Chemoselective Characterization of New Extracellular Matrix Deposition in Bioengineered Tumor Tissues

Abstract The extracellular matrix (ECM), present in nearly all tissues, provides extensive support to resident cells through structural, biomechanical, and biochemical means, and in return the ECM undergoes constant remodeling from interacting cells to adapt to the evolving tissue states. Bioengineered 3D tissues, commonly known as cell-ECM composites, are robust model systems to recapitulate and investigate native pathophysiology. Key to this engineered morphogenesis process are the intricate cell-ECM interactions reflected by how cells respond to and thereby modulate their surrounding microenvironments through their ongoing ECM secretome. However, investigating ECM-regulated new ECM production has been challenging due to the proteomic background from the pre-existing biomaterial ECM. To address this hindrance, here we present a chemoselective strategy to label, enrich, and characterize newly synthesized ECM (newsECM) proteins produced by resident cells, allowing distinction from the pre-existing ECM background. Applying our analytical pipeline to bioengineered tumor tissues, either built upon decellularized ECM (dECM-tumors) or as ECM-free tumor spheroids (tumoroids), we observed distinct ECM synthesis patterns that were linked to their extracellular environments. Tumor cells responded to the dECM presence with elevated ECM remodeling activities, mediated by augmented digestion of pre-existing ECM coupled with upregulated synthesis of tumor-associated ECM. Our findings highlight the sensitivity of newsECM profiling to capture remodeling events that are otherwise under-represented by bulk proteomics and underscore the significance of dECM support for enabling native-like tumor cell behaviors. We anticipate the described newsECM analytical pipeline to be broadly applicable to other tissue-engineered systems to probe ECM-regulated ECM synthesis and remodeling, both fundamental aspects of cell-ECM crosstalk in engineered tissue morphogenesis.