Epstein-Barr virus BOLF1-containing extracellular vesicles promote nasopharyngeal carcinoma metastasis via SPP1+ macrophages

Nasopharyngeal carcinoma (NPC) patients with elevated plasma Epstein-Barr virus (EBV) DNA levels are more susceptible to distant metastasis. However, the mechanisms underlying NPC progression mediated by EBV interactions with surrounding immune cells remain poorly understood. In this study, we investigated immune cell subsets within the tumor microenvironment of NPC patients, stratified by EBV-DNA load. We innovatively identified SPP1+ tumor-associated macrophages (TAMs), uniquely associated with EBV, as crucial prognostic indicators in NPC, within a retrospective cohort of 186 patients with a 10-year follow-up. Additionally, utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS), we confirmed the presence of EBV protein in plasma extracellular vesicles (EVs). Screening revealed that only BOLF1-EVs promote NPC metastasis by upregulating SPP1+ TAMs. Notably, BOLF1-EVs induced significant metabolic alterations in TAMs, characterized by increased glycolysis and spare respiratory capacity. Importantly, we identified the JAK1-STAT3 pathway as a key mediator of BOLF1-EV-induced upregulation of SPP1 in macrophages, a crucial mechanism underlying tumor metastasis promotion. These findings highlight a potential biomarker for predicting patient metastasis and offer valuable insights for therapeutic decision-making. Overall design: To assess the response of macrophages to BOLF1-EVs, we performed RNA sequencing (RNA-seq) on THP1-derived macrophages co-cultured with either an empty vector control (Vector-EVs) or BOLF1-EVs (100 ng/ml for 48 h)