Abstract Spatial biology techniques such as RNAscope™ and multi-omics in situ hybridization (ISH) offer critical insights into tumor biology and the tumor microenvironment by enabling spatially resolved gene expression analysis. However, conventional ISH methods often lack the sensitivity required to detect low-abundance transcripts, which can hold key mechanistic, prognostic, and therapeutic significance. In this study, we present an enhanced and optimised ISH approach that integrates a new specifically designed range of fluorophores for tyramide signal amplification (TSA) with RNAscope™ Multiplex Assay probes to improve the detection of immuno-oncology (IO)-related targets. These two new reagents targeted at 620nm and 690nm wavelengths increase the number of assessable targets and enable robust four-plex detection of heterogeneously expressed transcripts. Our findings demonstrate that the new bright TSA reagents significantly increase the sensitivity and dynamic range of transcript and protein detection, facilitating the reliable identification of targets across a spectrum of expression levels. The newly developed four-plex dye system further expands upon multiplexing capabilities, supporting comprehensive spatial profiling in complex tumor tissues. Citation Format: Marco T. Pinheiro, Paul Wood, Sean Goggins, Henry Lamparski, John Donaldson, Grant Esomonu, Renzo Adilardi, Emerald Doolittle, Rose Delvillar, Hannah Maple. Novel tyramide signal amplification dyes optimized for the detection of IO markers and four-plex detection of heterogeneously expressed targets abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 811.
Pinheiro et al. (Fri,) studied this question.
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