Abstract 386: Design and application of an 8-plex multiplex immunofluorescence panel for deep phenotypic profiling of the tumor microenvironment using InSituPlex technology
Authors: Wong Alexis , Koerner Mara , Patel Katir K. , Potier Anna , Hennek Stephanie , Abubakar Mustapha , Olanich Mary E. , Golubeva Yelena G. , Mutreja Karun , Lawrence Scott 
Published: September 2020

Abstract
Background: The tumor microenvironment is composed of a complicated network of different cell types and activation states, the exact composition of which is important for immuno-oncology research efforts to correlate treatment options and outcome. The number of biomarkers necessary to sufficiently profile the various cellular phenotypes and elucidate contextual relationships in situ is larger than can be accommodated by standard immunohistochemistry (IHC) approaches. Furthermore, conventional IHC requires more sections, potentially exhausting tissue sample availability. To overcome these limitations, Ultivue has developed InSituPlex® technology and UltiMapper™ assays that enable biomarker multiplexing in tissue samples with an automated workflow to support high sample throughput.
Methods: An 8-plex immune-profiling panel using the InSituPlex approach was designed and tested to detect and classify T cells, macrophages, and tumor cell populations along with PD-L1 checkpoint expression in multiple tumor types. This assay was designed, developed, and tested using UltiMapper reagents and corresponding protocols. The assay was run on multiple FFPE tissue samples from breast cancer, NSCLC, and colorectal cancer (CRC) cases. Each sample was stained for the following 8 markers: CD3, CD4, CD8, CD68, FoxP3, PD-1, PD-L1, and pan-CK. Staining was performed in a single step using the Leica Biosystems BOND RX autostainer. Whole slide imaging (WSI) was performed in two steps on the ZEISS Axio Scan.Z1 slide scanner, and image analysis was performed using HALO software from Indica Labs.
Results: The 8-plex immune-profiling panel demonstrated specific biomarker staining and was verified for staining reproducibility. The 8-plex workflow was compatible across multiple tumor tissue types. The samples were characterized by cell analysis and phenotyped based on their expression of specific biomarkers. The following cell phenotypes were detected: T cells (CD3+), cytotoxic T cells (CD3+/CD8+), regulatory T cells (CD4+/FOXP3+), exhausted T cells (CD3+/PD-1+), macrophages (CD68+), and tumor cells (CK+) with a dynamic range of PD-L1 expression levels.
Conclusion: The 8-plex panel revealed important immunophenotypes across multiple sample types. The assay was automated and optimized for a high-throughput workflow. By testing across multiple tissue types, this assay showcases the potential for broad application across additional cancer types. The ability to interrogate an 8-plex I/O panel on a single slide with WSI presents an attractive approach for the next generation of IHC, enabling profiling within the tumor microenvironment and sensitive detection of PD-L1 expression in cancer tissues.
Funded by NCI Contract No. 75N910D00024
Author affiliations for CGR staff: Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD 21702
Citation Format: Alexis Wong, Mara Koerner, Katir K. Patel, Anna Potier, Stephanie Hennek, Mustapha Abubakar, Mary E. Olanich, Yelena G. Golubeva, Karun Mutreja, Scott Lawrence. Design and application of an 8-plex multiplex immunofluorescence panel for deep phenotypic profiling of the tumor microenvironment using InSituPlex technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 386.