Abstract 5767: Multiplex in situ proximity ligation assay for functional studies of proteins and their interactions in patient samples

Abstract Detecting protein-protein interactions (PPIs) and their localization is essential for the understanding of protein function and enables deep-diving into processes such as signal transduction, synaptic signaling, regulation of gene expression in cancer, identification of diagnostic markers, patient stratification and more. In situ proximity ligation assay (isPLA) is a well-established tool for the study of PPIs in cell and tissue samples. It detects proteins located within 40 nm of each other via antibodies conjugated to oligonucleotides that generate amplified fluorescent or chromogenic signal. The method can be automated and/or combined with multiplexed immunofluorescence (mIF) staining of individual proteins to visualize tissue context e.g., the tumor microenvironment or the immune landscape. However, current isPLA technology has been limited to the detection of a single PPI at a time. We hereby present a new multiplexed mix-and-match approach to isPLA, which overcomes this limitation. We developed a 9-plex assay based on the Naveni® technological solution, suitable for the simultaneous detection of up to nine PPIs or single proteins (here collectively referred to as markers) of the user’s choice. Every marker is detected by two directly conjugated primary antibodies (Navenibodies) that participate in the isPLA reaction provided the proximity criterion is fulfilled, leading to specific and strong signal. Additional cycles incorporating other technologies such as mIF or HCR-FISH are also compatible with the method. For isPLA markers, users can choose from a set list of possibilities, including various signal transduction networks such as the RAS/MAPK pathway, the PD1/PD-L1 and PD1/PD-L2 receptor-ligand interactions, various B- and T-cell markers, cell adhesion proteins and more. Thanks to this flexible approach, it was possible to create a tailored 9-plex study, investigating interactions involving and downstream of PD1, as well as activation of nodes in the RAS/MAPK pathway. ERK activation downstream of KRAS mutations has been shown to increase PD-L1 expression in several cancers (e.g., lung and colorectal cancer), which in turn helps tumor cells evade immune detection by binding to PD1 on T-cells, inhibiting T-cell activity and promoting immune tolerance to the tumor. Our data could help indicate which patients would be the best candidates for immune checkpoint or MAPK inhibition therapy. In summary, our multiplex isPLA approach is a sensitive and specific method that visualizes low and high abundant targets in tissues and adds value by uncovering protein interplay in complex signaling networks with implications in translational research. Citation Format: Hampus Elofsson, Desirée Edén, Olof Hahne, Linda Arngården, Frida Tallqvist, Carl-Magnus Clausson, Axel Klaesson, Annica Önell, Sara Bodbin, Caroline Gallant, Agata Zieba Wicher. Multiplex in situ proximity ligation assay for functional studies of proteins and their interactions in patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5767.