Abstract A117: Development of multiplex immunofluorescence workflows for characterizing tumor-immune and stromal compartments for pharmacodynamic assessments of solid tumors

Abstract Micvotabart pelidotin (micvo) is an antibody–drug conjugate that targets the non-cellular extradomain-B of fibronectin, an extracellular matrix protein highly expressed in tumors compared to normal adult tissues. Micvo is designed to kill cancer cells, remodel the tumor microenvironment (TME), and mobilize an anti-tumor immune response. Micvo is currently in clinical development as a monotherapy (NCT05720117) and in combination with pembrolizumab for difficult-to-treat cancers (NCT06795412). Histological methods to characterize participants’ tumors before and after treatment with micvo are needed. Multiplex immunofluorescence (mIF) enables high-resolution phenotyping of tissue architecture and cellular interactions to inform biomarker development and pharmacodynamic assessments. This study aimed to establish mIF methodologies for integrated spatial assessment of tumor-immune interactions and stromal architecture to better characterize microenvironmental variation across tumor types for future use in biomarker discovery and characterization of pharmacodynamic responses to micvo. A practical method for staining six custom targets simultaneously was developed using Akoya’s Opal™ mIF kit, which employs iterative immunofluorescent labeling to visualize multiple markers within a single formalin-fixed, paraffin-embedded section while preserving spatial context. Utilizing this technology, two complementary mIF panels were designed, optimized and validated; one focused on tumor-immune interactions, and the other on key stromal features, including fibroblast subtypes and extracellular matrix components. Same-slide H&E and Masson’s trichrome staining were performed on mIF stained samples to provide additional context for tumor cell morphology and collagen distribution, respectively. A digital pathology workflow was developed to enable integrated spatial assessment of immune and stromal compartments by aligning and combining region annotations between sequential slides; positive-cell thresholds and region classifiers were guided by a board-certified pathologist. To validate this workflow for human clinical samples, each mIF panel was deployed and validated on commercially sourced whole-tissue slides of head and neck squamous cell carcinoma (HNSCC) and pancreatic ductal adenocarcinoma (PDAC), characterizing histologic and microenvironmental differences across indications. Digital pathology enabled precise spatial quantification, revealing differences in the composition and spatial organization of immune and stromal components of the TME between HNSCC and PDAC. These results demonstrate the feasibility of integrating tumor-immune and stromal mIF panels for high-resolution TME profiling, offering a scalable and customizable approach to investigate tumor heterogeneity and treatment response across solid tumors. The validated mIF workflow will be applied to clinical biopsies from micvo-treated participants to characterize pharmacodynamic changes in immune and stromal compartments. Citation Format: Justin Trickett, Sara Lewandowski, Krystal Watkins, Eila Crochiere, Victor Zota, Marsha Crochiere. Development of multiplex immunofluorescence workflows for characterizing tumor-immune and stromal compartments for pharmacodynamic assessments of solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A117.