Efficacy Evaluation of “Enhanced” Natural Killers with CISH and B2M Knockouts on Viability and Metabolic Status of 3D Glioblastoma Spheroid Cells in Patients

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. The aim of the study 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. Materials and Methods. The study used a primary culture of GBM7-Luc2-mKate2 human glioblastoma, a line of YT (YTwt) wild-type human NK cells, as well as lines created by us with overexpression of VAV1 protein with either CISH (YT–Vav1+CISH–/–) or B2M (YT–Vav1+B2M–/–) 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). Results. It was found that autofluorescence attenuation parameters of NAD(P)H coenzyme in human glioblastoma cells change significantly when exposed to both YT–Vav1+ CISH–/– and YT–Vav1+ B2M–/–, 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. Conclusion. 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. © 2025 Elsevier B.V., All rights reserved.