The characterization of tumors as either “hot” or “cold” is determined by intrinsic properties of the cancer cells, the characteristics of the tumor immune landscape, the composition of the tumor microenvironment (TME), and underlying signaling mechanisms. These biological factors are critical in defining the clinical outcomes and therapeutic responses observed in cancer patients. The TME of glioblastoma exemplifies a case of “cold” TME, which significantly hinders antitumor immunity. This constitutes the predominant rationale underlying the ineffectiveness of immunotherapy. This review provides a thorough analysis of contemporary immunotherapeutic strategies that have been developed for the purpose of altering the immunological characteristics of tumors, with a view to achieving their effective elimination. The core mechanisms of action and future clinical applications of immune checkpoint inhibitors, adoptive cellular therapy, and oncolytic viruses (OV) are delineated. A combination of preclinical and clinical evidence suggests that OV-based combinations could be an effective treatment strategy for “cold” tumors.

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.

BackgroundThe complex interplay between tumor cells and the stromal components of the glioma microenvironment necessitates the development of sophisticated in vitro models capable of modelling key aspects of cellular interactions that occur beyond the limitations of conventional monocultures.MethodologyThe development and characterization of homo- and heterotypic 3D spheroid models incorporating CCF-STTG1 astrocytes, HMC3 microglia, and U87MG glioma cells was undertaken. The assessment of morphological, molecular, and functional properties was performed via flow cytometry, cytokine arrays, ECM analysis and invasion assays (Matrigel™/gelatin).ResultsHeterotypic spheroids have been observed to spontaneously self-assemble into a spatially polarized architecture, with microglia and glioma cells segregating into distinct compartments, a pattern suggestive of the cellular topology at the invasive front. The morphological, molecular, and functional properties of the generated 3D models recapitulated several established features associated with in vivo tumors, including growth, invasion, resistance to chemotherapy, and metabolic reprogramming alongside the expression of stemness markers, and key pro-invasive mediators (MMPs, SDF-1α, VEGF). Secretome profiling revealed a marked, non-additive upregulation of chemokines (IP-10, MIP-1α) and the emergence of novel correlations (HGF/SDF-1α, MCP-1/LIF), indicating potential modulation of paracrine networks involved in immune cell trafficking in the heterotypic setting. The initial formation of a rigid ECM matrix appears to be initiated by microglia, while the supply of fibronectin and laminin may be linked to astrocytes exhibiting some features of reactive gliosis, which could help organize invasion pathways.ConclusionThese heterotypic 3D spheroid models offer a stroma-enriched, reproducible platform for the analysis of stromal contributions to glioma progression and for exploratory preclinical evaluation of therapeutic strategies.

Abstract Boron neutron capture therapy (BNCT) is one of the promising treatment methods for cancer. BNCT is based on the unique high ability of the non-radioactive boron-10 ( 10 B) nucleus to absorb thermal neutrons. The absorption of a neutron by boron results in a nuclear reaction 10 B(n,α) 7 Li with 84% of the energy being released within the cell, thereby inducing cancer cell death. We propose a novel approach based on using lithium instead of boron in neutron capture therapy: the neutron capture reaction cross-section for lithium is 4 times smaller than that of boron, while the energy release is 2 times higher, and the primary advantage of lithium is that 100% of the energy is released inside the cell. A series of in vivo neutron irradiation experiments using the murine B16 skin melanoma model demonstrated the efficacy of lithium neutron capture therapy (LiNCT). Higher lithium concentrations in the tumor achieved through intraperitoneal administration, compared to the oral route, contributed to a significant increase in animal survival and a significant decrease in tumor growth. The results of our study provide entirely new opportunities for the development of neutron capture therapy, both with lithium and with a possible lithium-boron combination.

The recreation of the tumor microenvironment remains a significant challenge in the development of experimental cancer models. The present study constitutes an investigation into the interconnection between tumor, endothelial and stromal cells in heterotypic breast cancer spheroids. The generation of models was achieved through the utilization of MCF7, MDA-MB-231, and SK-BR-3 tumor cell lines, in conjunction with endothelial TIME-RFP cells and either cancer-associated (BrC4f) or normal (BN120f) fibroblasts, within ultra-low attachment plates. It was established that stromal cells, most notably fibroblasts, were conducive to the aggregation of tumor cells into spheroids and the formation of pseudovessels in close proximity to fibroblast bands. In contrast to the more aggressive tumor models MDA-MB-231 and SK-BR-3, microenvironment cells do not influence the migration ability of MCF7 tumor cells. Heterotypic spheroids incorporating CAFs demonstrated a more aggressive and immunosuppressive phenotype. Multiplex immunoassay analysis of cytokines, followed by STRING cluster analysis, was used to identify key processes including angiogenesis, invasion, stem cell maintenance, and immunosuppression. Furthermore, a cluster of cytokines (LIF, SDF-1, HGF, SCGFb) was identified as potentially involved in the regulation of PD-L1 expression by tumor cells. This finding reveals a potential mechanism of immune evasion and suggests new avenues for therapeutic investigation.

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.

Due to the complexity of modeling tumor-host interactions within the tumor microenvironment in vitro, we developed a 3D heterotypic cellular breast cancer (BC) model. We generated spheroid models using MCF7, MDA-MB-231, and SK-BR-3 cell lines alongside cancer-associated (BrC4f) and normal (BN120f) fibroblasts in ultra-low attachment plates. Stromal spheroids (3Df) were formed using a liquid overlay technique (graphical abstract). The YT cell line and peripheral blood NK (PB-NK) cells were used as immune components in our 3D model. In this study, we showed that stromal cells promoted tumor cell aggregation into spheroids, regardless of the initial proliferation rates, with NK cells accumulating in fibroblast-rich regions. The presence of CAFs within the model induced alterations in the expression levels of MICA/B and PD-L1 by tumor cells within the 3D-2 model. The feasibility of utilizing a 3D cell BC model in combination with cytokines and PB-NKs was evaluated. We observed that IL-15 and IL-2 enhanced NK cell activity within spheroids, whereas TGFβ had varying effects on proliferation depending on the cell type. Stimulation with IL-2 and IL-15 or TGFβ1 altered PB-NK markers and stimulated their differentiation into ILC1-like cells in 3D models. These findings underscore the regulatory function of CAFs in shaping the response of the tumor microenvironment to immunotherapeutic interventions.

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.

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.

Cancer-associated fibroblasts (CAFs) constitute a heterogeneous population of cells within the tumor microenvironment and are associated with cancer development and drug resistance. The absence of a universal classification for CAFs hinders their research and therapeutic targeting. To define CAF phenotypes, we developed patient-derived cell cultures of breast cancer (BC) and validated and characterized four distinct CAF subtypes (S1-S4) by Costa's classification. Three out of five primary cell cultures of BC demonstrated different functional features rather than fixed cellular states due to the plasticity of the CAF phenotype. CAF crosstalk with cancer cells supported their survival in the presence of anticancer drugs. Based on the analysis of the cytotoxic effect of doxorubicin, cisplatin and tamoxifen, it was demonstrated that CAF-S4 and CAF-S1 cells were sensitive to the action of all drugs investigated, despite the fact that they possessed different mechanisms of action. CAF-S2 cells exhibited the highest level of resistance to the antitumour agents. Homotypic and heterotypic spheroids with CAFs could be used to model the fibrotic area of BC in vitro. The patient-derived cell cultures of CAFs formed spheroids. Hypoxia-activated CAF-S4 have been shown to stimulate the metastatic potential of triple-negative BC cells in a heterotypic spheroid model. Consequently, this study could be a starting point for the development of novel therapeutic strategies that target CAFs and their interactions with cancer cells.

Cancer treatment has transitioned from traditional chemotherapy to the molecular medicine era, emphasizing personalized therapy at the molecular level. Aptamers, also known as 'chemical antibodies', play a pivotal role in advancing molecular medicine. Utilizing the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique, these aptamers exhibit exceptional affinity for a wide range of targets, ranging from picomolar to nanomolar levels. Their exceptional characteristics, including ease of preparation, small size, low immunogenicity, remarkable chemical stability, and convenient modification, make them highly versatile for precise cancer therapy. Notably, aptamers have been successfully combined with therapeutic agents, such as small interfering RNAs (siRNAs), microRNAs (miRNAs), and small molecule toxins for diverse research purposes. This review article will primarily focus on recent progress in aptamer-based targeted therapy for cancer, offering readers a comprehensive insight into the latest developments in aptamer-based cancer treatment.

Numerous studies over three decades have confirmed the significant role of S100A4/FSP1 in the development of metastasis, the formation of the cellular and inflammatory components of the tumor microenvironment, and the development of fibrosis. S100A4 is a promising biomarker whose detection is associated with predicting overall survival in cancer patients. The action of S100A4 is mediated by extra- and intracellular signaling pathways involving targets currently used in the development of therapeutic agents, including monoclonal antibodies and drugs for targeted protein degradation. This review is devoted to the analysis of publications from the perspective of developing diagnostic predictive platforms and modern targeted antitumor therapy aimed at inhibiting the effects of S100A4, which allows avoiding the development of side effects and effectively modulates the tumor microenvironment to overcome immunosuppression and chemoresistance.

Aptamers are short oligonucleotides that bind specifically to various ligands and are characterized by their low immunogenicity, thermostability, and ease of labeling. Many biomedical applications of aptamers as biosensors and drug delivery agents are currently being actively researched. Selective affinity selection with exponential ligand enrichment (SELEX) allows to discover aptamers for a specific target, but it only provides information about the sequence of aptamers; hence other approaches are used for determining aptamer structure, aptamer-ligand interactions and the mechanism of action. The first one is in silico modelling that allows to infer likely secondary and tertiary structures and model their interactions with a ligand. The second approach is to use instrumental methods to study structure and aptamer-ligand interaction. In silico modelling and instrumental methods are complimentary and their combined use allows to eliminate some ambiguity in their respective results. This review examines both the advantages and limitations of in silico modelling and instrumental approaches currently used to study aptamers, which will allow researchers to develop optimal study designs for analyzing aptamer structure and ligand interactions.

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.

Glioblastoma is one of the most malignant and aggressive tumors of the central nervous system. Despite the standard therapy consisting of maximal surgical resection and chemo- and radiotherapy, the median survival of patients with this diagnosis is about 15 months. Oncolytic virus therapy is one of the promising areas for the treatment of malignant neoplasms. In this review, we have focused on emphasizing recent achievements in virotherapy, both as a monotherapy and in combination with other therapeutic schemes to improve survival rate and quality of life among patients with glioblastoma.

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.

Oncolytic viral therapy is a relatively young but rapidly developing area in cancer therapy. Some viruses can be used to combat cancer cells due to their cytotoxic action, but it is desirable to inhibit the influence of the human immune system on them. Aptamers, short oligonucleotides capable of specifically binding to their molecular targets, can perform a dual role: binding to oncoviruses and blocking their receptors through which the immune system attacks them, and also delivering them to another target - cancer cells, to increase the effectiveness of the virus. For such a complex task, it is critically important to know the three-dimensional structure of such bifunctional molecules as aptamers. This work presents a study of the NV14t_56 aptamer to the oncolytic virus VV-GMCSF-Lact using the small-angle X-ray scattering method in solution, determination of the structural characteristics extracted using this method, and validation of the molecular model constructed on the basis of the predicted secondary structure.

Virotherapy is one of the perspective technologies in the treatment of malignant neoplasms. Previously, we have developed oncolytic vaccinia virus VV-GMCSF-Lact and its high cytotoxic activity and antitumor efficacy against glioma was shown. In this work, using immortalized and patient-derived cells with different sensitivity to VV-GMCSF-Lact, we evaluated the cytotoxic effect of chemotherapy agents. Additionally, we studied the combination of VV-GMCSF-Lact with temozolomide which is the most preferred drug for glioma treatment. Experimental results indicate that first adding temozolomide and then the virus to the cells is inherently more efficient than dosing it in the reverse order. Testing these regimens in the U87 MG xenograft glioblastoma model confirmed this effect, as assessed by tumor growth inhibition index and histological analysis. Moreover, VV-GMCSF-Lact as monotherapy is more effective against U87 MG glioblastoma xenografts comparing temozolomide.

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.

RL2 (recombinant lactaptin 2), a recombinant analogon of the human milk protein Κ-Casein, induces mitophagy and cell death in breast carcinoma cells. Furthermore, RL2 was shown to enhance extrinsic apoptosis upon long-term treatment while inhibiting it upon short-term stimulation. However, the effects of RL2 on the action of chemotherapeutic drugs that induce the intrinsic apoptotic pathway have not been investigated to date. Here, we examined the effects of RL2 on the doxorubicin (DXR)-induced cell death in breast cancer cells with three different backgrounds. In particular, we used BT549 and MDA-MB-231 triple-negative breast cancer (TNBC) cells, T47D estrogen receptor alpha (ERα) positive cells, and SKBR3 human epidermal growth factor receptor 2 (HER2) positive cells. BT549, MDA-MB-231, and T47D cells showed a severe loss of cell viability upon RL2 treatment, accompanied by the induction of mitophagy. Furthermore, BT549, MDA-MB-231, and T47D cells could be sensitized towards DXR treatment with RL2, as evidenced by loss of cell viability. In contrast, SKBR3 cells showed almost no RL2-induced loss of cell viability when treated with RL2 alone, and RL2 did not sensitize SKBR3 cells towards DXR-mediated loss of cell viability. Bioinformatic analysis of gene expression showed an enrichment of genes controlling metabolism in SKBR3 cells compared to the other cell lines. This suggests that the metabolic status of the cells is important for their sensitivity to RL2. Taken together, we have shown that RL2 can enhance the intrinsic apoptotic pathway in TNBC and ERα-positive breast cancer cells, paving the way for the development of novel therapeutic strategies.