Interferon gamma (IFNg) is a pleiotropic cytokine that can potentially reprogram the tumor microenvironment; however, the antitumor immunomodulatory properties of IFNg still need to be validated due to variable therapeutic outcomes in preclinical and clinical studies. We developed a replication-deficient Semliki Forest virus vector expressing IFNg (SFV/IFNg) and evaluated its immunomodulatory antitumor potential in vitro in a model of 3D spheroids and in vivo in an immunocompetent 4T1 mouse breast cancer model. We demonstrated that SFV-derived, IFN-g-stimulated bone marrow macrophages can be used to acquire the tumoricidal M1 phenotype in 3D nonattached conditions. Coculturing SFV/IFNg-infected 4T1 spheroids with BMDMs inhibited spheroid growth. In the orthotopic 4T1 mouse model, intratumoral administration of SFV/IFNg virus particles alone or in combination with the Pam3CSK4 TLR2/1 ligand led to significant inhibition of tumor growth compared to the administration of the control SFV/Luc virus particles. Analysis of the composition of intratumoral lymphoid cells isolated from tumors after SFV/IFNg treatment revealed increased CD4⁺ and CD8⁺ and decreased T-reg (CD4⁺/CD25⁺/FoxP3⁺) cell populations. Furthermore, a significant decrease in the populations of cells bearing myeloid cell markers CD11b, CD38, and CD206 was observed. In conclusion, the SFV/IFNg vector induces a therapeutic antitumor T-cell response and inhibits myeloid cell infiltration in treated tumors.

Viral vectors have been widely investigated as tools for cancer immunotherapy. Although many preclinical studies demonstrate significant virus-mediated tumour inhibition in synergy with immune checkpoint molecules and other drugs, the clinical success of viral vector applications in cancer therapy currently is limited. A number of challenges have to be solved to translate promising vectors to clinics. One of the key elements of successful virus-based cancer immunotherapy is the understanding of the tumour immune state and the development of vectors to modify the immunosuppressive tumour microenvironment (TME). Tumour-associated immune cells, as the main component of TME, support tumour progression through multiple pathways inducing resistance to treatment and promoting cancer cell escape mechanisms. In this review, we consider DNA and RNA virus vectors delivering immunomodulatory genes (cytokines, chemokines, co-stimulatory molecules, antibodies, etc.) and discuss how these viruses break an immunosuppressive cell development and switch TME to an immune-responsive “hot” state. We highlight the advantages and limitations of virus vectors for targeted therapeutic programming of tumour immune cell populations and tumour stroma, and propose future steps to establish viral vectors as a standard, efficient, safe, and non-toxic cancer immunotherapy approach that can complement other promising treatment strategies, e.g., checkpoint inhibitors, CAR-T, and advanced chemotherapeutics.

The development and testing of antimicrobial coatings continues to be a crucial approach, considering the ongoing emergence of antibiotic-resistant bacteria and the rapid transmission of highly pathogenic viruses. In this study, three types of coatings—pure metallic copper (Cu), zinc oxide (ZnO), and a three-layer zinc oxide and copper mixed coating (ZnO/Cu/ZnO)—were deposited by magnetron sputtering on polyethylene terephthalate substrates to evaluate their antimicrobial potential using various microorganisms, including viruses. Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria were used for the assessment of antibacterial properties. Antiviral testing was performed using MS2 bacteriophage and replication-deficient Semliki Forest virus, both representing single-stranded RNA-containing viruses. The samples’ ability to cause reactive oxygen species formation was measured, and the effect on bacterial metabolic activity was evaluated. Cu-coated samples showed high inhibitory activity (>95%) against E. coli and S. aureus bacteria, as well as against tested viruses (SFV and MS2). The antibacterial and antiviral properties of ZnO/Cu/ZnO and ZnO coatings were not significant. Although ZnO/Cu/ZnO and ZnO caused inhibition of the metabolic activity of the bacteria, it was insufficient for complete bacteria eradication. Furthermore, significant reactive oxygen species (ROS) production was detected only for single Cu-coated samples, correlating with the strong bacteria-killing ability. We suppose that the ZnO layer exhibited a low release of Zn ions and prevented contact of the Cu layer with bacteria and viruses in the ZnO/Cu/ZnO coating. We conclude that current ZnO and Cu-ZnO-layered coatings do not possess antibacterial and antiviral activity.

Reprogramming of tumor-associated macrophages (TAMs) is a promising strategy for cancer immunotherapy. Several studies have shown that cancer cells induce/support the formation of immunosuppressive TAMs phenotypes. However, the specific factors that orchestrate this immunosuppressive process are unknown or poorly studied. In vivo studies are expensive, complex, and ethically constrained. Therefore, 3D cell interaction models could become a unique framework for the identification of important TAMs programming factors. In this study, we have established and characterized a new in vitro 3D model for macrophage programming in the presence of cancer cell spheroids. First, it was demonstrated that the profile of cytokines, chemokines, and surface markers of 3D-cultured macrophages did not differ conceptually from monolayer-cultured M1 and M2-programmed macrophages. Second, the possibility of reprogramming macrophages in 3D conditions was investigated. In total, the dynamic changes in 6 surface markers, 11 cytokines, and 22 chemokines were analyzed upon macrophage programming (M1 and M2) and reprogramming (M1→M2 and M2→M1). According to the findings, the reprogramming resulted in a mixed macrophage phenotype that expressed both immunosuppressive and anti-cancer immunostimulatory features. Third, cancer cell spheroids were shown to stimulate the production of immunosuppressive M2 markers as well as pro-tumor cytokines and chemokines. In summary, the newly developed 3D model of cancer cell spheroid/macrophage co-culture under free-floating conditions can be used for studies on macrophage plasticity and for the development of targeted cancer immunotherapy.

The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and β-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications.

A set of styrylpyridinium (SP) compounds was synthesised in order to study their spectroscopic and cell labelling properties. The compounds comprised different electron donating parts (julolidine, p-dimethylaminophenyl, p-methoxyphenyl, 3,4,5-trimethoxyphenyl), conjugated linkers (vinyl, divinyl), and an electron-withdrawing N-alkylpyridinium part. Geminal or bis-compounds incorporating two styrylpyridinium (bis-SP) moieties at the 1,3-trimethylene unit were synthesised. Compounds comprising a divinyl linker and powerful electron-donating julolidine donor parts possessed intensive fluorescence in the near-infrared region (maximum at ~760 nm). The compounds had rather high cytotoxicity towards the cancerous cell lines HT-1080 and MH-22A; at the same time, basal cytotoxicity towards the NIH3T3 fibroblast cell line ranged from toxic to harmful. SP compound 6e had IC₅₀ values of 1.0 ± 0.03 µg/mL to the cell line HT-1080 and 0.4 µg/mL to MH-22A; however, the basal toxicity LD₅₀ was 477 mg/kg (harmful). The compounds showed large Stokes’ shifts, including 195 nm for 6a,b, 240 nm for 6e, and 325 and 352 nm for 6d and 6c, respectively. The highest photoluminescence quantum yield (PLQY) values were observed for 6a,b, which were 15.1 and 12.2%, respectively. The PLQY values for the SP derivatives 6d,e (those with a julolidinyl moiety) were 0.5 and 0.7%, respectively. Cell staining with compound 6e revealed a strong fluorescent signal localised in the cell cytoplasm, whereas the cell nuclei were not stained. SP compound 6e possessed self-assembling properties and formed liposomes with an average diameter of 118 nm. The obtained novel data on near-infrared fluorescent probes could be useful for the development of biocompatible dyes for biomedical applications.