Background Understanding the tumor microenvironment (TME) is crucial for evaluating the efficacy of new therapeutic targets. The TME is a dynamic network of tumor cells, immune cells, and stromal components, influencing tumor progression and therapy response. Therefore, high multiplex immunofluorescence (mIF) assays are essential to detail complex spatial relationships, critical for identifying and validating therapeutic targets. As of today, mIF assays for more than 12 targets lack either single cell resolution or sufficient sensitivity for low abundance targets.

Here, InSituPlex® (ISP) assays enable an efficient workflow for creating 24-plex panels with pre-validated targets (OmniVUE™ panels), covering a broad range of biomarkers for a comprehensive view of the TME with high sensitivity and single cell detection. Analysis is carried out using the highly scalable cloud-based STARVUE™ image data science platform, enabling high-throughput, accurate quantification of cellular phenotypes and spatial distributions.

Methods Slides were stained with a cocktail of 24 primary antibodies using a Leica Biosystems BOND RX autostainer, amplified simultaneously, then imaged on the Zeiss AxioScan.Z1 using multiple rounds of detection. Images were co-registered and exported for downstream analysis using Ultivue’s STARVUE^TM image data science platform. Serial sections were stained with the same antibodies in single-plex, and concordance was assessed by quantifying percent difference in cellular density from 1-plex to 24-plex.

Results Comprehensive 24-plex immunophenotyping showed a high degree of concordance with the single-plex assays. Anticipated levels and patterns of expression were observed for every biomarker with minimal variation observed in cellular density between single-plex and multiplex formats. Spatial analysis facilitated by Ultivue’s STARVUE™ software enabled deep immune cell phenotyping and detection of unique cell phenotypes.

Conclusions Our streamlined and well-established workflow for InSituPlex® (ISP) assays now supports up to 24-plex multiplex immunofluorescence (mIF), representing a significant advancement in tissue immunophenotyping. The workflow boasts highly sensitive multiplex IF and scalable AI-based image analysis powered by STARVUE™. The high concordance between single-plex and multiplex assays confirms the robustness and accuracy of this method. The resultant phenotypic and spatial data enabled by this workflow can profoundly impact the understanding of the TME, guiding the development of targeted therapies and personalized treatment strategies.