Abstract 4336: Automation of proximity ligation immunoassay for interaction between PDL1 and PD1 detection in the tumor microenvironment using microfluidic based system
Authors: Elofsson Hampus , Wicher Agata Zieba , Sepulveda Carolina Oses , Ullman Tony , Procopio Maria-Giuseppina , Faillétaz Alix , Dupouy Diego G. , Stadler Charlotte 
Published: April 2023

Abstract
The PD-1/PD-L1 signaling pathway is essential for immune control and maintaining immune system balance. PD-1 and PD-L1 proteins exert most functions in cells and tissues by undergoing modifications and forming dynamic complexes – effects that cannot be explored by genomics, transcriptomics, or conventional immunostaining methods. Numerous cancer therapies are being developed that affect PD-1/PD-L1 signaling, and tools to study the PD-1/PD-L1 axis are, therefore, essential. One prerequisite for successfully establishing diagnostic assays is reproducibility, standardization, and, ideally, high sample throughput via automation. Here, we aimed to introduce a new technological solution to allow for the simultaneous detection of PD-L1 and PD-1 interaction and the adjacent immune cell context in tissue samples on the Lunaphore’s automated microfluidic staining platform. The assay for detecting PD-1-PD-L1 interaction was developed and technically validated by Navinci Diagnostics. Although the detection of interaction alone provided important information on the immune status in the tumor microenvironment, we believed that PD-1/PD-L1 readout would be most powerful in the context of its cellular ecosystem. Therefore, the PD-1/PD-L1 assay was combined with the analysis of immune and cancer cells to determine their involvement in this signaling. The feasibility of combining multiplex immunofluorescence with proximity ligation assay was conducted and confirmed on human tonsils. The spatial profiling panel included immune markers for all T-cells (CD3), helper T-cell (CD4), cytotoxic T-cells (CD8), B-cells (CD19/20) and activation/exhaustion markers granzyme B (activated cytotoxic T-cell), Ki67 (proliferating cells), and LAG3 (malignant B cells, CD8 T cells, CD4 Tregs). We evaluated, implemented, and optimized the assays on the Lunaphore staining instrument to reduce labor time and cost for the end users. In the next phase, the potential of our assay to be benchmarked as a diagnostic tool in clinical praxis will be thoroughly tested and validated on diagnostic tissue samples from NSCLC patient cohorts. We believe this approach will enable spatial and functional studies of the interface between the tumor and the immune system and provide necessary information about signaling pathway activation in situ, the latest representing a novel state-of-the-art in tissue diagnostics.
Citation Format: Hampus Elofsson, Agata Zieba Wicher, Carolina Oses Sepulveda, Tony Ullman, Maria-Giuseppina Procopio, Alix Faillétaz, Diego G. Dupouy, Charlotte Stadler. Automation of proximity ligation immunoassay for interaction between PDL1 and PD1 detection in the tumor microenvironment using microfluidic based system. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4336.