Abstract 6035: The role of EBV+ nasopharyngeal carcinoma in shaping double-negative B cells to form a pro-metastatic niche

Abstract Introduction: B cells can play an anti-tumor role in the tumor microenvironment (TME) by secreting antibodies and activating other immune cells. In our previous study, IgD-CD27- double-negative (DN) B cells were associated with worse prognoses in nasopharyngeal carcinoma (NPC), a malignancy often associated with Epstein-Barr virus (EBV) infection, implying a potential role in NPC disease progression. Methods: To characterize DN B cells and identify features associated with NPC clinical outcomes, we analyzed single-cell RNA-sequencing data (scRNA-seq) from NPC tumors, blood samples, and normal nasopharynx tissues (n = 49, 15, and 7), as well as bulk RNA-sequencing (RNA-seq) data from two cohorts of NPC patients with clinical data (n = 77 and 100) respectively. Multiplex Immunohistochemical (mIHC) staining of tumor slides from 40 NPC patients was analyzed for the spatial identification of tumor-infiltrating B cells. To understand the in vitro interactions between NPC cancer cells and B cells, tonsil-derived IgD+CD27- naïve B cells were cocultured with EBV-positive (EBV+) or negative (EBV-) NPC43 cells, followed by Flow Cytometry and scRNA-seq for quantifying DN B cells and investigating their interactions with cancer cells. Results: Trajectory analysis identified DN B cells and Plasma B cells as potential terminal B cell development states in NPC, with both populations enriched in tumors. DN B cells could be classified into two major subpopulations in NPC: DN Branch 1 and DN Branch 2, with the former potentially being influenced by EBV infection and the latter being under stress. RNA-seq data revealed that B-cell-related biological pathways were downregulated in metastatic NPC, while mIHC staining showed that most DN B cells were in NPC tumor nests and were enriched in metastatic NPC. Flow cytometry on our co-culture experiments detected increased DN B cell content in EBV+ NPC43 co-culture (40.0%) compared to EBV- NPC43 (25.9%), supporting our hypothesis that EBV+ NPC malignant cells may be involved in promoting DN B cell development in NPC. Detailed cell-cell interaction analysis indicated that the EGFR-TGFB1, LTBR-LTB, and MIF-TNFRSF14 are potential key interactions between EBV+ NPC cells and naïve B cells, likely contributing to DN B cell development and tumour growth. Conclusions: DN B cells in NPC may be in a stable development state, with their correlation with distant metastasis suggesting their involvement in B cell dysregulation involved in poor NPC prognosis. Taken together, our results suggest EBV+ NPC cells may receive pro-tumor signals from naïve B cells and promote the development of DN B cells to form a pro-metastatic niche. Future studies are needed for detailed validations of these findings. Acknowledgments: This study is funded by Theme-based Research Scheme (TBRS) (T12-703/22-R), Health and Medical Research Fund (HMRF) (08192296) from Hong Kong (SAR), P.R. China. Citation Format: Michael King Yung Chung, Ngar Woon Kam, Yingpei Zhang, Zhaozheng Hou, Kazi Anisha Islam, Yu Zhang, Gong Lanqi, Xin-Yuan Guan, Wei Dai. The role of EBV+ nasopharyngeal carcinoma in shaping double-negative B cells to form a pro-metastatic niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6035.