Background Tumors and their tumor microenvironment (TME) can present in various forms, and this heterogeneity can impact the severity of the disease, treatment options, and treatment effectiveness. To account for some of these differences, cancers are typically categorized based on specific molecular, morphological, and clinical characteristics. However, classical methods like low plex IHC do not allow for a comprehensive spatial profiling of the TME. Multiplex immunofluorescence (mIF) is a powerful tool that enables deep phenotyping of immune and cancer cells across a wide variety of tissue types. The initial effort and cost required to develop mIF panels is significant and poses a considerable hurdle for adoption of spatial biology applications. To streamline mIF assay development, we leveraged assay panels compatible with CellScape™, an automated end-to-end cyclic mIF platform for precise spatial multiplexing. Multiplex assay development involved a multi-stage, iterative process to evaluate antibodies for sensitivity, specificity, and reproducibility. These assay panels are formulated as ready-to-use VistaPlex™ assay kits and are intended to enable rapid, reliable spatial phenotyping of key immune and tumor cell populations. Furthermore, VistaPlex assay kits are modular and can be combined with additional panels to interrogate specific sample or cell types.

Materials and Methods To evaluate their effectiveness and versatility, we used the VistaPlex Human FFPE Spatial Immune Profiling Kit and paired it with a panel of breast cancer markers to stain and image breast cancer tissue samples on the CellScape platform. The CellScape workflow involves mounting tissues in enclosed fluidic chambers and conducting on-instrument staining, imaging, and signal erasure in iterative rounds. The two panels contain antibodies against a total of 23 common biomarkers used in breast cancer immuno-oncology applications. These include classical breast cancer markers HER2, ER, and PR, as well as immune cell lineage markers CD3, CD4, CD8, CD20, and CD45. PD-1 and PD-L1 are included to interrogate immune checkpoints, and markers that inform about cell proliferation state and activation are included to enable comprehensive profiling of the TME. Spatial phenotyping was performed using unsupervised clustering analysis and AI-assisted cluster labelling, and biomarker distributions were studied in the context of cellular neighborhoods and other proximity analyses.

Results Overall, we identified more than 20 distinct cell phenotypes with varying frequencies and spatial arrangements across tissue samples.

Conclusions Our data were robust and demonstrated that VistaPlex Assay kits for high-plex spatial profiling present an efficient and cost-effective solution for users that want to break into spatial biology.

A. Christians: A. Employment (full or part-time); Significant; Canopy Biosciences. J. Boog: A. Employment (full or part-time); Significant; Canopy. C.E. Jackson: A. Employment (full or part-time); Significant; Canopy Biosciences. M.H. Ingalls: A. Employment (full or part-time); Significant; Canopy Biosciences. A. Northcutt: A. Employment (full or part-time); Significant; Canopy Biosciences. K.E. Cashion: A. Employment (full or part-time); Significant; Canopy Biosciences. J. Rauer: A. Employment (full or part-time); Significant; Canopy Biosciences. A. Brix: A. Employment (full or part-time); Significant; Canopy Biosciences. K. Kwarta: A. Employment (full or part-time); Significant; Canopy Biosciences. J.S. Schwarz: A. Employment (full or part-time); Significant; Canopy Biosciences. T. Campbell: A. Employment (full or part-time); Significant; Canopy Biosciences. O. Braubach: A. Employment (full or part-time); Significant; Canopy Biosciences.