Epstein-Barr virus (EBV) infection of endothelial cells via endocytosis is associated with a poor prognosis in nasopharyngeal carcinoma

ABSTRACT Epstein-Barr virus (EBV) infection is strongly associated with several malignancies, including nasopharyngeal carcinoma (NPC), Burkitt lymphoma, and certain gastric cancers, though its potential to infect endothelial cells (ECs) and the consequent pathological implications remain poorly understood. This study demonstrates through analysis of 99 NPC clinical samples (primary tumors) that Epstein-Barr virus-encoded small RNAs (EBERs) positivity in ECs significantly correlates with N stage (lymphatic metastasis, P < 0.05), M stage (distant metastasis, P < 0.01), and advanced clinical stage ( P < 0.01), while immunological profiling reveals concomitant reductions in B-cell proportions ( P < 0.01) in patients with EBERs-positive ECs. Through comprehensive in vitro modeling employing EBV particle infection, Transwell coculture, and direct-contact systems with EBV-positive Akata and Raji cells, we observed that human lymphatic endothelial cells actively internalize infected lymphocytes, with RT-PCR confirming expression of EBV oncogenes (EBNA1, LMP1, and LMP2)—particularly in direct-contact conditions—alongside significant upregulation of endocytosis-related genes Rab5a and EHD1 and ultrastructural evidence of phagosome formation. These findings collectively suggest that phagocytic uptake of EBV-infected lymphocytes or their components may serve as a primary infection mechanism for ECs, potentially establishing an immunosuppressive microenvironment that contributes to tumor progression and poor clinical outcomes, though the exact molecular pathways require further elucidation. IMPORTANCE Clinical investigations of NPC specimens identified EBERs-positive endothelial cells as clinically significant biomarkers, demonstrating robust correlations with lymphatic metastasis ( P < 0.05), distant metastasis ( P < 0.01), and advanced tumor staging ( P < 0.01), while immunological profiling of affected patients revealed concomitant reductions in B-cell populations ( P < 0.01), collectively indicative of systemic immunoregulatory status. Furthermore, integrative experimental approaches incorporating live-cell dynamic imaging, single-cell transcriptional profiling, and ultrastructural electron microscopy provided compelling evidence that endothelial phagocytosis of lymphocytes serves as a principal route for EBV cellular entry and subsequent modulation of the NPC tumor microenvironment.