Abstract 7672: Automated, flexible multiplex immunofluorescence for tumor microenvironment profiling using HCR™ Gold IF and clinically-relevant antibody clones.

Abstract Background: Multiplexed immunofluorescence (mIF) is central to oncology research but is often limited by fixed panels, antibody modification requirements, harsh stripping, and workflows that risk epitope damage. HCR™ Gold IF enabled by HCR™ HiFi Encoders addresses these constraints by providing enzyme-free amplification with universal compatibility to unmodified rabbit IgG and mouse IgG1 primaries. Here, we evaluated a complete, interoperable workflow combining automated HCR™ Gold IF on Leica Biosystems’ BOND RX staining instrument, validated Abcam antibodies, and quantitative image analysis using Indica Lab’s HALO platform for tumor microenvironment (TME) profiling. Methods: FFPE tissue sections from healthy tissues and tumors were stained via HCR™ Gold IF on the BOND RX staining instrument. Panels included epithelial, proliferation, immune, and checkpoint markers (e.g., panCK, Ki67, CD3, CD8, CD68, PD-1, PD-L1). All primary antibodies were obtained from Abcam and qualified in a single-plex experiment prior to multiplex assembly. Slides were imaged with whole-slide scanners and analyzed in the HALO® platform to quantify biomarker expression and interrogate spatial relationships between cell populations. Results: The automated, protease-free workflow generated high signal-to-background with crisp subcellular localization and clean channel separation across tissues and targets. Universal compatibility with unmodified Abcam antibodies streamlined panel design and minimized assay development time. Single-plex to multiplex concordance met predefined acceptance criteria, indicating minimal epitope masking or channel cross-talk. Across tumor cohorts, we observed expected and quantifiable TME shifts versus matched healthy tissues, including elevated PD-L1 on tumor/myeloid compartments and increased CD8+ and CD68+ densities at tumor-stroma interfaces. Conclusions: Integrating HCR™ Gold IF, powered by the HiFi Encoders, on the BOND RX staining instrument and analyzing in the HALO platform delivers an automated, plug-and-play mIF solution that couples superior, enzyme-free amplification with true bring-your-own-antibody flexibility. This end-to-end workflow lowers barriers to custom, high-plex TME studies, preserves tissue integrity, and enables standardized generation of spatial biomarkers to inform immune-oncology research and patient stratification. The approach is readily extensible to larger panels and to combined RNA/protein co-detection for deeper, multi-omic context. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Wudy Yang, Randy Chen, Harry Choi, Aneesh Acharya, Anne Hellebust, Will Howat. Automated, flexible multiplex immunofluorescence for tumor microenvironment profiling using HCR™ Gold IF and clinically-relevant antibody clones 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 7672.