Yuzhakova D V, Sachkova D A, Shirmanova M V, Shcheslavskiy V I, Mozherov A M, Dashinimaev E B, Baklaushev V P, Yusubalieva G M
PhD, Senior 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; Senior Researcher, Laboratory of Cellular Technologies; Federal Scientific and Clinical Center of the Federal Medical Biological Agency of Russia, 28 Orekhovy Blvd., Moscow, 115682, 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; PhD Student, Department of Biophysics, Institute of Biology and Biomedicine; National Research Lobachevsky State University of Nizhny Novgorod, 23 Prospekt Gagarina, Nizhny Novgorod, 603022, Russia.
Sovrem Tekhnologii Med. 2025;17(1):109-118. doi: 10.17691/stm2025.17.1.10. Epub 2025 Feb 28.
One of the alternative approaches to glioblastoma treatment is cellular immunotherapy based on natural killer cells (NK cells). To enhance their cytotoxic effect on tumor cells, new NK cell lines are being created using genetic engineering techniques. was to evaluate the impact efficacy of "enhanced" NK cells on early metabolic rearrangements and the viability of glioblastoma cells in a patient using a tumor spheroid model.
The study used a primary culture of GBM7-Luc2-mKate2 human glioblastoma, a line of YT (YTwt) wildtype human NK cells, as well as lines created by us with overexpression of VAV1 protein with either (YT-Vav1CISH) or (YT-Vav1B2M) knockouts. Tumor spheroids were produced in round-bottomed, low-adhesive plates. 100 thousand immune cells were added to each spheroid, and spheroids viability was evaluated at several time points applying fluorescence staining using a live/dead cell viability assay kit; autofluorescence of metabolic coenzyme nicotinamide adenine dinucleotide (phosphate), or NAD(P)H, was visualized in spheroids using an LSM 880 laser scanning microscope (Carl Zeiss, Germany) with a FLIM module (Becker & Hickl GmbH, Germany).
It was found that autofluorescence attenuation parameters of NAD(P)H coenzyme in human glioblastoma cells change significantly when exposed to both YT-Vav1CISH and YT-Vav1B2M, indicating occurrence of an early metabolic shift in tumor cells towards a less aggressive oxidative phenotype, and this is consistent with dead cells fraction increase and living cells fraction decrease in spheroid composition.
The data obtained on enhanced cytotoxic activity of new modified NK cell lines against human glioblastoma spheroids are important to understand interaction mechanisms between tumor and immune cells and the development of glioblastoma adoptive cell therapy.
胶质母细胞瘤治疗的替代方法之一是基于自然杀伤细胞（NK细胞）的细胞免疫疗法。为了增强它们对肿瘤细胞的细胞毒性作用，正在使用基因工程技术创建新的NK细胞系。本研究旨在使用肿瘤球体模型评估“增强型”NK细胞对胶质母细胞瘤患者早期代谢重排和细胞活力的影响效果。
本研究使用了GBM7-Luc2-mKate2人胶质母细胞瘤的原代培养物、YT（YTwt）野生型人NK细胞系，以及我们创建的过表达VAV1蛋白且带有CISH敲除（YT-Vav1CISH）或β2微球蛋白（B2M）敲除（YT-Vav1B2M）的细胞系。在圆底、低粘附板中生成肿瘤球体。向每个球体中加入10万个免疫细胞，并使用活/死细胞活力检测试剂盒通过荧光染色在几个时间点评估球体活力；使用带有FLIM模块（德国贝克尔&希克尔有限公司）的LSM 880激光扫描显微镜（德国卡尔蔡司公司）在球体中观察代谢辅酶烟酰胺腺嘌呤二核苷酸（磷酸）即NAD(P)H的自发荧光。
发现当暴露于YT-Vav1CISH和YT-Vav1B2M时，人胶质母细胞瘤细胞中NAD(P)H辅酶的自发荧光衰减参数发生显著变化，表明肿瘤细胞发生了早期代谢转变，向侵袭性较小的氧化表型转变，这与球体组成中死细胞比例增加和活细胞比例降低一致。
关于新的修饰NK细胞系对人胶质母细胞瘤球体增强的细胞毒性活性所获得的数据，对于理解肿瘤与免疫细胞之间的相互作用机制以及胶质母细胞瘤过继性细胞治疗的发展具有重要意义。
