Chemoselective Characterization of New Extracellular Matrix Deposition in Bioengineered Tumor Tissues

In both native and engineered tissues, the extracellular matrix (ECM) supports and regulates nearly all aspects of cellular pathophysiology, and in response, cells extensively remodel their surrounding extracellular environments through new ECM protein deposition. Understanding this intricate bi‐directional cell‐ECM interaction is key to tissue engineering, but it remains challenging to investigate. This is partly due to the limited sensitivity of conventional proteomics to capture low‐abundance newly synthesized ECM (newsECM). This study presents a glycosylation‐enabled, chemoselective strategy to label, enrich, and characterize newsECM proteins with augmented specificity and sensitivity. Applying newsECM profiling to bioengineered tumor tissues, either built upon decellularized ECM materials (dECM‐tumor) or as ECM‐free tumoroids, revealed distinct ECM synthesis patterns. Tumor cells cultured within dECM scaffold present elevated ECM remodeling activities, mediated by augmented digestion of pre‐existing ECM coupled with upregulated synthesis of tumor‐associated ECM components. These 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. The newsECM profiling described here is anticipated to be applicable to a wide range of engineered tissue models and pathophysiological processes to deliver fundamental insights regarding the mutual cell‐ECM crosstalk.