Abstract 2069: Simultaneous, high-throughput detection of RNA and protein biomarkers using HCR™ Gold and the OrionTM imaging system for comprehensive analysis of tumor samples in clinical trials

Background: Understanding the tumor microenvironment (TME) during cancer progression and treatment has been aided by multiomic analysis of tissues collected at initial patient screening and after treatment. Cyclic staining methods have recently demonstrated multiomic capabilities on a single tissue, but with drawbacks that can include tissue degradation and processing times up to several days. The OrionTM imaging system overcomes these limitations through single round staining and scanning of whole tissue samples of up to 18 biomarkers simultaneously. When paired with Molecular Instruments’ HCR™ Gold RNA-FISH, a novel product featuring a truly protease-free workflow with next-generation amplification technology, transcriptomic and proteomic multiplex immunofluorescence (mIF) analysis can be carried out in parallel on a single tissue section in a straightforward, high-throughput approach to provide comprehensive insight into tumor biology and immune response. Methods: A 16-plex multiomic assay featuring 5 RNA markers and 11 protein markers was developed to interrogate formalin-fixed paraffin-embedded (FFPE) tissue samples. Following dewaxing and antigen retrieval, HCR™ HiFi Probes against RNA targets of interest were incubated on the tissue sample followed by fluorescently-labeled HCR™ Gold amplifiers for signal generation. Subsequently, ArgoFluorTM-conjugated antibodies directed to proteins of interest were incubated in a single cocktail on the same tissue, streamlining the staining procedure and decreasing downsides inherent to some amplification-based IF methods. Following coverslipping, whole tissues were scanned on OrionTM in one pass. Images were processed and interrogated with a custom analysis pipeline. Results: HCR™ Gold RNA-FISH and mIF protein data for 16 biomarkers were obtained from human colon, lung, gastric, and pancreatic cancer tissues after a single round of staining and scanning. Immune cells were characterized within the TME based on biomarker expression. HCR™ Gold RNA-FISH was used to probe secreted factors, such as the cytokines IL-6, TNFα, and IFNγ, which were found to be colocalized with tumor-associated cells identified by protein mIF. Colocalization of RNA and protein markers differed across tissues. Conclusions: The speed and versatility of Orion™ combined with the 1-step protease- and enzyme-free HCR™ Gold amplification enables development of custom assays to detect both protein and RNA markers on the same tissue in one cycle of staining and scanning. This powerful combination overcomes the limitations of low-plex cyclic staining and long protocol RNA-ISH methods to provide researchers and clinicians with a high-throughput method of translating basic research discoveries into assays for use in clinical trials and diagnostics. Citation Format: Jon J. Ladd, Adam Maddox, Aneesh Acharya, Arturo Ramirez. Simultaneous, high-throughput detection of RNA and protein biomarkers using HCR™ Gold and the OrionTM imaging system for comprehensive analysis of tumor samples in clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2069.