Abstract The use of multiplex immunofluorescence (mIF) to study the tumor microenvironment (TME) has significantly advanced our understanding of spatial dynamics within tumors. This technique has emerged as a valuable tool for identifying biomarkers and therapeutic targets. Despite its growing adoption, mIF protocols remain complex and technically demanding. Their manual execution and reliance on dedicated reagents make them time-consuming and expensive. Additionally, concerns persist regarding their reproducibility and transferability across different tissue types. Ready-to-use, validated antibody panels, such as the SPYRE™ Core Panels, help address these challenges. In this study, we demonstrate the development and validation of two new antibody panels covering relevant stromal and vessel biomarkers to enable spatial analysis of the TME on the COMET™ platform across various tissue types as well as two additional antibodies against SOX10 and S100B optimized for melanoma studies. The stroma panel enables simultaneous detection of Vimentin, E-Cadherin, Collagen I, and FAP, while the vessel panel contains CD31, CD34, Podoplanin, and LYVE-1. Formalin-fixed paraffin-embedded human tissue sections from a 24-cores multi-organ tissue microarray and whole-section melanoma samples were stained on COMET™ by fully automated sequential immunofluorescence (seqIF™, PMID: 37813886). Staining and detection are done via indirect immunofluorescence using unlabeled primary antibodies and fluorophore conjugated secondary antibodies. Both panels were developed and validated on several tumoral and non-tumoral tissues at the same time. The sections retrieved from COMET™ after seqIF™, were stained by a histology facility with standard immunohistochemistry (IHC) established for pathological diagnosis to compare seqIF™ and IHC staining patterns and verify antibody specificity. All markers demonstrate accurate detection with specific seqIF™ staining, comparable to gold-standard IHC counterparts, as well as robust performance across multiple tissues. Protocols were optimized to achieve high staining quality for all ten markers in terms of sensitivity and signal-to-background ratio. The repeatability and reproducibility of the automated stainings on the COMET™ platform was verified by day-to-day tests on one instrument and tests among multiple ones. Our validated SPYRE™ Stroma and Vessel Focus panels, along with melanoma-specific antibodies, deliver highly specific and reproducible results across diverse tissues. Ready-to-use on the COMET™ platform and designed as modular extensions of the SPYRE™ Core Panels, they enable quantitative marker detection, combination with custom antibodies, and empower researchers with robust and scalable workflows for advanced spatial biology studies. Citation Format: Paula Juricic, Francois Rivest, Clara Sinthon, Jade Nguyen, Isabelle Blanc, Bastian Nicolai, Alexandre Kehren, Saska Brajkovic. Validation of two melanoma biomarkers and two novel ready-to-use multiplex immunofluorescence panels for spatial profiling of the stromal and vascular compartments in the tumor microenviroment 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 6665.
Juricic et al. (Fri,) studied this question.