Abstract 6463: Interrogating the tumor microenvironment of nasopharyngeal carcinoma by single-cell spatial transcriptomics

Abstract Introduction: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignancy characterized by a dense lymphocyte infiltrate and a complex tumor microenvironment (TME). This heterogenous ecosystem influences tumor progression, immune evasion and the variable treatment outcomes observed in NPC patients. Conventional approaches such as bulk RNA sequencing lack the resolution to capture the complex heterogeneity of the TME. Spatial transcriptomics allows us to uncover the heterogeneity and cellular interactions at a single-cell resolution, providing insights into the tumor biology of NPC. Methods: Recent formalin-fixed, paraffin-embedded (FFPE) NPC tissue samples were selected for analysis with the NanoString CosMx Spatial Molecular Imaging platform. Quality control, dimension reduction and unsupervised clustering at a resolution of 0.6 using the Louvain/Leiden algorithm were performed using Seurat package (v5.1.0). Cluster level annotation was performed using the singleR package using the human primary cell atlas reference. Cell type identities were validated by comparing cluster specific marker genes with canonical markers of known cell types. Results: Six NPC tissue biopsies were fully interrogated comprising 254 ROIs. Post-filtering, 162, 290 cells were examined. A total of 7 clusters were identified, broadly comprising immune, stromal and epithelial cells. Tumor composition was heterogenous (B cell 4.68-50.4%, T cell 0.74-8.78%, endothelial cells 0.91-3.66%, epithelial cells 28.5-66.8%, fibroblasts 1.16%-23.9%, macrophages 6.43%-29.1%, pro B cell CD34+ 0.01-7.1%) despite similar clinical stages III-IV. Conclusion: We describe the single-cell spatial landscape of nasopharyngeal carcinoma, providing insights into the heterogeneity of the TME. We will evaluate epithelial - immune cell interactions, differential biological processes at the cellular level and compare them with deep gene expression profiles from our larger laser-capture microdissection mRNA-Seq cohort of patients to glean further insights. Citation Format: Chee Yit Lim, Yi Ren, Wei Keat Teo, Chor Hiang Siow, Kwok Seng Loh, Raymond KY Tsang, Bingcheng Wu, Joshua K. Tay. Interrogating the tumor microenvironment of nasopharyngeal carcinoma by single-cell spatial transcriptomics [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 6463.