# The Immunomodulatory Effects of Fluorescein-Mediated Sonodynamic Treatment Lead to Systemic and Intratumoral Depletion of Myeloid-Derived Suppressor Cells in a Preclinical Malignant Glioma Model

## TL;DR

This study shows that sonodynamic therapy using fluorescein can delay glioma growth and improve survival by reducing immune-suppressing cells in mice.

## Contribution

The study is the first to evaluate fluorescein-mediated sonodynamic therapy in immunocompetent glioma mice and its effects on the immune microenvironment.

## Key findings

- Fluorescein-mediated sonodynamic therapy (FL-SDT) depletes myeloid-derived suppressor cells and increases CD8+ T cell infiltration in gliomas.
- FL-SDT significantly delays glioma progression and improves survival in mice.
- Treatment with FL-SDT or fluorescein alone correlates with better survival and a favorable immune profile.

## Abstract

Sonodynamic therapy (SDT) is emerging as a promising innovative technique for treating malignant gliomas in a noninvasive fashion. The use of fluorescein, instead of 5-aminolevulinic acid, could extend the application of sonodynamic therapy to intracranial tumors other than gliomas. Our aim was to investigate the feasibility of sonodynamic therapy with fluorescein in an intracranial mouse model of malignant glioma, its efficacy, and its effects on the immune microenvironment.

Fluorescein-mediated sonodynamic therapy (FL-SDT) is an extremely promising approach for glioma treatment, resulting from the combination of low-intensity focused ultrasound (FUS) with a sonosensitizer. In the present study, we evaluated the efficacy and immunomodulation of SDT with fluorescein as the sonosensitizer in immunocompetent GL261 glioma mice for the first time. In vitro studies demonstrated that the exposure of GL261 cells to FL-SDT induced immunogenic cell death and relevant upregulation of MHC class I, CD80 and CD86 expression. In vivo studies were then performed to treat GL261 glioma-bearing mice with FL-SDT, fluorescein alone, or FUS alone. Perturbation of the glioma-associated macrophage subset within the immune microenvironment was induced by all the treatments. Notably, a relevant depletion of myeloid-derived suppressor cells (MDSCs) and concomitant robust infiltration of CD8+ T cells were observed in the SDT-FL-treated mice, resulting in a significant radiological delay in glioma progression and a consequent improvement in survival. Tumor control and improved survival were also observed in mice treated with FL alone (median survival 41.5 days, p > 0.0001 compared to untreated mice), reflecting considerable modulation of the immune microenvironment. Interestingly, a high circulating lymphocyte-to-monocyte ratio and a very low proportion of MDSCs were predictive of better survival in FL- and FL-SDT-treated mice than in untreated and FUS-treated mice, in which elevated monocyte and MDSC frequencies correlated with worse survival. The immunostimulatory potential of FL-SDT treatment and the profound modulation of most immunosuppressive components within the microenvironment encouraged the exploration of the combination of FL-SDT with immunotherapeutic strategies.