Tumor-derived Extracellular Vesicles Induce ER Stress to Drive Tolerogenic Dendritic Cell Development in the Tumor Microenvironment

Abstract

The efficacy of immune checkpoint blockade relies on the robust priming of T cells by immunostimulatory dendritic cells (DCs). However, the tumor microenvironment (TME) frequently drives DCs into a dysfunctional, pro-tolerogenic state governed by aberrant metabolic rewiring, creating a barrier to durable antitumor immunity. While tumor-derived extracellular vesicles (EVs) are abundant in the TME, their specific role in orchestrating this immunosuppressive metabolic reprogramming remains poorly understood. This study provides insight into the signaling axes through which tumor-derived EVs alter DC function and evaluates the therapeutic potential of targeting these pathways to overcome immunotherapy resistance.Tumor models were engineered to express EV fluorescent markers to track tumor EV uptake in vivo. Bulk and single-cell RNA sequencing was integrated with multi-parameter flow cytometry to characterize the reprogramming of tumor EV-educated DCs both in vitro and in vivo. Western blotting, quantitative real-time polymerase chain reaction (qrt-PCR) assays, various cellular metabolic assays, as well as T cell-based immunologic studies were utilized to characterize the underlying mechanisms of tumor EV-mediated DC reprogramming. DC-specific Ppara-deficient mice were developed to verify these mechanisms in vivo. PPARα targeted inhibitors were evaluated based on their ability to overcome checkpoint inhibitor resistance in an autochthonous model of melanoma. Tumor-derived EVs were found to promote tumor progression by suppressing host immunity. Further studies reveal that tumor-derived EVs induce a tolerogenic 'mregDC' transcriptional signature characterized by the upregulation of immunoregulatory molecules in DCs both in vitro and in vivo. These tumor EV-educated DCs exhibit an impaired capacity for CD8+ T cell priming, while demonstrating a proficiency for promoting CD4+FoxP3+ regulatory T cell differentiation. Mechanistically, tumor EVs concurrently trigger the unfolded protein response (UPR) via the PERK-ATF4 and IRE1α-XBP1s signaling axes, subsequently activating the SREBP2 and PPARα transcription factors, respectively. This process drives both aberrant lipid accumulation and fatty acid oxidation (FAO) in DCs residing within the TME. DC-restricted ablation of PPARα significantly reversed the pro-tolerogenic effect of tumor EVs in vivo while pharmacologic targeting of PPARα overcomes anti-PD-1 resistance and augments CD8+ T cell infiltration in an autochthonous model of BRAFV600E melanoma. Tumor EVs contribute to the development and pro-tolerogenic function of mregDCs in the TME by triggering the UPR pathway. Aberrant lipid metabolism involving enhanced FAO are common characteristics associated with DC dysfunction in the TME. Strategies to interrupt these pathways represent promising approaches for reversing immune tolerance and enhancing tumor-targeted CD8+ T cell responses.