Abstract 1488: Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer
Authors: Bhargava Mahak , Kaur Jaspreet , Saini Geetanjali , Zheng Junjun , Xu Yitian , Chen Shu-Hsia , Pan Ping-Ying , Rewcastle Emma , Kiraz Umay , Janssen Emiel Adrianus , Aneja Ritu 
Published: March 2024

Abstract
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer associated with high rates of recurrence and metastasis, which contribute to poor overall survival. The challenges in treating TNBC stem from its high intratumor heterogeneity and lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 druggable targets. The tumor microenvironment (TME) plays a crucial role in disease progression. Within the TME, complex spatial interactions among tumor cells, immune cells, stroma, angiogenesis- and hypoxia-inducing factors collectively contribute to the advancement and spread of TNBC. In-depth understanding of the TME and interactions of the various components within can elucidate newer therapeutic targets, enhancing the prognosis of TNBC patients. Imaging mass cytometry (IMC) enables detailed analysis of cell-cell and cell-matrix interactions within the TME at the single-cell level. In this study, we aim to characterize the spatial relationships among various cell types within the TNBC TME using IMC, while also investigating differences in the TME of TNBC patients who developed distant metastasis (DM) compared to those who remained non-metastatic (NM). IMC was performed on formalin-fixed paraffin-embedded TNBC primary resection tissues (n= 16; DM= 7, NM= 9) from Stavanger University Hospital, Norway, using a panel of 25 metal-conjugated antibodies. Regions of interest were marked by the pathologist and the slides were laser ablated using Hyperion Imaging System. DeepCell was used to segment single cells based on surface markers. Mean intensity values of all markers for each cell were extracted and consolidated in R scripts for unsupervised clustering. Cell clusters were annotated based on phenotypic markers. Normalized expression of markers was compared for each cell type across DM and NM patients, including markers for cell proliferation, angiogenesis, hypoxia, and cell migration. Neighborhood analysis examined spatial associations between different cell types, highlighting differences in DM and NM patients. The IMC panel successfully provided spatial insights into the TME, including the characterization of tumor cells, immune cells (B cells, T cells, macrophages), endothelial cells, and proliferating cells. We identified 18 unique phenotypic cell clusters that were present at different frequencies in DM and NM patients. These clusters demonstrated different expression profiles of functional markers. t-SNE plots revealed distinct patterns of cell type distribution and unique protein expression profiles, highlighting significant differences between the two groups. Our data suggest a distinct spatial organization of the TME in TNBC patients who develop metastasis. This warrants further investigations to determine the role and interplay of TME features in predicting the risk of distant metastasis in TNBC.
Citation Format: Mahak Bhargava, Jaspreet Kaur, Geetanjali Saini, Junjun Zheng, Yitian Xu, Shu-Hsia Chen, Ping-Ying Pan, Emma Rewcastle, Umay Kiraz, Emiel Adrianus Janssen, Ritu Aneja. Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer [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 1488.