Abstract 2831: Developing and validating a high-throughput 9-color immunofluorescence assay for translational studies in immuno-oncology research
Authors: Remeniuk Bethany , Coltharp Carla , Roman Kristin , Wang Chichung , Neumeister Veronique , Hoyt Clifford 
Published: September 2020

Abstract
Introduction: Advancing our understanding of the underlying mechanisms driving cancer progression is critical to developing new therapeutic regimens and improving patient outcomes. Recent studies utilizing multiplex immunofluorescence have revealed complex cell-to-cell interactions within the tumor microenvironment, however, further examination of the biology comprising these interactions, including cellular composition and spatial interactions, require higher levels of multiplexing. With the rapidly increasing number of available multiplexing approaches, there is an inherent tradeoff between capability and throughput.
Here, we have developed and optimized a 9-color assay and validated its reproducibility across two imaging platforms. This translational workflow offers an optimal balance between multiplexing and sample throughput to facilitate the research and support translational studies on whole formalin-fixed paraffin-embedded (FFPE) tissue.
Methods: Serial sections of FFPE lung cancer tissue were stained using Opal™ reagents. Tissues were stained against the following key immuno-oncology markers of interest: CD8, CD68/CD163, FoxP3, Ki67, PD-1, PD-L1, ATPase/CD45LcA/ CD33, CK, and DAPI nuclear counterstain. The panel was designed on Akoya's Mantra 2 Quantitative Workstation multispectral microscope, which allows for rapid assessment of staining performance. Once optimized, multispectral images were acquired on both the Mantra 2 and Vectra Polaris of the same tissue regions and analyzed to show equivalence between the two platforms. Cell counts, densities, and spatial parameters were generated using Akoya's inForm image analysis software and the R script package phenoptrReports, to produce outputs of the image analysis data. These analyses were also used to evaluate reproducibility of all markers when run in a high-throughput capacity.
Results: Analysis of multispectral images showed concordance between the Mantra 2 and Vectra Polaris-based measurements was generally &gt;95% when comparing cellular expression signals and cell counts based on cell phenotyping classifiers. Dynamic range of detected positive cells for all markers on both imaging platforms was demonstrated an average median of 200:1. Cross talk was undetectable after spectral unmixing despite significant spectral overlap inherent in a 9-color assay. Reproducibility of analyzed regions between platforms was generally &lt;10% coefficient of variation for all markers in the assay, supporting this translational workflow.
Conclusions: We successfully established a standardized process for 9-color multiplexing that offers a balance between elucidating the intricate cellular biology driving disease progression and therapeutic responsiveness within the tumor microenvironment while simultaneously providing a practical and reliable assay that can be implemented to support translational, high-throughput studies in clinical research.
Citation Format: Bethany Remeniuk, Carla Coltharp, Kristin Roman, Chichung Wang, Veronique Neumeister, Clifford Hoyt. Developing and validating a high-throughput 9-color immunofluorescence assay for translational studies in immuno-oncology research [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 2831.