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

Our plain-English summary of EZH2 Inhibition in Mesothelioma Cells Increases the Release of Extracellular Vesicles That Skew Neutrophils Toward a Protumor Phenotype, by G. Pinton, E. Bari, S. Fallarini, et al (International Journal of Molecular Sciences, 2025).

The study

• Authors: G. Pinton, E. Bari, S. Fallarini, V. Gigliotti, V. De Giorgis, F. Chiazza, M.L. Torre, M. Manfredi, L. Moro
• Journal: International Journal of Molecular Sciences
• Published: 2025;26(21)
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This was a laboratory study of one pleural mesothelioma cell line, MSTO-211H, which the authors describe as biphasic (mixed type). The cells were grown as small three-dimensional clusters called spheroids. No asbestos exposure was studied. The spheroids were treated for 72 hours with 10 µM of the drug EPZ-6438 (tazemetostat), which blocks a protein called EZH2, or were left untreated. Of 881 proteins measured, 33 changed with treatment. These included RAB27b and CD63, which are linked to the release of tiny particles called extracellular vesicles (EVs). Treated spheroids released more than double the amount of EVs per cell. The authors then exposed neutrophils, a type of white blood cell from healthy blood donors, to these EVs. About 60% of the EV-exposed neutrophils carried mesothelin, a mesothelioma marker, on their surface. EVs from treated cells raised the markers CD66b, PD-L1 and CXCR4. EV-exposed neutrophils held back T-cell multiplication, more strongly with EVs from treated cells, and they promoted spheroid growth. The authors concluded that mesothelioma-derived EVs can promote immunosuppressive neutrophils. They noted that only one cell line was used and that the findings need checking in tissue biopsies.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) experiment on a cultured mesothelioma cell line grown as spheroids, with protein analysis (proteomics) and co-culture experiments using blood cells from healthy donors
• Who was studied: A single biphasic pleural mesothelioma cell line (MSTO-211H), obtained from the Istituto Scientifico Tumori cell bank in Genoa, Italy; neutrophils and T cells were isolated from blood (buffy coats) of healthy voluntary donors. Number of donors not stated.
• Exposure: No asbestos or fiber exposure was studied. The exposure was the EZH2-inhibiting drug EPZ-6438 (tazemetostat) at 10 µM for 72 hours, applied to mesothelioma spheroids. Neutrophils were then exposed to EVs released by treated or untreated spheroids.
• Main results:
  • In treated spheroids, 881 proteins were identified and quantified. Of these, 33 were differentially expressed: 21 upregulated and 12 downregulated.
  • Among the most upregulated proteins in treated spheroids were RAB27b (fold change 4.8, p = 1.3 × 10^-5) and CD63 (fold change 2, p = 1.2 × 10^-5).
  • EV content, normalized by cell number, was more than double for treated spheroids compared with untreated (p < 0.00001).
  • Mean EV size was 145.4 ± 4.6 nm from untreated spheroids and 133.8 ± 4.8 nm from treated spheroids. The authors report no significant differences in diameter measures.
  • About 60% of healthy-donor neutrophils treated with EVs from the mesothelioma cells expressed mesothelin (MSLN) on their surface.
  • EVs from untreated cells increased PD-L1 and CXCR4 and decreased CD62L on neutrophils. EVs from EPZ-6438-treated cells increased CD66b and significantly increased PD-L1 and CXCR4.
  • Neutrophils exposed to EVs significantly suppressed proliferation of activated T cells from healthy donors. The effect was more significant with EVs from EPZ-6438-treated cells.
  • When EV-exposed neutrophils were cocultured with mesothelioma spheroids, they promoted cell growth.
  • In the EVs themselves, 391 proteins were identified and 54 were differentially expressed (37 more and 17 less concentrated in EVs from treated spheroids).
  • MSLN was enriched in EVs from treated spheroids (fold change 2), but PD-L1 was not identified in the EV cargo.
• The authors' conclusion: The authors concluded that tazemetostat treatment increases EV release from mesothelioma cells. They concluded that these EVs shift neutrophils toward a protumor, immunosuppressive phenotype that inhibits T-cell immunity, and that MSLN can be transferred from tumor cells to immune cells via EVs. They described this as possible unintended consequences of strategies targeting EZH2.
• Limitations the authors state:
  • Only a single cell line of mixed histotype was used, which may limit how far the findings apply to other mesothelioma subtypes.
  • The findings have not been checked in tissue biopsies or in living patients (in vivo).

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, BAP1, Pleural Mesothelioma, CDKN2A, EZH2 Inhibitor, PD-L1, Mesothelin.

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