Spatial immune profiling of solid tumors by complementing H&E with a novel 17-plex immunofluorescence approach

Background: Spatial biology has increasingly leveraged “spatial neighborhoods” to integrate how cells are organized and interact, yet standards for defining and identifying these neighborhoods are still developing. Multiplex fluorescence immunohistochemistry (fIHC) offers detailed profiling of immune and tumor cell states, while hematoxylin and eosin (H&E) staining remains a gold standard for visualizing tissue and cellular morphology. We validated a 17-plex fIHC assay on the Orion™ (RareCyte) platform and aligned the fIHC with same slide H&E images to improve fidelity of cellular neighborhoods identification across breast, lung, prostate, melanoma, and colorectal cancers. Methods: We developed a generalizable workflow to align same-slide H&E and 17-plex fIHC Orion images for assessment of cellular neighborhoods through integration cell phenotyping (via fIHC) with tissue architecture (via H&E). The 17-plex fIHC assay includes antibody-fluorophore conjugates targeting Granzyme B, Ki67, CD3e, CD20, CD4, CD163, CD8a, PD-L1, NKp46, PD-1, LAG3, FOXP3, CD11b, CD138, SOX10, Cytokeratin, and Hoechst nuclear stain. Slides were first stained and imaged by fIHC, followed by H&E staining and imaging on the Orion. Cell phenotyping and slide registration were performed on HALO® (Indica Labs). Results: Integrating H&E with multiplex fIHC supported identification of spatial themes, including regions with enriched tumor-infiltrating lymphocytes, immune deserts, and mixed/transitional zones populating the tumor/stroma interface. This approach improves interpretability of these patterns by connecting cell phenotypes with tissue architecture morphology, supporting identification of microenvironments with greater consistency. Conclusions: Combining H&E with multiplex fIHC enables anatomically informed mapping of cellular neighborhoods across multiple tumor types. This discovery-oriented workflow improves interpretability of spatial patterns, is adaptable to diverse datasets and pipelines, and may yield new insights into the immune microenvironment to inform next-generation therapies. TM and ® are trademarks or registered trademarks of their respective owners. All rights are reserved by their respective owners.