Optimizing Adjuvant Combinations to Potentiate a Microparticulate Breast Cancer Vaccine

Document Type

Poster Presentation

Publication Date

4-17-2026

Keywords

fsc2026

Abstract

Objective: Determine the immunogenicity of varying concentrations of the adjuvants Poly (I:C) and granulocyte-macrophage colony-stimulating factor (GM-CSF) when used in combination with a microparticulate breast cancer vaccine. The combination that produces the best immune response will be tested in further studies.

Methods: Murine dendritic cells (DC2.4) were seeded in two six-well plates at 5 x 105 cells per well with 4 mL of complete RPMI media and incubated at 37°C/5% CO2 for 24 hours. The cells were then treated using the following adjuvant combinations: (a) PIC (6.25 ug/mL) + GM-CSF (25 pg/mL), (b) PIC (12.5 ug/mL) + GM-CSF (37.5 pg/mL), (c) PIC (25 ug/mL) + GM-CSF (50 pg/mL), (d) PIC (25 ug/mL) + GM-CSF (25 pg/mL), and (e) PIC (6.25 ug/mL) + GM-CSF (50 pg/mL). Each combination was tested in duplicate with either vaccine or placebo particles. Two wells were used as negative controls with cells only. The plates were incubated at 37°C/5% CO2 for 24 hours. Next, murine T-lymphocyte cells (EL4.IL2) were introduced at 5 x 105 cells per well and co-cultured in a combination of complete RPMI and complete DMEM for 24 hours at 37°C/5% CO2. After 24 hours, T cells were removed by centrifugation (1000 rpm for 10 minutes), and the pellets were quantified in duplicate using a cell counter. The supernatants were stored at -20°C to be later analyzed via enzyme-linked immunosorbent assays (ELISA) for tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), interleukins 1ꞵ, 2, 6, and 12 (IL-1ꞵ, IL-2, IL-6, IL-12). Results were analyzed with a UV plate reader at 450 nm.

Results: A statistically significant increase in secretion of TNF-alpha was observed in the vaccine + PIC (6.25 ug/mL) + GM-CSF (25 pg/mL) treatment group compared to vaccine groups containing higher levels of GM-CSF (p< 0.05). No other statistically significant difference was observed among vaccine groups containing the following combinations: PIC (6.25 ug/mL) + GM-CSF (25 pg/mL)], PIC (12.5 ug/mL) + GM-CSF (37.5 pg/mL), PIC (25 ug/mL) + GM-CSF (50 pg/mL). A statistically significant increase in secretion of IL-1β was observed in the vaccine + PIC (25 ug/mL) + GM-CSF (25 pg/mL) group compared to vaccine + PIC (6.25 ug/mL) + GM-CSF (50 pg/mL) (p< 0.05). A statistically significant increase in secretion of IL-12 was observed in the vaccine + PIC (25 ug/mL) + GM-CSF (50 pg/mL) treatment group compared to vaccine + PIC (25 ug/mL) + GM-CSF (25 pg/mL) and vaccine + PIC (6.25 ug/mL) + GM-CSF (50 pg/mL) (p< 0.05). A statistically significant increase in secretion of IL-12 was also observed in the vaccine + PIC (6.25 ug/mL) + GM-CSF (25 pg/mL) treatment group compared to vaccine + PIC (6.25 ug/mL) + GM-CSF (50 pg/mL) (p< 0.05). No statistically significant differences between vaccine treatment groups were detected for T-cell quantification, IFN-gamma, IL-2, or IL-6.

Conclusion: The combination of Poly(I:C) and GM-CSF showed no significant concentration-dependent response for T-cell expansion or ELISA assays for IFN-gamma, IL-1β, IL-2, IL-6, or IL-12, indicating a lack of synergistic activity and potential immunosuppressive effects at higher concentrations of GM-CSF. Immunosuppressive effects are further supported by the significantly higher levels of TNF-alpha produced in the vaccine group treated with low concentrations of both adjuvants compared to other vaccine treatment groups with higher levels of GM-CSF. The combination of PIC (6.25 ug/mL) + GM-CSF (25 pg/mL) with the vaccine will be tested in further studies.

Comments

Poster presented at the 2026 Fisher Showcase, St. John Fisher University, April 17, 2026.

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