Targeted delivery of chemotherapeutic agents with aptamers is a very effective method increasing therapeutic index compared to non-targeted drugs. Objective. To study the effectiveness of in vitro therapeutic effect of covalently conjugated GR20 DNA aptamer with doxorubicin on glioblastoma cells compared to reference culture of human fibroblasts. Material and methods. A Sus/fP2 cell culture was obtained from glioblastoma tissue sample to analyze the effectiveness of conjugate. A linear culture of human dermal fibroblasts (mesenchymal stem cells) DF1 was used as a control. To assess antiproliferative activity of covalently conjugated GR20 aptamer with doxorubicin, we used the MTS test. The Cell Index was measured using the xCelligence S16 cell analyzer assessing viability of cell cultures by recording changes in real time. Results. Human glioblastoma Sus/fP2 cells reduce own proliferative potential by 80% when exposed to doxorubicin (0.5 µM, 72 hours, MTS test), by 9% when exposed to GR20 aptamer (10 µM, 72 hours, MTS test) and by 26% when exposed to covalently conjugated DOX-GR20 (0.5 µM, 72 hours, MTS test). A long-term study of proliferative potential of Sus/fP2 cells on the xCelligence S16 analyzer revealed a significant decrease in the number of cells under the effect of doxorubicin and covalently conjugated DOX-GR20. Effectiveness of covalently conjugated DOX-GR20 is halved. GR20 aptamer at a concentration of 10 μM and its conjugate with doxorubicin DOX-GR20 at a concentration of 1 μM have no negative effect on cells of the control culture of DF1 fibroblasts, while doxorubicin is toxic for these cells. MTS test and xCelligence S16 cell analyzer found no decrease in metabolic activity of DF1 cells and their ability to proliferate. Conclusion. We established obvious antiproliferative effect of covalent conjugate DOX-GR20 on continuous human glioblastoma cell culture Sus/fP2 without toxic effect on the reference culture (dermal fibroblasts DF1).

Background/Objectives: Glioblastoma is an aggressive brain tumour with a poor prognosis and limited therapy. The truncated aptamer GR20, derived from aptamer U31, was developed to retain EGFR binding while reducing size. This study evaluated how aptamer truncation, while maintaining its tertiary structure, affects its antiproliferative activity against human glioma cells. Methods: Five transplantable human glioblastoma cell cultures (BU73, G22, G23, G01, Sus/fP2) were used. Aptamer binding was assessed by flow cytofluorimetry, intracellular accumulation was tracked by confocal microscopy. Changes in stemness and malignancy gene expression were evaluated by real-time PCR. Protein expression levels were assessed by immunocytochemistry. Proliferative activity was measured using the MTS assay. Results: GR20 retained EGFR binding and demonstrated more than two-fold higher binding efficiency to glioblastoma cells than antibodies or the U31 aptamer. Both aptamers accumulated in cells after 2 h of incubation. GR20 significantly reduced the expression of stemness genes (CD133, OCT4) and malignancy genes (EGFR, PDGFRα, TP53) but showed modest antiproliferative effects (9–11% reduction). U31 exhibited stronger antiproliferative activity, reducing Sus/fP2 viability by 31% and 58%. Molecular dynamics showed that U31 maintains a “multi-loop” conformational state absent in GR20. Conclusions: Although the truncated GR20 still binds to the EGFR and is taken up by cells, its antiproliferative activity is reduced compared to U31. The multi-loop conformation present in U31 but missing in GR20 appears to enhance activity, identifying this motif as a promising target for future aptamer optimization.

Aptamers can increase human glioblastoma cell death rate after exposure to radiotherapy. Cell cultures derived from human glioblastomas allow for selection of effective combined therapy. Objective. To analyze viability of human glioblastoma cell cultures exposed to EGFR-specific aptamers U31, GR20, and GR200 compared to combination of these aptamers and radiotherapy (20 Gy). Methodology. G22 and Sus\fP2 cell cultures were harvested from tumor tissue. They were exposed to aptamers (10 μM, 72 hours) and irradiated once with 20 Gy. We analyzed viability using flow cytometry. Results. In G22 cell culture, aptamer U31 reduces apoptosis by 45.8%, but increases necrosis by 67.5%, while GR20 and GR200 increase two types of death (apoptosis and necrosis). In Sus\fP2 culture, all three aptamers significantly increased apoptosis while proportion of necrosis decreased. The most significant decrease in the number of necrotic cells was observed for aptamer U31 (by 77.7%). Radiotherapy combined with aptamers was followed by the following effects for G22 cell culture: U31 and GR200 enhance apoptosis (by 59.4%) and necrosis (by 57.7%), aptamer GR20 increases necrosis by 28.3%. In Sus\fP2 culture, U31 aptamer enhances apoptosis by 132.9% and necrosis by 52.9%, GR20 — 125.4% and 17.7%, GR200 — 27.4% and 30.6%, respectively. Conclusion. Aptamers have different effects on glioblastoma cell cultures. For Sus\fP2, a combination of radiotherapy with GR20 aptamer is optimal. Either a single application of U31 aptamer or combination of radiotherapy and GR200 aptamer is optimal for G22 cell culture. This result demonstrates the importance of combined therapy and preliminary studies in cell cultures for individualized approach to each CNS tumor.

Differentiation of high-grade gliomas (WHO grades III and IV) is still a pressing issue despite advances of molecular biology in tumor stratification. Even with a comprehensive approach to differential diagnosis, uncertainty sometimes arises in classifying a glioma into a particular grade. This can complicate prognosis and selection of appropriate treatment. Objective. To develop an effective method for differentiating high-grade gliomas based on marker gene expression analysis. Material and methods. We analyzed expression of 31 marker genes in high-grade glioma samples (Grade III and IV) diagnosed with isocitrate dehydrogenase (IDH)-mutated oligodendroglioma, IDH-mutated astrocytoma and IDH-wildtype glioblastoma. Real-time polymerase chain reaction was used. Spearman rank correlation method was applied to select genes whose expression was most closely related to glioma grade. Gene expression and their ratios were compared in groups of Grade III and IV malignant gliomas using the Mann-Whitney test. Results. Expression ratios of three genes (CD44, CIRBP, and SOX2) were selected to assess malignancy grade. Expression ratios for these genes distinguishes IDH wild-type glioblastomas from IDH-mutated astrocytomas and oligodendrogliomas. They also have a stronger correlation with glioma grade than expression of each individual marker gene. Conclusion. Original method based on marker gene expression ratios does not replace standard diagnostic protocols, but may be an additional tool for optimized diagnostic process. This approach will be valuable to minimize errors and personalize therapeutic strategies. The last one is critical for prognosis.