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  1. Synthetic matrices with dynamic presentation of cell guidance cues are needed for the development of physiologically relevant in vitro tumor models. Towards the goal of mimicking prostate cancer progression and metastasis, we engineered a tunable hyaluronic acid-based hydrogel platform with protease degradable and cell adhesive properties employing bioorthogonal tetrazine ligation with strained alkenes. The synthetic matrix was first fabricated via a slow tetrazine-norbornene reaction, then temporally modified via a diffusion-controlled method using trans-cyclooctene, a fierce dienophile that reacts with tetrazine with an unusually fast rate. The encapsulated DU145 prostate cancer single cells spontaneously formed multicellular tumoroids after 7 days of culture. In situ modification of the synthetic matrix via covalent tagging of cell adhesive RGD peptide induced tumoroid decompaction and the development of cellular protrusions. RGD tagging did not compromise the overall cell viability, nor did it induce cell apoptosis. In response to increased matrix adhesiveness, DU145 cells dynamically loosen cell-cell adhesion and strengthen cell-matrix interactions to promote an invasive phenotype. Characterization of the 3D cultures by immunocytochemistry and gene expression analyses demonstrated that cells invaded into the matrix via a mesenchymal like migration, with upregulation of major mesenchymal markers, and down regulation of epithelial markers. The tumoroids formed cortactin positive invadopodia like structures, indicating active matrix remodeling. Overall, the engineered tumor model can be utilized to identify potential molecular targets and test pharmacological inhibitors, thereby accelerating the design of innovative strategies for cancer therapeutics. 
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    Free, publicly-accessible full text available August 1, 2024
  2. Abstract

    Toward the goal of establishing an engineered model of the vocal fold lamina propria (LP), mesenchymal stem cells (MSCs) are encapsulated in hyaluronic acid (HA)‐based hydrogels employing tetrazine ligation with strained alkenes. To mimic matrix stiffening during LP maturation, diffusion‐controlled interfacial bioorthogonal crosslinking is carried out on the soft cellular construct using HA modified with a ferocious dienophile,trans‐cyclooctene (TCO). Cultures are maintained in MSC growth media for 14 days to afford a model of a newborn LP that is homogeneously soft (nLP), a homogeneously stiffened construct zero (sLP0) or 7 days (sLP7) post cell encapsulation, and a mature LP model (mLP) with a stiff top layer and a soft bottom layer. Installation of additional HA crosslinks restricts cell spreading. Compared to the nLP controls, sLP7 conditions upregulate the expression of fibrous matrix proteins (Col I, DCN, andFN EDA), classic fibroblastic markers (TNC, FAP, andFSP1), and matrix remodeling enzymes (MMP2, TIMP1, andHAS3). Day 7 stiffening also upregulates the catabolic activities, enhances ECM turnover, and promotesYAPexpression. Overall, in situ delayed matrix stiffening promotes a fibroblast transition from MSCs and enhances YAP‐regulated mechanosensing.

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