Abstract 3769: Spatial biology in situ hybridization and multiplexed immunohistochemistry detection compatibility using a modified protocol

Abstract

Abstract In the field of spatial-omics imaging, combining mRNA in situ hybridization (ISH) detection with multiplexed protein immunohistochemistry detection (IHC) is desirable. We present a simplified workflow in FFPE tissue sections where ISH is detected using branched ssDNA signal amplification visible by brightfield imaging combined with multiplexed IHC using directly labeled antibodies detected by fluorescence. In multiple studies mRNA expression of PCNA (Proliferating Cell Nuclear Antigen) shows moderate to high nuclear positivity observed in most cancer cells, with gliomas and lymphomas being the few negative exceptions. In contrast, expression of PCNA in adult non-cancerous tissue has low to no expression, with tonsil and bone marrow being the exceptions. We first show the relationship between proliferation as measured by EdU detection, PCNA antibody and PCNA mRNA expression in the embryonic mouse model where PCNA expression is prevalent in developing tissue. Then we demonstrate a simplified working protocol for combining ISH and IHC in FFPE human cancer tissue sections for the co-detection of PCNA mRNA together with IHC using a panel of six protein markers relevant to the tumor microenvironment. For IHC, we directly labeled validated unconjugated antibodies with fluorophores using amine-reactive chemistry and select the degree of labeling (DOL) and titer for optimal signal/background most useful for multiplexing on FFPE tissue. We found important protocol steps to consider for compatibility between IHC and ISH detection method. Loss of IHC protein epitope due to ISH protocol use of proteases and loss of ISH signal due to presence of RNase commonly present in IHC antibody relevant reagents can prevent combining IHC with ISH. Stabilizing IHC through refixation prior to ISH workflow is also required. Modifying the protocols to avoid these incompatible treatments and using directly labeled antibodies provides useful strategies for multiplexing IHC with ISH. Citation Format: Scott T. Clarke, Mae Voeun, Leticia Montoya, Chris Vonnegut, Bhaskar Mandavilli. Spatial biology in situ hybridization and multiplexed immunohistochemistry detection compatibility using a modified protocol [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 3769.