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Strilakou, Athina, Apostolos Perelas, Andreas Lazaris, Asteria Papavdi, Petros Karkalousos, Ioanna Giannopoulou, Anastasios Kriebardis, Ioannis Panayiotides, and Charis Liapi. "Immunohistochemical determination of the extracellular matrix modulation in a rat model of choline-deprived myocardium: the effects of carnitine." Fundamental & Clinical Pharmacology 30, no. 1 (December 8, 2015): 47–57. http://dx.doi.org/10.1111/fcp.12163.
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Xiao, Zebin, Leslie A. Hopper, Meghan C. Kopp, Emily McMillan, Yue Li, Richard L. Barrett, and Ellen Puré. "Abstract C009: Disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells enhances anti-tumor immunity and immunotherapy." Cancer Research 82, no. 22_Supplement (November 15, 2022): C009. http://dx.doi.org/10.1158/1538-7445.panca22-c009.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease due to the poor response to current therapeutic treatments. A major barrier to effective treatment of PDAC is the extensive remodeling of tumor stroma characterized by accumulation of cancer associated fibroblasts (CAFs) and extracellular matrix which forms a physical barrier that limits access of the drugs to the cancer cells, suppresses the immune system, and attenuates efficacy of immunotherapies. Fibroblast activation protein (FAP) is highly expressed in a pro-tumorigenic subset of CAFs in PDAC. We hypothesized that depletion of FAP+-CAFs would deplete extracellular matrix (ECM) and reduce the immune suppressive function of the stroma and thereby enhance the efficacy of tumor antigen targeted CAR T cell therapy in PDAC. Using real-time tumor fragment-based 2-photon microscopy, multiparametric flow cytometry and multiplexed immunofluorescence staining, we showed that FAP targeted CAR T cells (FAP-CAR T) efficiently traffic into tumors compared with tumor-antigen (mesothelin) targeted CAR (Meso-CAR) T cells which were trapped in the stroma-rich or matrix-dense areas and led to depletion of immunosuppressive FAP+ cells and reprogrammed the fibrillar collagen network surrounding tumor nests, advancing the infiltration of FAP-CAR T cells into tumor nests. Strikingly, FAP-CAR T cell-mediated depletion for FAP+ cells also rendered the tumor microenvironment permissive to the infiltration and anti-tumor activity of tumor antigen meso-CAR T cells. Moreover, ablation of FAP+ cells markedly enhanced endogenous T cell infiltration which further enhanced anti-tumor immunity and immunotherapy in PDAC models. Thus, our findings established that FAP-CAR T cell-mediated ablation of immunosuppressive FAP+-CAFs and disruption of the desmoplastic stroma they generate, can enhance accumulation and functionality of endogenous anti-tumor immunity and CAR-T cell therapy in the context of highly desmoplastic solid tumors. Citation Format: Zebin Xiao, Leslie A. Hopper, Meghan C. Kopp, Emily McMillan, Yue Li, Richard L Barrett, Ellen Puré. Disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells enhances anti-tumor immunity and immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C009.
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Chia, Jean-San, Chiou-Yueh Yeh, and Jen-Yang Chen. "Identification of a Fibronectin Binding Protein from Streptococcus mutans." Infection and Immunity 68, no. 4 (April 1, 2000): 1864–70. http://dx.doi.org/10.1128/iai.68.4.1864-1870.2000.
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ABSTRACT The interaction of viridans streptococci with components of the extracellular matrix (ECM) plays an important role in the pathogenesis of infective endocarditis. We have identified a surface protein ofStreptococcus mutans which binds the ECM constituent fibronectin (Fn). Initially, we found that S. mutans could adsorb soluble Fn in plasma, but with lower efficiency thanStreptococcus pyogenes. In addition, S. mutanscould bind immobilized Fn in a dose-dependent manner when tested using an enzyme-linked immunosorbent assay. Crude extracts of cell wall-associated proteins or extracellular proteins from S. mutans MT8148 specifically bound Fn through a protein with the molecular mass of ca. 130 kDa, as detected by far-Western immunoblotting. The candidate Fn binding protein (FBP-130) was purified to near homogeneity by using Fn coupled Sepharose 4B affinity column chromatography. A rabbit polyclonal antibody against FBP-130 reacted specifically with a protein of molecular mass of ca. 130 kDa in both cell wall and extracellular fractions, and the abundance of FBP was higher in the former than in the latter fractions. The purified FBP bound specifically to immobilized Fn, whereas the binding of soluble Fn to coated FBP could only be detected in the presence of high concentrations of Fn. The purified FBP, as well as anti-FBP immunoglobulin G, inhibited the adherence of S. mutans to immobilized Fn and endothelial cells (ECV304) in a dose-dependent manner. These results demonstrated that FBP-130 mediated the adherence of S. mutans specifically to Fn and endothelial cells in vitro. The characteristics of S. mutans and FBP-130 in binding Fn confirmed that viridans streptococci adopt different strategies in their interaction with ECM.
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Eieland, Alexander K., Kjersti R. Normann, Arvind Y. M. Sundaram, Tuula A. Nyman, Kristin A. B. Øystese, Tove Lekva, Jens P. Berg, Jens Bollerslev, and Nicoleta C. Olarescu. "Distinct Pattern of Endoplasmic Reticulum Protein Processing and Extracellular Matrix Proteins in Functioning and Silent Corticotroph Pituitary Adenomas." Cancers 12, no. 10 (October 14, 2020): 2980. http://dx.doi.org/10.3390/cancers12102980.
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Functioning (FCA) and silent corticotroph (SCA) pituitary adenomas act differently from a clinical perspective, despite both subtypes showing positive TBX19 (TPIT) and/or adrenocorticotropic hormone (ACTH) staining by immunohistochemistry. They are challenging to treat, the former due to functional ACTH production and consequently hypercortisolemia, and the latter due to invasive and recurrent behavior. Moreover, the molecular mechanisms behind their distinct behavior are not clear. We investigated global transcriptome and proteome changes in order to identify signaling pathways that can explain FCA and SCA differences (e.g., hormone production vs. aggressive growth). In the transcriptomic study, cluster analyses of differentially expressed genes revealed two distinct groups in accordance with clinical and histological classification. However, in the proteomic study, a greater degree of heterogeneity within the SCA group was found. Genes and proteins related to protein synthesis and vesicular transport were expressed by both adenoma groups, although different types and a distinct pattern of collagen/extracellular matrix proteins were presented by each group. Moreover, several genes related to endoplasmic reticulum protein processing were overexpressed in the FCA group. Together, our findings shed light on the different repertoires of activated signaling pathways in corticotroph adenomas, namely, the increased protein processing capacity of FCA and a specific pattern of adhesion molecules that may play a role in the aggressiveness of SCA.
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Cardoso, I., M. Brito, and M. J. Saraiva. "Extracellular Matrix Markers for Disease Progression and Follow-Up of Therapies in Familial Amyloid Polyneuropathy V30M TTR-Related." Disease Markers 25, no. 1 (2008): 37–47. http://dx.doi.org/10.1155/2008/549872.
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Familial Amyloidotic Polyneuropathy (FAP) is a disorder characterized by the extracellular deposition of fibrillar Transthyretin (TTR) amyloid, with a special involvement of the peripheral nerve. Several extracellular matrix proteins have been found elevated in tissues from FAP patients, namely metalloproteinase-9 (MMP-9), neutrophil gelatinase associated lipocalin (NGAL) and biglycan. In this work we assessed the levels of MMP-9, tissue inhibitor of metalloproteinase 1 (TIMP-1), NGAL, biglycan and chondroitin sulphate (CSPG) in an FAP V30M TTR-related transgenic mouse model at different stages of TTR deposition and after two different treatment approaches to remove fibrillar deposits. Immunohistochemistry or RT-PCR analysis showed that biglycan was already increased in animals presenting TTR deposited in a non-fibrillar state, whereas MMP-9, TIMP-1, NGAL and CSPG were elevated only in mice with TTR amyloid deposits. Mice treated with doxycycline, a TTR fibril disrupter, presented lower levels of MMP-9, TIMP-1 and NGAL, suggestive of matrix recovery. Mice immunized with TTR Y78F to remove TTR deposition showed significantly lower levels of all the five tested markers, suggesting removal of fibrillar and non-fibrillar deposits. Cellular studies using oligomeric TTR showed induction of MMP-9 when compared to soluble TTR, large aggregates or fibrils. Furthermore, this induction was neutralized by an anti-receptor for advanced glycation end products (RAGE) antibody, indicating RAGE engagement in this process. Further studies in a larger number of tissue samples will indicate the application of these ECM markers in parallel with Congo Red staining in tissue characterization of pre-clinical and clinical stages in FAP and other amyloidoses.
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Comba, Andrea, Syed M. Faisal, Maria Luisa Varela, Anna Argento, Patrick Dunn, Clifford Abel, Todd Hollon, et al. "TMIC-62. INHIBITION OF TUMOR-ASSOCIATED COL1A1 MATRIX ARRESTS GLIOMA MESENCHYMAL TRANSFORMATION AND REPROGRAMS THE TUMOR MICROENVIRONMENT." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii285. http://dx.doi.org/10.1093/neuonc/noac209.1106.
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Abstract Tumor mesenchymal transformation (MT) is a hallmark of high-grade gliomas. The mesenchymal state is associated with specific changes related to cell adhesion, migration, and the extracellular matrix. Collagen 1a1 (COL1A1) is a main component of the extracellular matrix in gliomas, and its expression correlates inversely with patient survival. However, the cellular and molecular mechanisms of the tumor-associated COL1A1 matrix in gliomas remains elusive. Our study integrates histopathological features, spatially resolved transcriptomics, cellular dynamics and microenvironment alterations associated with MT in high-grade gliomas. Using deep learning analysis of mouse and human glioma histological samples we identified that the density of areas of MT, named oncostreams, correlates with tumor aggressiveness. Spatial transcriptomics analysis, using laser capture microdissection, identified a signature enriched in extracellular matrix related proteins, in which COL1A1 appeared as a key determinant of mesenchymal organization. Correspondingly, human and mouse high-grade gliomas showed prominent alignment of collagen fibers along these mesenchymal fascicles and higher COL1A1 expression compared to low-grade gliomas. Moreover, RNA fluorescent multiplex assays identified at single cell level that different cells within glioma tumors contribute to COL1A1 expression, including neoplastic cells and perivascular non-neoplastic cells such as ACTA2+, CYR61+ and FAP+. Inhibition of COL1A1 using genetically engineered mouse models decreased areas of mesenchymal transformation and increased survival. COL1A1 downregulation impaired tumor cell proliferation and remodeled the tumor microenvironment by reducing CD68+ macrophages/microglia cells, CD31+ endothelial cells, ACTA2+, CYR61+ and FAP+ perivascular cells, and increased GFAP+ astrocytes infiltration withing the tumor mass. Further studies, using ex-vivo glioma explants demonstrated that CO1A1 downregulation decreased collective invasion of the normal brain, supporting its importance in tumor progression. We propose that COL1A1 expression is a valuable marker for diagnosis, and COL1A1 depletion within glioma tumors is a promising direct or complementary therapeutic approach to reprogram mesenchymal transformation, and halt tumor growth.
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Shi, Yixin, Ziren Kong, Penghao Liu, Guozhu Hou, Jiaming Wu, Wenbin Ma, Xin Cheng, and Yu Wang. "Oncogenesis, Microenvironment Modulation and Clinical Potentiality of FAP in Glioblastoma: Lessons Learned from Other Solid Tumors." Cells 10, no. 5 (May 10, 2021): 1142. http://dx.doi.org/10.3390/cells10051142.
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Currently, glioblastoma (GBM) is the most common malignant tumor of the central nervous system in adults. Fibroblast activation protein (FAP) is a member of the dipeptidyl peptidase family, which has catalytic activity and is engaged in protein recruitment and scaffolds. Recent studies have found that FAP expression in different types of cells within the GBM microenvironment is typically upregulated compared with that in lower grade glioma and is most pronounced in the mesenchymal subtype of GBM. As a marker of cancer-associated fibroblasts (CAFs) with tumorigenic activity, FAP has been proven to promote tumor growth and invasion via hydrolysis of molecules such as brevican in the extracellular matrix and targeting of downstream pathways and substrates, such as fibroblast growth factor 21 (FGF21). In addition, based on its ability to suppress antitumor immunity in GBM and induce temozolomide resistance, FAP may be a potential target for immunotherapy and reversing temozolomide resistance; however, current studies on therapies targeting FAP are still limited. In this review, we summarized recent progress in FAP expression profiling and the understanding of the biological function of FAP in GBM and raised the possibility of FAP as an imaging biomarker and therapeutic target.
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Dendl, Katharina, Stefan A. Koerber, Clemens Kratochwil, Jens Cardinale, Rebecca Finck, Mardjan Dabir, Emil Novruzov, et al. "FAP and FAPI-PET/CT in Malignant and Non-Malignant Diseases: A Perfect Symbiosis?" Cancers 13, no. 19 (September 30, 2021): 4946. http://dx.doi.org/10.3390/cancers13194946.
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A fibroblast activation protein (FAP) is an atypical type II transmembrane serine protease with both endopeptidase and post-proline dipeptidyl peptidase activity. FAP is overexpressed in cancer-associated fibroblasts (CAFs), which are found in most epithelial tumors. CAFs have been implicated in promoting tumor cell invasion, angiogenesis and growth and their presence correlates with a poor prognosis. However, FAP can generally be found during the remodeling of the extracellular matrix and therefore can be detected in wound healing and benign diseases. For instance, chronic inflammation, arthritis, fibrosis and ischemic heart tissue after a myocardial infarction are FAP-positive diseases. Therefore, quinoline-based FAP inhibitors (FAPIs) bind with a high affinity not only to tumors but also to a variety of benign pathologic processes. When these inhibitors are radiolabeled with positron emitting radioisotopes, they provide new diagnostic and prognostic tools as well as insights into the role of the microenvironment in a disease. In this respect, they deliver additional information beyond what is afforded by conventional FDG PET scans that typically report on glucose uptake. Thus, FAP ligands are considered to be highly promising novel tracers that offer a new diagnostic and theranostic potential in a variety of diseases.
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Xiao, Zebin, Leslie A. Hopper, Meghan C. Kopp, Emily McMillan, Yue Li, Richard L. Barrett, and Ellen Puré. "Abstract 2475: Visualization of cell to cell and cell to matrix interactions in the disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 2475. http://dx.doi.org/10.1158/1538-7445.am2022-2475.
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Abstract The efficacy of chimeric antigen receptor (CAR) T-cell therapy in solid tumors is limited by inefficient T-cell infiltration, localization and hypofunctionality within the tumor microenvironment. Prior studies demonstrated that stromal cell targeted CAR T cells, in particular CAR T cells that target fibroblast activation protein (FAP), can effectively infiltrate, and inhibit tumor growth in mouse models of pancreatic ductal adenocarcinomas (PDAC) due to their capacity to deplete stromal cells and extracellular matrix (ECM) that otherwise present a barrier to adoptive cell therapies. However, the kinetics of tumor infiltration by FAP-CAR T cells, and the cell-cell and cell-matrix interactions that contribute to their anti-tumor activity are poorly understood. A better understanding FAP-CAR T-cell kinetics and intratumoral interactions should provide insights into the requirements for successful adoptive cell therapies in solid tumors. By combining immunostaining and real-time two-photon microscopy in tumor fragments of PDAC, we found that stromal cell targeted FAP-CAR T cells can efficiently traffic into tumors. Initially (days 1-3) FAP-CAR T cells were enriched in the stroma surrounding tumor nests but excluded from the tumor nests per se. While associated with the stroma rich region, the CAR T cells appeared activated and T cell motility was observed in loose collagen regions, but T cells migrated poorly in dense matrix areas. Aligned collagen fibers in the tumor boarder dictated the migratory trajectory of T cells and restricted them from entering tumor nests. By day 5 we observed depletion of immunosuppressive stromal cells, dissolution of the fibrillar collagen network surrounding tumor nests and infiltration of FAP-CAR T cells into tumor nests. Treatment with hyaluronidase increased the ability of T cells to infiltrate the tumor nest. Thus, FAP-CAR T cell-mediated ablation of immunosuppressive FAP+ stromal cells and disruption of the desmoplastic stroma they generate, can enhance accumulation and functionality of CAR-T cell therapy in the context of highly desmoplastic solid tumors. Citation Format: Zebin Xiao, Leslie A. Hopper, Meghan C. Kopp, Emily McMillan, Yue Li, Richard L. Barrett, Ellen Puré. Visualization of cell to cell and cell to matrix interactions in the disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2475.
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Hoffmann, Daniel B., Daniela Fraccarollo, Paolo Galuppo, Stefan Frantz, Johann Bauersachs, and Jochen Tillmanns. "Genetic ablation of fibroblast activation protein alpha attenuates left ventricular dilation after myocardial infarction." PLOS ONE 16, no. 3 (March 5, 2021): e0248196. http://dx.doi.org/10.1371/journal.pone.0248196.
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Introduction Regulating excessive activation of fibroblasts may be a promising target to optimize extracellular matrix deposition and myocardial stiffness. Fibroblast activation protein alpha (FAP) is upregulated in activated fibroblasts after myocardial infarction (MI), and alters fibroblast migration in vitro. We hypothesized that FAP depletion may have a protective effect on left ventricular (LV) remodeling after MI. Materials and methods We used the model of chronic MI in homozygous FAP deficient mice (FAP-KO, n = 51) and wild type mice (WT, n = 55) to analyze wound healing by monocyte and myofibroblast infiltration. Heart function and remodeling was studied by echocardiography, morphometric analyses including capillary density and myocyte size, collagen content and in vivo cell-proliferation. In non-operated healthy mice up to 6 months of age, morphometric analyses and collagen content was assessed (WT n = 10, FAP-KO n = 19). Results Healthy FAP-deficient mice did not show changes in LV structure or differences in collagen content or cardiac morphology. Infarct size, survival and cardiac function were not different between FAP-KO and wildtype mice. FAP-KO animals showed less LV-dilation and a thicker scar, accompanied by a trend towards lower collagen content. Wound healing, assessed by infiltration with inflammatory cells and myofibroblasts were not different between groups. Conclusion We show that genetic ablation of FAP does not impair cardiac wound healing, and attenuates LV dilation after MI in mice. FAP seems dispensable for normal cardiac function and homeostasis.
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Rujchanarong, Denys, Julia Lefler, Janet E. Saunders, Sarah Pippin, Laura Spruill, Jennifer R. Bethard, Lauren E. Ball, et al. "Defining the Tumor Microenvironment by Integration of Immunohistochemistry and Extracellular Matrix Targeted Imaging Mass Spectrometry." Cancers 13, no. 17 (September 1, 2021): 4419. http://dx.doi.org/10.3390/cancers13174419.
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Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization. The clinical relevance of collagen HYP site modifications in cancer processes remains undefined due to technical issues accessing collagen from formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a targeted approach for investigating collagen and other extracellular matrix proteins from FFPE tissue. Here, we hypothesized that immunohistochemistry staining for fibroblastic markers would not interfere with targeted detection of collagen stroma peptides and could reveal peptide regulation influenced by specific cell types. Our initial work demonstrated that stromal peptide peak intensities when using MALD-IMS following IHC staining (αSMA, FAP, P4HA3 and PTEN) were comparable to serial sections of nonstained tissue. Analysis of histology-directed IMS using PTEN on breast tissues and TMAs revealed heterogeneous PTEN staining patterns and suggestive roles in stromal protein regulation. This study sets the foundation for investigations of target cell types and their unique contribution to collagen regulation within extracellular matrix niches.
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RIPPMANN, Jörg F., Klaus PFIZENMAIER, Ralf MATTES, Wolfgang J. RETTIG, and Dieter MOOSMAYER. "Fusion of the tissue factor extracellular domain to a tumour stroma specific single-chain fragment variable antibody results in an antigen-specific coagulation-promoting molecule." Biochemical Journal 349, no. 3 (July 25, 2000): 805–12. http://dx.doi.org/10.1042/bj3490805.
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Solid tumours growing beyond a size of 1–2mm in diameter induce supporting connective tissue structures, the tumour stroma, comprising activated fibroblasts and newly formed blood vessels, embedded in an extracellular matrix. The selective destruction of this tissue or the inhibition of its function (e.g. tumour neoangiogenesis) may result in the destruction of tumour nodules, thus providing novel opportunities for tumour therapy. Our approach aims at an antibody-mediated induction of coagulation in tumour nodules to cut off their blood supply. As a target structure the fibroblast activation protein (FAP) is used, which is specifically and abundantly expressed on the activated fibroblasts of the tumour stroma. We constructed a fusion protein comprising a single-chain module of a FAP-specific humanized antibody [single-chain fragment variable (scFv) OS4] and the extracellular domain of human tissue factor. The fusion protein, designated TFOS4, was produced in the Proteus mirabilis protoplast expression system with a yield of 15µg/ml. Biochemical characterization of TFOS4 revealed high-affinity binding to cellular FAP. Further, TFOS4 bound to factor VIIa and also exerted allosteric activation of factor VIIa. A complex of TFOS4 and factor VIIa bound to FAP-expressing cells efficiently generated activated factor X. Finally, cell-bound TFOS4 selectively induced plasma coagulation, implying its activity under physiological conditions, notably with relevant concentrations of coagulation factors and their natural inhibitors. These findings suggest that TFOS4 has the potential to increase the procoagulant state in a cell-type-specific fashion. No systemic coagulation or side effects were observed when TFOS4 was injected intravenously into normal mice, indicating the biosafety and specificity of the recombinant protein.
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Wang, Leshan, Chaoyang Li, Qianglin Liu, Yuxia Li, Peidong Gao, Xujia Zhang, Matthew Welborn, and Xing Fu. "PSV-A-10 Single-Cell Atlas of Bovine Skeletal Muscle Identifies Mechanisms Regulating Intramuscular Adipogenesis and Fibrogenesis." Journal of Animal Science 100, Supplement_3 (September 21, 2022): 257–58. http://dx.doi.org/10.1093/jas/skac247.466.
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Abstract Human and mouse studies have shown that fibro/adipogenic progenitors (FAPs) are a major source of intramuscular fat (IMF) and extracellular matrix (ECM) proteins. IMF and ECM proteins directly influence the palatability of beef, suggesting an essential role of FAPs in beef quality determination, which is still largely unexplored. We performed single-cell RNAseq (scRNAseq) using cells isolated from full blood Wagyu and Brahman cattle and Wagyu/Brahman cross cattle, which identified 21 cell clusters representing FAPs, several endothelial cell types, vascular smooths muscle cells, satellite cells, muscle fibers, and multiple immune cell types. More abundant FAPs were identified in the muscle of Brahman cattle, while a larger number of endothelial cells were identified in the muscle of Wagyu cattle. Further analysis of FAPs identified multiple FAP subpopulations with distinct gene expression profiles and anatomic locations. GSEA analysis revealed adipogenic and fibrogenic FAP subpopulations. A comparison of FAP subpopulations among different breeds showed higher complement system activity in the adipogenic FAP subpopulation of Wagyu cattle. Forced activation of the complement system in FAPs enhanced their adipogenic efficiency in vitro. In addition, cell-cell communication analysis identified active interactions between FAPs and other cell types through direct contact and secreted factors, many of which may affect FAP activities. In conclusion, our study revealed the single-cell atlas of bovine skeletal muscle and identified mechanisms regulating bovine intramuscular adipogenesis and fibrogenesis.
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Oster, Christoph, Kathy Keyvani, Tobias Blau, Teresa Schmidt, Jonas Feldheim, Lazaros Lazaridis, Christoph Kleinschnitz, et al. "TMIC-66. FIBROBLAST ACTIVATION PROTEIN: TISSUE EXPRESSION, POSITRON-EMISSION TOMOGRAPHY UPTAKE AND ITS PROGNOSTIC SIGNIFICANCE IN GLIOSARCOMAS AND GLIOBLASTOMAS." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii286. http://dx.doi.org/10.1093/neuonc/noac209.1110.
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Abstract BACKGROUND Although histologically distinct, gliosarcomas and glioblastomas are treated according to the same standards. Fibroblast-activation-protein (FAP) is a component of a tumor-specific subpopulation of fibroblasts that play a critical role in tumor growth and invasion. Few case studies suggest an elevated expression of FAP in glioblastoma and a particularly strong expression in gliosarcoma, possibly for its predominant mesenchymal differentiation. However, the prognostic impact of FAP in glioblastoma and gliosarcoma is unclear. Question:What is the clinical relevance of FAP-expression in gliosarcoma and glioblastoma and how does it correlate with FAPI46-PET-uptake? METHODS In a retrospective analysis, patients diagnosed with glioblastoma and gliosarcoma were enrolled. Histological examination was performed using immunohistochemical FAP-staining and quantitative analysis with standardized FAP-Score (0 to 3,with higher values indicating stronger expression). In a subset of patients, FAPI46-PETs have been performed. The SUV values are planned be correlated with FAP-expression in the tumor tissue. Data obtained from immunohistochemical analysis and SUV values are planned to be brought into perspective with clinically relevant prognostic factors, including survival estimates. RESULTS A total of 45 patients have been enrolled. On immunohistochemical staining, gliosarcomas expressed significantly more FAP (average FAP-Score,2.3) than glioblastomas (average FAP-Score,0.7,p< 0.0001). While in glioblastoma FAP-expression was confined to the perivascular space, in gliosarcoma the neoplastic cells themselves -in addition to the perivascular extracellular matrix- expressed FAP. Initial analyses indicate that tissue FAP-expression is correlated with tracer uptake on FAPI46-PET. Further studies on clinical relevance using correlation to clinical data are currently underway. CONCLUSIONS This study indicates that FAP-expression is much more abundant in gliosarcomas as opposed to glioblastomas. This could open not only a diagnostic but also a therapeutic gap. The prognostic role and the association of FAP-tissue expression with FAPI46-PET tracer uptake will be reported at the time of abstract presentation.
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Cho, Nathan, Shadi E. Razipour, and Megan L. McCain. "Featured Article: TGF-β1 dominates extracellular matrix rigidity for inducing differentiation of human cardiac fibroblasts to myofibroblasts." Experimental Biology and Medicine 243, no. 7 (March 4, 2018): 601–12. http://dx.doi.org/10.1177/1535370218761628.
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Cardiac fibroblasts and their activated derivatives, myofibroblasts, play a critical role in wound healing after myocardial injury and often contribute to long-term pathological outcomes, such as excessive fibrosis. Thus, defining the microenvironmental factors that regulate the phenotype of cardiac fibroblasts and myofibroblasts could lead to new therapeutic strategies. Both chemical and biomechanical cues have previously been shown to induce myofibroblast differentiation in many organs and species. For example, transforming growth factor beta 1, a cytokine secreted by neutrophils, and rigid extracellular matrix environments have both been shown to promote differentiation. However, the relative contributions of transforming growth factor beta 1 and extracellular matrix rigidity, two hallmark cues in many pathological myocardial microenvironments, to the phenotype of human cardiac fibroblasts are unclear. We hypothesized that transforming growth factor beta 1 and rigid extracellular matrix environments would potentially have a synergistic effect on the differentiation of human cardiac fibroblasts to myofibroblasts. To test this, we seeded primary human adult cardiac fibroblasts onto coverslips coated with polydimethylsiloxane of various elastic moduli, introduced transforming growth factor beta 1, and longitudinally quantified cell phenotype by measuring expression of α-smooth muscle actin, the most robust indicator of myofibroblasts. Our data indicate that, although extracellular matrix rigidity influenced differentiation after one day of transforming growth factor beta 1 treatment, ultimately transforming growth factor beta 1 superseded extracellular matrix rigidity as the primary regulator of myofibroblast differentiation. We also measured expression of POSTN, FAP, and FSP1, proposed secondary indicators of fibroblast/myofibroblast phenotypes. Although these genes partially trended with α-smooth muscle actin expression, they were relatively inconsistent. Finally, we demonstrated that activated myofibroblasts incompletely revert to a fibroblast phenotype after they are re-plated onto new surfaces without transforming growth factor beta 1, suggesting differentiation is partially reversible. Our results provide new insights into how microenvironmental cues affect human cardiac fibroblast differentiation in the context of myocardial pathology, which is important for identifying effective therapeutic targets and dictating supporting cell phenotypes for engineered human cardiac disease models. Impact statement Heart disease is the leading cause of death worldwide. Many forms of heart disease are associated with fibrosis, which increases extracellular matrix (ECM) rigidity and compromises cardiac output. Fibrotic tissue is synthesized primarily by myofibroblasts differentiated from fibroblasts. Thus, defining the cues that regulate myofibroblast differentiation is important for understanding the mechanisms of fibrosis. However, previous studies have focused on non-human cardiac fibroblasts and have not tested combinations of chemical and mechanical cues. We tested the effects of TGF-β1, a cytokine secreted by immune cells after injury, and ECM rigidity on the differentiation of human cardiac fibroblasts to myofibroblasts. Our results indicate that differentiation is initially influenced by ECM rigidity, but is ultimately superseded by TGF-β1. This suggests that targeting TGF-β signaling pathways in cardiac fibroblasts may have therapeutic potential for attenuating fibrosis, even in rigid microenvironments. Additionally, our approach can be leveraged to engineer more precise multi-cellular human cardiac tissue models.
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Shishkova, D. K., A. V. Sinitskaya, M. Yu Sinitsky, V. G. Matveeva, E. A. Velikanova, V. E. Markova, and A. G. Kutikhin. "Spontaneous endothelial-to-mesenchymal transition in human primary umbilical vein endothelial cells." Complex Issues of Cardiovascular Diseases 11, no. 3 (October 12, 2022): 97–114. http://dx.doi.org/10.17802/2306-1278-2022-11-3-97-114.
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Highlights. Spontaneous endothelial-to-mesenchymal transition of primary human umbilical vein endothelial cells (HUVEC) is characterized by an acquired expression of SNAI2 and TWIST1 genes, loss of endothelial markers and transcription factors (CD31/PECAM1, VE-cadherin, and ERG transcription factor), pronounced expression of S100A4 and ACTA2 genes, and active production of type I collagen, a major component of the extracellular matrix.An optimal algorithm to detect endothelial-to-mesenchymal transition includes gene expression profiling of endothelial lineage markers (PECAM1, CDH5, VWF, ERG), SNAI2 and TWIST1 transcription factors, mesenchymal specification markers (FAP, S100A4, ACTA2) and markers of extracellular matrix synthesis (COL1A1, COL1A2) along with the subsequent negative staining for CD31/PECAM1, VE-cadherin, or ERG and positive staining for intracellular type I collagen.Aim. To develop an algorithm and tools to determine endothelial-to-mesenchymal transition (EndoMT) in vitro.Methods. We examined two batches of human umbilical vein endothelial cells (HUVEC) where the first cell batch had a conventional endothelial morphology and the second cell batch underwent a spontaneous EndoMT. Human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) were used as the negative control for EndoMT. Molecular profile was assessed by means of reverse transcription-quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining with the further confocal microscopy.Results. In contrast to HUVEC with the physiological profile and arterial ECs, HUVEC undergoing EndoMT lost the expression of endothelial lineage markers (PECAM1, CDH5, VWF, ERG) and acquired the expression of EndoMT transcription factors (SNAI2, TWIST1), mesenchymal markers (FAP, S100A4, ACTA2), and extracellular matrix components (COL1A1, COL1A2) while retaining expression of the common vascular markers (HES1, NRP1). Western blotting analysis confirmed the loss of endothelial markers (CD31/PECAM1, VE-cadherin/CDH5, ERG) and demonstrated retained expression of abovementioned vascular markers. Negligible expression of MYH11 and SMTN genes encoding specific contractile markers (smooth muscle myosin heavy chain and smoothelin) in combination with the acquired expression of ACTA2 gene encoding less specific contractile marker alpha smooth muscle actin indicated the phenotypic identity of EndoMT-transformed HUVEC to myofibroblasts but not contractile vascular smooth muscle cells. Loss of immunofluorescence staining of endothelial markers (CD31/PECAM-1, VE-cadherin, and ERG transcription factor) and pronounced intracellular staining of type I collagen testified to the ongoing EndoMT.Conclusion. An algorithm to assess EndoMT implies measurement of the expression of PECAM1, CDH5, VWF, ERG, SNAI2, TWIST1, FAP, S100A4, ACTA2, COL1A1, and COL1A2 genes in combination with the respective immunofluorescence staining for CD31/PECAM-1, VE-cadherin, or ERG transcription factor and type I collagen.
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Moshal, Karni S., Mahavir Singh, Utpal Sen, Dorothea Susanne E. Rosenberger, Brooke Henderson, Neetu Tyagi, Hong Zhang, and Suresh C. Tyagi. "Homocysteine-mediated activation and mitochondrial translocation of calpain regulates MMP-9 in MVEC." American Journal of Physiology-Heart and Circulatory Physiology 291, no. 6 (December 2006): H2825—H2835. http://dx.doi.org/10.1152/ajpheart.00377.2006.
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Hyperhomocysteinemia (HHcy) is associated with atherosclerosis, stroke, and dementia. Hcy causes extracellular matrix remodeling by the activation of matrix metalloproteinase-9 (MMP-9), in part, by inducing redox signaling and modulating the intracellular calcium dynamics. Calpains are the calcium-dependent cysteine proteases that are implicated in mitochondrial damage via oxidative burst. Mitochondrial abnormalities have been identified in HHcy. The mechanism of Hcy-induced extracellular matrix remodeling by MMP-9 activation via mitochondrial pathway is largely unknown. We report a novel role of calpains in mitochondrial-mediated MMP-9 activation by Hcy in cultured rat heart microvascular endothelial cells. Our observations suggested that calpain regulates Hcy-induced MMP-9 expression and activity. We showed that Hcy activates calpain-1, but not calpain-2, in a calcium-dependent manner. Interestingly, the enhanced calpain activity was not mirrored by the decreased levels of its endogenous inhibitor calpastatin. We presented evidence that Hcy induces the translocation of active calpain from cytosol to mitochondria, leading to MMP-9 activation, in part, by causing intramitochondrial oxidative burst. Furthermore, studies with pharmacological inhibitors of calpain (calpeptin and calpain-1 inhibitor), ERK (PD-98059) and the mitochondrial uncoupler FCCP suggested that calpain and ERK-1/2 are the major events within the Hcy/MMP-9 signal axis and that intramitochondrial oxidative stress regulates MMP-9 via ERK-1/2 signal cascade. Taken together, these findings determine the novel role of mitochondrial translocation of calpain-1 in MMP-9 activation during HHcy, in part, by increasing mitochondrial oxidative tress.
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GORRELL, Mark D. "Dipeptidyl peptidase IV and related enzymes in cell biology and liver disorders." Clinical Science 108, no. 4 (March 22, 2005): 277–92. http://dx.doi.org/10.1042/cs20040302.
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DP (dipeptidyl peptidase) IV is the archetypal member of its six-member gene family. Four members of this family, DPIV, FAP (fibroblast activation protein), DP8 and DP9, have a rare substrate specificity, hydrolysis of a prolyl bond two residues from the N-terminus. The ubiquitous DPIV glycoprotein has proved interesting in the fields of immunology, endocrinology, haematology and endothelial cell and cancer biology and DPIV has become a novel target for Type II diabetes therapy. The crystal structure shows that the soluble form of DPIV comprises two domains, an α/β-hydrolase domain and an eight-blade β-propeller domain. The propeller domain contains the ADA (adenosine deaminase) binding site, a dimerization site, antibody epitopes and two openings for substrate access to the internal active site. FAP is structurally very similar to DPIV, but FAP protein expression is largely confined to diseased and damaged tissue, notably the tissue remodelling interface in chronically injured liver. DPIV has a variety of peptide substrates, the best studied being GLP-1 (glucagon-like peptide-1), NPY (neuropeptide Y) and CXCL12. The DPIV family has roles in bone marrow mobilization. The functional interactions of DPIV and FAP with extracellular matrix confer roles for these proteins in cancer biology. DP8 and DP9 are widely distributed and indirectly implicated in immune function. The DPL (DP-like) glycoproteins that lack peptidase activity, DPL1 and DPL2, are brain-expressed potassium channel modulators. Thus the six members of the DPIV gene family exhibit diverse biological roles.
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Moshynets, Olena V., Ianina Pokholenko, Olga Iungin, Geert Potters, and Andrew J. Spiers. "eDNA, Amyloid Fibers and Membrane Vesicles Identified in Pseudomonas fluorescens SBW25 Biofilms." International Journal of Molecular Sciences 23, no. 23 (December 1, 2022): 15096. http://dx.doi.org/10.3390/ijms232315096.
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Pseudomonas fluorescens SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air–liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.
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Miranda-Alarcón, Yoliem S., Alexandra M. Brown, Anthony M. Santora, and Ipsita A. Banerjee. "Growth of Self-Assembled Bio-Organic Nanomatrices for Skin Tissue Engineering — An in vitro Study." Nano LIFE 06, no. 01 (March 2016): 1650002. http://dx.doi.org/10.1142/s1793984416500021.
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In this paper, we have developed self-assembled nanoscale assemblies that were prepared by conjugating furan-2-carboxylic acid-3-aminopropyl amide with the short peptide sequence Gly-His (abbreviated Gly-His-FCAP). To mimic the extracellular matrix of mammalian fibroblasts and keratinocytes, the assemblies were then conjugated with Type I collagen. We then integrated the collagen bound Gly-His-FCAP assemblies with a short peptide sequence derived from salamander skin into the nanoscale assemblies for the first time to impart regenerative and wound healing properties to the composites. The antioxidant, antimicrobial and biodegradable properties were examined and results indicate that the nanocomposites displayed antioxidant properties as displayed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The biodegradability was found to be gradual. The nanocomposites were also found to inhibit the growth of the fungus Rhizopus sporangia over an 18[Formula: see text]h growth period. As proof of concept, to demonstrate the development of three-dimensional (3D) engineered skin in vitro, 3D printed PLA scaffolds of 2.5[Formula: see text]mm thickness were submerged in media containing nanocomposites and co-cultures of dermal fibroblasts with epidermal keratinocytes mimicking three dimensional skin substitute was examined. Our results indicated that the nanocomposites adhered to and supported cell proliferation and mimicked the components of skin and may have potential applications in skin tissue regeneration.
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Azaman, Farah Alwani, Keran Zhou, María del Mar Blanes-Martínez, Margaret Brennan Fournet, and Declan M. Devine. "Bioresorbable Chitosan-Based Bone Regeneration Scaffold Using Various Bioceramics and the Alteration of Photoinitiator Concentration in an Extended UV Photocrosslinking Reaction." Gels 8, no. 11 (October 28, 2022): 696. http://dx.doi.org/10.3390/gels8110696.
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Bone tissue engineering (BTE) is an ongoing field of research based on clinical needs to treat delayed and non-union long bone fractures. An ideal tissue engineering scaffold should have a biodegradability property matching the rate of new bone turnover, be non-toxic, have good mechanical properties, and mimic the natural extracellular matrix to induce bone regeneration. In this study, biodegradable chitosan (CS) scaffolds were prepared with combinations of bioactive ceramics, namely hydroxyapatite (HAp), tricalcium phosphate-α (TCP- α), and fluorapatite (FAp), with a fixed concentration of benzophenone photoinitiator (50 µL of 0.1% (w/v)) and crosslinked using a UV curing system. The efficacy of the one-step crosslinking reaction was assessed using swelling and compression testing, SEM and FTIR analysis, and biodegradation studies in simulated body fluid. Results indicate that the scaffolds had comparable mechanical properties, which were: 13.69 ± 1.06 (CS/HAp), 12.82 ± 4.10 (CS/TCP-α), 13.87 ± 2.9 (CS/HAp/TCP-α), and 15.55 ± 0.56 (CS/FAp). Consequently, various benzophenone concentrations were added to CS/HAp formulations to determine their effect on the degradation rate. Based on the mechanical properties and degradation profile of CS/HAp, it was found that 5 µL of 0.1% (w/v) benzophenone resulted in the highest degradation rate at eight weeks (54.48% degraded), while maintaining compressive strength between (4.04 ± 1.49 to 10.17 ± 4.78 MPa) during degradation testing. These results indicate that incorporating bioceramics with a suitable photoinitiator concentration can tailor the biodegradability and load-bearing capacity of the scaffolds.
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Yan, Ming, Ke Di Xu, Xiao Xiang Zheng, Zheng Jian Chen, and Hong Liang Jiang. "Enhanced Cellular Function of Human Vascular Endothelial Cell on Poly (ε-caprolactone)/Gelatin Coaxial-Electrospun Scaffold." Applied Mechanics and Materials 138-139 (November 2011): 900–906. http://dx.doi.org/10.4028/www.scientific.net/amm.138-139.900.
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An extracellular matrix-like poly (ε-caprolactone) (PCL)/gelatin core-shell nanofibrous scaffold with high hydrophilicity was developed by coaxial-elecrospinning. However, the behavior of vascular endothelial cells (ECs) on the modified scaffold remains limited. In this study, human umbilical vein ECs (HUVECs) were seeded onto PCL scaffolds with or without gelatin. Morphological changes of HUVECs were observed under confocal laser scanning microscopy (LSCM). HUVECs’ adhesion, proliferation and apoptosis were detected by MTT assay and flow cytometry (FCM). Our results showed that HUVECs on PCL/gelatin scaffolds with identical polygonal and cobblestone-like characteristics reached confluence after 7 days. Modification of PCL nanofibers significantly promoted the attachment of HUVECs onto scaffolds within 1 hour. Compared to pristine PCL, a two-fold increase in proliferation of HUVECs was also observed after 7 days, whereas the apoptosis of HUVECs was obviously reduced by 40% on the modified scaffolds. In summary, these results indicated modified PCL/gelatin scaffold developed by coaxial-elecrospinning can increase the adhesion, proliferation, and suppress apoptosis of HUVECs, suggesting it has a great potential and promising vascular graft in tissue engineering applications.
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Chivu-Economescu, Mihaela, Laura G. Necula, Lilia Matei, Denisa Dragu, Coralia Bleotu, Andrei Sorop, Vlad Herlea, Simona Dima, Irinel Popescu, and Carmen C. Diaconu. "Collagen Family and Other Matrix Remodeling Proteins Identified by Bioinformatics Analysis as Hub Genes Involved in Gastric Cancer Progression and Prognosis." International Journal of Molecular Sciences 23, no. 6 (March 16, 2022): 3214. http://dx.doi.org/10.3390/ijms23063214.
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Gastric cancer has remained in the top five cancers for over ten years, both in terms of incidence and mortality due to the shortage of biomarkers for disease follow-up and effective therapies. Aiming to fill this gap, we performed a bioinformatics assessment on our data and two additional GEO microarray profiles, followed by a deep analysis of the 40 differentially expressed genes identified. PPI network analysis and MCODE plug-in pointed out nine upregulated hub genes coding for proteins from the collagen family (COL12A1, COL5A2, and COL10A1) or involved in the assembly (BGN) or degradation of collagens (CTHRC1), and also associated with cell adhesion (THBS2 and SPP1) and extracellular matrix degradation (FAP, SULF1). Those genes were highly upregulated at the mRNA and protein level, the increase being correlated with pathological T stages. The high expression of BGN (p = 8 × 10−12), THBS2 (p = 1.2 × 10−6), CTHRC1 (p = 1.1 × 10−4), SULF1 (p = 3.8 × 10−4), COL5A1 (p = 1.3 × 10−4), COL10A1 (p = 5.7 × 10−4), COL12A1 (p = 2 × 10−3) correlated with poor overall survival and an immune infiltrate based especially on immunosuppressive M2 macrophages (p-value range 4.82 × 10−7–1.63 × 10−13). Our results emphasize that these genes could be candidate biomarkers for GC progression and prognosis and new therapeutic targets.
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Liu, Xiaoli, Na Xu, Chengyun Pan, Xuan Zhou, Qifa Liu, Ru Feng, Jing Sun, and Hongsheng Zhou. "Emerging Potential Roles of Lysyl-Oxidase-like 2 Stimulates Cancer Associated Fibroblasts Promoting Myelofibrosis of Myeloproliferative Neoplasms." Blood 128, no. 22 (December 2, 2016): 5495. http://dx.doi.org/10.1182/blood.v128.22.5495.5495.
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Abstract Backgroud and objective: Myeloproliferative neoplasms (MPNs) are a group of clonal haematological disorders that characteristic with a multipotent haematopoietic stem cell transformation, often associating with the progression of myelofibrosis in the evolution course of disease. Progressive myelofibrosis finally turned out a high risk factor of transformation to leukemia and bone marrow failure. Cancer Associated Fibroblasts (CAFs) are recently thought to be a critical mediator in several hematological malignancies tumor microenvironment and associate with fibrosis. Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that promote the crosslinking of collagens or elastins in the extracellular matrix and mediate epithelial-mesenchymal transition (EMT). Here, we want to verify CAFs mediating myelofibrosis and explore the potential role of LOXL2 association with CAFs in simulated vivo microenvironment. Patients and methods:For bone marrow specimens, normal samples (n=19) and patients with polycythemia vera (PV) (n=21), essential thrombocythemia (ET) (n=32), and primary myelofibrosis (PMF) (n=9) were contrasted. Markers of CAFs including α-smooth actin(α-SMA), fibroblast activation protein(FAP), transforming growth factor-β1(TGF-β1) and LOXL2 were detected by quantitative reverse transcription-PCR(RT-PCR). we also detected α-SMA, FAP, LOXL2 and reticulin protein by western blot and immunohistochemical staining. For cell lines, α-SMA and FAP were measured after cocultured mesenchymal stem cell(MSCs) with recombinant human lysyl oxidase homolog 2 Protein(rhLOXL2) in hypoxic niche for 24, 48, 72 and 96 hours, respectively. Results: Markers of CAFs displayed a differential pattern of expression in MPNs especially in PMF(P<0.010) compared with normal samples(P=0.023) according to RT-PCR. Among these markers, the expression of α-SMA is the highest. For western blot and immunohistochemical staining, we discovered the level of α-SMA, FAP and LOXL2 expression was associated with the grading of reticulin fibrosis in bone marrows. we also founded that α-SMA and FAP were significantly expressed after cocultured MSCs with rhLOXL2 in hypoxia for 96 hours, but there were no significant increase in gene and protein level expression with the prolonged exposure time. Conclusion: Over expression of CAFs played a critical role in promoting bone marrow fibrosis. LOXL2 may be a key factor in stimulating MSC to CAFs and further promoting disease progression, which may provide a new target spot for further excavation of the pathogenesis of MPNs and looking for more effective targeted therapies. Disclosures No relevant conflicts of interest to declare.
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Lai, W. Michael, Van C. Mow, Daniel D. Sun, and Gerard A. Ateshian. "On the Electric Potentials Inside a Charged Soft Hydrated Biological Tissue: Streaming Potential Versus Diffusion Potential." Journal of Biomechanical Engineering 122, no. 4 (February 28, 2000): 336–46. http://dx.doi.org/10.1115/1.1286316.
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The main objective of this study is to determine the nature of electric fields inside articular cartilage while accounting for the effects of both streaming potential and diffusion potential. Specifically, we solve two tissue mechano-electrochemical problems using the triphasic theories developed by Lai et al. (1991, ASME J. Biomech Eng., 113, pp. 245–258) and Gu et al. (1998, ASME J. Biomech. Eng., 120, pp. 169–180) (1) the steady one-dimensional permeation problem; and (2) the transient one-dimensional ramped-displacement, confined-compression, stress-relaxation problem (both in an open circuit condition) so as to be able to calculate the compressive strain, the electric potential, and the fixed charged density (FCD) inside cartilage. Our calculations show that in these two technically important problems, the diffusion potential effects compete against the flow-induced kinetic effects (streaming potential) for dominance of the electric potential inside the tissue. For softer tissues of similar FCD (i.e., lower aggregate modulus), the diffusion potential effects are enhanced when the tissue is being compressed (i.e., increasing its FCD in a nonuniform manner) either by direct compression or by drag-induced compaction; indeed, the diffusion potential effect may dominate over the streaming potential effect. The polarity of the electric potential field is in the same direction of interstitial fluid flow when streaming potential dominates, and in the opposite direction of fluid flow when diffusion potential dominates. For physiologically realistic articular cartilage material parameters, the polarity of electric potential across the tissue on the outside (surface to surface) may be opposite to the polarity across the tissue on the inside (surface to surface). Since the electromechanical signals that chodrocytes perceive in situ are the stresses, strains, pressures and the electric field generated inside the extracellular matrix when the tissue is deformed, the results from this study offer new challenges for the understanding of possible mechanisms that control chondrocyte biosyntheses. [S0148-0731(00)00604-X]
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Kaps, Leonard, and Detlef Schuppan. "Targeting Cancer Associated Fibroblasts in Liver Fibrosis and Liver Cancer Using Nanocarriers." Cells 9, no. 9 (September 3, 2020): 2027. http://dx.doi.org/10.3390/cells9092027.
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Cancer associated fibroblasts (CAF) and the extracellular matrix (ECM) produced by them have been recognized as key players in cancer biology and emerged as important targets for cancer treatment and drug discovery. Apart from their presence in stroma rich tumors, such as biliary, pancreatic and subtypes of hepatocellular cancer (HCC), both CAF and certain ECM components are also present in cancers without an overt intra-tumoral desmoplastic reaction. They support cancer development, growth, metastasis and resistance to chemo- or checkpoint inhibitor therapy by a multitude of mechanisms, including angiogenesis, ECM remodeling and active immunosuppression by secretion of tumor promoting and immune suppressive cytokines, chemokines and growth factors. CAF resemble activated hepatic stellate cells (HSC)/myofibroblasts, expressing α-smooth muscle actin and especially fibroblast activation protein (FAP). Apart from FAP, CAF also upregulate other functional cell surface proteins like platelet-derived growth factor receptor β (PDGFRβ) or the insulin-like growth factor receptor II (IGFRII). Notably, if formulated with adequate size and zeta potential, injected nanoparticles home preferentially to the liver. Several nanoparticular formulations were tested successfully to deliver dugs to activated HSC/myofibroblasts. Thus, surface modified nanocarriers with a cyclic peptide binding to the PDGFRβ or with mannose-6-phosphate binding to the IGFRII, effectively directed drug delivery to activated HSC/CAF in vivo. Even unguided nanohydrogel particles and lipoplexes loaded with siRNA demonstrated a high in vivo uptake and functional siRNA delivery in activated HSC, indicating that liver CAF/HSC are also addressed specifically by well-devised nanocarriers with optimized physicochemical properties. Therefore, CAF have become an attractive target for the development of stroma-based cancer therapies, especially in the liver.
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Manuela, Besser, Khosravani Milad, Severing Anna-Lena, Rembe Julian-Dario, and Stuermer Ewa Klara. "Acute and Chronic Wound Fluid Inversely Influence Wound Healing in an in-Vitro 3D Wound Model." Journal of Tissue Repair and Regeneration 1, no. 1 (December 12, 2017): 1–11. http://dx.doi.org/10.14302/issn.2640-6403.jtrr-17-1818.
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If a wound progressively heals or the healing process is impaired is basically influenced by the surrounding milieu. This is reflected by the wound fluid. Its specific composition triggers the migration, proliferation and differentiation of dermal and epidermal cells which so far was not sufficiently examined in 2D cell culture models. The influence of the different wound entities was analyzed on a newly implemented three dimensional in-vitro model, which improved the transferability to the in-vivo situation. The influence of pooled wound fluids from patients suffering from acute or chronic wounds were investigated within a time period of 10 days after wound application. Histological and immunohistochemical analyses were performed addressing the impact of AWF and CWF on regeneration, such as cell proliferation, fibroblast activity and cell migration. AWF slightly stimulated fibroblast migration while CWF inhibited their activation and migration. The CXCR4- immunopositive population was continuously decreased compared to the control and AWF treatment. The expression of FAP was enhanced under AWF and medium. In keratinocytes CWF massively stimulated cell proliferation initiating on day six after injury. The presence of 10% CWF inhibited fibroblast activation and migration and induced the degradation of the collagen matrix. Keratinocytes were stimulated to proliferate, resulting in healing inhibiting hyperplasia. Transferred to human wounds, no effective wound closure would be achieved because of the de-regulation of pro-proliferative and migration-stimulating factors and a degraded extracellular matrix. This newly implemented 3D study model represents a novel appropriate in-vitro system for studying healing mechanisms and potential therapeutic applications.
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Lu, Yingzhi, Zhenxin Wang, Ling Zhou, Zhaoming Ma, Jianguo Zhang, Yan Wu, Yan Shao, and Yunyun Yang. "FAT1 and PTPN14 Regulate the Malignant Progression and Chemotherapy Resistance of Esophageal Cancer through the Hippo Signaling Pathway." Analytical Cellular Pathology 2021 (October 19, 2021): 1–9. http://dx.doi.org/10.1155/2021/9290372.
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Background. Esophageal cancer (EC) is a common malignant tumor, which brings heavy economic burden to patients and society. Therefore, it is important to understand the molecular mechanism of recurrence, metastasis, and drug resistance of esophageal cancer. Methods. Human esophageal cancer cell line TE13 (poorly differentiated squamous cell carcinoma) and normal human esophageal epithelial cell line het-1a were selected for aseptic culture. At the same time, 6 bottles of TE13 cell line were inoculated in logarithmic phase. Cell apoptosis was analyzed by flow cytometry (FCM). Cell clone formation assay was used to analyze the proliferation. Fibronectin-coated dishes were used to detect the characteristics of cell adhesion to extracellular matrix. The Transwell method was used to detect the cell invasion ability. Western blot was used to analyze the expression of Yap1, PTPN14, FAT1, and Myc. Results. Results showed that FAT1 and PTPN14 were downregulated, while Yap1 was upregulated in esophageal cancer tissues. FAT1 inhibited the proliferation, adhesion, and invasion of human esophageal cancer cell lines, which might be associated with the upregulation of PTPN14 and the inhibition of Yap1 and Myc. Conclusion. The results suggested that PTPN14 and FAT1 could regulate malignant progression and chemotherapy resistance of esophageal cancer based on the Hippo signaling pathway.
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Zhang, Sijia, Danielle S. Bassett, and Beth A. Winkelstein. "Stretch-induced network reconfiguration of collagen fibres in the human facet capsular ligament." Journal of The Royal Society Interface 13, no. 114 (January 2016): 20150883. http://dx.doi.org/10.1098/rsif.2015.0883.
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Biomaterials can display complex spatial patterns of cellular responses to external forces. Revealing and predicting the role of these patterns in material failure require an understanding of the statistical dependencies between spatially distributed changes in a cell's local biomechanical environment, including altered collagen fibre kinematics in the extracellular matrix. Here, we develop and apply a novel extension of network science methods to investigate how excessive tensile stretch of the human cervical facet capsular ligament (FCL), a common source of chronic neck pain, affects the local reorganization of collagen fibres. We define collagen alignment networks based on similarity in fibre alignment angles measured by quantitative polarized light imaging. We quantify the reorganization of these networks following macroscopic loading by describing the dynamic reconfiguration of network communities, regions of the material that display similar fibre alignment angles. Alterations in community structure occur smoothly over time, indicating coordinated adaptation of fibres to loading. Moreover, flexibility, a measure of network reconfiguration, tracks the loss of FCL's mechanical integrity at the onset of anomalous realignment (AR) and regions of AR display altered community structure. These findings use novel network-based techniques to explain abnormal collagen fibre reorganization, a dynamic and coordinated multivariate process underlying tissue failure.
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Nadav, Liat, Ben-Zion Katz, Shoshana Baron, Lydia Lydia, Aaron Polliack, Varda Deutsch, Benjamin Geiger, and Elizabeth Naparstek. "The Location of Multiple Myeloma Adhesion Variants in Diverse Niches within the Bone Marrow Affects Plasma Cells Enumeration." Blood 108, no. 11 (November 16, 2006): 5063. http://dx.doi.org/10.1182/blood.v108.11.5063.5063.
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Abstract Background - The diagnosis of multiple myeloma (MM) is based on clinical and laboratory criteria combined with bone marrow (BM) plasmocytosis, estimated by inspection of bone marrow aspirates. Recent advances in flow-cytometry (FCM) have provided an additional tool for the diagnosis of MM and for monitoring response to therapy. However, significant discrepancy has been reported regarding the enumeration of plasma cells in marrow samples of MM patients using these two methods. Aims - In this study we compared the bone marrow plasmocytosis by microscopic examination of BM aspirates, to the flow cytometry results in samples obtained form MM patients. We tested whether the noted discrepancy between these two methods applies only to MM, or represents a trend in other hematopoietic malignancies as well. We defined this discrepancy and explained it. Methods - The number of plasma cells or blasts from BM aspirates of 41 MM or seven acute myeloid leukemia (AML) patients respectively were analyzed simultaneously by morphological evaluation and by FCM. Each sample was assessed independently by two qualified laboratory specialists and/or hemato-pathologist. In MM we found plasma cell fractions that were characterized by FCM and gene expression profile. Results - In MM it was evident that FCM under-estimated the number of BM plasma cells samples by an average of 60%, compared with conventional morphological evaluation. On the other hand in AML there was a good correlation between the morphological and FCM assessments of the blast cell population, indicating that the discrepancy observed in the MM BM samples may be related to unique characteristics of the malignant plasma cells. Since flow cytometry is performed on the bone marrow fluid which is depleted of fat tissue-adhesive plasma cells, we disrupted spicules from MM BM samples (by repeated passages through 21g needle) and found a 40% increase in plasma cell compared with the fluid of the same BM samples. In order to determine the FCM profile of the cells in these two fractions, we isolated BM derived spicules from aspirates of MM patients and treated them with extracellular matrix (ECM) degrading enzymes followed by mechanical shearing. This combination released the highly adhesive plasma cells from the spicules. The released myeloma cells displayed a different FCM profile and in particular had a higher level of CD138 expression. Gene expression profile, which was performed on similar adhesion variants of cultured MM cells, demonstrated distinct oncogenic and transcriptional programs. Summary - We have shown a major discrepancy between the percentage of MM cells obtained by routine BM morphology and flow cytometry counts. It is possible that this discrepancy is partially attributable to the two distinct microenvironmental components occupied by MM cells in the BM sample - the lipid spicules, and the fluid phase. MM cells located in different niches of the BM also differ in their FCM and gene expression profile. This study indicates that multiple myeloma patients contain heterogeneous populations of malignant plasma cells. These sub-populations may play distinct roles in the biological and clinical manifestations of the disease and differ in their response to anti-myeloma therapy.
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Du, Guoqing, Yi Song, Lei Wei, Linghui Li, Xuezong Wang, Qinguang Xu, Hongsheng Zhan, Yuelong Cao, Yuxin Zheng, and Daofang Ding. "Osthole Inhibits Proliferation and Induces Catabolism in Rat Chondrocytes and Cartilage Tissue." Cellular Physiology and Biochemistry 36, no. 6 (2015): 2480–93. http://dx.doi.org/10.1159/000430208.
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Background/Aims: Cartilage destruction is thought to be the major mediator of osteoarthritis. Recent studies suggest that inhibition of subchrondral bone loss by anti-osteoporosis (OP) drug can protect cartilige erosion. Osthole, as a promising agent for treating osteoporosis, may show potential in treating osteoarthritis. The purpose of this study was to investigate whether Osthole affects the proliferation and catabolism of rat chondrocytes, and the degeneration of cartilage explants. Methods: Rat chondrocytes were treated with Osthole (0 μM, 6.25 μM, 12.5 μM, and 25 μM) with or without IL1-β (10ng/ml) for 24 hours. The expression levels of type II collagen and MMP13 were detected by western Blot. Marker genes for chondrocytes (A-can and Sox9), matrix metalloproteinases (MMPs), aggrecanases (ADAMTS5) and genes implicated in extracellular matrix catabolism were evaluated by qPCR. Cell proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and fluorescence activated cell sorter. Wnt7b/β-catenin signaling was also investigated. Cartilage explants from two-week old SD rats were cultured with IL-1β, Osthole and Osthole plus IL-1β for four days and glycosaminoglycan (GAG) synthesis was assessed with toluidine blue staining and Safranine O/Fast Green FCF staining, collagen type II expression was detected by immunofuorescence. Results: Osthole reduced expression of chondrocyte markers and increased expression of MMP13, ADAMTS5 and MMP9 in a dose-dependent manner. Catabolic gene expression levels were further improved by Osthole plus IL-1β. Osthole inhibited chondrocyte proliferation. GAG synthesis and type II collagen were decreased in both the IL-1β groups and the Osthole groups, and significantly reduced by Osthole plus IL-1β. Conclusions: Our data suggested that Osthole increases the catabolism of rat chondrocytes and cartilage explants, this effect might be mediated through inhibiting Wnt7b/β-catenin pathway.
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Gao, Mei, Charles J. Bailey, Megan M. Harper, Michael J. Cavnar, Prakash Pandalai, Shadi A. Qasem, and Joseph Kim. "Abstract A029: Identification of tumor microenvironment components in patient-derived pancreatic ductal adenocarcinoma organoids." Cancer Research 82, no. 10_Supplement (May 15, 2022): A029. http://dx.doi.org/10.1158/1538-7445.evodyn22-a029.
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Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly drug resistant with no change in therapeutic approach for the last decade. The PDAC tumor microenvironment (TME) is highly desmoplastic and associated with altered treatment response. Our group previously identified 9 distinct cell types in human PDAC tumors using single-cell RNA-sequencing, demonstrating a diverse TME. However, the TME of in vitro organoid PDAC models remains underexplored. We initiated characterization of the TME from PDAC patient derived organoids (PDOs) to establish a working model for accurate drug sensitivity testing. Methods: We performed immunofluorescence (IF) staining on whole-mount early passage (<P4) PDO lines and corresponding paraffin-embedded primary PDAC specimens to evaluate for activated cancer-associated fibroblasts (CAFs) using antibodies against α-smooth muscle actin (α-SMA) and fibroblast activated protein (FAP); and to evaluate for T-regulatory (Treg) cells with antibodies against interleukin-2 receptor α (IL-2Rα) and forkhead box protein P3 (FOXP3). Results: CAFs promote an immunosuppressive TME in PDAC and have recently been subclassified into inflammatory fibroblasts and myofibroblasts, which can both restrain and promote tumor progression. In this study, we identified three distinct subtypes of CAFs in PDOs, which are consistent with the subtypes found in matched clinical tumor specimens: α-SMA+, which has been reported to be indicative of pancreatic stellate cell activation and associated with increased desmoplasia; FAP+, which influences cancer cell motility, invasion, and cycle progression, extracellular matrix deposition, and angiogenesis within the TME; and α-SMA+/FAP+, which is a myofibroblast subtype. We additionally identified Treg populations in PDOs with peripheral IL-2Rα and central FOXP3 expression in the organoid spheres. Tregs have previously been shown to be overexpressed in the PDAC TME, making this finding clinically relevant. Conclusions: In this preliminary study, we show that PDAC PDOs demonstrate TME components that mirror primary tumor specimens, including CAFs and Tregs. The demonstration of a tumor-matched, immunocompetent TME in PDAC PDOs provides a more clinically relevant in vitro tumor model for patient-matched drug sensitivity testing. Citation Format: Mei Gao, Charles J. Bailey, Megan M. Harper, Michael J. Cavnar, Prakash Pandalai, Shadi A. Qasem, Joseph Kim. Identification of tumor microenvironment components in patient-derived pancreatic ductal adenocarcinoma organoids [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A029.
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Darpolor, Josephine Kebbeh, Xiaofeng Zheng, Sujuan Yang, Hikaru Sugimoto, Julienne Carstens, Pedro Correa de Sampiao, Valerie LeBleu, and Raghu Kalluri. "4078 Identification of distinct fibroblast populations with unique roles in pancreatic cancer progression and tumor immunity." Journal of Clinical and Translational Science 4, s1 (June 2020): 96. http://dx.doi.org/10.1017/cts.2020.301.
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OBJECTIVES/GOALS: The desmoplastic reaction in PDAC involves a significant accumulation of immune cells and fibroblasts.The functional diversity of carcinoma associated fibroblasts (CAFs) remains largely unknown, and identification of immune regulating subsets would have a substantial impact in augmentation of immunotherapy efficacy. METHODS/STUDY POPULATION: Employing histology, FACs, multiplex immunohistochemistry, single cell RNA sequencing (sc-RNA-seq) and genetically engineered mouse models, we demonstrate that aSMA+ cells are a dominant CAF population in PDAC with tumor restraining properties (TS-CAFs), as opposed those of the FAP+ CAFs, which demonstrate tumor promoting activity (TP-CAFs). RESULTS/ANTICIPATED RESULTS: Analysis of bulk tumor depleted of either TS-CAFs or TP-CAFs showed that TS-CAFs predominantly modulate extracellular matrix (ECM) production, facilitate cell-ECM adhesion and regulate adaptive immunity, while TP-CAFs exhibit a lineage that is skewed towards a pro-inflammatory, chemokine secreting phenotype. Further, scRNA-Seq analyses demonstrate that CAFs share distinct gene expression profiles characteristic of lymphocytic and myeloid lineages. Together our data distinguish two populations of CAFs, one which is tumor suppressing with roles in ECM remodeling and another which is tumor promoting with roles in cytokine production, both with immune modulating capabilities. DISCUSSION/SIGNIFICANCE OF IMPACT: Our study identifies a complex network of functionally heterogeneous fibroblasts during PDAC progression with significant immunotherapeutic implication. The identification of distinct fibroblast subsets will allow us to discriminately target fibroblast populations to augment immunotherapy efficacy in pancreatic cancer.
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Lopes, Kelvin Saldanha, Francisco Willyego Holanda Maciel, Roque Soares Martins Neto, Vilana Maria Adriano Araújo, Juscelino de Freitas Jardim, and Mardonio Rodrigues Pinto. "Aplicações e possibilidades terapêuticas do uso do biomaterial quitosana para a odontologia: revisão de literatura." ARCHIVES OF HEALTH INVESTIGATION 9, no. 6 (April 20, 2020): 587–91. http://dx.doi.org/10.21270/archi.v9i6.4782.
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A quitosana, polissacarídeo linear obtido a partir do exoesqueleto de crustáceos e artrópodes, tem sido pesquisada em Odontologia por suas diversas propriedades terapêuticas. O objetivo do presente estudo foi realizar uma revisão da literatura sobre as aplicações atuais e as possibilidades terapêuticas da quitosana na odontologia. A busca foi realizada através do banco de dados eletrônico do Pubmed, utilizando os descritores Quitosana, Odontologia e Biomateriais. Foram incluídas pesquisas científicas utilizando quitosana em diversas áreas da odontologia e excluídas revisões de literatura e estudos não odontológicos, sendo selecionados 13 artigos. A quitosana induz resposta transcricional e anti-inflamatória em fibroblastos gengivais sobre citocinas inflamatórias, fatores transformadores do crescimento (TGF -β) e fatores de crescimento tumoral (TNF-α) que estão diretamente relacionados à atividade patológica periodontal. Nas infecções endodônticas persistentes, a substância atua criando ligações de hidrogênio e liberação de íons cálcio, o que potencializa a ação dos irrigadores intracanal, além de causar menos estresse oxidativo. Para a odontologia restauradora, a quitosana demonstrou eficácia como auxiliar no condicionamento da dentina e mostrou potencial para induzir a migração de odontoblastos na proteção do complexo dentino-pulpar. A substância atua como uma cura de feridas orais devido à sua capacidade de estimular a formação de fibroblastos e novos vasos sanguíneos, além de células anti-inflamatórias.
Descritores: Biopolímeros; Biomateriais; Biotecnologia.
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Ke, B. J., S. Abdurahiman, F. Biscu, S. Verstockt, B. Verstockt, G. de Hertogh, S. Vermeire, and G. Matteoli. "DOP33 Single-cell analysis identifies pathological fibroblasts as a new therapeutic target to prevent intestinal fibrosis in Crohn’s disease." Journal of Crohn's and Colitis 17, Supplement_1 (January 30, 2023): i96—i97. http://dx.doi.org/10.1093/ecco-jcc/jjac190.0073.
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Abstract Background Recurrent episodes of intestinal inflammation and tissue remodeling progressively result in fibro-stenosis and bowel obstruction in Crohn’s disease (CD). This irreversible end-stage complication is the main indication for surgical intervention but is often associated with postoperative recurrence of inflammation and repeated tissue resection. Methods To understand transmural inflammation-induced fibro-stenosis, full-thickness ileum from CD patients (n=10) undergoing ileocecal resection were profiled using single-cell RNA sequencing (scRNA seq) on 10x Genomics platform. Our tissue sampling strategy aimed at recapitulating different disease stages from the same patient including normal ileum, chronic active inflammation and stenotic ileum, allowing a better definition of cell heterogeneity, inter-cellular communication, and tissue organization in CD. Healthy ileum from CRC patients (n=5) undergoing right hemicolectomy were included as external control. Flow cytometry (FACS) was carried out to confirm scRNA seq findings. For functional validation, intestinal fibroblasts were co-cultured with NicheNet-predicted cytokines and FACS-sorted myeloid supernatants. Finally, the chronic dextran sulfate sodium (DSS)-induced colitis model was used to validate the transcription regulation in activated fibroblasts. Results Using scRNA seq, we identified a specific subset of activated fibroblasts during chronic inflammation and fibrosis. FACS showed increasing FAP+ fibroblasts in both inflamed and stenotic ileum, compared to control ileum and proximal ileum (p<0.01). Computational methods predicting ligand-receptor signaling suggest that fibroblast activation is mostly mediated by myeloid cell-derived inflammatory signals resulting in collagen deposition and tissue fibrosis. Intestinal fibroblasts co-cultured with inflammatory monocyte supernatant and predicted ligands expressed higher protein levels of FAP, COL3A1 and showed signs of EMT transformation. Furthermore, stimulated intestinal fibroblasts secreted higher levels of ECM proteins, including COL1 (p<0.001), and COL3A1 (p<0.005). After inhibiting an EMT-related transcription factor identified in the activated fibroblasts, collagen expression and extracellular matrix accumulation were decreased in fibroblasts. Finally, inhibition of EMT transformation in chronic DSS colitis resulted in reduced ECM deposition, compared to vehicle mouse (p<0.05). Conclusion Our findings suggest that inflammatory monocytes mediate local activation of fibroblasts promoting excessive collagen deposition. We furthermore show that targeting activated fibroblasts was able to reduce tissue remodeling and may therefore prevent fibrostenosis in CD.
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Rio, Danila Del, Valentina Caprara, Ilenia Masi, Francesca Spadaro, Sara Giannitelli, Alberto Rainer, Anna Bagnato, and Laura Rosanò. "Abstract 6137: Tumor-derived endothelin-1 recruits and activates fibroblasts to support tumor aggressiveness." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6137. http://dx.doi.org/10.1158/1538-7445.am2022-6137.
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Abstract The recruitment of fibroblasts to tumor and their activation to cancer-associated fibroblast (CAF) is an exploited strategy used by tumor to guide matrix remodeling, supporting cancer invasion and metastatic disease. CAFs are the major effectors of extracellular matrix (ECM) remodelling through secretion of collagens, cross-linking enzymes and proteases, and in turn engagement of integrins. Although several tumor cell-secreted factors have been identified in generating tumor-promoting stroma, there has been a great interest to define factors and associated molecular mechanisms involved in promoting CAF premetastatic function. Tumor-related endothelin-1 (ET-1) has an important role in the crosstalk between cancer and stromal cells supporting serous ovarian cancer (SOC) progression. By binding its receptors (ETAR and ETBR), ET-1 favours the recruitment of the protein β-arrestin-1 and the generation of signaling complexes regulating also cytoskeleton reorganization, thereby fine-tuning SOC cell invasion and metastasis. However, how the integration of ET-1 receptors might “educate” stroma to become permissive for invasion, supplying an altered ECM governing metastasis needs to be investigated. In this study, we identified endothelin-1 as a key factor in priming CAF conversion in SOC. We demonstrate that primary ovarian fibroblasts express ETA and ETB, along with β-arrestin1, and secrete ET-1; b) the autocrine and cancer-secreted ET-1 promotes fibroblast proliferation, and an increase in the expression of CAF markers (αSMA, vimentin and FAP), secretion of proinflammatory cytokines, collagen contraction ability and cell motility. Moreover, ET-1 induces activation of b1 integrin and downstream signaling. Mechanistically, ET1R/intb1 signaling allows ECM remodeling, either by promoting and invasive protrusion formation, invadosome, and collagen secretion. Importantly, therapeutical inhibition of ET-1 receptors with Ambrisentan and intβ1 with ATN-116 markedly reduced the ability of SOC cells to adhere in the intraperitoneal organs and to metastasize. Our findings support a model in which ET-1 educates the stromal fibroblasts to become permissive for tumor invasion and demonstrate for the time the significance of an ET-1-dependent integrin signaling in this process. Citation Format: Danila Del Rio, Valentina Caprara, Ilenia Masi, Francesca Spadaro, Sara Giannitelli, Alberto Rainer, Anna Bagnato, Laura Rosanò. Tumor-derived endothelin-1 recruits and activates fibroblasts to support tumor aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6137.
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Wang, S. X., J. Wang, E. Ozhegov, R. Srinivasan, O. O. Olowokure, A. X. Qu, B. Aronow, and V. Bogdanov. "Identification of unique gene expression signatures in colon cancer stroma using microarray technology." Journal of Clinical Oncology 29, no. 4_suppl (February 1, 2011): 447. http://dx.doi.org/10.1200/jco.2011.29.4_suppl.447.
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447 Background: Microarrays are widely used to study gene expression patterns in cancer. In colorectal cancer, it has proven useful to predict recurrence. The majority of expression profiles are generated from the cancer itself. Given the increasing evidence of importance of the microenvironment for tumor invasion, progression and metastasis, we explored tumor stroma gene signature using microarrays. Methods: Four formalin-fixed paraffin embedded (FFPE) colon cancer specimen carrying a pathological stage of T3-4/N0-2 were retrieved. Tumor stroma and normal stroma were separated using microdissection technology. Random sampling was used to minimize sampling bias. Total RNA was extracted, amplified, and labeled using Nugene FFPE kit, with array analysis using Affymetrix Human Gene 1.0. Eight samples, four normal stroma and four tumor stroma, were analyzed and compared. Array data were balanced and analyzed using standard software. To identify gene signature differences in tumor vs normal stroma, ComparativeMarker analysis and unsupervised cluster analysis were carried out. Results: We identified a 969 Affymetrix probe set as a signature that is highly expressed in tumor stroma. The top 117 genes were further analyzed to carry out a pathway analysis. We found a strong signature evident in tumor stroma, and much of this signature comprised the genes of the extracellular matrix. The pathway analysis revealed evidence of the generalized IGF1/TGFbeta/CTGF/activin mediated effect on the stroma, raising the possibility that some of this derives from, or is accompanied by, angiotensin receptor signaling. Through literature search, we found that several upregulated genes (e.g., FAP) were reported to be associated with stroma activation in vitro and in vivo. Conclusions: In this study, we successfully applied microarrays to study reactive colon tumor stroma in FFPE samples. We identified a specific gene signature reflecting stromal reaction to tumor invasion. We further identified the potential pathway that was activated in the reactive tumor stroma. We provide evidence that microarrays are useful in stroma analysis and may help identify new stromal pathways with potential diagnostic and therapeutic value. No significant financial relationships to disclose.
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Carvalho, J. O., R. Sartori, P. L. Monteiro, L. O. Leme, and M. A. N. Dode. "69 Can Bovine Sperm Interaction with the Oviduct Cells After Artificial Insemination Affect the Transcriptome Profile of the Oviduct?" Reproduction, Fertility and Development 30, no. 1 (2018): 173. http://dx.doi.org/10.1071/rdv30n1ab69.
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During their transit through the female genital tract, spermatozoa bind to oviduct cells, where they are maintained alive for a long period. Although some studies have been carried out to evaluate the effects of the oviduct on spermatozoa, less is known about the effect of spermatozoa on the oviduct. The aim of this study was to evaluate the effect of sperm on the transcriptome profile of the bovine oviduct after fixed-time AI (FTAI). Nellore cows submitted to FTAI were randomly inseminated using 8 × 106 frozen–thawed spermatozoa (sperm group, n = 9) or saline solution (control group, n = 9). Eighteen hours after FTAI, the cows were slaughtered, and the cells from oviducal isthmus were collected and stored in liquid nitrogen until transcriptome analysis. For RNA extraction, 3 pools of 3 oviducts/cows from each group were formed. Total RNA was extracted and the RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The transcription profiling from each group was performed by HiSEqn 2500 (Illumina Inc., San Diego, CA, USA). After sequencing, the data were analysed by edgeR software, and differential gene expression analysis between sperm and control group was performed using the generalized linear model (P = 0.05). The P-value was adjusted by false discovery rate (FDR) Benjamini-Hochberg method, to avoid false positives. Moreover, using mRNA expression levels (log fold change and P-values) as coefficients, functional enrichment analysis was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. A total of 9,374 genes were found to be expressed in bovine oviducal isthmus, of which 219 genes were differentially expressed between sperm and control group. Differentially expressed genes genes were classified according to 3 major GO classifications, with 10 gene categories in molecular function, 3 gene categories in cellular component, and zero categories in biological process. In the molecular function category, genes related to calcium ion binding (n = 113), molecular transducer activity (n = 111), and receptor activity (n = 111), were predominant. For cellular component, the highest number of genes (n = 176) was related to extracellular space. Moreover, KEGG assignments were used to classify the functional annotations of the pathways genes to further understand the biological functions of the expressed genes. It was shown that steroid hormone biosynthesis, arachidonic acid metabolism, calcium signalling, cytokine-cytokine receptor interaction, neuroactive ligant-receptor interaction, extracellular matrix-receptor interaction, cell adhesion molecules, coagulation and complement cascades, ovarian steroidogenesis, protein digestion and absorption and mineral absorption pathways had genes with higher or lower expression than the median of gene expression out of the tested category. The results suggested that bovine sperm affect the transcriptome profile of the bovine oviduct after FTAI. Moreover, based on the functional enrichment analysis, an effect of the sperm-oviduct interaction on some pathways analysed by GO and KEGG was identified. This research was financially supported by FAP-DF, FAPESP, CNPq.
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Ding, Jingyi, Yanxi Liu, and Yu Lai. "Identifying MMP14 and COL12A1 as a potential combination of prognostic biomarkers in pancreatic ductal adenocarcinoma using integrated bioinformatics analysis." PeerJ 8 (November 23, 2020): e10419. http://dx.doi.org/10.7717/peerj.10419.
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Background Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant neoplasm. It is necessary to improve the understanding of the underlying molecular mechanisms and identify the key genes and signaling pathways involved in PDAC. Methods The microarray datasets GSE28735, GSE62165, and GSE91035 were downloaded from the Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified by integrated bioinformatics analysis, including protein–protein interaction (PPI) network, Gene Ontology (GO) enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The PPI network was established using the Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape software. GO functional annotation and KEGG pathway analyses were performed using the Database for Annotation, Visualization, and Integrated Discovery. Hub genes were validated via the Gene Expression Profiling Interactive Analysis tool (GEPIA) and the Human Protein Atlas (HPA) website. Results A total of 263 DEGs (167 upregulated and 96 downregulated) were common to the three datasets. We used STRING and Cytoscape software to establish the PPI network and then identified key modules. From the PPI network, 225 nodes and 803 edges were selected. The most significant module, which comprised 11 DEGs, was identified using the Molecular Complex Detection plugin. The top 20 hub genes, which were filtered by the CytoHubba plugin, comprised FN1, COL1A1, COL3A1, BGN, POSTN, FBN1, COL5A2, COL12A1, THBS2, COL6A3, VCAN, CDH11, MMP14, LTBP1, IGFBP5, ALB, CXCL12, FAP, MATN3, and COL8A1. These genes were validated using The Cancer Genome Atlas (TCGA) and Genotype–Tissue Expression (GTEx) databases, and the encoded proteins were subsequently validated using the HPA website. The GO analysis results showed that the most significantly enriched biological process, cellular component, and molecular function terms among the 20 hub genes were cell adhesion, proteinaceous extracellular matrix, and calcium ion binding, respectively. The KEGG pathway analysis showed that the 20 hub genes were mainly enriched in ECM–receptor interaction, focal adhesion, PI3K-Akt signaling pathway, and protein digestion and absorption. These findings indicated that FBN1 and COL8A1 appear to be involved in the progression of PDAC. Moreover, patient survival analysis performed via the GEPIA using TCGA and GTEx databases demonstrated that the expression levels of COL12A1 and MMP14 were correlated with a poor prognosis in PDAC patients (p < 0.05). Conclusions The results demonstrated that upregulation of MMP14 and COL12A1 is associated with poor overall survival, and these might be a combination of prognostic biomarkers in PDAC.
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Scharff, Bibi F. S. S., Signe Modvig, Maria Thastrup, Mette Levinsen, Matilda Degn, Lars Peter Ryder, Kjeld Schmiegelow, Claus René Lykke Christensen, and Hanne Vibeke Marquart. "A Comprehensive Study of Human Integrins in Pediatric Lymphoblastic Leukemia Supports a Role of CD49f (Integrin α6) in the Localization to Bone Marrow but Not Spinal Fluid." Blood 134, Supplement_1 (November 13, 2019): 5205. http://dx.doi.org/10.1182/blood-2019-126701.
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Introduction: The overall survival of children with ALL (Acute Lymphoblastic Leukemia) has improved markedly over the last decade, yet relapse occurs in about 20% of patients. The spread of leukemic blasts to the Central Nervous System (CNS) and increased resistance to therapy due to cell adherence within the Bone Marrow (BM) or CNS constitutes major challenges to treatment. For these reasons, adhesion molecules governing the homing and adhesion of leukemic cells are perceived to be of extraordinary importance, both as potential biomarkers and therapeutic targets. Integrins constitute a large family of heterodimeric receptors composed of alpha and beta subunits, which play important roles during homing and migration of normal leucocytes by facilitating adhesion to both stromal cells and components of the extracellular matrix. Increased expression of CD49d (integrin subunit α4) is a marker for adverse prognosis in ALL and recently, CD49f (integrin subunit α6) was shown to facilitate metastasis of ALL xenografts to the central nervous system in mice (Yao H et al., Nature 2018). Methods Previous studies of integrins in BCP-ALL have focused on individual alpha integrins in xenograft models and were based on limited numbers of clinical samples. The present study was based on a large number of Danish pediatric BCP-ALL patients stratified between 2012-2018 using Minimal Residual Disease (MRD) according to the Nordic NOPHO-2008 protocol (Toft N et al., Eur J Haematol 2013). Diagnostic BM samples were subjected to flowcytometric analysis (FCM) of CD49f (n=246) and CD49d (n=135), using a backbone of lineage-specific B-cell markers (CD45, CD10, CD19, CD20). Leukemic blasts were detected in Cerebrospinal Fluid (CSF) using high-sensitivity FCM and the following markers CD45, CD10, CD19, CD20, CD34 and CD38 (n = 246, with matching BM and CSF samples). Results Our data provided us with a unique possibility to identify the role of CD49d and CD49f with respect to minimal residual disease (MRD) at the end of induction therapy (day 29), which is considered the most important prognostic factor in paediatric lymphoblastic leukemia. We found that CD49f was more highly expressed in patients with MRD ≥ 0,1% at day 29 than patients with MRD < 0,1% (p = 0,01), whereas no difference was seen with respect to CD49d. We also investigated the correlation between white blood cell (WBC) and surface expression of CD49d and CD49f in diagnostic BM blasts with respect to different cytogenetic subtypes. A Kruskall-Wallis test showed that the expression varies according to genetic subtypes (p‹0.0001). We found that the expression of CD49d was highest among the high hyperdiploid and iAMP21 (intrachromosomal amplification of chromosome 21) patients, whereas the expression of CD49f was highest among the t12,21 and iAMP21 patients. Notably, the expression of CD49f was inversely correlated to WBC (r=0,17, p=0.01), which was most pronounced among the patients in the B-other cytogenetic subgroup defined as leukaemia that could not be classified into the existing cytogenetic groups (r=0,42, P‹0.0001). In case of strong adherence to BM, lower levels of leukemic blasts might be expected in circulation resulting in high MRD but low WBC. Therefore, both MRD and WBC data are consistent with a prominent adhesive role of CD49f within BM. In contrast, we found significantly lower CD49f surface expression in diagnostic BM samples in patients with leukemic blasts within CSF (p=0.0297). Conclusions: Recently, Yao et al. (2018) showed that ALL cells in circulation are unable to breach the blood-brain barrier in mice and instead employ CD49f (integrin α6) to migrate into the CNS along vessels that connect vertebral or calvarial bone marrow and the subarachnoid space. Potentially, this mechanism could account for cases of ALL relapse within CNS. Our work shows a strong association between high MRD and the expression of CD49f which is a function that would have been anticipated for CD49d due to previous works with ALL and CLL. Furthermore, we found significantly lower CD49f surface expression in leukemic blasts within the BM in patients with CSF involvement and therefore no support for the recently proposed role of CD49f in facilitating the spread of leukemic cells to the CNS. Disclosures No relevant conflicts of interest to declare.
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Fein, Daniel EC, Nicole Traugh, Gat Rauner, Youssof Mal, Colin Trepicchio, and Charlotte Kuperwasser. "Abstract P5-06-07: Selective inhibition of fibroblast-specific domain discoidin receptor 1 (DDR1) results in reduced collagen deposition and immunomodulatory cytokine release: A potential target to modulate the activity of breast cancer associated fibroblasts." Cancer Research 82, no. 4_Supplement (February 15, 2022): P5–06–07—P5–06–07. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-06-07.
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Abstract Background: Cancer-Associated Fibroblasts (CAFs) are a major cell type within breast tumor microenvironment (TME) which have emerged as a new target for cancer therapy given their contributions to tumor growth, metastasis, and suppression of the anti-tumor immune response. In the breast, CAFs are predominantly formed from conversion of tissue-resident mammary fibroblasts through mechanisms including increasing extracellular matrix (ECM) stiffness, ECM composition, hypoxia, and activating signals TGFβ and IL-6. Domain Discoidin Receptor 1 (DDR1) is a cell surface tyrosine kinase receptor expressed by epithelial and stromal cells which is activated by collagen within the TME. DDR1 expression and activity has been implicated in the development of fibrosis within the breast as well as chemotherapy resistance in several tumor types. Thus, we set out to examine whether selective inhibition of DDR1 would affect CAF immunomodulatory function to generate an immune-permissive breast microenvironment and result in reduction in stromal desmoplasia. Results: Using a human cell model of CAFs, we studied the effects of DDR1 inhibition (DDR1i) on the expression of immune modulating factors and CAF functions and features. Using a multiplex cytokine array, we observed high expression levels of key immunomodulatory cytokines IL-6, IL-8, and MCP-1 in conditioned media collected from CAF-like cells cultured in 3D collagen gels which were consequently downregulated in the presence of DDR1i. Downregulation of IL-6, IL-8, and MCP-1 induced by DDR1i was also by observed by RT-qPCR, with enhanced downregulation seen in cells cultured with increasing concentrations of collagen. Downregulation of CAF markers αSMA and FAP as well as Collagen type 1 were observed with selective DDR1 inhibition, an effect seen also with CAF-like cells stimulated with recombinant TGFβ1. Additionally, we observed reduced contraction of CAF-like cells in 3D collagen gels in the presence of DDR1i. In vivo DDR1i resulted in tissue remodeling and reduced collagen deposition as well as changes in immunomodulatory cytokine release within the mammary gland in a murine model. We observed downregulation of IL-6, as well as downregulation of Collagens type I, type IV, and MMP9. RT-qPCR also revealed reduced expression of potent chemokines CXCL1 and CXCL12 in mammary gland tissue of mice treated with DDR1i. Consistent with this, multiplexed flow cytometry demonstrated trends of increased CD45.2+ immune cell infiltration within the mammary glands and reduction of Ly6G+/Ly6C- neutrophil infiltration within the mammary gland and spleens in mice treated with DDR1i. Conclusions: Collectively, these findings suggest that breast fibroblast-specific DDR1 mediates collagen deposition and immunomodulatory function within the mammary gland and warrants further investigation as a potential target for CAF-modulating therapy in breast cancer. Citation Format: Daniel EC Fein, Nicole Traugh, Gat Rauner, Youssof Mal, Colin Trepicchio, Charlotte Kuperwasser. Selective inhibition of fibroblast-specific domain discoidin receptor 1 (DDR1) results in reduced collagen deposition and immunomodulatory cytokine release: A potential target to modulate the activity of breast cancer associated fibroblasts [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-07.
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Apollonio, Benedetta, Nicole S. Nicholas, Lesley-Ann Sutton, Jon Salisbury, Piers E. Patten, Shireen Kassam, Stephen Devereux, Rose Marie Amini, Richard Rosenquist, and Alan G. Ramsay. "Diffuse Large B-Cell Lymphoma (DLBCL) Tumor Cells Reprogram Lymphatic Fibroblasts into Cancer-Associated Fibroblasts (CAFs) That Contribute to Tumor Microenvironment (TME)-Driven Immune Privilege." Blood 126, no. 23 (December 3, 2015): 1474. http://dx.doi.org/10.1182/blood.v126.23.1474.1474.
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Abstract There is a clinical need to identify novel treatments for relapsed/refractory DLBCL. Cancer cells engage in novel associations with stromal and immune cells in the TME that provide crucial contributions to disease progression, immune evasion and therapeutic response. However, these hallmark capabilities have been understudied in DLBCL. Given tumor cell genetic complexity, targeting the TME has become a compelling therapeutic strategy. Immune checkpoint blockade therapy (ICB) (e.g. anti-PD-1), which can activate anti-tumor immunity, has provided a new weapon against cancer and serves as an illustrative example of therapeutically re-educating the TME. Clinical results indicate that only a fraction of DLBCL patients currently respond to ICB. Understanding ill-defined TME-driven immune suppression should help optimise ICB and identify novel therapeutic opportunities. Gene expression studies of DLBCL have identified molecular signatures present in both GCB and ABC subtypes related to the TME that correlated with outcome. The prognostically favorable stromal-1 signature reflects reprogrammed stromal cells, extracellular matrix (ECM) and an active immune response. The less favorable stromal-2 signature indicates elevated angiogenesis and blood vessel density. CAFs promote ECM remodelling and angiogenesis in solid cancers. We hypothesized that CAFs play an important role in the pathogenesis of DLBCL including the regulation of subverted host anti-tumor immunity. To assess whether DLBCL tumor cells induce a CAF phenotype in previously healthy stromal cells, we established a co-culture system with subsequent imaging of conditioned cells. Primary human lymphatic fibroblasts (HLFs) were co-cultured for 5 days in direct contact with a panel of GCB (SU-DHL4, SU-DHL6, DOHH2) and ABC (OCI-LY10, RIVA, U2932) DLBCL cell lines or healthy control B-cells. Quantitative analysis revealed a strong induction of CAF molecular marker expression including FAPα and α-SMA in all DLBCL-educated stromal cells compared to healthy B-cell exposed fibroblasts (P<.01). DLBCL-educated HLFs exhibited dramatic cytoskeletal changes including increased stress fibres. More significantly, the ability of DLBCL-educated HLFs to contract collagen gels, a measure of their matrix remodelling functional capacity, significantly increased compared to control HLFs (P<.01). We next investigated the potential immunomodulatory capacity of DLBCL-educated CAFs using 2-part functional assays. First, healthy T cells were co-cultured (24 h) with either DLBCL-educated HLFs or control HLFs. Second, these T cells were purified and used in subsequent immunologic assays. Exposure to DLBCL-educated HLFs resulted in significant impairment of proliferation of CD4+ and CD8+ T cells in response to anti-CD3/-CD28 (P<.01). The ability of T cells to recognize target tumor cells requires formation of the immunological synapse. We utilized the immune synapse bioassay to examine CD8+ T cell interactions with DLBCL tumor cells. We show that prior co-culture with DLBCL-educated HLFs significantly decreased the formation and strength of CD8+ T cell F-actin immune synapses compared with control HLF co-culture (P<.01). Flow cytometric analysis of FAP+ CAFs revealed markedly increased surface expression of the immune checkpoint ligand PD-L1. The up-regulation of PD-L1 led to the pre-treatment of DLBCL-educated HLFs with an anti-PD-L1 blocking antibody that increased T cell synapse activity. Current experiments are investigating this TME checkpoint axes using primary patient DLBCL tumor cells and T cells. IHC/IF image analysis revealed that PD-L1+ stromal cells reside in the DLBCL TME (archival biopsies, n=20). TME biopsies showed increased expression of α-SMA and FAPα in both GCB and ABC subtypes compared to reactive lymph node samples. CAFs were interspersed within the TME and in close proximity to CD20+ DLBCL tumor cells. In conclusion, our results establish the ability of DLBCL tumor cells to reprogram HLFs into CAFs that acquire functional capabilities to modulate the TME. Notably, activated CAFs show a compensatory inhibitory response by up-regulating PD-L1 expression that may represent an important TME-driven immunosuppressive mechanism. We believe this data contributes to the understanding of the biology that underlies stromal signatures in the DLBCL TME, in particular the contribution of CAFs to immune privilege. Disclosures Ramsay: Celgene: Research Funding; MedImmune: Research Funding.
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Luo, Xin, Dezhi Geng, Qirong Zhang, Tao Ye, Yifan Zhang, Ziyi Li, Yadong Huang, and Qi Xiang. "Recombinant expression a novel fibronectin—collage fusion peptide modulating stem cell stemness via integrin β3." Applied Microbiology and Biotechnology, May 20, 2022. http://dx.doi.org/10.1007/s00253-022-11965-4.
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Abstract Constructing bionic extracellular matrix (ECM) is an attractive proposition for tissue engineering and clinical regeneration therapy involving the stemness of stem cells. Here, a novel recombinant protein fibronectin-collagen peptide (FCP) was designed to modulate the function of ECM expressed by Picha. pastoris strain X33. This FCP promotes cell migration and adhesion and maintains rBMSC stemness by binding integrin β3. Its effects were blocked by both integrin β3 siRNA and the integrin β3 inhibitor Cilengitide. A template-independent ab initio prediction modeling approach is the best approach to construct a stable FCP protein model, which predicts the binding sites between FCP and integrin β3. FCP may be used in the in vitro culture and clinical regeneration of stem cells that highly express integrin β3, such as hematopoietic stem cells. The study provides information on the molecular structure of FCP and its bioactivity, which can be used to design new compounds. Key points • Design a novel recombinant fibronectin-collagen peptide biomimetic ECM. • FCP promotes cell adhesion, migration, and proliferation. • Predicted and verified FCP structure and affinity with integrin β3. • FCP binds integrin β3 to maintain rBMSC stemness.
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Leibovitzh, Haim, Sun-Ho Lee, Juan Antonio Raygoza Garay, Osvaldo Espin-Garcia, Mingyue Xue, Anna Neustaeter, Ashleigh Goethel, et al. "Immune response and barrier dysfunction-related proteomic signatures in preclinical phase of Crohn’s disease highlight earliest events of pathogenesis." Gut, February 14, 2023, gutjnl—2022–328421. http://dx.doi.org/10.1136/gutjnl-2022-328421.
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ObjectiveThe measure of serum proteome in the preclinical state of Crohn’s disease (CD) may provide insight into biological pathways involved in CD pathogenesis. We aimed to assess associations of serum proteins with future CD onset and with other biomarkers predicting CD risk in a healthy at-risk cohort.DesignIn a nested case–control study within the Crohn’s and Colitis Canada Genetics Environment Microbial Project (CCC-GEM) cohort, which prospectively follows healthy first-degree relatives (FDRs), subjects who developed CD (n=71) were matched with four FDRs remaining healthy (n=284). Using samples at recruitment, serum protein profiles using the Olink Proximity Extension Assay platform was assessed for association with future development of CD and with other baseline biomarkers as follows: serum antimicrobial antibodies (AS: positive antibody sum) (Prometheus); faecal calprotectin (FCP); gut barrier function using the fractional excretion of lactulose-to-mannitol ratio (LMR) assay.ResultsWe identified 25 of 446 serum proteins significantly associated with future development of CD. C-X-C motif chemokine 9 (CXCL9) had the highest OR with future risk of CD (OR=2.07 per SD, 95% CI 1.58 to 2.73, q=7.9e-5), whereas matrix extracellular phosphoglycoprotein had the lowest OR (OR 0.44, 95% CI 0.29 to 0.66, q=0.02). Notably, CXCL9 was the only analyte significantly associated with all other CD-risk biomarkers with consistent direction of effect (FCP: OR=2.21; LMR: OR=1.67; AS: OR=1.59) (q<0.05 for all).ConclusionWe identified serum proteomic signatures associated with future CD development, reflecting potential early biological processes of immune and barrier dysfunction.
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Mentlein, Rolf, Kirsten Hattermann, Charles Hemion, Achim A. Jungbluth, and Janka Held-Feindt. "Expression and role of the cell surface protease seprase/fibroblast activation protein-α (FAP-α) in astroglial tumors." Biological Chemistry 392, no. 3 (March 1, 2011). http://dx.doi.org/10.1515/bc.2010.119.
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Abstract Seprase or fibroblast activation protein-α (FAP-α) is a cell-surface serine protease that was previously described nearly exclusively on reactive and tumor stromal fibroblasts and thought to be involved in tissue remodeling. We investigated the expression and significance of FAP-α in astrocytomas/glioblastomas. As shown by quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohisto-chemistry, FAP-α was elevated in whole glioblastoma tissues and in particular in most glioma cells in situ and in vitro. In glioma stem-like cells (gliospheres), FAP-α was detected at low levels; however, FAP-α was considerably induced upon differentiation with 10% fetal calf serum. To explore its functional role, FAP-α was silenced by siRNA transfection. In Boyden chamber assays, FAP-α silenced cells migrated similar as control cells through non-coated or Matrigel (basal lamina)-coated porous membranes, but significantly slower through membranes coated with gelatin or brevican, a major component of brain extracellular matrix. Furthermore, FAP-α-silenced glioma cells migrated through murine brain slices much slower under the conditions tested than differentially fluorescent-labeled control cells. Thus, FAP-α is highly expressed on the surface of glioma cells and contributes to diffuse glioma invasion through extracellular matrix components.
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Ateshian, Gerard A., Kevin D. Costa, Evren U. Azeloglu, Barclay Morrison, and Clark T. Hung. "Continuum Modeling of Biological Tissue Growth by Cell Division, and Alteration of Intracellular Osmolytes and Extracellular Fixed Charge Density." Journal of Biomechanical Engineering 131, no. 10 (September 1, 2009). http://dx.doi.org/10.1115/1.3192138.
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A framework is formulated within the theory of mixtures for continuum modeling of biological tissue growth that explicitly addresses cell division, using a homogenized representation of cells and their extracellular matrix (ECM). The model relies on the description of the cell as containing a solution of water and osmolytes, and having a porous solid matrix. The division of a cell into two nearly identical daughter cells is modeled as the doubling of the cell solid matrix and osmolyte content, producing an increase in water uptake via osmotic effects. This framework is also generalized to account for the growth of ECM-bound molecular species that impart a fixed charge density (FCD) to the tissue, such as proteoglycans. This FCD similarly induces osmotic effects, resulting in extracellular water uptake and osmotic pressurization of the ECM interstitial fluid, with concomitant swelling of its solid matrix. Applications of this growth model are illustrated in several examples.
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Guo, Miao, Siqi Tan, Junli Zhu, Aihua Sun, Peng Du, and Xiaoxiang Liu. "Genes Involved in Biofilm Matrix Formation of the Food Spoiler Pseudomonas fluorescens PF07." Frontiers in Microbiology 13 (June 6, 2022). http://dx.doi.org/10.3389/fmicb.2022.881043.
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The extracellular matrix is essential for the biofilm formation of food spoilers. Pseudomonas fluorescens PF07 is a previous isolate from spoiled marine fish; however, the genes involved in the extracellular matrix formation of PF07 biofilms remain poorly defined. In this study, PF07 formed a wrinkled macrocolony biofilm through the high production of extracellular matrix. The genes involved in biofilm matrix formation and regulation were screened and identified by RNA-seq-dependent transcriptomic analysis and gene knock-out analysis. The macrocolony biofilms of PF07 grown for 5 days (PF07_5d) were compared with those grown for 1 day (PF07_1d). A total of 1,403 genes were significantly differentially expressed during biofilm formation. These mainly include the genes related to biofilm matrix proteins, polysaccharides, rhamnolipids, secretion system, biofilm regulation, and metabolism. Among them, functional amyloid genes fapABCDE were highly upregulated in the mature biofilm, and the operon fapA-E had a –24/–12 promoter dependent on the sigma factor RpoN. Moreover, the RNA-seq analyses of the rpoN mutant, compared with PF07, revealed 159 genes were differentially expressed in the macrocolony biofilms, and fapA-E genes were positively regulated by RpoN. In addition, the deletion mutants of fapC, rpoN, and brfA (a novel gene coding for an RpoN-dependent transcriptional regulator) were defective in forming mature macrocolony biofilms, solid surface-associated (SSA) biofilms, and pellicles, and they showed significantly reduced biofilm matrices. The fap genes were significantly downregulated in ΔbrfA, as in ΔrpoN. These findings suggest that the functional amyloid Fap is the main component of PF07 biofilm matrices, and RpoN may directly regulate the transcription of fap genes, in conjunction with BrfA. These genes may serve as potential molecular targets for screening new anti-biofilm agents or for biofilm detection in food environments.
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Feng, Yewen, Yumin Zhao, Yao Li, Teng Peng, Yu Kuang, Xingming Shi, Gang Wang, Fu Peng, and Chenghao Yu. "Inhibition of Fibroblast Activation in Uterine Leiomyoma by Components of Rhizoma Curcumae and Rhizoma Sparganii." Frontiers in Public Health 9 (March 1, 2021). http://dx.doi.org/10.3389/fpubh.2021.650022.
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Background: The herbs Rhizoma Curcumae and Rhizoma Sparganii (RCRS) are often used in traditional Chinese medicine for the treatment of uterine leiomyoma (UL). The effectiveness of RCRS for the treatment of UL has been confirmed in our previous studies.Purpose: This study aimed to investigate the molecular mechanism by which RCRS inhibits the activation of fibroblast activation protein (FAP) and prevents UL in rats.Study Design and Methods: A Sprague Dawley (SD) rat model of UL was established via estrogen and progesterone load combined with external stimulation. Histological analyses, enzyme-linked immunosorbent assays, and western blotting were performed to evaluate the effect of RCRS on UL and elucidate its mechanism of action.Results: Our data showed that the treatment of SD rats with RCRS significantly reduced the expression of extracellular matrix component collagen, FAP, and transforming growth factor beta (a FAP-activating factor) and the phosphorylation of the cell proliferation pathway-related signaling factors AKT/MEK/ERK.Conclusion: Our results suggest that RCRS is effective in the prevention and treatment of UL in rats, and RCRS may exert its functions by inhibiting the activation of tumor-associated fibroblasts and cell proliferation and by improving the tumor extracellular matrix.
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Xin, Lei, Jinfang Gao, Ziliang Zheng, Yiyou Chen, Shuxin Lv, Zhikai Zhao, Chunhai Yu, Xiaotang Yang, and Ruiping Zhang. "Fibroblast Activation Protein-α as a Target in the Bench-to-Bedside Diagnosis and Treatment of Tumors: A Narrative Review." Frontiers in Oncology 11 (August 19, 2021). http://dx.doi.org/10.3389/fonc.2021.648187.
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Fibroblast activation protein-α (FAP) is a type II integral serine protease that is specifically expressed by activated fibroblasts. Cancer-associated fibroblasts (CAFs) in the tumor stroma have an abundant and stable expression of FAP, which plays an important role in promoting tumor growth, invasion, metastasis, and immunosuppression. For example, in females with a high incidence of breast cancer, CAFs account for 50–70% of the cells in the tumor’s microenvironment. CAF overexpression of FAP promotes tumor development and metastasis by influencing extracellular matrix remodeling, intracellular signaling, angiogenesis, epithelial-to-mesenchymal transition, and immunosuppression. This review discusses the basic biological characteristics of FAP and its applications in the diagnosis and treatment of various cancers. We review the emerging basic and clinical research data regarding the use of nanomaterials that target FAP.
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Nicolela Geraldo Martins, C., L. R. Paim, L. M. Da Silva, G. Scriptori, A. A. Bau, A. Coy-Cangucu, R. Yamaguti, et al. "Subclinical myocardial injury in ATTR-related familial amyloid polyneuropathy patients without clinical signs of cardiomyopathy." European Heart Journal 43, Supplement_2 (October 1, 2022). http://dx.doi.org/10.1093/eurheartj/ehac544.259.
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Abstract Background ATTR-related Familial Amyloid Polyneuropathy (FAP) is a hereditary disease that primarily affects peripheral nerve function. Few studies have investigated cardiac involvement and myocardial tissue remodeling in FAP. Aim To investigate subclinical myocardial tissue remodeling in FAP patients without cardiomyopathy using a multiparametric CMR protocol. Results Thirty-one FAP patients (46.9±16.1 years, 57% female, 60% Val30Met mutation) and 33 healthy controls (41.3±13.7 years, 58% female) were enrolled, undergoing a multiparametric CMR protocol for assessment of ventricular morphology and function, native myocardial T1, extracellular volume fraction (ECV) and intracellular lifetime of water). Cardiopulmonary exercise capacity was evaluated with a cycle ergometer. Cardiac high-sensitive troponin T (cTnT) and NT-proBNP were measured to assess for cardiac injury. The majority of ATTR-PN patients were in stage 1 (70%) with mild symptoms of sensory, motor and autonomic neuropathy. Adjusted maximum oxygen consumption was reduced among FAP patients compared to healthy controls (FAP: 22.2±8.2 mL/kg/min vs. controls: 30.3±10.2 mL/kg/min, p<0.001). Although none of FAP patients reported heart failure symptoms, NT-proBNP (FAP: 251.240±624.446 ng/dL, vs. controls: 34.3±29 ng/dL, p<0.005) and cTnT (FAP: 13.2 [3.0, 19.0] ng/dL, vs. controls: 3.6 [3.0, 6.0] ng/dL, p<0.005) were elevated, and both correlated with ECV (cTnT: R=0.81, P<0.001; NT-proBNP: R=0.61, P=0.001, Fig. 1). While LVEF was preserved among FAP patients (FAP: 67.9±8.2% vs. controls: 65.4±4.3%, p=NS), LVmass index was increased compared to control subjects (FAP: 58.5±18.8 vs. and controls: 42±9.2 g/m2, p<0.005). Both native T1 (FAP: 1,303.924±120.152, vs. controls: 1,212.78±76.01 ms, p<0.05) and ECV (FAP: 0.36±0.1, vs. controls: 0.26±0.02, p<0.001) were markedly elevated among FAP patients. In contrast the intracellular lifetime of water, a validated marker of cardiomyocyte size was reduced in the FAP group (FAP: 0.082±0.04 vs. controls: 0.14±0.05, p<0.001). There was a trend for ECV to increase linearly with FAP stage, and native T1 trended higher in stage 1 and 2 patients compared to stage 0. Both ECV (R=0.89, p<0.001) and native T1 (R=0.62, p<0.001) were correlated with LVmass index (Fig 2). Conclusion In FAP without clinical signs of cardiac involvement, significant extracellular matrix expansion was present. The increase of LV mass in these patients is associated with expansion of the extracellular matrix, possibly as a result of diffuse replacement fibrosis, and below-normal cardiomyocyte diameter. These findings from serum biomarkers and CMR tissue phenotyping provide evidence of sub-clinical cardiac involvement through adverse myocardial tissue remodeling in FAP patients presenting with mostly mild symptoms of peripheral neuropathy. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Pfizer