laboratory of Genomics of Adaptive Antitumor Immunity, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia., Lukina MM; Researcher, Laboratory of Molecular Oncology; Federal Research and Clinical Center of Physical and Chemical Medicine, Federal Medical and Biological Agency, 1a Malaya Pirogovskaya St., Moscow, 119435, Russia; Researcher, Laboratory of Fluorescent Bioimaging; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia., Sachkova DA; Master Student, Department of Biophysics; National Research Lobachevsky State University of Nizhni Novgorod, 23 Prospekt Gagarina, Nizhny Novgorod, 603950, Russia; Laboratory Assistant, Laboratory of Fluorescent Bioimaging, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia., Yusubalieva GM; Senior Researcher, Laboratory of Cell Technologies; Federal Research and Clinical Center, Federal Medical and Biological Agency, 28 Orekhovy Blvd., Moscow, 115682, Russia; Senior Researcher, Laboratory of Molecular Mechanisms of Regeneration and Aging; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova St., Moscow, 119991, Russia., Baklaushev VP; Deputy General Director for Research and Medical Technologies; Federal Research and Clinical Center, Federal Medical and Biological Agency, 28 Orekhovy Blvd., Moscow, 115682, Russia; Head of the Laboratory of Molecular Mechanisms of Regeneration and Aging; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova St., Moscow, 119991, Russia., Mozherov AM; Junior Researcher, Laboratory of Optical Spectroscopy and Microscopy, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia., Dudenkova VV; Researcher, Laboratory of Optical Spectroscopy and Microscopy, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia., Gavrina AI; Junior Researcher, Laboratory of Molecular Biotechnologies, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia., Yashin KS; Oncologist, Neurosurgeon, Department of Oncology and Neurosurgery, Institute of Traumatology and Orthopedics, University Сlinic; Assistant, Department of Traumatology and Neurosurgery named after M.V. Kolokoltsev; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia., Shirmanova MV; Deputy Director for Science, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia.
Jazyk:	angličtina
Zdroj:	Sovremennye tekhnologii v meditsine [Sovrem Tekhnologii Med] 2023; Vol. 15 (2), pp. 28-38. Date of Electronic Publication: 2023 Mar 29.
DOI:	10.17691/stm2023.15.2.03
Abstrakt:	Patient-specific in vitro tumor models are a promising platform for studying the mechanisms of oncogenesis and personalized selection of drugs. In case of glial brain tumors, development and use of such models is particularly relevant as the effectiveness of such tumor treatment remains extremely unsatisfactory. The aim of the study was to develop a model of a 3D tumor glioblastoma spheroid based on a patient's surgical material and to study its metabolic characteristics by means of fluorescence lifetime imaging microscopy of metabolic coenzymes. Materials and Methods: The study was conducted with tumor samples from patients diagnosed with glioblastoma (Grade IV). To create spheroids, primary cultures were isolated from tumor tissue samples; the said cultures were characterized morphologically and immunocytochemically, and then planted into round-bottom ultra low-adhesion plates. The number of cells for planting was chosen empirically. The characteristics of the growth of cell cultures were compared with spheroids from glioblastomas of patients with U373 MG stable line of human glioblastoma. Visualization of autofluorescence of metabolic coenzymes of nicotinamide adenine dinucleotide (phosphate) NAD(P)H and flavin adenine dinucleotide (FAD) in spheroids was performed by means of an LSM 880 laser scanning microscope (Carl Zeiss, Germany) with a FLIM module (Becker & Hickl GmbH, Germany). The autofluorescence decay parameters were studied under normoxic and hypoxic conditions (3.5% О 2 ). Results: An original protocol for 3D glioblastoma spheroids cultivation was developed. Primary glial cultures from surgical material of patients were obtained and characterized. The isolated glioblastoma cells had a spindle-shaped morphology with numerous processes and a pronounced granularity of cytoplasm. All cultures expressed glial fibrillary acidic protein (GFAP). The optimal seeding dose of 2000 cells per well was specified; its application results in formation of spheroids with a dense structure and stable growth during 7 days. The FLIM method helped to establish that spheroid cells from the patient material had a generally similar metabolism to spheroids from the stable line, however, they demonstrated more pronounced metabolic heterogeneity. Cultivation of spheroids under hypoxic conditions revealed a transition to a more glycolytic type of metabolism, which is expressed in an increase in the contribution of the free form of NAD(P)H to fluorescence decay. Conclusion: The developed model of tumor spheroids from patients' glioblastomas in combination with the FLIM can serve as a tool to study characteristics of tumor metabolism and develop predictive tests to evaluate the effectiveness of antitumor therapy. Competing Interests: Conflicts of interest. The authors declare no conflicts of interest.
Databáze:	MEDLINE
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