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Directing cell differentiation and tissue morphogenesis using a bioorthogonal hydrogel platform
Date
2024
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
The extracellular matrix (ECM) is an important determinant of tissue specificity. The ECM provides cell instructive signals to induce phenotypic changes and promote cell growth, differentiation, and tissue morphogenesis. Complex biological processes are highly dynamic. During embryonic organ development, the surrounding matrix is constantly remodeled. Similarly stromal ECM surrounding aggressive tumors is continually altered. The functions of cells and tissues evolve over time and space through intricate cell-cell and cell-ECM interactions. Towards the goal of mimicking the native ECM, I demonstrated the utility of a hyaluronic acid (HA)-based hydrogel with protease degradability and cell adhesive properties for modeling prostate cancer and for engineering salivary gland. The initial hydrogel was fabricated via a slow tetrazine-norbornene reaction, then temporally modified via a diffusion-controlled method using trans-cyclooctene (TCO), a fierce dienophile that reacts with tetrazine with an unusually fast rate. Using an array of biologically relevant tool molecules, hydrogel was established to maintain the desired phenotype and cell behavior in vitro. ☐ Towards the goal of mimicking prostate cancer progression and metastasis in vitro, I cultured DU145 prostate cancer cells in the HA gels with integrin binding RGD peptide introduced 7 days post cell encapsulation. I hypothesized that temporal conjugation of the RGD ligand during cell culture will dynamically remodel the synthetic ECM to induce epithelial-to-mesenchymal transition (EMT). DU145 prostate cancer cells encapsulated in RGD-free HA gels spontaneously formed tumoroids post 7 days. In situ RGD tagging triggered downregulation of E-cadherin and formation of vimentin and cortactin positive invadopodia like protrusive structures. In non-adhesive hydrogels, cells remained as compact spheroids without signs of dissemination into the matrix. Overall, prostate cancer cells cultured in the HA gels exhibited migratory behaviors that were reminiscent of cancer progression. ☐ During tumorigenesis, cytokines control communication between different stromal and cancer cells to remodel the tumor microenvironment. One such chemokine, transforming growth factor beta-1 (TGF-β1), has been implicated in prostate cancer disease progression. I hypothesize that TGF-β1 exerts its oncogenic effects to induce cellular plasticity in synergy with integrin binding ligands in the cancer stroma. Here, DU145 cells were cultured in HA gels in TGF-β1-conditioned media. DU145 cells encapsulated in gels with low RGD concentration formed compact tumoroids with minute protrusions at the border of the cell aggregates. Upon addition of soluble TGF-β1, the cells underwent significant morphological changes to form extended cellular networks with individual cells exhibiting well-defined F-actin rich fibers. In non-adhesive gels, cells remained as compact spheroids even in the presence of TGF-β1. In cell-adhesive hydrogels, TGF-β1 activated TGF-β1 signaling via nuclear localization of SMAD2/3. The activation of TGF-β signaling led to the upregulation of a wide range of TGF target genes. Prolonged exposure to TGF-β1 eventually induced EMT, with loss of cytokeratins and attainment of vimentin in DU145 cells. My findings highlight the need for integrin engagement for the activation of TGF-β signaling, leading to the acquisition of aggressive phenotype to promote tumor progression. ☐ The HA-based hydrogels can also be used to recapitulate developmental processes to stimulate tissue growth. Using the same tools molecules, I attempted to engineer a functional salivary gland tissue that can be implanted in patients to treat hyposalivation, a common side effect from radiation therapy for head and neck cancers. The salivary gland is intimately integrated with the vasculature, which provides cell instructive signals during development to guide epithelial morphogenesis. I hypothesize that co-culture of human salivary gland stem/progenitor cells (hS/PCs) and human umbilical vein endothelial cells (HUVECs) in the HA gels in a tissue mimetic configuration will facilitate the development of pro-acinar phenotype in hS/PCs. Two-dimensional monoculture experiments showed that EGM2, a commercial formulation designed for the maintenance of HUVECs, was appropriate for co-culture studies. Using non-adhesive agarose microwells, I cultured hS/PC in EGM2 for 7 days. The resultant epithelial spheroids were then encapsulated in the HA hydrogels. Subsequently, HUVECs were seeded on top of hS/PC laden construct and the coculture was maintained in EGM2 for 14 days. hS/PC spheroids formed a distinct lobular structure with or without the cocultured HUVECs. Under coculture conditions, hS/PCs expressed higher mRNA levels of stem/progenitor biomarkers (KRT5, KRT14, ETV5), acinar biomarkers (AMY, AQP3, SLC12A2) and ductal biomarkers (KRT7, TFCP2L1), as compared to the corresponding monoculture. Finally, HA gels loaded with EGM2 were implanted in partially resected parotid gland in rats. Animals with gel implants showed reduced fibrosis and more organized acinar structures compared to their resection only counterparts. Since radiation therapy causes tissue fibrosis and acinar damage, these findings were significant. Collectively, endothelial cues promote pro-acinar phenotype in-vitro and reduce tissue fibrosis in-vivo. ☐ In summary, I successfully developed hydrogel-derived tissues and tissue models using the same hydrogel building blocks. The hydrogel platform recapitulates spatial heterogeneity and temporal dynamics seen in native ECM, allowing precise in-situ control of cell phenotype, fate, and behavior during live cell culture. My investigations shed critical insight into the roles of integrin engagement and TGF-β1 signaling in cancer progression. My research also highlights the importance of recapitulating the vascular niches to promote ex vivo assembly of salivary gland from adult progenitor cells.
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Keywords
Biomaterials, Cell phenotype, Hydrogel, Tissue engineering, Tissue morphogenesis