Abstract LB199: Multiplexed <i>in situ</i> detection of protein and glycan expression using CFP Fluor cleavable TSA fluorophores
Authors: Nishinki Muthumuni, Jia Guo, Jing Zhou, Xiaoshan Wang, Shuhui Chen, Erika Leonard
Journal: Cancer Research (2025)

Abstract Multiplexed protein analysis in native cellular contexts holds great promise for uncovering the composition, interactions, and functions of distinct cell types in complex biological systems. However, current multiplexed protein imaging technologies often face limitations in detection sensitivity or involve technically demanding protocols. To address these issues, we have developed an ultra-sensitive, multiplexed in situ protein profiling approach that combines reiterative staining with off-the-shelf antibodies and the newly developed Cleavable Fluorescent Probe (CFP™) Fluor cleavable TSA fluorophores. Target proteins are recognized by primary antibodies followed by secondary antibodies conjugated with horseradish peroxidase (HRP), which catalyzes the covalent deposition of CFP fluorophores on or close to the target proteins. After imaging, the fluorophores are chemically cleaved, and antibodies stripped, enabling subsequent rounds of staining, imaging, cleavage, and stripping. This approach allows for the interrogation of many proteins in individual cells while maintaining spatial resolution. In addition to protein analysis, glycan modifications can also be profiled using this approach. Glycan modifications also play critical roles in cell function in both healthy and diseased states from controlling proliferation to modulating the immune response. Lectins are part of a broader group called glycan-binding proteins that recognize glycan chains and mediate their functionality. Their glycan-specific nature makes them important tools to profile, characterize, and capture complex glycans in biological systems. We demonstrated the compatibility of CFP Fluor cleavable TSA fluorophores with lectins developed by Vector Laboratories. This assay facilitates the exploration of glycan complexity, enabling the detection of major glycan motifs, including sialylation, fucosylation, and galactosylation, for characterizing glycan distributions in target specimens. These methods are highly effective for detecting multiplexed proteins and glycans in the same biological samples, including FFPE tissues. We developed a novel protocol which enabled us to stain more than 10 protein markers on the same tissue section of human tonsil FFPE samples using the cleavable TSA fluorophores. We have also applied this approach to stain 7 neural cell markers and 4 varied glycans together on 5 normal and 5 Alzheimer’s human hippocampus FFPE tissues. The obtained results enable us to explore the altered glycosylation in individual cells of Alzheimer’s brains and/or other tissues across various disease indications such as cancers. Citation Format: Nishinki Muthumuni, Jia Guo, Jing Zhou, Xiaoshan Wang, Shuhui Chen, Erika Leonard. Multiplexed in situ detection of protein and glycan expression using CFP Fluor cleavable TSA fluorophores [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB199.