# Extracellular vesicles derived from irradiated tumor cells foster immunosuppressive macrophages formation to promote esophageal squamous cell carcinoma immune evasion

## TL;DR

Radiation therapy in esophageal cancer triggers tumor cells to release molecules that turn immune cells into suppressors, helping the tumor avoid immune detection.

## Contribution

Identifies DYNLL1-AS1 as a novel lncRNA in extracellular vesicles that reprograms macrophages to promote tumor immune evasion.

## Key findings

- Irradiated tumor-derived EVs enriched with DYNLL1-AS1 convert macrophages into PD-L1⁺ immunosuppressive TAMs.
- DYNLL1-AS1 binds SEC22B and FOXP1 to activate PD-L1 transcription, suppressing CD8⁺ T cell function.
- High DYNLL1-AS1 expression in ESCC correlates with poor immunotherapy response and reduced patient survival.

## Abstract

Background: Radiotherapy (RT) remodels the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are key mediators of TME, yet how RT reprograms TAMs toward a programmed death ligand- 1(PD-L1)⁺ immunosuppressive phenotype remains unclear.

Materials and Methods: Esophageal squamous cell carcinoma (ESCC) subcutaneous xenografts in immunodeficient mice received localized RT or sham treatment. Tumor-infiltrating PD-L1⁺ TAMs were quantified via multiplex immunofluorescence and flow cytometry. Extracellular vesicles (EVs) derived from irradiated ESCC cells (IR-EVs) were isolated and characterized by nanoparticle tracking analysis and transmission electron microscopy. Functional assays included co-culture of IR-EVs-educated macrophages with autologous CD8⁺ T cells. RNA sequencing identified DYNLL1-AS1 as the most upregulated lncRNA in IR-EVs. Mechanistic studies employed RNA pull-down, mass spectrometry, RNA immunoprecipitation, and dual-luciferase reporter assays. Clinical validation utilized ESCC specimens for RNA in situ hybridization. Prognostic significance was assessed via Kaplan-Meier and Cox regression analyses.

Results: RT triggered ESCC cells to secrete DYNLL1-AS1-enriched EVs, which reprogrammed macrophages into PD-L1⁺ immunosuppressive TAMs. IR-EVs-educated macrophages suppressed CD8⁺ T cell proliferation and IFN-γ/ Granzyme B secretion. Mechanistically, DYNLL1-AS1 bound SEC22B, enabling its interaction with FOXP1 to activate PD-L1 transcription via promoter binding. In vivo, EVs carrying DYNLL1-AS1 counteract anti-PD-L1 therapy by suppressing CD8+ T cell function and promoting tumor growth. In ESCC patients, high DYNLL1-AS1 expression correlated with PD-L1⁺ TAM density, poor immunotherapy response, and reduced survival. Multivariate analysis confirmed DYNLL1-AS1 as an independent prognostic factor.

Conclusions: Radiation-induced DYNLL1-AS1 in ESCC EVs drives PD-L1⁺ TAMs immunosuppression via SEC22B/ FOXP1 signaling. Combining DYNLL1-AS1 inhibition with PD-L1 blockade may reverse RT-induced immunosuppression, offering a transformative strategy for ESCC radio-immunotherapy.