Development of a specific fluorescent probe to detect advanced glycation end products (AGEs).

Advanced glycation end products (AGEs) play a pivotal role in the aging process, regarded as a hallmark of aging. Despite their significance, the absence of adequate monitoring tools has hindered the exploration of the relationship between AGEs and aging. Here, we present a novel AGE-selective probe, AGO, for the first time. AGO exhibited superior sensitivity in detecting AGEs compared to the conventional method of measuring autofluorescence from AGEs. Furthermore, we validated AGO's ability to detect AGEs based on kinetics, demonstrating a preference for ribose-derived AGEs. Lastly, AGO effectively visualized glycation products in a collagen-based mimicking model of glycation. We anticipate that this study will enhance the molecular tool sets available for comprehending the physiological processes of AGEs during aging.

Metallization of Targeted Protein Assemblies in Cell-Derived Extracellular Matrix by Antibody-Guided Biotemplating.

Biological systems are composed of hierarchical structures made of a large number of proteins. These structures are highly sophisticated and challenging to replicate using artificial synthesis methods. To exploit these structures in materials science, biotemplating is used to achieve biocomposites that accurately mimic biological structures and impart functionality of inorganic materials, including electrical conductivity. However, the biological scaffolds used in previous studies are limited to stereotypical and simple morphologies with little synthetic diversity because of a lack of control over their morphologies. This study proposes that the specific protein assemblies within the cell-derived extracellular matrix (ECM), whose morphological features are widely tailorable, can be employed as versatile biotemplates. In a typical procedure, a fibrillar assembly of fibronectin-a constituent protein of the ECM-is metalized through an antibody-guided biotemplating approach. Specifically, the antibody-bearing nanogold is attached to the fibronectin through antibody-antigen interactions, and then metals are grown on the nanogold acting as a seed. The biomimetic structure can be adapted for hydrogen production and sensing after improving its electrical conductivity through thermal sintering or additional metal growth. This study demonstrates that cell-derived ECM can be an attractive option for addressing the diversity limitation of a conventional biotemplate.

Multi-targeted therapy resistance via drug-induced secretome fucosylation.

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here, we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.

Glycation-mediated tissue-level remodeling of brain meningeal membrane by aging.

Collagen is a prominent target of nonenzymatic glycation, which is a hallmark of aging and causes functional alteration of the matrix. Here, we uncover glycation-mediated structural and functional changes in the collagen-enriched meningeal membrane of the human and mouse brain. Using an in vitro culture platform mimicking the meningeal membrane composed of fibrillar collagen, we showed that the accumulation of advanced glycation end products (AGEs) in the collagen membrane is responsible for glycation-mediated matrix remodeling. These changes influence fibroblast-matrix interactions, inducing cell-mediated ECM remodeling. The adherence of meningeal fibroblasts to the glycated collagen membrane was mediated by the discoidin domain-containing receptor 2 (DDR2), whereas integrin-mediated adhesion was inhibited. A-kinase anchoring protein 12 (AKAP12)-positive meningeal fibroblasts in the meningeal membrane of aged mice exhibited substantially increased expression of DDR2 and depletion of integrin beta-1 (ITGB1). In the glycated collagen membrane, meningeal fibroblasts increased the expression of matrix metalloproteinase 14 (MMP14) and less tissue inhibitor of metalloproteinase-1 (TIMP1). In contrast, the cells exhibited decreased expression of type I collagen (COL1A1). These results suggest that glycation modification by meningeal fibroblasts is intimately linked to aging-related structural and functional alterations in the meningeal membrane.

Bi-directional crosstalk between cells and extracellular matrix leads to network morphogenesis in multi-layered tissues.

Cell-generated mechanical forces drive many cellular and tissue-level movements and rearrangements required for the tissue or organ to develop its shape1, 2, 3, 4, 5. The prevalent view of tissue morphogenesis relies on epithelial folding resulting in compressed epithelial monolayers, overlooking the involvement of stroma in morphogenesis1, 4, 6, 7. Here, we report a giant web-like network formation of stromal cells in the epithelium-stroma interface, resulting from a multi-scale mechano-reciprocity between migrating cells and their extracellular environment. In multi-layered tissues, surface wrinkles form by a stromal cell-mediated tensional force exerted at the basement membrane. The topographical cue is transmitted to the stromal cell, directing its protrusion and migration along the wrinkles. This inductive movement of the cells conveys traction forces to its surrounding extracellular matrix, remodeling the local architectures of the stroma. In this manner, stromal cells and wrinkles communicate recursively to generate the cellular network. Our observation provides a rational mechanism for network formation in living tissues and a new understanding of the role of cellular-level tensional force in morphogenesis.

Assessing Spatial Distribution of Multicellular Self-Assembly Enables the Prediction of Phenotypic Heterogeneity in Glioblastoma.

Phenotypic heterogeneity of glioblastomas is a leading determinant of therapeutic resistance and treatment failure. However, functional assessment of the heterogeneity of glioblastomas is lacking. We developed a self-assembly-based assessment system that predicts inter/intracellular heterogeneity and phenotype associations, such as cell proliferation, invasiveness, drug responses, and gene expression profiles. Under physical constraints for cellular interactions, mixed populations of glioblastoma cells are sorted to form a segregated architecture, depending on their preference for binding to cells of the same phenotype. Cells distributed at the periphery exhibit a reduced temozolomide (TMZ) response and are associated with poor patient survival, whereas cells in the core of the aggregates exhibit a significant response to TMZ. Our results suggest that the multicellular self-assembly pattern is indicative of the intertumoral and intra-patient heterogeneity of glioblastomas, and is predictive of the therapeutic response.

Re-engineered cell-derived extracellular matrix as a new approach to clarify the role of native ECM.

An extracellular matrix (ECM) has both biochemical and mechanophysical characteristics obtained from multiple components, which provides cells a dynamic microenvironment. During reciprocal interactions with ECM, the cells actively remodel the matrix, including synthesis, degradation, and chemical modification, which play a pivotal role in various biological events such as disease progression or tissue developmental processes. Since a cell-derived decellularized ECM (cdECM) holds in vivo-like compositional heterogeneity and interconnected fibrillary architecture, it has received much attention as a promising tool for developing more physiological in vitro model systems. Despite these advantages, the cdECM has obvious limitations to mimic versatile ECMs precisely, suggesting the need for improved in vitro modeling to clarify the functions of native ECM. Recent studies propose to tailor the cdECM via biochemically, biomechanically, or incorporation with other systems as a new approach to address the limitations. In this chapter, we summarize the studies that re-engineered the cdECM to examine the features of native ECM in-depth and to increase physiological relevancy.

Tris(2-carboxyethyl)phosphine-Mediated Nanometric Extracellular Matrix-Coating Method of Mesenchymal Stem Cells.

Human iPSC-derived mesenchymal stem cells (iMSCs) are an alternative to primary mesenchymal stem cells (MSCs), which have been a limited supply, and have attracted a great deal of interest as a promising cell source in cell-based therapy. However, despite their enormous therapeutic potential, it has been difficult to translate this potential into clinical applications due to the short viability duration of transplanted iMSCs. Therefore, to maximize the therapeutic effects of iMSCs, it is extremely important to extend their retention rate during and even after the transplantation. In this study, we developed a new extracellular matrix (ECM)-coating method involving the mild reduction of the cell surface. The reduction of disulfide bonds around the cell membrane enhanced the coating efficiency without a decrease in the viability and differentiation potential of iMSCs. We then induced ECM-coated single iMSCs to form three-dimensional spheroids via self-assembly of the aggregates within a physically confined microenvironment. The spheroids exhibited longer maintenance of the survival rate. Nanometric ECM coating of the cell membrane is a new approach as a key for resolving the conventional challenges of cell-based therapy.