Letícia Andrade Savério

Conference 2024 Live Talk

Talk Title

Glioblastoma mRNA vaccine: the possible modulation of tumor mRNA on the immune system

Authors and Affiliations

Letícia Andrade Savério, Tiago Francisco da Silva, Jaqueline Vaz de Oliveira, Célia Regina Pinto Pizzo, Sandra Márcia Muxel, José Alexandre Marzagão Barbuto.
Institute of Biomedical Sciences, University of São Paulo (ICB / USP). São Paulo – SP, Brazil.

Abstract

Background

Glioblastoma (GBM) is the most aggressive and frequent type of tumor in the central nervous system, and the standard treatment for this disease still falls short of expectations. One approach that has shown promise is immunotherapy, which, in our laboratory, has been explored through therapeutic vaccination using monocyte-derived dendritic cells (moDC) from healthy donors fused with autologous tumor cells from patients. However, producing this vaccine requires significant amounts of tumor, making it challenging to sustain treatment over extended periods. Additionally, the preparation process involves an invasive procedure. One strategy to address these challenges and enhance antigen presentation by moDC is to use total mRNA from tumor cells. This mRNA can be amplified in vitro and transfected into moDC.

Methods

In this project, we evaluated the transfection of mRNA obtained from the U-87 MG glioblastoma cell line into moDC. We used 5ug of total mRNA and tested different transfection times (6, 12, and 24 hours) with two transfection methods (passive transfection and lipofection) under two moDC maturation conditions (immature and mature). Transfection efficiency was assessed by GFAP protein expression. We also determined the phenotype of transfected moDC using flow cytometry, evaluating the expression of molecules involved in antigen presentation (HLA-DR and HLA-ABC), co-stimulation (CD80 and CD83), and regulation of the immune response (CD274 – PD-L1).

Results

We observed the highest frequency of GFAP expression in immature dendritic cells (iDC) after 6 hours of lipofection with tumor mRNA. For mature dendritic cells (mDC), the highest frequency occurred at the same time but after passive transfection. Subsequent tests revealed a loss of GFAP expression in all conditions, potentially due to antigen processing activity within the cells.At these time points, moDC transfected with total mRNA from the glioblastoma cell line, regardless of the transfection method, exhibited reduced expression of PD-L1 compared to controls. This reduction was more significant in the iDC group. Interestingly, lipofection of total mRNA in iDC maintained the intensity of expression and frequency of CD83+ cells.

Conclusions

These results indicate that moDC can translate non-constitutive proteins through mRNA delivery. Additionally, our analysis of the phenotype of transfected moDC suggests that these cells retain the ability to stimulate lymphocyte responses after mRNA delivery. The specific lymphocyte response profile will be the focus of our next studies.