Glioblastoma is an aggressive primary brain tumour characterised by pronounced cellular heterogeneity, infiltrative growth, and frequent disease recurrence despite multimodal treatment. Glioblastoma stem cells represent an important component of tumour heterogeneity, which may differ from differentiated glioblastoma cells in their biological properties and response to treatment. Electroporation represents a potential approach for local glioblastoma treatment; however, the influence of differences between stem and differentiated phenotypes and of the three-dimensional organisation of tumour cells on the response to electric pulses remains insufficiently understood.
In this master’s thesis, we investigated the response of three-dimensional spheroids formed from glioblastoma stem cells and their corresponding differentiated cells, derived from the tumour tissue of three patients, to high-frequency biphasic electric pulses. The response was evaluated by assessing cell membrane permeabilization, metabolic activity, cytotoxicity, and direct counting of viable cells. We also determined cell and spheroid sizes, and in GSC NIB216 spheroids we examined how the initial cell number and centrifugation time affected spheroid size, cell membrane permeabilization, and metabolic activity after electroporation. A numerical model was used to analyse the effects of spheroid size and position on the electric field distribution within the electroporation cuvette, while electric current, voltage, and temperature changes during pulse delivery were measured experimentally. An additional numerical model was used to investigate the effects of membrane permeability and cellular volume fraction on bleomycin transport through the spheroid and on the resulting intracellular concentration. The effects of electrochemotherapy with bleomycin were also evaluated experimentally.
The results demonstrated differences in response both among individual cell cultures and between stem and differentiated phenotypes. In the directly compared NIB216 and NIB237 cultures, glioblastoma stem cells exhibited more pronounced cell membrane permeabilization at lower electric field strengths, while direct cell counting at higher field strengths also showed lower survival than in the corresponding differentiated cells. Glioblastoma stem cells formed larger spheroids from the same initial number of cells; however, spheroid size alone did not explain the observed differences in response. During the experimental work, differentiated spheroids appeared more compact and were more difficult to dissociate, while experiments with modified spheroid preparation conditions suggested that structural properties other than size may also influence the response. The numerical electric field model showed that spheroid size had only a minor effect on the local electric field, whereas spheroid position within the cuvette had a greater influence. Pronounced sample heating was observed at the highest applied voltage. Heating was greater in the medium used for differentiated cells; nevertheless, these cells remained less sensitive to electroporation than the corresponding glioblastoma stem cells, indicating that the observed differences between phenotypes cannot be explained solely by thermal effects. The transport model showed the formation of a bleomycin concentration gradient across the spheroid and a pronounced effect of membrane permeability on the achieved intracellular concentration. The addition of bleomycin further reduced survival in some glioblastoma stem cell cultures, with a statistically significant effect observed in GSC NIB237, whereas no additional reduction in survival was confirmed in differentiated cells.
Overall, the results demonstrate that the response of glioblastoma spheroids to electroporation depends on cellular phenotype, individual cell culture characteristics, three-dimensional spheroid organisation, and the physical conditions of exposure. These findings highlight the importance of using patient-derived cell cultures, three-dimensional models, and accounting for glioblastoma heterogeneity when developing electroporation-based therapeutic approaches.
|
