Abstract 1156: A systematic comparison of common multiplex immunohistochemistry (mIHC) methodologies to the novel SignalStarTM assay in the characterization of the tumor microenvironment (TME)
Authors: Ziello Jennifer , Papalegis Derek , Vu Lily , Goldman Kelsey , Loebelenz Jean , Sponaugle Shleby , Tkachev Sasha , Klein Sarah 
Published: March 2024

Abstract
The emergence of an increasing number of immunotherapy biomarkers and their importance within the spatial context of the TME has resulted in a concomitant need for reliable and accurate mIHC assays. Many of the currently available mIHC technologies do not offer amplification, resulting in the loss of detection of low expressing cells, while others require deposition and cycling of antibodies, which can lead to epitope masking and degradation, respectively. The SignalStar mIHC assay from Cell Signaling Technology is a novel methodology used to label, amplify and visualize up to 8 targets within the same formalin fixed, paraffin-embedded carcinoma (FFPE) tissue section without the need for fluorophore deposition or antibody cycling.
In this study, the SignalStar mIHC assay was used to simultaneously label 8 targets including TIM-3, PD-1, PD-L1, LAG3, CD3, CD68, CD8 and Pan-Keratin in FFPE carcinoma tissue. A network of complementary, fluorescently-labeled oligonucleotides was used to amplify the signal of the first 4 antibody-oligo conjugates followed by tissue imaging. The fluorescent signal was then removed, and the process repeated.The resulting two 4-plex images were then computationally aligned with QuPath. This methodology was compared to the Tyramide Signal Amplification (TSA) deposition assay, as well as to indirect and direct immunofluorescent detection with respect to mean fluorescence intensity (MFI) per cell and percent positivity of cells in matched regions of interest in serial sections. All mIHC strategies were in turn compared to the canonical chromogenic IHC assay. Furthermore, the fluorescent signal from the 8-plex SignalStar assay was removed and the same tissue was stained with fluorescent direct conjugates to achieve even higher plex.
Our data demonstrates that the SignalStar assay detected similar percentages of cells when compared to chromogenic and TSA staining. Alternatively, the SignalStar assay often outperformed indirect and direct immunofluorescent detection. SignalStar was also successfully combined with a panel of direct Alexa Fluor® dye conjugates, showing the complementarity of these assays in such a way that both plex and flexibility can be increased. Ultimately, we demonstrate that SignalStar mIHC is a useful tool in the characterization and analysis of the complex TME. SignalStar mIHC outshines existing technologies as it does not require deposition in order to amplify the signal of multiple colocalizing targets, including low abundance proteins, while also maintaining dynamic range.
Citation Format: Jennifer Ziello, Derek Papalegis, Lily Vu, Kelsey Goldman, Jean Loebelenz, Shleby Sponaugle, Sasha Tkachev, Sarah Klein. A systematic comparison of common multiplex immunohistochemistry (mIHC) methodologies to the novel SignalStarTM assay in the characterization of the tumor microenvironment (TME) [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 1156.