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

Background

Melanoma, the most lethal form of skin cancer, arises from transformed melanocytes and is characterized by high metastatic potential and marked immunogenicity [1]. Despite its substantial tumor mutational burden and responsiveness to immune checkpoint inhibitors, up to two-thirds of patients fail to benefit or eventually relapse following initial responses, highlighting the knowledge gaps in treatment resistance and disease progression [2].

Recent studies suggest that ECM remodeling in melanoma, among other cancers, plays a central role in invasion, metastasis, and immune regulation. Alterations such as increased matrix stiffness, collagen fiber alignment, and crosslinking, mediated by enzymes such as lysyl oxidases (LOX) and matrix metalloproteinases (MMPs), promote cancer cell dissemination and can physically hinder immune cell infiltration [3]. These changes not only facilitate melanoma progression but also contribute to phenotype switching and resistance to targeted or immune-based therapies. Preclinical studies targeting ECM components, such as integrins, hyaluronan, and MMPs, have demonstrated potential synergy with immunotherapy, pointing to the ECM as a promising therapeutic co-target in melanoma [4]. Therefore, the advancement of new tools to study ECM in melanoma can advance melanoma research and serves as a basis for the development of new treatments for melanoma.

The composition and organization of ECM proteins vary across tissues, reflecting their unique properties and functional requirements [5]. Additionally, the ECM undergoes dynamic changes over time, adapting to tissue needs and the embedded cells within each organ [6]. In cancer, the ECM plays a pivotal role, usually supporting tumor development and progression. Unlike normal tissues, the ECM of solid tumors exhibits significant alterations in protein composition and structure [7]. Changes in the ECM occur not only in the primary tumor but also in distant organs [8]. ECM remodeling in distant organs creates a supportive environment for disseminated cancer cells, creating a pre-metastatic microenvironment, allowing cancer cells to home to such sites and colonize there. These ECM alterations are driven by factors secreted by the primary tumor, such as extracellular vesicles and LOX, and involve interactions among resident stromal cells and recruited immune cells [9].

Current in vitro ECM models, including collagen gels, Matrigel, and synthetic hydrogels, do not reflect the diverse ECM compositions of melanoma across anatomical or metastatic sites [10,11]. While tissue-derived hydrogels generated from human or porcine organs provide more physiological ECM environments [12], they are limited by availability, batch variability, and lack of compatibility with murine experimental systems. Mouse-derived ECM hydrogels would enable species-matched studies, facilitate integration with in vivo perturbations, and support mechanistic work in genetically engineered or syngeneic models. However, their use has been restricted by the small size of murine tissues, which often yields insufficient material for conventional decellularization and hydrogel preparation [13].

This protocol addresses these limitations by establishing a miniaturized, tissue-specific workflow for generating ECM hydrogels from a small quantity of murine skin, lung, and melanoma tumors. The method preserves native ECM cues and supports downstream applications for modeling melanoma–ECM–immune interactions, metastatic niche conditions, and therapy-induced ECM remodeling [14,15]. Beyond melanoma, the same workflow can be adapted to other solid tumor types; we have successfully applied similar decellularization steps to LLC, 4T1, and PyMT tumors, indicating broad applicability across murine cancer models.