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Optogenetic control of integrin-matrix interaction
摘要： Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix–OptoIntegrin system may serve as a blueprint for rendering matrix–receptor interactions amendable to precise control with light.
关键词： Optogenetics,Mechanosensing,Extracellular matrix,Cell adhesion,Integrin
更新于2025-11-21 11:24:58
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Infrared Spectroscopic Imaging Visualizes a Prognostic Extracellular Matrix-Related Signature in Breast Cancer
摘要： Molecular analysis techniques such as gene expression analysis and proteomics have contributed greatly to our understanding of cancer heterogeneity. In prior studies, gene expression analysis was shown to stratify patient outcome on the basis of tumor-microenvironment associated genes. A specific gene expression profile, referred to as ECM3 (Extracellular Matrix Cluster 3), indicated poorer survival in patients with grade III tumors. In this work, we aimed to visualize the downstream effects of this gene expression profile onto the tissue, thus providing a spatial context to altered gene expression profiles. Using infrared spectroscopic imaging, we identified spectral patterns specific to the ECM3 gene expression profile, achieving a high spectral classification performance of 0.87 as measured by the area under the curve of the receiver operating characteristic curve. On a patient level, we correctly identified 20 out of 22 ECM3 group patients and 19 out of 20 non-ECM3 group patients by using this spectroscopic imaging-based classifier. By comparing pixels that were identified as ECM3 or non-ECM3 with H&E and IHC images, we were also able to observe an association between tissue morphology and the gene expression clusters, showing the ability of our method to capture broad outcome associated features from infrared images.
关键词： extracellular matrix,prognostic signature,gene expression,breast cancer,infrared spectroscopic imaging
更新于2025-09-23 15:21:01
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Lens-free microscopy for 3D + time acquisitions of 3D cell culture
摘要： Thanks to a novel three-dimensional imaging platform based on lens-free microscopy, it is possible to perform multi-angle acquisitions and holographic reconstructions of 3D cell cultures directly into the incubator. Being able of reconstructing volumes as large as ~5 mm3 over a period of time covering several days, allows us to observe a broad range of migration strategies only present in 3D environment, whether it is single cell migration, collective migrations of cells and dispersal of cells. In addition we are able to distinguish new interesting phenomena, e.g. large-scale cell-to-matrix interactions (>1 mm), fusion of cell clusters into large aggregate (~10,000 μm2) and conversely, total dissociation of cell clusters into clumps of migrating cells. This work on a novel 3D + time lens-free microscopy technique thus expands the repertoire of phenomena that can be studied within 3D cell cultures.
关键词： cell migration,holographic reconstruction,extracellular matrix,3D cell culture,lens-free microscopy
更新于2025-09-23 15:21:01
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Selective Stiffening of Fibrin Hydrogels with Micron Resolution Via Photocrosslinking
摘要： Fibrin hydrogels are used as a model system for studying cell-ECM biophysical interactions. Bulk mechanical stiffness of these hydrogels has been correlated to mechanotransduction and downstream signaling. However, stiffness values proximal to cells can vary by orders of magnitude at the length scale of microns. Patterning of matrix stiffness at this spatial scale can be useful in studying such interactions. Here we present and evaluate a technique to selectively stiffen defined regions within a fibrin hydrogel. Laser scanning illumination activates ruthenium-catalyzed crosslinking of fibrin tyrosine residues, resulting in tunable stiffness changes spanning distances as small as a few microns and a localized compaction of the material. As probed by active microrheology, stiffness increases by as much as 25X, similar to previously observed stiffness changes around single cells in 3D culture. In summary, our method allows for selective modification of fibrin stiffness at the micron scale with the potential to create complex patterns, which could be valuable for the investigation of mechanotransduction in a biologically meaningful way.
关键词： Fibrin,Tissue Mechanics,Microrheology,Hydrogel,Stiffness,Extracellular Matrix
更新于2025-09-19 17:15:36
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<i>VCAN</i> Canonical Splice Site Mutation is Associated With Vitreoretinal Degeneration and Disrupts an MMP Proteolytic Site
摘要： PURPOSE. To gain insight into the pathophysiology of vitreoretinal degeneration, the clinical course of three family members with Versican Vitreoretinopathy (VVR) is described, and a canonical splice site mutation in the gene encoding for versican (VCAN) protein was biochemically analyzed. METHODS. A retrospective chart review, human eye histopathology, Sanger DNA sequencing, protein structural modeling, and in vitro proteolysis assays were performed. RESULTS. The proband (II:1), mother (I:2), and younger sibling (II:2) suffered retinal degeneration with foveal sparing and retinal detachments with proliferative vitreoretinopathy, features that were confirmed on histopathologic analysis. All affected members carried a heterozygous adenine to guanine variant (c.4004-2A>G) predicted to result in exon 8 skipping or the deletion of 13 amino acids at the beginning of the GAGb chain (VCAN p.1335-1347). This deleted region corresponded to a putative MMP cleavage site, validated using fluorescence resonance energy transfer (FRET)-based proteolysis assays. Proteomic network analysis identified 10 interacting partners in the human vitreous and retina linked to retinal detachment and degeneration. CONCLUSIONS. VVR causes significant ocular disease, including retinal detachment and retinal dystrophy. The intronic VCAN mutation removes an MMP cleavage site, which alters versican structure and results in abnormal vitreous modeling. Disruption of a versican protein network may underlie clinicopathologic disease features and point to targeted therapies.
关键词： versican,VCAN,extracellular matrix,MMP-2,genetics,erosive vitreoretinopathy,retinal detachment,gelatinase,vitreous,wagner disease,MMP-9
更新于2025-09-19 17:15:36
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Three-Dimensional Multi-layered Microstructure using Laser Direct-Writing System
摘要： Tissue engineering is an essential component of developing effective regenerative therapies. Here, we introduce a promising method to create scaffold-free three-dimensional (3D) tissue engineered multi-layered microstructures from cultured cells using the “3D tissue fabrication system” (Regenova?, Cyfuse, Japan). This technique utilizes the adhesive nature of cells. When cells are cultured in non-adhesive wells, they tend to aggregate and form a spheroidal structure. The advantage of this approach is that cellular components can be mixed into one spheroid, thereby promoting the formation of extracellular matrices, such as collagen and elastin. This system enables one to create a pre-designed 3D structure composed of cultured cells. We found the advantages of this system to be: (1) the length, size, and shape of the structure were designable and highly reproducible because of the computer controlled robotics system, (2) the graftable structure could be created within a reasonable period (8 days), and (3) the constructed tissue did not contain any foreign material, which may avoid the potential issues of contamination, biotoxicity, and allergy. The utilization of this robotic system enabled the creation of a 3D multi-layered microstructure made of cell based spheres with a satisfactory mechanical properties and abundant extracellular matrix during a short period of time. These results suggest that this new technology will represent a promising, attractive, and practical strategy in the field of tissue engineering.
关键词： three-dimensional,scaffold free,bio-fabrication,tissue engineering,extracellular matrix
更新于2025-09-16 10:30:52
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Collagenase Encapsulated pH-Responsive Nanoscale Coordination Polymers for Tumor Microenvironment Modulation and Enhanced Photodynamic Nanomedicine
摘要： The abundant tumor extracellular matrix (ECM) could result in the insufficient tumor retention and ineffective intra-tumor penetration of therapeutic agents, as well as acidic & hypoxic tumor microenvironment (TME), further leading to the unsatisfactory therapeutic outcomes for many types of therapies. Therefore, developing strategies to modulate the TME by selectively degrading the condensed ECM may be helpful to improve existing cancer therapies. Herein, collagenase (CLG) encapsulated nanoscale coordination polymers (NCPs) are synthesized based on Mn2+ and an acid-sensitive benzoic-imine organic linker (BI-linker), and then modified by polyethylene glycol (PEG). Upon intravenous (i.v.) injection, these CLG@NCP-PEG nanoparticles show efficient accumulation within the tumor, in which CLG would be released due to the collapse of NCP structures within the acidic TME. The released CLG enzyme could then specifically degrade collagens, the major component of ECM, leading to loosened ECM structure, enhanced tumor perfusion and relieved hypoxia. As the results, the second-wave of nanoparticles, chlorin e6 (Ce6)-loaded liposomes (Liposome@Ce6), would exhibit enhanced retention and penetration within the tumor. Such phenomena together with relieved tumor hypoxia could then lead to greatly enhanced photodynamic therapeutic effect of Liposome@Ce6 for mice pretreated with CLG@NCP-PEG. Our work thus presents a unique strategy for TME modulation using pH-responsive NCPs as smart enzyme carriers.
关键词： Nanoscale coordination polymers,Extracellular matrix,Collagenase,Photodynamic therapy,Tumor microenvironment
更新于2025-09-10 09:29:36
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Metallic Nanoparticles-Based Biochip with Multi-Channel for Immunoassay
摘要： Osteogenesis is the cellular and molecular foundation of skeletal development and bone regeneration related to fracture healing, revitalization of bone graft and therapies for osteoporosis. A hallmark of osteogenesis is the mineralization of extracellular matrix. Because of its potential impact on developmental biology and human health, understanding and regulation of osteogenesis are subjects of intensive study. Although significant advancements have been made over the past decades, there are still unsolved puzzles in regulations of osteogenesis. Angiogenesis generally refers to new blood vessels branching out from established vasculature. Besides of fundamental physiology, angiogenesis involves in pathology, such as growth of cancer, and is essential for the repair of virtually all types of tissues. It has long been recognized that osteogenesis and angiogenesis are coupling events during bone formation. The classic osteogenic and angiogenic pathways intertwine and cross-talk during bone formation. To better understand the signal pathways and coupling factors of osteogenesis and angiogenesis is critically important for enhancing bone regeneration and tissue engineering of bone. The conventional biological models, however, have very limited capacity of isolating angiogenic and osteogenic events from the cascade of bone regeneration, and precisely quantifying the effects of angiogenic and osteogenic factors on bone formation at a molecular level. Biochips and tissue chips provide a powerful tool to simulate and quantify angiogenic and osteogenic events on the chips and effectively untangle these biologically important and clinically relevant molecular events during bone formation.
关键词： Angiogenesis,Osteogenesis,Extracellular matrix,Regeneration,Blood vessels
更新于2025-09-09 09:28:46
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Tissue-derived microparticles reduce inflammation and fibrosis in cornea wounds
摘要： Biological materials derived from the extracellular matrix (ECM) of tissues serve as scaffolds for rebuilding tissues and for improved wound healing. Cornea trauma represents a wound healing challenge as the default repair pathway can result in fibrosis and scar formation that limit vision. Effective treatments are needed to reduce inflammation, promote tissue repair, and retain the tissue’s native transparency and vision capacity. Tissue microparticles derived from cornea, cartilage and lymph nodes were processed and screened in vitro for their ability to reduce inflammation in ocular surface cells isolated from the cornea stroma, conjunctiva, and lacrimal gland. Addition of ECM particles to the media reduced expression of inflammatory genes and restored certain tear film protein production in vitro. Particles derived from lymph nodes were then applied to a rabbit lamellar keratectomy corneal injury model. Application of the tissue particles in a fibrin glue carrier decreased expression of inflammatory and fibrotic genes and scar formation as measured through imaging, histology and immunohistochemistry. In sum, immunomodulatory tissue microparticles may provide a new therapeutic tool for reducing inflammation in the cornea and ocular surface and promoting tissue repair.
关键词： ocular surface,inflammation,Extracellular matrix,corneal wound healing,corneal fibrosis
更新于2025-09-04 15:30:14
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FTIR imaging of MCF-7 colonies and their vicinity in Matrigel-embedded 3D cultures
摘要： BACKGROUND: It is now recognized that tumor cells can actively alter their microenvironment and that this remodelled microenvironment can subsequently play a critical role in cancer progression and in?uence therapeutic responses. To date, the molecular heterogeneity within a 3D cancer cell colony and its in?uence on the extracellular matrix have not been studied by infrared imaging. OBJECTIVE: The objective of this study is to investigate by mid-infrared imaging 3D Matrigel-embedded colonies of pure MCF-7 human mammary adenocarcinoma cell line and the surrounding microenvironment after undergoing formalin ?xation and paraf?n embedding (FFPE). METHODS: In order to better reproduce the procedure used for preservation and storage of clinical tissue specimens for pathologic analysis, 7- and 10-day MCF-7 colonies embedded and grown in Matrigel were FFPE-treated; 4-μm-thick sections were cut, mounted on barium ?uoride window and deparaf?nized. The Fourier transform infrared (FTIR) images of 4096 spectra were collected in transmission mode using a FPA-based FTIR imaging system. They were pre-processed and analysed by principal component and K-mean cluster analyses. RESULTS: At 1654 cm?1 and 1234 cm?1, the intensity of absorption in the colonies is signi?cantly higher than in Matrigel. It can be also noted, on the one hand, that there is a spectral heterogeneity in the intracolonial distribution of the absorbances at 1654, 1644, 1640 and 1634 cm?1 (Amide I range) possibly due to changes in protein secondary structures. On the other hand, we observe that Matrigel close to MCF-7 colonies appears altered with respect to more distant Matrigel matrix. CONCLUSIONS: FTIR imaging allowed us to highlight changes in the chemical content in MCF-7 colonies and their direct vicinity in Matrigel-embedded 3D cultures.
关键词： extracellular matrix,FTIR imaging,3D cell culture
更新于2025-09-04 15:30:14