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.

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.

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 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.

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.