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.

Comparison of Glioblastoma Cell Culture Platforms Based on Transcriptional Similarity with Paired Tissue.

No standardized in vitro cell culture models for glioblastoma (GBM) have yet been established, excluding the traditional two-dimensional culture. GBM tumorspheres (TSs) have been highlighted as a good model platform for testing drug effects and characterizing specific features of GBM, but a detailed evaluation of their suitability and comparative performance is lacking. Here, we isolated GBM TSs and extracellular matrices (ECM) from tissues obtained from newly diagnosed IDH1 wild-type GBM patients and cultured GBM TSs on five different culture platforms: (1) ordinary TS culture liquid media (LM), (2) collagen-based three-dimensional (3D) matrix, (3) patient typical ECM-based 3D matrix, (4) patient tumor ECM-based 3D matrix, and (5) mouse brain. For evaluation, we obtained transcriptome data from all cultured GBM TSs using microarrays. The LM platform exhibited the most similar transcriptional program to paired tissues based on GBM genes, stemness- and invasiveness-related genes, transcription factor activity, and canonical signaling pathways. GBM TSs can be cultured via an easy-to-handle and cost- and time-efficient LM platform while preserving the transcriptional program of the originating tissues without supplementing the ECM or embedding it into the mouse brain. In addition to applications in basic cancer research, GBM TSs cultured in LM may also serve as patient avatars in drug screening and pre-clinical evaluation of targeted therapy and as standardized and clinically relevant models for precision medicine.

Rebuilding the microenvironment of primary tumors in humans: a focus on stroma.

Conventional tumor models have critical shortcomings in that they lack the complexity of the human stroma. The heterogeneous stroma is a central compartment of the tumor microenvironment (TME) that must be addressed in cancer research and precision medicine. To fully model the human tumor stroma, the deconstruction and reconstruction of tumor tissues have been suggested as new approaches for in vitro tumor modeling. In this review, we summarize the heterogeneity of tumor-associated stromal cells and general deconstruction approaches used to isolate patient-specific stromal cells from tumor tissue; we also address the effect of the deconstruction procedure on the characteristics of primary cells. Finally, perspectives on the future of reconstructed tumor models are discussed, with an emphasis on the essential prerequisites for developing authentic humanized tumor models.

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.

Brain physiome: A concept bridging in vitro 3D brain models and in silico models for predicting drug toxicity in the brain.

In the last few decades, adverse reactions to pharmaceuticals have been evaluated using 2D in vitro models and animal models. However, with increasing computational power, and as the key drivers of cellular behavior have been identified, in silico models have emerged. These models are time-efficient and cost-effective, but the prediction of adverse reactions to unknown drugs using these models requires relevant experimental input. Accordingly, the physiome concept has emerged to bridge experimental datasets with in silico models. The brain physiome describes the systemic interactions of its components, which are organized into a multilevel hierarchy. Because of the limitations in obtaining experimental data corresponding to each physiome component from 2D in vitro models and animal models, 3D in vitro brain models, including brain organoids and brain-on-a-chip, have been developed. In this review, we present the concept of the brain physiome and its hierarchical organization, including cell- and tissue-level organizations. We also summarize recently developed 3D in vitro brain models and link them with the elements of the brain physiome as a guideline for dataset collection. The connection between in vitro 3D brain models and in silico modeling will lead to the establishment of cost-effective and time-efficient in silico models for the prediction of the safety of unknown drugs.

Targeting extracellular matrix glycation to attenuate fibroblast activation.

The extracellular matrix (ECM) of the tumor microenvironment undergoes constant remodeling that alters its biochemical and mechano-physical properties. Non-enzymatic glycation can induce the formation of advanced glycation end-products (AGEs), which may cause abnormal ECM turnover with excessively cross-linked collagen fibers. However, the subsequent effects of AGE-mediated matrix remodeling on the characteristics of stromal cells in tumor microenvironments remain unclear. Here, we demonstrate that AGEs accumulated in the ECM alter the fibroblast phenotype within a three-dimensional collagen matrix. Both the AGE interaction with its receptor (RAGE) and integrin-mediated mechanotransduction signaling were up-regulated in glycated collagen matrix, leading to fibroblast activation to acquire a cancer-associated fibroblast (CAF)-like phenotype. These effects were blocked with neutralizing antibodies against RAGE or the inhibition of focal adhesion (FA) signaling. An AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711), also reduced the transformation of fibroblasts into the CAF-like phenotype because of its dual inhibitory role in the AGE-modified matrix. Apart from targeting the AGE-RAGE interaction directly, the decreased matrix stiffness attenuated fibroblast activation by inhibiting the downstream cellular response to matrix stiffness. Our results suggest that indirect/direct targeting of accumulated AGEs in the ECM has potential for targeting the tumor stroma to improve cancer therapy. STATEMENT OF SIGNIFICANCE: Advanced glycated end-products (AGEs)-modified extracellular matrix (ECM) is closely associated with pathological states and is recognized as a critical factor that precedes tumorigenesis. While increased matrix stiffness is known to induce fibroblast activation, less is known about how both biochemical and mechano-physical changes in AGE-mediated matrix-remodeling cooperate to produce a myofibroblastic cancer-associated fibroblast (CAF)-like phenotype. For the first time, we found that both the AGE interaction with its receptor (RAGE) and integrin-mediated mechanotransduction were up-regulated in glycated collagen matrix, leading to fibroblast activation. We further demonstrated that an AGE cross-link breaker, ALT-711, reduced the CAF-like transformation because of its dual inhibitory role in the AGE-modified matrix. Our findings offer promising extracellular-reversion strategies targeting the non-enzymatic ECM glycation, to regulate fibroblast activation.

Cancer Cell-Sticky Hydrogels to Target the Cell Membrane of Invading Glioblastomas.

Owing to their remarkable infiltrative traits, glioblastomas develop unclear tumor margins toward the brain, hampering the complete resection. Since the remaining invasive cells tend to have resistance to therapeutics and cause recurrence around the surgical voids, this has been a major challenge for glioblastoma treatment. Thus, we design a cancer cell-sticky hydrogel (CSH) that interacts with the glioblastoma cells to impede their invasive motility by modifying the cell membrane with active thiol-enriched interfaces. Highly reactive thiols at the cell surface can make the infiltrated cancer cells adhere to the hydrogel, resulting in increased cell adhesion and decreased motility. Cotreatment with the CSH and chemical inhibitors of the major proinvasive molecules, focal adhesion kinase and hyaluronic acid synthase, maximized the invasion-inhibitory effect. In addition, a significant decrease in tumor mass was achieved via CSH implantation in mouse models. Overall, our results highlight the use of the CSH to inhibit the aggressive invasion as a novel therapeutic strategy against glioblastoma.

Extracellular Matrix-Based Hydrogels to Tailoring Tumor Organoids.

When embedded into a three-dimensional (3D) matrix, cancer stem cells (or cancer-initiating cells) can grow into self-organizing organotypic structures called tumor organoids. During organoid formation, the matrix not only provides structural support but also delivers biochemical signals. Although increasing evidence indicates that the extracellular matrix (ECM) is an essential component of the tumor microenvironment during tumor development and progression, the influence of the ECM on organoid formation has been largely ignored; the ECM has only recently been recognized to play a role in the regulation of cancer cell phenotypes. We reviewed ECM-based hydrogels to tailoring tumor organoids and highlight the potential role of the ECM in the development of recapitulating malignant/invasive tumor organoids with enhanced capacity for in vitro representation of ECM-regulated tumor progression.

Matrix stiffness epigenetically regulates the oncogenic activation of the Yes-associated protein in gastric cancer.

In many cancers, tumour progression is associated with increased tissue stiffness. Yet, the mechanisms associating tissue stiffness with tumorigenesis and malignant transformation are unclear. Here we show that in gastric cancer cells, the stiffness of the extracellular matrix reversibly regulates the DNA methylation of the promoter region of the mechanosensitive Yes-associated protein (YAP). Reciprocal interactions between YAP and the DNA methylation inhibitors GRHL2, TET2 and KMT2A can cause hypomethylation of the YAP promoter and stiffness-induced oncogenic activation of YAP. Direct alteration of extracellular cues via in situ matrix softening reversed YAP activity and the epigenetic program. Our findings suggest that epigenetic reprogramming of the mechanophysical properties of the extracellular microenvironment of solid tumours may represent a therapeutic strategy for the inhibition of cancer progression.

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.

Crosstalk between GBM cells and mesenchymal stemlike cells promotes the invasiveness of GBM through the C5a/p38/ZEB1 axis.

BACKGROUND: Mesenchymal stemlike cells (MSLCs) have been detected in many types of cancer including brain tumors and have received attention as stromal cells in the tumor microenvironment. However, the cellular mechanisms underlying their participation in cancer progression remain largely unexplored. The aim of this study was to determine whether MSLCs have a tumorigenic role in brain tumors. METHODS: To figure out molecular and cellular mechanisms in glioma invasion, we have cultured glioma with MSLCs in a co-culture system. RESULTS: Here, we show that MSLCs in human glioblastoma (GBM) secrete complement component C5a, which is known for its role as a complement factor. MSLC-secreted C5a increases expression of zinc finger E-box-binding homeobox 1 (ZEB1) via activation of p38 mitogen-activated protein kinase (MAPK) in GBM cells, thereby enhancing the invasion of GBM cells into parenchymal brain tissue. CONCLUSION: Our results reveal a mechanism by which MSLCs undergo crosstalk with GBM cells through the C5a/p38 MAPK/ZEB1 signaling loop and act as a booster in GBM progression. KEY POINTS: 1. MSLCs activate p38 MAPK-ZEB1 signaling in GBM cells through C5a in a paracrine manner, thereby boosting the invasiveness of GBM cells in the tumor microenvironment.2. Neutralizing of C5a could be a potential therapeutic target for GBM by inhibition of mesenchymal phenotype.

Astrocyte-Encapsulated Hydrogel Microfibers Enhance Neuronal Circuit Generation.

Astrocytes, the most representative glial cells in the brain, play a multitude of crucial functions for proper neuronal development and synaptic-network formation, including neuroprotection as well as physical and chemical support. However, little attention has been paid, in the neuroregenerative medicine and related fields, to the cytoprotective incorporation of astrocytes into neuron-culture scaffolds and full-fledged functional utilization of encapsulated astrocytes for controlled neuronal development. In this article, a 3D neurosupportive culture system for enhanced induction of neuronal circuit generation is reported, where astrocytes are confined in hydrogel microfibers and protected from the outside. The astrocyte-encapsulated microfibers significantly accelerate the neurite outgrowth and guide its directionality, and enhance the synaptic formation, without any physical contact with the neurons. This astrocyte-laden system provides a pivotal culture scaffold for advanced development of cell-based therapeutics for neural injuries, such as spinal cord injury.

Fluid-Matrix Interface Triggers a Heterogeneous Activation of Macrophages.

Spontaneous activation of macrophages in response to inflammation is a key part of innate immunity and host defense. Macrophages represent a heterogeneous population of cells with different phenotypic profiles performing distinct functions in host defense. Although a spectrum of macrophage activation stages exists in an inflamed region, the effect of local physical conditions on the heterotypic activation of macrophages is unknown. Here, we introduce an in vivo fluid-matrix interface analogous culture platform, an asymmetric microenvironment, facilitating the formation of macrophage aggregates (MAs). Macrophages were self-assembled to form MAs of ∼100 µm diameter at the collagen matrix-medium interface upon phorbol-12-myristate-13-acetate treatment. The macrophages within the half-embedded MAs into the matrix were heterogeneously activated, resulting in inhomogeneous cell-cell and cell-matrix interactions within the aggregates. Our demonstration may aid in a better understanding of the acquisition of macrophage heterogeneity in response to tissue-specific microenvironments.

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.

Hyaluronic acid-based extracellular matrix triggers spontaneous M2-like polarity of monocyte/macrophage.

Hyaluronic acid (HA) is found in various tumor tissues, and is considered tumor-associated extracellular matrix (ECM). Within this tumor-associated ECM, stromal cells, especially immune cells, are involved in tumor progression. However, the effects of tumor-associated ECM on the characteristics of immune cells remain unexplored. Therefore, we studied the triggering effect of HA on spontaneous M2-like polarity of monocytes/macrophages using HA-mixed collagen (HA-COL) matrix. In the presence of HA, expression of the HA receptor (CD44) and M2 polarity-related genes was upregulated in human monocytes (THP-1 cells). We confirmed the CD44-mediated activation of STAT3 in THP-1 cells cultured in an HA-rich environment. Furthermore, when we induced the THP-1 cells to differentiate into cells with M1 or M2 polarity within an HA-rich environment, the HA-rich environment influenced the direction of induction. Our findings might improve understanding of the crosstalk between immune cells and tumor-associated ECM, and facilitate development of tumor immunotherapy strategies.