Introduction: In 2020, cancer caused approximately ten million deaths globally despite extensive research. The disease's lethality stems from the rapid proliferation and invasion of abnormal cells into other body parts. Although surgery and chemotherapy can remove tumors and cancer cells, the associated side effects—such as pain and hair loss—disrupt patients' daily lives. Consequently, there is a significant demand for more effective cancer treatments. Immunotherapies like checkpoint inhibitors, CAR T-cell therapy, and cancer vaccines offer fewer side effects than traditional treatments. Notably, cancer vaccines have gained attention due to their favorable safety profile.
Cancer vaccines modulate dendritic cells by delivering DNA/RNA or antigen peptides. These cells present antigens and activate T cells using three distinct signals to target cancer cells specifically. We employ a strategy involving porous alginate hydrogels coated with chemokines like granulocyte-macrophage colony-stimulating factor (GM-CSF), which attract dendritic cells. Once these cells migrate to the hydrogel, the uptake of antigens and adjuvants further activates them.
Materials and
Methods: The DC-recruiting alginate hydrogel is fabricated by mixing the GM-CSF, porogen beads, and alginate hydrogel derived from medium viscous alginate salt. The porogen beads are then bleached at 37C, leaving pores inside Alginate hydrogel. The surface morphology of the fabricated porous hydrogel was analyzed using Scanning Electron Microscopy (SEM). For in vitro studies, bone marrow-derived DCs (BMDCs) were isolated and differentiated from C57BL/6 mice. The DCs are then transfected with Ovalbumin (OVA) mRNA by Lipofectamine 2000 in the porous alginate hydrogel for two days. The DC antigen presentation and activation are detected by anti-SIINFEKL and CD86 stainings, respectively. The DC population is then quantified through FACS assay. In addition, the OT-1 cells are prepared from the spleen of mice with mouse Tcra-V2 and Tcrb-V5 genes. The OVA mRNA transfected DCs were cocultured with CFSE-stained OT-1 cells, and the FACS Assay determined the OT-1 cell proliferation index. For in vivo studies, EG.7-OVA cells are subcutaneously injected into C57BL/6 as a tumor model. The GM-CSF-coated porous alginate hydrogel was also subcutaneously implanted for DC recruiting. The therapy efficacy was determined by comparing the tumor volume and survival rate between alginate hydrogel-treated and untreated groups for 90 days. In the prophylactic study, the porous alginate hydrogel with mRNA was subcutaneously implanted, and the immune response was quantified by analyzing peripheral blood mononuclear cells (PBMC).
Results, Conclusions, and Discussions: Result: First, the porosity of the Alginate hydrogel is verified by SEM image. The SEM image shows that the pore size of alginate hydrogel is around 80 micrometers, which fits the DC recruiting requirement. Then, we confirmed the OVA mRNA transfection leads to specific antigen presentation on the DC surface. The SIINFEKL-positive population of the best-performing group is 20% higher than the PBS and mRNA-only groups. Moreover, the OVA-mRNA-transfected DCs lead to a higher proliferation index of OT-1 cells than the non-treated DCs group. In peripheral blood mononuclear cells (PBMC) analysis, the T cell which was restimulated with SIINFEKL peptide showed a more robust IFN-gamma production 21 days after the hydrogel implantation. Overall, the unvaccinated group developed a large tumor mass, and all reached the endpoint after 30 days, while the fully vaccinated group maintained a 40% survival rate for more than 120 days.
Conclusions and Discussions: SEM images reveal the alginate hydrogel's interconnected porosity supports dendritic cell (DC) homing and modulation for specific antigen presentation. Quantitative analysis of this porosity could be further enhanced with additional SEM imagery and statistical evaluation. The material effectively recruits and modulates DCs in vivo, and identifying the subtypes of dendritic cells attracted to the biomaterial could provide deeper insights. These mRNA-based bioscaffolds have been shown to enhance therapy efficacy in the EG7.OVA tumor model. Additionally, assessing the treatment's effectiveness against the 4T1 neoantigen model could validate the therapy's broader potential.
