P01.05 Development of signal amplification for spatially-resolved, highly multiplexed biomarker analysis of human tumor tissues

Background Characterizing the complexities of the tumor microenvironment is fundamental to understanding cancer. Spatial relationships between infiltrating immune cells and the existing cellular matrix are now recognized as key determinants of tumor heterogeneity. Nevertheless, most available technologies for studying cells within the context of their tissue microenvironment, like traditional immunofluorescence (IF) and immunohistochemistry (IHC), are limited—allowing the visualization of only a few markers at a time. Materials and Methods CO-Detection by indEXing (CODEX®) technology has overcome this limitation through a DNA-based labeling strategy, involving the sequential addition and removal of dye-labeled oligonucleotide reporters to antibodies equipped with complementary oligonucleotide tags. In this manner, it is possible to visualize tens of antibodies in the same tissue, in situ and at cellular resolution. Additionally, CODEX® interfaces with existing inverted microscopes and provides a cost-effective, fully automated platform for ultra-high plex immunofluorescence imaging. We have expanded the CODEX® platform to include Tyramide Signal Amplification of weak fluorescent signals, i.e. from low-expression biomarkers. This approach was tested with key biomarkers used in routine analyses of the tumor microenvironment, including PD-L1, PD-1 and FOXP3. Results We demonstrate >50X amplification of PD-L1, PD-1 and FOXP3 signals when compared to control tissues. Moreover, we successfully included our amplification step in the CODEX® labeling/imaging workflow, so that it was possible to analyze amplified PD-L1, PD-1 and FOXP3 signals concurrently with a panel of 20+ additional antibodies. Analysis of our data also generated unique biological insights, including increased PD-L1 expression in Treg cells and other tumor and stromal regions. Conclusions Our findings demonstrate the feasibility of amplifying weak biomarker signals in the CODEX® workflow. Furthermore, our experiments were conducted on human formalin fixed paraffin embedded tumor tissues, thereby demonstrating the applicability of CODEX® analyses for clinical and translational research agendas. Disclosure Information O. Braubach: A. Employment (full or part-time); Significant; Akoya Biosciences. S. Mistry: A. Employment (full or part-time); Significant; Akoya Biosciences. G. Dakshinamoorthy: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Yuan: A. Employment (full or part-time); Significant; Akoya Biosciences. P. Noordam: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Kim: A. Employment (full or part-time); Significant; Akoya Biosciences. W. Lee: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Kennedy-Darling: A. Employment (full or part-time); Significant; Akoya Biosciences.