# The S1P/S1P1 Signaling Axis Plays Regulatory Functions in the Crosstalk Between Brain-Metastasizing Melanoma Cells and Microglia

## TL;DR

This study shows that blocking the S1P/S1P1 signaling pathway can shift microglia to fight melanoma in the brain, offering a new treatment approach.

## Contribution

The study reveals that S1P1 inhibition alters microglial function and reduces melanoma growth in the brain.

## Key findings

- S1P1 inhibition shifts microglia toward an anti-tumor state and reduces melanoma and microglia viability.
- Blocking S1P1 reduces immunosuppressive factors in microglia and enhances their pro-inflammatory response.
- Combining S1P1 inhibition with BRAF inhibitors improves treatment efficacy in melanoma cells.

## Abstract

Brain metastases are a frequent and lethal complication of melanoma. The tumor microenvironment is well recognized as a critical driver of cancer progression. Within the brain, melanoma cells engage in dynamic interactions with microglia, the resident immune cells. However, the mechanisms governing this crosstalk remain poorly understood. In our previous work, we showed that brain-metastasizing melanoma cells reprogram microglia, inducing molecular and functional changes, including upregulation of sphingosine-1-phosphate receptor 1 (S1PR1). Here we aimed to investigate whether this upregulation affects microglial phenotype and thereby contributes to microglia-mediated support of melanoma brain metastasis progression. We found that inhibition of S1P1 shifted microglia toward an anti-tumor state, which can facilitate infiltration of cytotoxic immune cells, and also attenuated the growth of both microglia and melanoma cells. These findings highlight the S1P1 pathway as a key player in the tumor–brain environment and suggest that its targeting could be a novel therapeutic approach against brain-metastasizing melanoma.

Background/Objectives: The interaction between brain-metastasizing melanoma cells and surrounding microglia shapes the immune tumor microenvironment and influences tumor progression. Gene expression analysis revealed that sphingosine-1-phosphate receptor 1 (S1PR1), encoding the S1P1 receptor, is upregulated in microglia upon interaction with melanoma cells. Here, we investigated the functions of S1P1 in microglia and its contribution to melanoma–microglia crosstalk. Methods: We examined the effects of S1P1 inhibition on microglia and four brain-metastasizing human melanoma cell lines in monocultures and co-cultures using the selective S1P1 antagonist NIBR0213 and S1PR1 gene knockdown. Results: We found that melanoma-secreted IL-6 upregulated S1PR1 expression in microglia. S1P1 inhibition increased expression of CD32, CD150, and CD163 in microglia; however, CD150 and CD163 upregulation was abolished in the presence of melanoma cells. S1P1 inhibition downregulated immunosuppressive and anti-inflammatory factors in microglia, including CD274, SOCS3, TGFBR1, TGFBR2, and JunB, promoting a pro-inflammatory phenotype. It also reduced viability of both melanoma and microglia cells, inducing apoptosis in melanoma-associated microglia, possibly via downregulation of CH25H, an upstream regulator of SREBPs. In co-cultures, melanoma cells were more sensitive than microglia to NIBR0213-induced growth arrest. In 3D spheroid cultures, NIBR0213 delayed melanoma–microglia aggregation. Combined treatment with the BRAF inhibitor Vemurafenib and NIBR0213 enhanced Vemurafenib efficacy in three of four melanoma lines. Conclusions: S1P1 contributes to the immunosuppressive phenotype of microglia. Inhibiting the S1P/S1P1 axis impairs viability and crosstalk between melanoma cells and tumor-activated microglia, offering a potential therapeutic strategy for melanoma brain metastases.