Mayya Alexandrovna Dymova

Background/Objectives: Oncolytic viruses are an immunotherapeutic approach that can modulate the tumor microenvironment (TME), transforming immunologically ‘cold’ tumors into ‘hot’ ones. Insertion of genes encoding immunomodulatory proteins can further enhance antitumor immune responses. In this study, we compared the antitumor and immunomodulatory effects of the double recombinant vaccinia virus VV-GMCSF-Lact, which carries the human GM-CSF gene, with those of recombinant human GM-CSF (rhGM-CSF) in an immunocompetent murine GL261 glioma model. Methods: The study was conducted using a subcutaneous GL261 glioma model in immunocompetent C57BL/6 mice, comparing intratumoral VV-GMCSF-Lact and rhGM-CSF treatments with evaluation of immune cell populations by flow cytometry, tumor morphology by H&E staining, and tumor transcriptome profiles by RNA sequencing. Results: Flow cytometry showed that VV-GMCSF-Lact reduced the number of immunosuppressive cells in the TME of subcutaneously transplanted gliomas, targeting different components of the TME depending on animal sex. The immunotherapeutic effects of rhGM-CSF were less pronounced and primarily affected peripheral immune cells. Histological analysis revealed a decrease in mitotic figures in tumors from female mice after viral therapy. Transcriptome profiling of GL261 tumors demonstrated divergent gene expression patterns and cellular compositions between treatment groups. VV-GMCSF-Lact treatment was associated with a decreased proportion of malignant GL261 cells and CD8⁺ T lymphocytes, while rhGM-CSF treatment increased proportions of MDSCs, macrophages, NK cells, and tumor-associated neutrophils. Conclusions: Taken together, our data demonstrate that VV-GMCSF-Lact induces antitumor immune responses in murine GL261 glioma in vivo and modulates the tumor microenvironment more effectively than rhGM-CSF alone, supporting its potential for developing new strategies for glioma treatment.

Introduction: Human cerebral organoids (COs) are sophisticated three-dimensional (3D) models that successfully replicate the 3D cytoarchitecture of human brain development in its early stages. Co-cultivation of COs obtained from human-induced pluripotent stem cells with 3D U-87 MG glioblastoma cell spheroids (glioblastoma–CO assembloid [GCOA]) represents a robust strategy for modeling brain cancer behavior. Objectives: This study aims to evaluate the feasibility of using GCOAs and 3D U-87 MG glioblastoma cell spheroids as in vitro cell models to evaluate the cytotoxic effect of the oncolytic recombinant vaccinia virus-granulocyte–macrophage colony-stimulating factor (VV-GM-CSF)-Lact compared to the VV-GMCSF-del virus. Methods: To assess the cytotoxicity of two virus strains, we used fluorescent and confocal microscopy, spectrophotometry, the MTT assay, and real-time polymerase chain reaction Results: Glioblastoma cells died faster in the presence of VV-GMCSF-Lact virus, and 3D spheroids infected with this virus contained more caspase-3-positive cells. On the 10th day in GCOA, glioblastoma cells began to invade the interior of the CO. Under conditions of 3D spheroid and GCOA infection with recombinant viruses, three differentially expressed genes – FSCN1, SCUBE2, and TNFRSF9 – associated with invasion were analyzed. Conclusion: These 3D models of glioblastoma can be used in the development of antitumor drugs to study their cytotoxic effects.

Background: With the rising incidence of cancer, there is a growing need for improved preclinical models to test new therapies. While patient-derived xenografts (PDX) in immunodeficient mice are the gold standard, they are costly and result in a complete absence of a functional immune system, limiting their utility for studying tumor–immune interactions. This study characterizes a pharmacological partial immunosuppression protocol in immunocompetent mice as a promising alternative, evaluating its impact on the immune system and demonstrating its efficacy for growing human tumor xenografts. Methods: Mice received a regimen of cyclosporine (20 mg/kg, i.p., every 48 h for 12 days), cyclophosphamide (60 mg/kg, i.p., every 48 h for 8 days), and ketoconazole (10 mg/kg, p.o., for 12 days). The dynamics of CD3⁺, CD4⁺, CD8⁺, and CD19⁺ lymphocyte subpopulations and the CD4/CD8 index were monitored via flow cytometry on days 1, 5, 8, 12, 16, and 21. The protocol’s utility was tested by orthotopic transplantation of human glioma and lung cancer cells, and subcutaneous transplantation of breast cancer cells (MCF7). Tumor engraftment and growth were assessed using in vivo microscopy, MRI, and histology. Results: The immunosuppressive protocol induced a significant but partial reduction in CD3⁺ T-cells and CD19⁺ B-cells by day 8 (p = 0.0277). A profound and progressive decrease in the CD4/CD8 index was observed, indicating a shift towards immunosuppression. Crucially, CD8⁺ and CD4⁺ T-cells populations recovered rapidly post-therapy, demonstrating that the protocol creates a temporary and modifiable immune window rather than inducing complete ablation. The protocol enabled successful engraftment and growth of all three tested tumors in a residual immune microenvironment, confirmed by in vivo imaging and histopathological analysis. Conclusions: This drug-induced partial immunosuppression protocol effectively creates a reproducible state of transient immunodeficiency in outbred mice, suitable for various human tumor xenograft models. It represents a cost-effective and flexible alternative to genetic models, with the distinct advantage of preserving a residual immune microenvironment, making it particularly valuable for preclinical studies that require a partially intact host immune system.

Virotherapy represents a promising approach for cancer treatment. However, when administered intravenously, oncolytic viruses are often hampered by neutralization from antibodies, reducing their antitumor efficacy. Aptamers offer a potential solution by shielding viral particles from neutralizing antibodies and prevent viral particle aggregation. In this study, human glioblastoma xenografts were orthotopically implanted in immunocompromised ICR mice. The therapeutic agents (VV-GMCSF-Lact-Apt1, VV-GMCSF-Lact-Apt2 or VV-GMCSF-Lact) were administered intravenously. Using magnetic resonance imaging, we observed a decrease in tumor growth in all experimental groups. Using the Kaplan-Meier method, it was shown that the mouse survival in the VV-GMCSF-Lact-Apt1, VV-GMCSF-Lact-Apt2 groups was higher than in the control group. The level of key cytokines (IL-1, IL-10, TNF-alpha) in mouse plasma was measured using ELISA. A study of cytokine levels (IL-1, IL-10, and TNF-α) showed that the aptamers shift the immune response from a pro-inflammatory systemic response, by reducing IL-1, to an anti-tumor one, with an increase in TNF-α. In histological analysis, the most destructive changes in tumor tissue were observed in the VV-GMCSF-Lact-Apt2 group, while the morphology of organ tissues (spleen, liver, kidneys and lungs) did not differ between the control and experimental groups. Thus, our results demonstrate a synergistic antitumor effect of the aptamer in combination with the oncolytic virus against glioblastoma tumor xenografts.

Virotherapy is a promising method for treating oncological diseases, including such aggressive and difficult-to-treat brain tumors such as glioblastoma. Recombinant vaccinia virus VV-GMCSF-Lact has previously shown high antitumor potential against tumor cells of varying histogenesis, including gliomas, and completed a Phase I clinical trial, demonstrating safety and good tolerability in patients with recurrent/refractory metastatic breast cancer. Investigating two types of VV-GMCSF-Lact delivery, intravenous and intratumoral, into orthotopically transplanted C6 glioma in rats, it was shown that intratumoral injection significantly increases tumor volumes in comparison with intravenous virus delivery and is accompanied by noticeable toxic effects. Extensive areas of necrotic decay of tumor tissue and its significant mixed-cell infiltration and peritumoral edema, affecting the tumor volume, were detected using H&E staining of C6 tumors after intratumoral injection of VV-GMCSF-Lact. However, only with intratumoral administration was a significant decrease in the level of the tumor cell proliferation marker Ki67 demonstrated by immunohistochemical staining. The observed toxic effects of VV-GMCSF-Lact with intratumoral administration revealed the need for dose selection, which was performed on a mouse GL261 glioma model. Results of the study allowed us to determine the viral dose that does not lead to toxic effects and can potentially increase life expectancy of mice. The data obtained show the need for careful selection of both the route of viral drug dose and administration.

High-grade gliomas are among the most aggressive malignant pathologies of the brain. The high invasive potential of tumor cells causes relapses of the disease even after radical resection of the tumor. The signatures of the genes associated with the invasion of glioma cells have now been identified. The expression products of these genes are involved in various signaling pathways, such as cellular protein catabolism, the p53 signaling pathway, transcription dysregulation, and the JAK-STAT signaling pathway. Therefore, they can indirectly modulate the invasive potential of tumor cells. Using RNA interference technology, it is possible to change the expression level of the detected genes and reduce the invasive and proliferative potentials of cancer cells. This review focuses on the use of this technology to influence various links in signaling pathways and, accordingly, the cellular processes associated with the invasion of glioblastoma cells. Furthermore, the review discusses the problems associated with delivering interfering RNAs into cells and ways to solve them.

Oncolytic virotherapy is a rapidly evolving approach to cancer treatment. Our group previously designed VV-GMCSF-Lact, a recombinant oncolytic vaccinia virus targeting solid tumors including gliomas. In this study, we used single-cell RNA sequencing to compare transcriptional responses in human glioma cells, non-malignant brain cells, and immortalized glioblastoma U87 MG cells following infection with this oncolytic virus. We found that proneural glioblastoma cells and microglia-like cells from patient-derived glioma cultures were the most susceptible to VV-GMCSF-Lact. Increased expressions of histones, translational regulators, and ribosomal proteins positively correlated with viral load at the transcript level. Furthermore, higher viral loads were accompanied by a large-scale downregulation of genes involved in mitochondrial translation, metabolism, and oxidative phosphorylation. Levels of early vaccinia virus transcripts are also positively correlated with infection intensity, suggesting that the fate of cells is determined at the early stage of infection.

Oncolytic virotherapy is a promising approach for cancer treatment. However, when introduced into the body, the virus provokes the production of virus-neutralizing antibodies, which can reduce its antitumor effect. To shield viruses from the immune system, aptamers that can cover the membrane of the viral particle are used. Aptamers that specifically bind to the JX-594 strain of the vaccinia virus were developed earlier. However, the parameters for binding to the recombinant virus VV-GMCSF-Lact, developed based on the LIVP strain of the vaccinia virus, may differ due its different repertoire of antigenic determinants on its membrane compared to JX-594. In this work, the spatial atomic structures of aptamers to JX-594 and bifunctional aptamers were determined using molecular modeling. The efficiency of viral particles binding to the aptamers (EC50), as well as the cytotoxicity and stability of the aptamers were studied. The synergistic effect of the VV-GMCSF-Lact combination with the aptamers in the presence of serum was investigated using human glioblastoma cells. This proposed approach allowed us to conduct a preliminary screening of sequences using in silico modeling and experimental methods, and identified potential candidates that are capable of shielding VV-GMCSF-Lact from virus-neutralizing antibodies.

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 μM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer–virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 10⁹ PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer–virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account.

Background. One of the promising methods of treating tumors is virotherapy, which is based on direct lysis of cancer cells by a virus and a virus-mediated antitumor immune response of the body. For the recombinant vaccinia virus strain VVGMCSF-Lact, producing human GMCSF and the oncotoxic protein lactaptin, cytotoxic and antitumor effects were shown in experiments in vitro and in vivo, respectively, when using adhesive cultures of U-87 MG human glioblastoma cells. 3D cultures are a more relevant tumor model than adhesive models, as they more fully reflect the realistic scenario of cancer development, as well as the response of the tumor to anticancer therapy.The aim. To evaluate the cytotoxic effect of the oncolytic virus VV-GMCSF-Lact against 3D cultures of human glioblastoma U-87 MG.Materials and methods. The following methods were used in the work: cultivation of 3D cell cultures, cytofluorometry, microscopic analysis, virus titration, statistical analysis.Results. U-87 MG cells were transduced with a lentiviral vector carrying the GFP reporter gene. The cytotoxicity of the VV-GMCSF-Lact virus (IC50) against the studied cells was 0.024 PFU/cell. U-87 MG cells were cultured under conditions for the formation of 3D structures. Microscopic analysis showed the oncolytic effect of the virus on the cells of 3D cultures as early as 24 hours after the start of incubation. Flow cytometry showed an increase in the granularity of glioblastoma cells under the action of the virus, which indicates active replication of the virus in the cells. The virus titer was 0.44 PFU/cell.Conclusions. The recombinant VV-GMCSF-Lact virus has a cytotoxic effect on 3D human glioblastoma U-87 MG cell cultures and actively replicates in them. In the future, to test the oncolytic effect of VV-GMCSF-Lact, it is planned to use not only 3D human glioblastoma cultures, but also cerebral organelles obtained in the process of cocultivation of glioblastoma cells and induced human pluripotent cells.