Abstract 5481: Spatial multiplex proteins profiling of tumor microenvironment to decipher de novo metastasis in nasopharyngeal carcinoma
Authors: Chow Larry Ka-Yue , Wang Ying , Gong Lanqi , Xu Siqi , Hou Zhaozheng , Guan Xinyuan , Kwong Dora Lai-Wan , Lee Victor Ho-Fun , ng Wai To , Lee Anne Wing-Mui , Liu Zhonghua , Xia Yunfei , Dai Wei 
Published: March 2024

Abstract
Background: Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southeast Asia, where more than 90% of NPC cases are associated with Epstein-Barr virus (EBV). Although NPC patients are generally sensitive to radiotherapy and chemotherapy, about 20-30% of patients have treatment failure due to the development of distant metastasis, i.e. de novo metastasis. In this study, we utilized GeoMx Digital Spatial Profiler (DSP), a multiplex and high-throughput sequencing platform designed for spatially- and morphologically resolved protein expression profiling to investigate the intra-tumoral and inter-tumoral heterogeneity of tumor microenvironment (TME) and identify the mechanisms underlying distant failure in NPC.
Methods: We utilized GeoMx Protein Assays consisting of 147 proteins from 15 functional modules to compare the difference between NPC cases developing distant failure (DF) (n=21) and stage-, age-, gender- and treatment-matched NPC cases without developing distant failure (noDF) (n=15) within five years of treatment. Two fluorescent morphological markers including PanCK and CD45, were used for delineating the structural architecture of the tissues, identifying cell types, and selecting regions of interest (ROIs), followed by sequencing for protein expression quantification. NanoString GeoMx DSP Analysis Suite was used for quality control and data normalization. The NPC TME heterogeneity was further investigated by the 10X Visum platform for spatial transcriptome. The public single-cell RNA sequencing (scRNA-Seq) and bulk RNA sequencing datasets were obtained for the downstream analyses.
Results: By analyzing protein expression profiles, the study revealed the intratumoral and intertumoral heterogeneity associated with EBV latent infection in the TME of NPC. Regions with high EBV latent gene expression exhibited a noticeable reduction in T cell infiltration, indicating an "immune cold" phenotype, whereas regions with low EBV latent gene expression displayed heavy T cell infiltration, indicating an "immune hot" phenotype. Moreover, the study identified a panel of autophagic and anti-apoptotic proteins that were significantly upregulated in the cancer cells of NPC cases developing distant failure after treatment. This observation, supported by the single-cell RNA sequencing datasets, further implicated EBV latent infection for the upregulation of autophagic and anti-apoptotic genes. Notably, the study also demonstrated that the genes upregulated in the immune cells in NPC cases with distant failure were associated with poor progression-free survival in an independent NPC cohort.
Conclusions: These findings shed light on the crucial role of EBV latent infection in TME heterogeneity and highlight the potential involvement of autophagy and anti-apoptosis in de novo metastasis in NPC.
Citation Format: Larry Ka-Yue Chow, Ying Wang, Lanqi Gong, Siqi Xu, Zhaozheng Hou, Xinyuan Guan, Dora Lai-Wan Kwong, Victor Ho-Fun Lee, Wai To ng, Anne Wing-Mui Lee, Zhonghua Liu, Yunfei Xia, Wei Dai. Spatial multiplex proteins profiling of tumor microenvironment to decipher de novo metastasis in nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5481.