Abstract 5308: RePROBE: A simple and customizable multiplex immunohistochemistry technique

Abstract Background: Multiplex immunostaining techniques have been actively developed and have recently become more prevalent in cancer research. However, most of these techniques require specialized instruments and customized primary antibodies. Here, we established a simple and versatile workflow for multiplex immunohistochemistry (IHC) named RePROBE (Repetitive Primary antibody Replacement and Overlaying images from a Broad range of fluorescence imaging Equipment) that can be readily adopted in many laboratories, providing deeper insight into tumor organization and spatial molecular biology in the tumor microenvironment (TME). Methods: Formalin-fixed paraffin-embedded (FFPE) sections of various tissue types, such as resected tumors of cancer patients, patient-derived xenografts, and syngeneic mouse tumors, were subjected to the RePROBE workflow. Commercially available primary and secondary antibodies were used for fluorescence immunostaining with routine indirect IHC methods. Images were acquired by multiple imaging platforms, including fluorescence microscopes, confocal microscopes, and whole slide image scanners. Antibody complexes were removed by heat-based stripping after imaging, and the slides were subjected to the next cycle. All image processing and analysis were performed by open-source software. Results: We confirmed that the RePROBE workflow worked across different types of tissue sections and imaging modalities and achieved up to 36-plex on human gastric cancer samples and more than 20-plex on syngeneic mouse tumors. The multiplex IHC images derived from the RePROBE workflow were able to be analyzed in downstream pipelines, such as QuPath, Cellpose, and MCMICRO. Heat-based stripping successfully removed most antibodies with some exceptions. Although some loss of cells was observed (less than 7%) after cycle 1, significant cell loss was not observed after cycle 2 up to cycle 10 in most of the samples. We also found that the cell loss was dependent on tissue type and samples. Conclusions: The RePROBE workflow enabled up to 36-plex whole-slide single-cell resolution imaging and spatial phenotyping analysis, revealing the cell population and spatial organization of tumor cells, CAFs, and immune cells in TME. Citation Format: Takashi Semba, Atsuko Yonemura, Huaitao Wang, Yilin Tong, Masaya Yamazaki, Takatsugu Ishimoto. RePROBE: A simple and customizable multiplex immunohistochemistry technique [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 5308.