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Introduction: Hepatocellular Carcinoma (HCC) is considered a prototype of inflammation-derived cancer arising from chronic liver injury. The cellular composition of the HCC tumor immune microenvironment (TiME) has a major impact on cancer biology as the TiME can influence tumor initiation, progress, and response to therapy. Mucosal-associated invariant T (MAIT) cells can represent the most abundant T cell subtype in the human liver and have been found to be impaired in both number and function in liver cancer. These innate-like T cells are assigned crucial roles in regulating immunity and inflammation in the context of infection, albeit their role in HCC remains elusive. Methods: High-dimensional flow cytometry was used to analyze MAIT cell phenotypic changes in murine and human liver cancer. Highly multiplexed immunofluorescence microscopy was used to quantify immune cell infiltration in primary human HCC samples. We developed and validated a comprehensive 37-plex antibody panel for immunofluorescence imaging of human fresh frozen HCC samples. We applied co-detection by indexing (CODEX) technology to simultaneously profile in situ expression of 37 proteins at sub-cellular resolution in 15 HCC patient samples using whole slide scanning. Initial image analysis was performed using HALO quantitative image analysis software. Finally, we established an image analysis pipeline to quantify the MAIT cell interaction network at the HCC invasive front. Results: Profiling of human and murine HCC using flow cytometry and highly multiplexed CODEX imaging revealed substantial dysregulation/aberrant activation of MAITs in liver cancer. In situ phenotyping of 4,500,000 single cells (including 1,500,000 CD45+ immune cells) allowed for the quantification of 20 distinct immune cell phenotype clusters, differential analysis of activation markers and spatial features of each individual cell. CODEX imaging revealed detailed composition of the MAIT cell niche in human liver cancer tissue allowing for further distinct spatial analysis including infiltration and nearest-neighbor analysis. Importantly, flow cytometry data of paired samples correlated well with image-based immune phenotyping. Beyond that, whole slide imaging revealed spatial relationships and interactions within the MAIT cell hub localized in distinct tissue regions. Conclusion: Here, we demonstrate that spatially resolved, single-cell analysis of human liver cancer tissue allows for in-depth characterization of interacting immune cellular programs underlying MAIT cell dysfunction in HCC. Citation Format: Benjamin Ruf, Noemi Kedei, Matthias Bruhns, Sepideh Babaei, Bernd Heinrich, Varun Subramanyam, Chi Ma, Simon Wabitsch, Benjamin Green, Kylynda C. Bauer, Yuta Myojin, Jonathan Qi, Amran Nur, Justin McCallen, Layla Greten, William G. Telford, Merrill K. Stovroff, Kesha Oza, Jiman Kang, Alexander Kroemer, Manfred Claassen, Firouzeh Korangy, Tim F. Greten. Spatially resolved immune cell atlas of human liver cancer identifies the cellular interaction network underlying mucosal-associated invariant T (MAIT) cell dysfunction in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2106.

Introduction: Hepatocellular Carcinoma (HCC) is a leading cause of cancer-related death and can be considered a prototype of inflammation-derived cancer arising from chronic liver injury. The cell composition of the HCC tumor immune microenvironment (TiME) has a major impact on cancer biology as the TME can have divergent capacities on tumor initiation, progress, and response to therapy. Recent development of multi-omics and single-cell technologies help us to comprehensively quantify the cellular heterogeneity and spatial organization of the TiME and to further our understanding of antitumor immunity. Methods: Multiplexed immunofluorescence microscopy and high-dimensional flow cytometry was used to analyze immune cell infiltration in primary human liver cancer samples. We developed and validated a comprehensive 37-plex antibody panel for immunofluorescence imaging of human fresh frozen HCC samples. We applied highly multiplexed co-detection by indexing (CODEX) technology to simultaneously profile in situ expression of 37 proteins at sub-cellular resolution in 15 HCC patient samples using whole slide scanning. We established an image analysis pipeline to quantify all major cell populations in the human liver using supervised manual gating and unsupervised clustering algorithms. Proximity and nearest neighbor calculations as well as infiltration analysis was performed using HALO quantitative image analysis software. Results: Using high-dimensional flow cytometry and parallel spatially resolved quantitative analysis of multiplexed immunofluorescence microscopy images, we profiled the single-cell pathology landscape for human HCC. The translation from raw images to numerical output was successfully established. In situ phenotyping of 4,500,000 single cells (including 1,500,000 CD45+ immune cells) allowed for the quantification of cell phenotype clusters, differential analysis of activation markers and spatial features of each individual cell. CODEX imaging revealed detailed composition of the immune cell niche in human liver cancer tissue allowing for further distinct spatial analysis including infiltration analysis and nearest-neighbor analysis. We found impaired infiltration of CD163+ macrophages, granulocytes, CD8+ T cells, NK cells and MAIT cells into human HCC tumors compared to unaffected liver tissue. whereas regulatory T cells accumulated in tumor tissue. Flow cytometry data correlated well with image-based immune phenotyping. Beyond that, whole slide imaging allowed for the identification of the tumor-to-liver interface as a unique site of immune cell inhibition. Conclusion: Here, we demonstrate that spatially resolved, single-cell analysis of human liver cancer tissue allows for the in-depth characterization of the immune cell composition of HCC. This tool can be used for biomarker research, to determine cellular functional states in intact tissue and to spatially and functionally quantify interactions between immune cells in the context of hepatocarcinogenesis. Citation Format: Benjamin Ruf, Noemi Kedei, Matthias Bruhns, Sepideh Babaei, Vanessa V. Catania, Simon Wabitsch, Chi Ma, Bernd Heinrich, Varun Subramanyam, Merrill K. Stovroff, Layla T. Greten, Alexander Kroemer, Manfred Claassen, Tim F. Greten, Firouzeh Korangy. Highly multiplexed spatial analysis of the HCC tumor immune microenvironment using CODEX imaging [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P021.