Core Matrisome Research Articles

An atlas of the aging mouse proteome reveals the features of age-related post-transcriptional dysregulation

To what extent and how post-transcriptional dysregulation affects aging proteome remains unclear. Here, we provide proteomic data of whole-tissue lysates (WTL) and low-solubility protein-enriched fractions (LSF) of major tissues collected from mice of 6, 15, 24, and 30 months of age. Low-solubility proteins are preferentially affected by age and the analysis of LSF doubles the number of proteins identified to be differentially expressed with age. Simultaneous analysis of proteome and transcriptome using the same tissue homogenates reveals the features of age-related post-transcriptional dysregulation. Post-transcriptional dysregulation becomes evident especially after 24 months of age and age-related post-transcriptional dysregulation leads to accumulation of core matrisome proteins and reduction of mitochondrial membrane proteins in multiple tissues. Based on our in-depth proteomic data and sample-matched transcriptome data of adult, middle-aged, old, and geriatric mice, we construct the Mouse aging proteomic atlas (https://aging-proteomics.info/), which provides a thorough and integrative view of age-related gene expression changes.

Preparation, physico-biochemical characterization, and proteomic analysis of highly transparent corneal extracellular matrices for lamellar keratoplasty and tissue-engineered cornea construction

Corneal opacity and deformation, which often require corneal transplantation for treatment, are among the leading causes of monocular blindness. To restore corneal clarity and integrity, there is a need for an artificial stroma that not only matches the transparency of donated human cornea but also effectively integrates to the corneal tissue. In this study, a transparent decellularized cornea was successfully developed using the high hydrostatic pressure method with processing conditions optimized for corneal decellularization. Biochemical analyses demonstrated the effective removal of cellular components from the transparent decellularized corneas, while preserving collagen and glycosaminoglycans. Proteome analysis also revealed that core matrisome and matrisome-associated proteins remained following decellularization, similar to the composition observed in untreated corneas. The light transmittance of the transparent decellularized corneas was 86.4 ± 1.5 % in the visible region, comparable to that of donated human corneas. No complications, such as angiogenesis, were observed following interlamellar corneal transplantation in rabbits. The grafts were almost imperceptible immediately following surgery and achieved complete transparency within a few days, becoming indistinguishable even under a microscope. The transparent decellularized cornea presented here has promising potential as a material for application in lamellar keratoplasty.

Atlas of mildly and highly insoluble matrisome driving liver fibrosis.

The excessive deposition and cross-linking of core matrisome components typically result in abnormal remodeling of the extracellular matrix (ECM), leading to increased liver stiffness and worsening liver fibrosis. Exploring the biochemical properties of the ECM scaffold can deepen our understanding of the pathological mechanisms driving liver fibrosis and potentially facilitate the identification of therapeutic targets. While traditional sodium dodecyl sulfate (SDS)-based liver decellularization followed by proteomics can uncover the matrisome components within the ECM scaffold, it lacks the ability to reveal physicochemical characteristics like solubility. In our present study, using adult mouse liver as an example, we introduced a novel two-step workflow that combines our previously enhanced SDS (ESDS) decellularization with the conventional SDS method, enabling the identification of matrisome members with mild and/or high solubilities. Through this approach, we visualized the atlas of the mildly and highly insoluble matrisome contents in the adult mouse liver, as well as the regulatory network of highly insoluble matrisome that largely governs liver stiffness. Given the strong correlation between increased matrisome insolubility and heightened ECM stiffness, we believe that this methodology holds promise for future research focused on liver stiffness.

Collagen-rich liver-derived extracellular matrix hydrogels augment survival and function of primary rat liver sinusoidal endothelial cells and hepatocytes

Liver sinusoidal endothelial cells (LSECs) are key targets for addressing metabolic dysfunction-associated steatotic liver disease (MASLD). However, isolating and culturing primary LSECs is challenging due to rapid dedifferentiation, resulting in loss of function. The extracellular matrix (ECM) likely plays a crucial role in maintaining the fate and function of LSECs. In this study, we explored the influence of liver-ECM (L-ECM) on liver cells and developed culture conditions that maintain the differentiated function of liver cells in vitro for prolonged periods. Porcine liver-derived L-ECM, containing 34.9 % protein, 0.045 % glycosaminoglycans, and negligible residual DNA (41.2 ng/mg), was utilized to culture primary rat liver cells in generated hydrogels. Proteomic analyses and molecular weight distribution of proteins of solubilized L-ECM revealed the typical diverse ECM core matrisome, with abundant collagens. L-ECM hydrogels showed suitable stiffness and stress relaxation properties. Furthermore, we demonstrated that collagen-rich L-ECM hydrogels enhanced LSECs' and hepatocytes' viability, and reduced the dedifferentiation rate of LSECs. In addition, hepatocyte function was maintained longer by culture on L-ECM hydrogels compared to traditional culturing. These beneficial effects are likely attributed to the bioactive macromolecules including collagens, and mechanical and microarchitectural properties of the L-ECM hydrogels.

Alterations of Matrisome Gene Expression in Naturally Aged and Photoaged Human Skin In Vivo.

The main component of human skin is a collagen-rich extracellular matrix (ECM), known as the matrisome. The matrisome is essential for maintaining the structural integrity and mechanical properties of the skin. Recently, we reported notable decreases in matrisome proteins in natural aging and photoaging human skin. This study aims to investigate the mRNA expression of the core matrisome proteins in human skin, comparing young versus aged and sun-protected versus sun-exposed skin by quantitative real-time PCR and immunostaining. Our findings reveal a notable decrease in core matrisome transcription in aged skin. The mRNA expression of the core matrisome, such as collagen 1A1 (COL1A1), decorin, and dermatopontin, is significantly reduced in aged skin compared to its young skin. Yet, the majority of collagen mRNA expression levels of aged sun-exposed skin are similar to those found in young sun-exposed skin. This discrepancy is primarily attributable to a substantial decrease in collagen transcription in young sun-exposed skin, suggesting early molecular changes in matrisome transcription due to sun exposure, which preceded the emergence of clinical signs of photoaging. These findings shed light on the mRNA transcript profile of major matrisome proteins and their alterations in naturally aged and photoaged human skin, offering valuable insights into skin matrisome biology.

Identification of a fibronectin-binding protein signature associated with idiopathic pulmonary fibrosis

The extracellular matrix (ECM) is a critical component of tissue where it provides structural and signaling support to cells. Its dysregulation and accumulation lead to fibrosis, a major clinical challenge underlying many diseases that currently has little effective treatment. An understanding of the key molecular initiators of fibrosis would be both diagnostically useful and provide potential targets for therapeutics. The ECM protein fibronectin (FN) is upregulated in fibrotic conditions and other ECM proteins depend on assembly of a FN foundational ECM for their matrix incorporation. We used cell culture and in vivo models to investigate the role of FN in the progression of lung fibrosis. We confirmed that normal human lung fibroblasts (NHLFs) treated with transforming growth factor-beta (TGF-β) to stimulate fibrotic gene expression significantly increased both FN expression and its assembly into a matrix. We found that levels of alternatively spliced EDA and EDB exons were proportional to the increase in total FN RNA and protein showing that inclusion of these exons is not enhanced by TGF-β stimulation. RNA-sequencing identified 43 core matrisome genes that were significantly up- or down-regulated by TGF-β treatment and a Luminex immunoassay demonstrated increased levels of ECM proteins in conditioned medium of TGF-β-treated NHLFs. Interestingly, among the regulated core matrisome genes, 16 encode known FN-binding proteins and, of these, insulin-like growth factor binding protein 3 (IGFBP3) was most highly up-regulated. To link the NHLF results with in vivo disease, we analyzed lung tissue and bronchoalveolar lavage fluid from bleomycin-treated mice and found dramatically higher levels of FN and the FN-binding proteins IGFBP3, tenascin-C, and type I collagen in fibrotic conditions compared to controls. Altogether, our data identify a set of FN-binding proteins whose upregulation is characteristic of IPF and suggest that FN provides the foundational matrix for deposition of these proteins as fibrosis develops.

Benefits and limits of decellularization on mass-spectrometry-based extracellular matrix proteome analysis of mouse kidney.

The extracellular matrix (ECM) is composed of collagens, ECM glycoproteins, and proteoglycans (also named core matrisome proteins) that are critical for tissue structure and function, and matrisome-associated proteins that balance the production and degradation of the ECM proteins. The identification and quantification of core matrisome proteins using mass spectrometry is often hindered by their low abundance and their propensity to form macromolecular insoluble structures. In this study, we aimed to investigate the added value of decellularization in identifying and quantifying core matrisome proteins in mouse kidney. The decellularization strategy combined freeze-thaw cycles and sodium dodecyl sulphate treatment. We found that decellularization preserved 95% of the core matrisome proteins detected in non-decellularized kidney and revealed few additional ones. Decellularization also led to an average of 59 times enrichment of 96% of the core matrisome proteins as the result of the successful removal of cellular and matrisome-associated proteins. However, the enrichment varied greatly among core matrisome proteins, resulting in a misrepresentation of the native ECM composition in decellularized kidney. This should be brought to the attention of the matrisome research community, as it highlights the need for caution when interpreting proteomic data obtained from a decellularized organ.

Assessing the effects of bladder decellularization protocols on extracellular matrix (ECM) structure, mechanics, and biology

Acellular matrices have historically been applied as biologic scaffolds in surgery, wound care, and tissue engineering, albeit with inconsistent outcomes. One aspect that varies widely between products is the selection of decellularization protocol, yet few studies assess comparative effectiveness of these protocols in preserving mechanics, and protein content. This study characterizes bladder acellular matrix (BAM) using two different detergent and enzymatic protocols, evaluating effects on nuclei and DNA removal (≥90%), structure, tensile properties, and maintenance of extracellular matrix proteins. Porcine bladders were decellularized with 0.5% Sodium Dodecyl Sulfate (SDS) or 0.25% Trypsin-hypotonic-Triton X-100 hypertonic (TT)-based agitation protocols, followed by DNase/RNase agents. Characterization of BAM included decellularization efficacy (DAPI, DNA quantification), structure (histology and scanning electron microscopy), tensile testing (Instron 345C-1 mechanical tester), and protein presence and diversity (mass spectrometry). SDS and TT data was directly compared to the same native bladder using two-tailed paired t-tests. Native, TT, and SDS cohorts for tensile testing were compared using one-way ANOVA; Tukey's post-hoc tests for among group differences. Effective nuclei removal was achieved by SDS- and TT-based protocols. However, target DNA removal was achieved with SDS but not TT. SDS more effectively maintained qualitative tissue architecture compared to TT. The tensile modulus of the TT cohort increased, and stretchability decreased after decellularization in both SDS and TT. UTS was unaffected by either protocol. Higher overall diversity and quantity of core matrisome and matrisome-associated proteins was maintained in the SDS vs TT cohort post-decellularization. The results indicated that detergent selection affects multiple aspects of the resultant BAM biologic product. In the selected protocols, SDS was superior to TT efficacy, and maintenance of gross tissue architecture as well as maintenance of ECM proteins. Decellularization increased scaffold resistance to deformation in both cohorts. Future studies applying biologic scaffolds must consider the processing method and agents used to ensure that materials selected are optimized for characteristics that will facilitate effective translational use.

Multiomic analysis of uterine leiomyomas in self-described Black and White women: molecular insights into health disparities

Black women are at an increased risk of developing uterine leiomyomas and experiencing worse disease prognosis than White women. Epidemiologic and molecular factors have been identified as underlying these disparities, but there remains a paucity of deep, multiomic analysis investigating molecular differences in uterine leiomyomas from Black and White patients. To identify molecular alterations within uterine leiomyoma tissues correlating with patient race by multiomic analyses of uterine leiomyomas collected from cohorts of Black and White women. We performed multiomic analysis of uterine leiomyomas from Black (42) and White (47) women undergoing hysterectomy for symptomatic uterine leiomyomata. In addition, our analysis included the application of orthogonal methods to evaluate fibroid biomechanical properties, such as second harmonic generation microscopy, uniaxial compression testing, and shear-wave ultrasonography analyses. We found a greater proportion of MED12 mutant uterine leiomyomas from Black women (>35% increase; Mann-Whitney U, P<.001). MED12 mutant tumors exhibited an elevated abundance of extracellular matrix proteins, including several collagen isoforms, involved in the regulation of the core matrisome. Histologic analysis of tissue fibrosis using trichrome staining and secondary harmonic generation microscopy confirmed that MED12 mutant tumors are more fibrotic than MED12 wild-type tumors. Using shear-wave ultrasonography in a prospectively collected cohort, Black patients had fibroids that were firmer than White patients, even when similar in size. In addition, these analyses uncovered ancestry-linked expression quantitative trait loci with altered allele frequencies in African and European populations correlating with differential abundance of several proteins in uterine leiomyomas independently of MED12 mutation status, including tetratricopeptide repeat protein 38. Our study shows that Black women have a higher prevalence of uterine leiomyomas harboring mutations in MED12 and that this mutational status correlates with increased tissue fibrosis compared with wild-type uterine leiomyomas. Our study provides insights into molecular alterations correlating with racial disparities in uterine leiomyomas and improves our understanding of the molecular etiology underlying uterine leiomyoma development within these populations.

Abstract 3906: Ex vivo breast tumors for understanding the patient specific tumor microenvironment remodeling and drug responses

Abstract Breast cancer is a heterogeneous disease driven by cell-intrinsic and cell-extrinsic signaling cues. Many of these cues derive from the tumor microenvironment (TME), which is composed of adipocytes, vasculature, stem cells, immune cells, and the extracellular matrix (ECM). Through these diverse components, the TME modulates cellular proliferation, survival, and ultimately response to therapy. Despite this high-level understanding of tumor-TME crosstalk, no existing pre-clinical models accurately capture the TME in vitro. We overcame that hurdle by developing a method to maintain healthy human breast tissue (HBT) alive ex vivo for at least 8 weeks. By culturing breast cancer cells in that HBT, we created ex vivo breast tumors (EVBTs) that retained patient-specific features relevant to breast cancer including menopause status, obesity status, and response to therapy. Using these EVBTs, we demonstrate breast cancer subtype-specific alterations in lipid accumulation, ECM remodeling, and cytokine profiles. RNA sequencing showed that breast cancer cell line signatures were retained in the EVBTs system at 1 week and 2 weeks in vitro. We demonstrated donor-specific transcriptional changes to the core matrisome gene set. Finally, we demonstrate that our EVBTs retained patient-specific heterogeneity in response to standard of care endocrine therapy and neoadjuvant chemotherapies. Our novel tumor model allows for 1) real-time examination of breast cancer interactions with HBT and the TME and 2) the isolation of breast cancer-specific factors vs. patient-specific factors in the development and progression of breast cancer. Citation Format: Elizabeth Martin, Megan Benz, Katie Hebert, Ethan Byrne, Frank Lau. Ex vivo breast tumors for understanding the patient specific tumor microenvironment remodeling and drug responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3906.

Abstract 7642: Activated FAP+ cancer-associated fibroblasts in non-muscle invasive bladder cancer are associated with progression to muscle-invasive disease

Abstract The majority of patients with bladder cancer initially present with non-muscle-invasive disease (NMIBC). While NMIBC may be cured with transurethral resection +/- intravesical therapies, approximately 20% of patients develop progression to muscle-invasive bladder cancer (MIBC) which requires life-altering treatments such as cystectomy and has a high risk of lethal metastatic recurrence. Current risk stratification for NMIBC relies on clinicopathologic features that overlook the potential role of the tumor microenvironment (TME) in bladder cancer pathogenesis. We sought to delineate the stromal and immune landscape alterations within the TME upon bladder cancer progression and to identify TME-related features that could enhance current prognostic approaches. Analyzing by single-cell RNA sequencing and multiplexed imaging of 22 untreated NMIBC and MIBC specimens, coupled with publicly available datasets, we discovered significant transcriptional variability among cancer-associated fibroblasts (CAFs) and macrophages across patients. This inter-patient variability was attributable to the extensive remodeling of these cell types during cancer progression. Notably, a specific subset of CAF, characterized by ITGA8+F3+ markers, was found to be highly enriched in NMIBC lesions and transcriptionally related to sub-urothelial fibroblasts of the healthy bladder, whereas a FAP+ CAF subset enriched for core matrisome related genes was dominant in MIBC lesions. We developed a FAP+ CAF signature which was then projected onto a large public dataset of bulk-RNAseq data from patients with NMIBC demonstrating that the FAP+ CAF signature was significantly associated with progression to MIBC independently of contemporary risk stratification scores. The presence of FAP+ CAFs in a subset of NMIBC specimens was orthogonally validated using immunohistochemistry. Importantly, areas with FAP staining were observed in close contact with individual or small groups of tumor cells exhibiting an invasive phenotype and partial epithelial-mesenchymal transition, likely explaining the higher risk of progression to MIBC. Our study improves our understanding of fibroblasts remodeling during bladder cancer progression and highlights the potential of integrating CAF-derived biomarkers into NMIBC risk stratification models. Citation Format: Abdenour Abbas, Shrisha Maskey, Evan Cheng, Ziao Li, Koushik Ganesh, Sudeh Izadmehr, Serena Janho dit Hreich, Benjamin Demaille, Jeremy Mesple, Philemon Sirven, Clementine Krucker, Jacqueline Fontugne, Isabelle Bernard-Pierrot, Yves Allory, Miriam Merad, Julien Adam, John Sfakianos, Ephraim Kenigsberg, Matthew Galsky, Helene Salmon. Activated FAP+ cancer-associated fibroblasts in non-muscle invasive bladder cancer are associated with progression to muscle-invasive disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7642.

Proteomic analysis of decellularized mice liver and kidney extracellular matrices

BackgroundThe extracellular matrix (ECM) is a three-dimensional network of proteins that encases and supports cells within a tissue and promotes physiological and pathological cellular differentiation and functionality. Understanding the complex composition of the ECM is essential to decrypt physiological processes as well as pathogenesis. In this context, the method of decellularization is a useful technique to eliminate cellular components from tissues while preserving the majority of the structural and functional integrity of the ECM.ResultsIn this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected.ConclusionThe identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell–matrix interactions and their contribution to a tissue-specific microenvironment.

High-Throughput Extracellular Matrix Proteomics of Human Lungs Enabled by Photocleavable Surfactant and diaPASEF.

The extracellular matrix (ECM) is a complex assembly of proteins that provide interstitial scaffolding and elastic recoil for human lungs. The pulmonary extracellular matrix is increasingly recognized as an independent bioactive entity, by creating biochemical and mechanical signals that influence disease pathogenesis, making it an attractive therapeutic target. However, the pulmonary ECM proteome ("matrisome") remains challenging to analyze by mass spectrometry due to its inherent biophysical properties and relatively low abundance. Here, we introduce a strategy designed for rapid and efficient characterization of the human pulmonary ECM using the photocleavable surfactant Azo. We coupled this approach with trapped ion mobility MS with diaPASEF to maximize the depth of matrisome coverage. Using this strategy, we identify nearly 400 unique matrisome proteins with excellent reproducibility that are known to be important in lung biology, including key core matrisome proteins.

Comprehensive Matrisome Profiling of Human Adipose Tissue for Soft Tissue Reconstruction.

For effective translation of research from tissue engineering and regenerative medicine domains, the cell-instructive extracellular matrix (ECM) of specific tissues must be accurately realized. As adipose tissue is gaining traction as a biomaterial for soft tissue reconstruction, with highly variable clinical outcomes obtained, a quantitative investigation of the adipose tissue matrisome is overdue. In this study, the human adipose tissue matrisome is profiled using quantitative sequential windowed acquisition of all theoretical fragment ion spectra - mass spectrometry (SWATH-MS)proteomics across a cohort of 13 fat-grafting patients, to provide characterization of ECM proteins within the tissue, and to understand human population variation. There are considerable differences in the expression of matrisome proteins across the patient cohort, with age and lipoaspirate collection technique contributing to the greatest variation across the core matrisome. A high abundance of basement membrane proteins (collagen IV and heparan sulfate proteoglycan) is detected, as well as fibrillar collagens I and II, reflecting the hierarchical structure of the tissue. This study provides a comprehensive proteomic evaluation of the adipose tissue matrisome and contributes to an enhanced understanding of the influence of the matrisome in adipose-related pathologies by providing a healthy reference cohort and details an experimental pipeline that can be further exploited for future biomaterial development.

Analysis of matrisome expression patterns in murine and human dorsal root ganglia.

The extracellular matrix (ECM) is a dynamic structure of molecules that can be divided into six different categories and are collectively called the matrisome. The ECM plays pivotal roles in physiological processes in many tissues, including the nervous system. Intriguingly, alterations in ECM molecules/pathways are associated with painful human conditions and murine pain models. Nevertheless, mechanistic insight into the interplay of normal or defective ECM and pain is largely lacking. The goal of this study was to integrate bulk, single-cell, and spatial RNA sequencing (RNAseq) datasets to investigate the expression and cellular origin of matrisome genes in male and female murine and human dorsal root ganglia (DRG). Bulk RNAseq showed that about 65% of all matrisome genes were expressed in both murine and human DRG, with proportionally more core matrisome genes (glycoproteins, collagens, and proteoglycans) expressed compared to matrisome-associated genes (ECM-affiliated genes, ECM regulators, and secreted factors). Single cell RNAseq on male murine DRG revealed the cellular origin of matrisome expression. Core matrisome genes, especially collagens, were expressed by fibroblasts whereas matrisome-associated genes were primarily expressed by neurons. Cell-cell communication network analysis with CellChat software predicted an important role for collagen signaling pathways in connecting vascular cell types and nociceptors in murine tissue, which we confirmed by analysis of spatial transcriptomic data from human DRG. RNAscope in situ hybridization and immunohistochemistry demonstrated expression of collagens in fibroblasts surrounding nociceptors in male and female human DRG. Finally, comparing human neuropathic pain samples with non-pain samples also showed differential expression of matrisome genes produced by both fibroblasts and by nociceptors. This study supports the idea that the DRG matrisome may contribute to neuronal signaling in both mouse and human, and that dysregulation of matrisome genes is associated with neuropathic pain.

Abstract 5957: Chemotherapy-induced extracellular matrix remodeling in HGSOC

Abstract In this study we explored the effects of chemotherapy on the extracellular matrix of high-grade serous ovarian cancer metastasis (HGSOC) at structural-textural protein level and transcriptionally, in order to identify the microenvironmental features that accompany a successful response. We studied two murine orthotopic, transplantable syngeneic models of HGSOC, one that displays excellent response to carboplatin/paclitaxel (60577) and one that is resistant to treatment (HGS2)1. Using immunohistochemistry, we stained omental tumors from both models for the ECM components fibronectin (FN1), collagen 1A1 (COL1A1) and versican (VCAN), also using Masson’s Trichrome to stain for collagen. We analyzed their abundance, textural features (Haralick features) and structure with the image analysis softwares, QuPath and TWOMBLI respectively. A compilation of 44 ECM metrics, which we named the “structure index”, correlated with response to chemotherapy, as measured by reduction in omental tumor weight. In parallel, we performed RNAseq on the omental tumors, identifying clusters of genes that change over time with chemotherapy treatment of the responsive model 60577, while the chemoresistant model HGS2 displayed limited alteration of gene expression. We integrated RNAseq and ECM structure and texture data, and showed the structure index was correlated with 144 core matrisome genes. To identify potential therapeutic targets, we then focused our study on genes that remained highly expressed in resistant tumors post chemotherapy, but were originally low or decreased with treatment in the sensitive tumors. Members of the LOX and P4HA family were found amongst these genes. We confirmed by immunohistochemical analysis that expression of LOX, LOXL2, P4HA1 and P4HA2 was downregulated by chemotherapy in the 60577 chemo-sensitive tumors while their levels remained relatively unaffected in the resistant HGS2 tumors. This study refines our understanding of the mechanisms of microenvironmental chemoresistance in HGSOC, highlighting putative therapeutic targets to increase the efficacy of platinum-based chemotherapy in ovarian cancer. 1 Maniati, E. et al. Mouse Ovarian Cancer Models Recapitulate the Human Tumor Microenvironment and Patient Response to Treatment. Cell Rep 30, 525-540.e527, doi:10.1016/j.celrep.2019.12.034 (2020). Citation Format: Panoraia Kotantaki, Florian Laforêts, Eleni Maniati, Chiara Berlato, Anna Malliouri, Michael John Devlin, Beatrice Malacrida, Samar Elorbany, Ranjit Manchanda, Frances R. Balkwill. Chemotherapy-induced extracellular matrix remodeling in HGSOC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5957.

Harnessing the synergy of perfusable muscle flap matrix and adipose-derived stem cells for prevascularization and macrophage polarization to reconstruct volumetric muscle loss.

Muscle flaps must have a strong vascular network to support a large tissue volume and ensure successful engraftment. We developed porcine stomach musculofascial flap matrix (PDSF) comprising extracellular matrix (ECM) and intact vasculature. PDSF had a dominant vascular pedicle, microcirculatory vessels, a nerve network, well-retained 3-dimensional (3D) nanofibrous ECM structures, and no allo- or xenoantigenicity. In-depth proteomic analysis demonstrated that PDSF was composed of core matrisome proteins (e.g., collagens, glycoproteins, proteoglycans, and ECM regulators) that, as shown by Gene Ontology term enrichment analysis, are functionally related to musculofascial biological processes. Moreover, PDSF-human adipose-derived stem cell (hASC) synergy not only induced monocytes towards IL-10-producing M2 macrophage polarization through the enhancement of hASCs' paracrine effect but also promoted the proliferation and interconnection of both human skeletal muscle myoblasts (HSMMs) and human umbilical vein endothelial cells (HUVECs) in static triculture conditions. Furthermore, PDSF was successfully prevascularized through a dynamic perfusion coculture of hASCs and HUVECs, which integrated with PDSF and induced the maturation of vascular networks in vitro. In a xenotransplantation model, PDSF demonstrated myoconductive and immunomodulatory properties associated with the predominance of M2 macrophages and regulatory T cells. In a volumetric muscle loss (VML) model, prevascularized PDSF augmented neovascularization and constructive remodeling, which was characterized by the predominant infiltration of M2 macrophages and significant musculofascial tissue formation. These results indicate that hASCs' integration with PDSF enhances the cells' dual function in immunomodulation and angiogenesis. Owing in part to this PDSF-hASC synergy, our platform shows promise for vascularized muscle flap engineering for VML reconstruction.

Bovine extracellular matrix proteins and potential role in meat quality: First in silico Bos taurus compendium

The extracellular matrix (ECM) is a three-dimensional set of proteins that binds tissues and defines their biomechanical properties. Among the ECM components known to be involved in beef sensory qualities, authors have often studied fibrillar collagens but also, although less often, proteoglycans and some glycoproteins. The ECM contains many other proteins. Our hypothesis is that to deepen the role of ECM proteins in beef qualities and to identify new ones among the vast amount of data generated by high throughput methods, it is necessary to have a list of proteins of this matrix to refer to for the bovine species. We have therefore defined the Bos taurus matrisome known as the set of genes encoding ECM (core matrisome proteins and matrisome-associated proteins). We have used orthology, as a reference method, and a bioinformatic approach based on a computational pipeline previously published for Homo sapiens, Mus musculus and Danio rerio for the definition of their respective matrisome. We have reported here that the Bos taurus matrisome is composed of 1022 genes that we have classified according to the different matrisome categories. This list is the only matrisome of a livestock species to be defined to date. SignificanceIn this study, we provide the first definition of matrisome of a livestock species, the Bos taurus. We believe that the Bos taurus matrisome will be of great interest for several reasons. It comes as a complement to the matrisomes of several other species such as Homo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster and Caenorabditis elegans previously defined by other authors. It could be used to identify matrisome molecules among the vast amount of data generated by the high throughput methods. It thus can be used in addition to the other matrisomes as a model by the scientific community to study cell behavior and mechanotransduction and could lead to the identification of novel biomarkers for several diseases and cancers in which ECM is involved. Moreover, in the domain of studies on livestock, the dataset that we have provided here can be used in the context of product quality studies, especially meat quality, but also, for example, for lactation studies.

Deciphering the Kidney Matrisome: Identification and Quantification of Renal Extracellular Matrix Proteins in Healthy Mice.

Precise characterization of a tissue's extracellular matrix (ECM) protein composition (matrisome) is essential for biomedicine. However, ECM protein extraction that requires organ-specific optimization is still a major limiting factor in matrisome studies. In particular, the matrisome of mouse kidneys is still understudied, despite mouse models being crucial for renal research. Here, we comprehensively characterized the matrisome of kidneys in healthy C57BL/6 mice using two ECM extraction methods in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS), protein identification, and label-free quantification (LFQ) using MaxQuant. We identified 113 matrisome proteins, including 22 proteins that have not been previously listed in the Matrisome Database. Depending on the extraction approach, the core matrisome (structural proteins) comprised 45% or 73% of kidney ECM proteins, and was dominated by glycoproteins, followed by collagens and proteoglycans. Among matrisome-associated proteins, ECM regulators had the highest LFQ intensities, followed by ECM-affiliated proteins and secreted factors. The identified kidney ECM proteins were primarily involved in cellular, developmental and metabolic processes, as well as in molecular binding and regulation of catalytic and structural molecules' activity. We also performed in silico comparative analysis of the kidney matrisome composition in humans and mice based on publicly available data. These results contribute to the first reference database for the mouse renal matrisome.

Unraveling the Relevance of Tissue-Specific Decellularized Extracellular Matrix Hydrogels for Vocal Fold Regenerative Biomaterials: A Comprehensive Proteomic and In Vitro Study.

Decellularized extracellular matrix (dECM) is a promising material for tissue engineering applications. Tissue-specific dECM is often seen as a favorable material that recapitulates a native-like microenvironment for cellular remodeling. However, the minute quantity of dECM derivable from small organs like the vocal fold (VF) hampers manufacturing scalability. Small intestinal submucosa (SIS), a commercial product with proven regenerative capacity, may be a viable option for VF applications. This study aims to compare dECM hydrogels derived from SIS or VF tissue with respect to protein content and functionality using mass spectrometry-based proteomics and in vitro studies. Proteomic analysis reveals that VF and SIS dECM share 75% of core matrisome proteins. Although VF dECM proteins have greater overlap with native VF, SIS dECM shows less cross-sample variability. Following decellularization, significant reductions of soluble collagen (61%), elastin (81%), and hyaluronan (44%) are noted in VF dECM. SIS dECM contains comparable elastin and hyaluronan but 67% greater soluble collagen than VF dECM. Cells deposit more neo-collagen on SIS than VF-dECM hydrogels, whereas neo-elastin (~50 μg/scaffold) and neo-hyaluronan (~ 6 μg/scaffold) are comparable between the two hydrogels. Overall, SIS dECM possesses reasonably similar proteomic profile and regenerative capacity to VF dECM. SIS dECM is considered a promising alternative for dECM-derived biomaterials for VF regeneration.