T cell therapies require the removal and culture of T cells ex vivo to expand several thousandfold. However, these cells often lose the phenotype and cytotoxic functionality necessary to mediate an effective therapeutic response. The extracellular matrix has been used to preserve and augment cell phenotype; however, it has not been applied to cellular immunotherapies.Here we engineered a hyaluronic acid (HA)-based hydrogel to present the two stimulatory signals required for T-cell activation-termed an artificial T cell stimulating matrix (aTM).We found that biophysical properties of the aTM-stimulatory ligand density, stiffness, and extracellular matrix (ECM) proteins-potentiate T cell signaling and skew phenotype of both murine and human T cells. Importantly, it was only the combination of the ECM environment and mechanically sensitive TCR signaling from the aTM that produced nearly 4-times the number of rare, antigen-specific CD8+ T cells. Adoptive transfer of these tumor-specific cells significantly suppressed tumor growth and improved animal survival as compared with T cells stimulated by traditional methods. Beyond immediate immunotherapeutic applications, demonstrating that the environment influences the cellular therapeutic product delineates the importance of the ECM and provides a case study of how to engineer ECM-mimetic materials for therapeutic immune stimulation in the future.
Vascular morphogenesis is the formation of endothelial lumenized networks. Cluster-based vasculogenesis of endothelial progenitor cells (EPCs) has been observed in animal models, but the underlying mechanism is unknown. Here, using O2-controllabe hydrogels, we unveil the mechanism by which hypoxia, co-jointly with matrix viscoelasticity, induces EPC vasculogenesis. When EPCs are subjected to a 3D hypoxic gradient ranging from <2 to 5%, they rapidly produce reactive oxygen species that up-regulate proteases, most notably MMP-1, which degrade the surrounding extracellular matrix. EPC clusters form and expand as the matrix degrades. Cell-cell interactions, including those mediated by VE-cadherin, integrin-β2, and ICAM-1, stabilize the clusters. Subsequently, EPC sprouting into the stiffer, intact matrix leads to vascular network formation. In vivo examination further corroborated hypoxia-driven clustering of EPCs. Overall, this is the first description of how hypoxia mediates cluster-based vasculogenesis, advancing our understanding toward regulating vascular development as well as postnatal vasculogenesis in regeneration and tumorigenesis.
BackgroundN-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis.MethodWeb based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3β phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses.ResultsInvasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/β-catenin signaling resulting from inactivation of GSK3β.ConclusionAberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.
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