Abstract 72: A spatial interactomics approach reveals interplay between the tumor microenvironment and T cell activation
Authors: Edén Desirée , Vennberg Jonas , Büsgen Tanja , Raykova Doroteya , Zieba-Wicher Agata 
Published: March 2024

Abstract
Background: T cells play a fundamental role in immune response to tumors. However, the tumor microenvironment (TME) - a complex network of cells, signaling molecules, and extracellular matrix components - employs various strategies to evade immune surveillance and suppress T cell activity. Among the many molecular interactions taking place in the TME, the role of CD8/MHC I, LAG3/MHC II and PD1/PDL1 is pivotal in modulating anti-cancer response and the escape routes that cancer in turn uses to withstand immune defense. LAG3/MHC II and PD1/PDL1 interactions are inhibitory, causing T cell dysfunction and exhaustion, while the CD8/MHC I interaction is essential for initiating an effective anti-tumor response. Here, we used a cutting-edge in situ proximity ligation strategy combined with immunofluorescence (IF) to reveal these key interactions and TME biomarkers in hepatocellular carcinoma and Hodgkin lymphoma. Since the method does not compromise the structural integrity of tissues, communication between proteins can be studied in their native milieu.
Method: Protein-protein interactions (PPIs) were detected with the NaveniFlex Tissue Atto647N kit in human FFPE tissues according to the manufacturer’s protocol. This technique can detect proteins located within interaction range (&lt;40 nm) via oligonucleotide-antibody conjugates that create amplified fluorescent signal. Parallel IF co-staining for α-fetoprotein, Ki67, CD30 and CD3 was performed during the Naveni® detection step. Slides were mounted and imaged on the Olympus V200 automated scanner.
Results: Tumor cells were identified by α-fetoprotein/Ki67 (hepatocellular carcinoma) or CD30 (Reed-Sternberg in Hodgkin lymphoma) IF staining, and tumor-infiltrating lymphocytes were visualized by CD3 staining. With the help of the Naveni® assay, we observed abundant interaction between CD8/MHC I in hepatocellular carcinoma and Hodgkin lymphoma. Suppressive immune checkpoint interactions such as LAG3/MHC II and PD1/PDL1 were also detected. In the case of Hodgkin lymphoma, the CD8/MHC I, LAG3/MHC II and PD1/PDL1 interactions were largely seen in the same areas, and specifically between T cells and Reed-Sternberg cells, indicating a crosstalk between the TME and the immune landscape in the cancer tissues.
Conclusion: The interactomics approach, employing in situ proximity ligation for observing protein function as opposed to mere expression, allows the detection of key PPIs in the TME. Understanding the intricate crosstalk between them is crucial in developing effective immunotherapies. Targeting these pathways simultaneously or in combination, such as using immune checkpoint inhibitors against PD1/PDL1 while exploring strategies to modulate LAG3/MHC II and CD8/MHC I interactions, holds promise in reinvigorating T cell responses against cancer and improving the efficacy of immunotherapies within the complex landscape of the TME.
Citation Format: Desirée Edén, Jonas Vennberg, Tanja Büsgen, Doroteya Raykova, Agata Zieba-Wicher. A spatial interactomics approach reveals interplay between the tumor microenvironment and T cell activation [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 72.