Anastasia Leonteva

Mounting evidence indicates that the tumor microenvironment plays a decisive role in cancer progression. However, the quantitative assessment of how individual stromal components contribute to cytokine secretion in 3D models remains poorly understood, especially when comparing tumours of different origins. The development of 16 groups of 3D spheroid cultures, encompassing stromal monocultures, two-component models (tumor + endothelium/glia), and three-component models (tumor + endothelium + fibroblast; glioma + astrocytes + microglia), was conducted. These models represent breast cancer and glioma cell culture models. The secretion of 32 distinct cytokines was measured using xMAP technology. Induction was defined as log2FC ≥ 1 with bootstrap-estimated 95% confidence intervals. The induced cytokines were subsequently classified into two operational categories: “emergent” (stromal monoculture level below 3D-2) or “stroma-contributing” (stromal level ≥ 3D-2). In the breast cancer model, a consensus across all cell lines identified seven cytokines (HGF, IL-10, LIF, MCP-1, MCP-3, MIP-1α, and SCGF-β). Cancer-associated fibroblasts (CAFs) were associated with a broader spectrum than normal fibroblasts (28 vs. 23), exhibiting a complete overlap in their repertoire. Triple-negative breast cancer (MDA-MB-231) showed minimal secretion (7–8 cytokines), with decreased secretion of GM-CSF and IP-10; b-FGF levels were reduced in six of eight comparisons. In the glioma model based on U87MG cells, the addition of astrocytes (CCF-STTG1) coincided with increased secretion of 13 different cytokines, whereas microglia (HMC3) were associated with 8 cytokines. The cross-tumor intersection comprised 14 cytokines (7 chemokines, 5 growth factors, and 2 immunoregulatory cytokines), with maximal production observed for LIF, IL-8, MCP-3, MIP-1α, and IL-6. These findings indicate that the addition of stromal cells is associated with both common and tissue-specific changes in the cytokine profile, and that CAFs display a broader secretory repertoire than normal fibroblasts in this model.

Both oncolytic virotherapy and immune checkpoint blockade are being actively explored as immunotherapy treatments for glioblastoma, one of the most lethal malignancies. Nevertheless, the antitumor molecular mechanisms of these therapies remain poorly understood, particularly whether they induce overlapping or distinct transcriptional programs. No comprehensive transcriptome-wide characterization and direct comparison of the responses induced by VV-GMCSF-Lact and anti-PD-1 therapy in glioma have been reported. To address this question, we performed whole-transcriptome profiling of GL261 tumors from immunocompetent C57BL/6 mice treated with VV-GMCSF-Lact, anti-PD-1 antibodies, or their combination. We analyzed therapy-associated changes in gene expression, signaling pathways, and tumor composition. Our findings indicate that anti-PD-1 and combination treatment led to distinctive activation of interferon-gamma response, JAK/STAT signaling, and TNF-alpha/NF-kB pathways. In contrast, VV-GMCSF-Lact preferentially activated T, B, and natural killer T cell-associated programs and reduced the relative abundance of malignant cells. Notably, combination therapy was associated with unique molecular patterns, including induction of monocyte and granulocyte chemotaxis-associated genes, downregulation of PP2A-regulated signaling, and suppression of Rap1-related pathways. This suggests that VV-GMCSF-Lact and anti-PD-1 therapy induce transcriptionally distinct, yet complementary, immune reprogramming and support further evaluation of their combination for glioma immunotherapy.

The interaction of biomaterial surface with cells is a pivotal factor in tissue engineering and three-dimensional (3D) modeling. This paper presents an approach to modifying polystyrene surface by plasma-enhanced chemical vapor deposition of thin plasma-polymerized hexamethyldisiloxane (pp-HMDSO) films to enhance biocompatibility and stimulate the formation of 3D cellular structures. The coatings were characterized by SEM, EDS, XPS, FTIR, AFM, contact angle measurements, and surface free energy (SFE) analysis. A hydrophobic surface initiates 3D structure formation by ensuring uniform cell repulsion and stimulating intercellular interactions. Biological evaluation was performed on U-87 MG (glioblastoma) and HMC3 (microglia) cell lines. For U-87 MG, the pp-HMDSO layer proved critical: cell death and atypical adhesion occurred on untreated plastic, whereas stable spheroids formed on the modified surface. HMC3 cells formed small spheroids even on unmodified surfaces, but on pp-HMDSO coatings, the process was more intense and the structures more uniform due to surface hydrophobicity. These results demonstrate the potential of plasma-polymerized HMDSO films as a scalable platform for creating biomaterials with controlled properties for 3D culturing.

Singlet oxygen (¹O₂) is a key mediator in photodynamic therapy (PDT), and its generation and reactivity in biological systems have been extensively studied. It has been shown that laser radiation at near-infrared (NIR) regions can be used to directly generate ¹O₂. In this work, we investigated photosensitizer-free ¹O₂ generation using an original all-fiber pulsed laser operating at 1066 nm and 1241 nm and evaluated its impact on mitochondrial activity in U-87 MG glioblastoma cells. Singlet oxygen was evaluated using the 1,3-diphenylisobenzofuran (DPBF) chemical probe and confirmed with argon-purging controls, demonstrating clear oxygen- and wavelength-dependent effects. Laser irradiation of glioblastoma cells demonstrated distinct effects depending on the wavelength, although decrease in cellular metabolic activity was observed in both cases. Interestingly, some inhibitory effect was also observed when the culture medium was pre-irradiated at 1241 nm and subsequently added to intact cells. These results demonstrate that laser radiation at both studied wavelengths can elicit measurable biological effects, although the relative efficiency in chemical versus cellular systems varies. Collectively, these findings provide a foundation for further systematic studies of wavelength-specific NIR interactions with cellular and molecular components in biological environments.

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.

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.

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.

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.

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.