Layla T. Greten

Abstract 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.

Introduction The cellular composition of the hepatocellular carcinoma (HCC) tumor microenvironment (TME) has major impact on tumor initiation, progress, and therapy response. Mucosal-associated invariant T (MAIT) are an abundant T cell subtype in the human liver and play a crucial role in regulating immunity and inflammation. Yet, their role in HCC and their potential for cancer immunotherapy remains elusive.

<h3>Background</h3> 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 influences 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 are assigned crucial roles in regulating immunity and inflammation in the context of infection, albeit their role in HCC remains elusive. <h3>Methods</h3> Study design is displayed in figure 1. High-dimensional flow cytometry (n=37) and scRNA sequencing (n=8) was used to analyze MAIT cell phenotypic changes in patient tissue samples. Highly-multiplexed immunofluorescence microscopy was used to quantify immune cell infiltration in paired human HCC samples. We developed and validated a 37-plex antibody panel and applied CODEX technology to simultaneously profile in situ expression of 37 proteins at sub-cellular resolution in n=15 HCC patient samples using whole slide scanning. We established an image analysis pipeline using a machine learning (ML) algorithm (S³-CIMA) to quantify the MAIT cell interaction network at the HCC invasive front. Murine models of orthotopic HCC using transgenic mouse strains were used for <i>in vivo</i> validation, a co-culture system using sorted MAITs from primary human liver cancer tissue was established to gain further mechanistic insight. <h3>Results</h3> Hepatic MAIT cells in n=37 patient samples are characterized by impaired infiltration (p&lt;0.001) into tumor lesions, increasing dysfunction (e.g. upregulation of PD-1 (p&lt;0.05) &amp; reduced IFN-γ (p&lt;0.01) within the HCC TiME and show previously underappreciated heterogeneity as seen by scRNA-seq. CODEX imaging revealed the distinct cellular composition of the MAIT neighborhood in human HCC tissue. This allowed for in-depth characterization of cellular interaction networks underlying the MAIT cell dysfunction in HCC. S³-CIMA, a novel ML method, to analyze our spatially resolved immune cell atlas of human liver cancer identified interactions of CSF1R⁺PD-L1⁺ tumor-associated macrophages (TAMs) and MAIT cells localized in the adjacent (non-tumor) liver as key regulatory elements of MAIT cell dysfunction. Finally, perturbation of this detrimental cell-cell interaction using PD-L1 and CSF1R blocking strategies or depletion of TAMs using <i>Lysm^Cre x Csf1r^LsL-DTR</i> transgenic mice increased MAIT cell infiltration (p&lt;0.05) into murine HCC lesions and reinvigorated the cytotoxic MAIT cell phenotype (p&lt;0.01). <h3>Conclusions</h3> This work provides evidence that MAIT antitumor immunity and response to ICB therapies relies on organized, spatially nuanced interactions between MAITs and PD-L1+CSF1R+ TAMs within the tumor immune microenvironment. <h3>Acknowledgements</h3> B.R. was supported by the International Liver Cancer Association (ILCA) Fellowship Award 2021, M.B. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany9s Excellence Strategy – EXC number 2064/1. S.B. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany9s Excellence Strategy – EXC number 2180. N.K was supported by the Intramural Research Program of the NIH, NCI (ZIC BC 011434). S.W. was funded by the Deutsche Forschungsgemeinschaft (WA-4610/1-1), A.K. acknowledges funding support from the National Institute of Allergy and Infectious Diseases (R01AI132389; R21AI130800). X.W.W. was supported by the Intramural Research program of the NIH, NCI (ZIA BC 010313). T.F.G. was supported by the Intramural Research Program of the NIH, NCI (ZIA BC 011345). <h3>Ethics Approval</h3> All patients gave informed consent for collection of clinical information, tissue acquisition under the Institutional Review Board (IRB)-approved protocol IRB #2017-0365 and the material transfer agreement (M.T.A. #43655-18) between the GUH and the U.S. National Cancer Institute (NCI).

Abstract 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.