Study on the Modes of Cell Death in Lung Cancer Epithelial Cells During Acute <scp>EBV</scp> Infection Using a Co‐Culture Model

OBJECTIVE: Pulmonary lymphoepithelial carcinoma is an EBV-associated malignancy; however, the pathogenesis of virus-associated cancers remains incompletely elucidated. In particular, the early pathological changes and fate decisions of pulmonary epithelial cells following acute EBV infection have yet to be fully elucidated. This study aimed to investigate cell death modes of lung epithelial cells upon acute EBV infection. METHODS: An acute infection model was established by co-culturing EBV-positive Akata cells with lung cancer cells. Following infection, we evaluated morphological alterations and modes of cell death using immunofluorescence staining and confocal laser scanning microscopy. RESULTS: The co-culture system successfully established a 72-h acute EBV infection in lung cancer cells, with ~10.3% of epithelial cells exhibiting EBV-associated GFP fluorescence. Under these conditions, the majority of epithelial cells underwent cell death. More than half of the epithelial cells died by day 5 of co-culture, and mortality progressively increased. At 72 h post-infection, immunofluorescence analysis of pMLKL, cGSDMD, and cCASP3 revealed a significant upregulation of pMLKL protein expression (p < 0.001), whereas no significant differences were observed in cGSDMD (p > 0.05) or cCASP3 (p > 0.05) levels. ~15.1% of the remaining epithelial cells following B-cell removal exhibited "cell-in-cell" structures. The internalized B cells underwent various forms of death, including apoptosis, pyroptosis, and necroptosis. CONCLUSION: Acute EBV infection drives lung cancer epithelial cell death through pMLKL-mediated necroptosis, without significant involvement of apoptosis or pyroptosis. Internalized B cells within "cell-in-cell" structures exhibit multiple death modalities. These findings provided novel insights into cellular fate decisions in EBV-infected epithelium prior to latency.