The Generation of Tissue-Specific ECM Hydrogels From Melanoma and Associated Organs to Study Cancer Biology

The extracellular matrix (ECM) critically shapes melanoma progression and therapeutic response, yet commonly used matrices such as Matrigel fail to capture tissue- and disease-specific ECM properties. This protocol provides a streamlined and scalable method for generating murine, tissue-specific ECM hydrogels from skin, lung, and melanoma tumors, therefore overcoming the restricted materials of mouse-derived ECM. The workflow integrates tissue-tailored decellularization, lyophilization, mechanical fragmentation, pepsin digestion, and physiological polymerization to produce hydrogels that reliably preserve fibrillar collagen architecture and organ-specific ECM cues. Decellularization efficiency and ECM integrity are validated by DNA quantification, H&E staining, and Picrosirius Red staining analysis. These hydrogels provide a species- and tissue-matched platform for studying melanoma-ECM-immune interactions, pre-metastatic niche features, and therapy-induced ECM remodeling. Overall, this protocol offers a reproducible and physiologically relevant ECM model that expands experimental capabilities for melanoma biology and treatment-resistance research and that can be easily extended to other tumors and tissues. Key features • A miniaturized, tissue-specific workflow for generating ECM hydrogels from small murine skin, lung, and melanoma tissues, overcoming size limitations of existing protocols. • Preservation of native ECM architecture using tailored decellularization steps validated by DNA quantification, H&E, and Picrosirius Red staining. • A standardized digestion-gelation process optimized for heterogeneous and lipid-rich murine tissues, enabling reproducible hydrogel formation at defined ECM concentrations. • A physiologically relevant platform capturing melanoma- and organ-specific ECM cues for studying ECM-tumor-immune interactions and therapy-induced remodeling.