# EZH2 Inhibition in Mesothelioma Cells Increases the Release of Extracellular Vesicles That Skew Neutrophils Toward a Protumor Phenotype

## TL;DR

EZH2 inhibition in mesothelioma cells increases extracellular vesicles that change neutrophils to support tumor growth.

## Contribution

This study reveals a novel mechanism by which EZH2 inhibition alters neutrophil function through extracellular vesicles.

## Key findings

- EZH2 inhibition increases extracellular vesicles containing RAB27b and CD63.
- EVs from treated cells skew neutrophils toward a pro-tumor phenotype with high PD-L1 and MSLN.
- EV-elicited neutrophils suppress T cell proliferation and enhance tumor growth.

## Abstract

We previously demonstrated that in BAP1-proficient pleural mesothelioma cells, CDKN2A is critical for mediating the response to selective EZH2 inhibition and highlighted a complex interplay between epigenetic regulation and the tumor immune microenvironment. In this study, we employed a quantitative proteomic mass spectrometry approach to assess alterations in protein expression following EZH2 inhibition in BAP1- and CDKN2A-proficient mesothelioma cells cultured as spheroids. Additionally, we analyzed extracellular vesicles (EVs), which were isolated through tangential flow filtration. Flow cytometric analysis and co-culture systems were used to characterize the effects of EVs on neutrophils. Upon EZH2 inhibition, we demonstrated RAB27b and CD63 upregulation and increased release of extracellular vesicles. We found that a brief exposure to EVs derived from EZH2 inhibitor-treated cells skewed naïve neutrophils toward a pro-tumor phenotype characterized by high levels of PD-L1 and MSLN (Mesothelin) expression on the surface. These EV-elicited neutrophils suppressed T cell proliferation while enhancing tumor cell growth. Moreover, we observed changes in the EV cargo derived from EZH2 inhibitor-treated spheroids. Our findings highlight the significant role of EVs in creating an immunosuppressive microenvironment, and underscore the urgent need for further investigation into the regulation of neutrophil biology and function in the PM.