Abstract Background: Analyzing interactions and the microanatomical distribution of the tumor immune microenvironment (TIME) is essential for a comprehensive understanding of tumor progression. Imaging Mass Cytometry (IMC) is a high-dimensional tissue imaging system that enables the deeper and multiparametric in situ exploration of tumor microenvironments at a single-cell level. This study describes a pipeline for analyzing 39-plex IMC images of human HPV(+) oropharyngeal squamous cell carcinoma (OPSCC) tumor sections, elucidating immune cells' nature, functions, and interactions with tumor cells. The IMC data analysis provides valuable information for clinical studies that could be used for the identification of prognostic biomarkers and mechanisms of resistance to current immunotherapies. Methods: A retrospective study was conducted on (N=20) patients (10 progressors and 10 non-progressors) with HPV(+) OPSCC. The dataset comprises paired hematoxylin and eosin (H&E) images with IMC data from multiple Regions of Interest (ROIs) (N=82), enabling the in-situ profiling of 39 proteins in primary tumor tissue. Cell segmentation was followed by employing original images and their segmentation masks for cell phenotype identification using a probabilistic neural network method to spatially profile cancer cells, macrophages, NK-M cells, B-cells, and distinct T-cell populations. We identified protein expression at single-cell resolution and investigated the impact of multicellular spatial organization on the disease progression. The spatial neighborhood interaction score of tumor cells with various other cells was computed for all the ROIs across the two groups. Results: The markers showed varying expression levels of intensity distribution, likely due to differences in tissue sample composition. Specifically, CD3, CD4, CD8, CD19, CD20, and CD68 showed significantly higher intensity in non-progressors. Interestingly, FoxP3 also had a significantly higher intensity level in non-progressors. Cell phenotyping emphasized that non-progressors generally have increased levels of NK-M cell markers. Spatial analysis revealed a significant increase in interaction between CD3 cells and tumor cells in non-progressors (p-value=0.07, Mann-Whitney U test). Furthermore, a significant rise in the interaction between B-cells and FoxP3+ cells was observed in non-progressors (p-value<0.05, Mann-Whitney U test) suggesting a coordinated suppression of tumor growth. Conclusion: We developed a deep learning-based pipeline to analyze spatial relationships in the TIME of IMC data, demonstrating its clinical relevance in predicting disease progression. Our findings emphasize the importance of spatial relationships in the TME for response and suggest that proximity between either CD3+ T-cells to cancer cells or B-cells to FoxP3+ cells are candidate biomarkers. Citation Format: Sumanth Reddy Nakkireddy, Routman David, Kathleen Bartemes, Daniel Ma, Tae Hyun Hwang, Kathryn Van Abel, Chadi Nimeh Abdel-Halim. AI-enhanced spatial proteomics reveals B-cells & FoxP3+ cells interactions in HPV(+) oropharyngeal squamous cell carcinoma Progression [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 5488.
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