Abstract 7517: Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging
Authors: Ullman Tony , Krantz David , Elofsson Hampus , Sepulveda Carolina Oses , Zieba-Wicher Agata , Östling Päivi , Ullén Anders , Basu Subham , Stadler Charlotte 
Published: March 2024

Abstract
Background: Programmed cell death protein 1 (PD1) and its ligand PD-L1 are pivotal components of the immune checkpoint machinery, playing a crucial role in the regulation of immune responses in cancer. The interaction between PD1 and PD-L1 is a significant therapeutic target in oncology, as its inhibitors have shown improved outcomes across a variety of malignancies. However, the treatment response to immunotherapies such as pembrolizumab has shown a modest response (~30%) in previous studies. This could be explained by the lack of molecular insight pertaining to the interaction of PD1 on T-cells and PD-L1 in the tumor cells in malignant tissues at the time of clinical diagnosis. The recent development of in situ Proximity Ligation Assay (isPLA), marks a significant advancement in the field. This method allows for the visualization of direct protein interactions within tissues, for example between PD1 and PD-L1. This adds an important layer of information compared to multiplexed immunofluorescence, which is useful for deep phenotyping of cell types in a tissue but lacks the ability to detect direct interactions between specific cells.
Methods: PD-L1-PD1 interactions in human FFPE tonsil and bladder cancer tissue were identified using the NaveniFlex Tissue Atto647N kit, following the guidelines provided by the manufacturer. This method is capable of detecting proteins within a proximity of less than 40 nm, utilizing oligonucleotide-antibody conjugates that generate an enhanced fluorescent signal. After completing the Naveni® detection process, we performed multiplexed immunofluorescence (IF) using the Phenocycler platform and a panel of 14 barcoded antibodies, to generate isPLA and multiplexed IF data on the same tissue section.
Results & Discussion: We have successfully established an automated workflow for the isPLA protocol. In this work, we aim to continue this effort and combine the isPLA, with downstream highly multiplexed immunofluorescence using the Phenocycler platform. We have generated proof of concept data for the successful integration of the two methods, using tonsil and bladder cancer tissue to detect protein interactions between PD1 and PD-L1, as well as phenotypic profiling of the tumor microenvironment with a 14 plex panel. This panel allows us to phenotype the relevant cell types (B cells, dendritic cells, T cells, macrophages, tumor cells and their proliferation status) and subsets of T-cells like cytotoxic CD8+ cells. The integration of the isPLA and multiplexed IF is poised to significantly advance our understanding of the TME and hopefully aid in patient stratification for immune checkpoint inhibition therapy. The clinical significance of this method integration will later be explored in a retrospective cohort of muscle invasive bladder cancer, receiving immune checkpoint inhibition therapy with pembrolizumab as a second line treatment.
Citation Format: Tony Ullman, David Krantz, Hampus Elofsson, Carolina Oses Sepulveda, Agata Zieba-Wicher, Päivi Östling, Anders Ullén, Subham Basu, Charlotte Stadler. Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging [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 7517.