VISTA expressed on tumor cells is regulated by m6A and influences immune microenvironment through STAT3/CCL22 in NSCLC

Background: Immunotherapy is playing an increasingly vital role in the treatment of non-small cell lung Cancer (NSCLC), yet the challenge of immune evasion remains prevalent. Therefore, investigating the regulatory mechanisms of the tumor immune microenvironment could yield significant benefits.

Methods: This study utilized differential expression analysis, WGCNA, and Lasso-Cox to analyze the GSE126044 dataset, identifying N6-methyladenosine (m6A)-related molecules that play significant roles in immunotherapy. The m6A modification of the molecule were validated through m6A dot blot, MeRIP, RNA pull-down and RNA stability assays. Their impact on the biological behavior of tumor cells (TCs) was evaluated by CCK-8 assays, wound healing assays, transwell assays, co-culture experiments and flow cytometry. Furthermore, RNA Sequencing was conducted to uncover downstream pathways and cytokines, followed by subsequent validation. Lastly, validation was carried out in vivo.

Results: Through bioinformatics analysis, we identified that V-domain Immunoglobulin Suppressor of T cell Activation (VISTA) serves a crucial role in immunotherapy and that its expression is regulated by m6A. It was subsequently confirmed that METTL3 enhances the m6A modification levels of VISTA mRNA, which is recognized by YTHDF1. This interaction improved the stability of VISTA mRNA, leading to increased expression of VISTA proteins on the surface of TCs. The elevated expression of VISTA boosted the cytotoxic response of CD8 + T cells toward TCs. RNA Sequencing analysis indicated that the JAK/STAT pathway significantly contributes to this process. Overexpression of VISTA on TCs decreased the phosphorylation of STAT3, which in turn reduced the expression and release of CCL22. This reduction alleviated immune suppression within the tumor microenvironment, resulting in increased enrichment and activity of CD8 + T cells and decreased enrichment of regulatory T cells (Tregs), ultimately enhancing their cytotoxic effects against TCs.

Conclusion: We conduct a pioneering systematic study on the expression, function, and molecular mechanisms of the VISTA molecule on NSCLC TCs. We propose that in the tumor immune microenvironment, VISTA functions as both an immune checkpoint and a regulator of CD8 + T cell activation/recruitment and Treg recruitment via its expression on TCs.

CCL22; Immunotherapy; METTL3; NSCLC; STAT3; VISTA; YTHDF1; m6A modification.