Single-Cell Sequencing Analysis and Weighted Co-Expression Network Analysis Based on Public Databases Identified That TNC Is a Novel Biomarker for Keloid.

Background: The pathophysiology of keloid formation is not yet understood, so the identification of biomarkers for kelod can be one step towards designing new targeting therapies which will improve outcomes for patients with keloids or at risk of developing keloids. Methods: In this study, we performed single-cell RNA sequencing analysis, weighted co-expression network analysis, and differential expression analysis of keloids based on public databases. And 3 RNA sequencing data from keloid patients in our center were used for validation. Besides, we performed QRT-PCR on keloid tissue and adjacent normal tissues from 16 patients for further verification. Results: We identified the sensitive biomarker of keloid: Tenascin-C (TNC). Then, Pseudotime analysis found that the expression level of TNC decreased first, then stabilized and finally increased with fibroblast differentiation, suggesting that TNC may play an potential role in fibroblast differentiation. In addition, there were differences in the infiltration level of macrophages M0 between the TNC-high group and the TNC-low group. Macrophages M0 had a higher infiltration level in low TNC- group (P<0.05). Conclusion: Our results can provide a new idea for the diagnosis and treatment of keloid.

Generation and characterization of dromedary Tenascin-C and Tenascin-W specific antibodies.

Tenascin-C (TNC) and tenascin-W (TNW), large hexameric glycoproteins overexpressed in the tumor microenvironment, are useful tumor biomarkers for theranostic applications. For now, polyclonal and monoclonal antibodies, as well as aptamers targeting TNC and TNW have been developed. However, the immunostaining sensitivity of antibodies is very heterogenous. The main aim of this study was to generate antibodies in dromedary that detect TNC and TNW, respectively. We show that immune sera from immunized dromedaries are able to specifically bind native TNC and TNW by ELISA and also to detect TNC and TNW in matrix tracks of mammary tumors by immunostaining. Furthermore, we demonstrate that purified IgG subtypes are able to interact specifically with TNC or TNW by ELISA and immunostaining. These camelid antibodies are a good basis to develop tools for the detection of TNC and TNW in the tumor microenvironment and could potentially have a broader application for early diagnosis of solid cancers.

Tenascin-C Is Increased in Inflammatory Bowel Disease and Is Associated with response to Infliximab Therapy.

Tenascin-C (TNC) is an extracellular matrix glycoprotein expressed in response to inflammation and tissue damage. The role of TNC in patients with inflammatory bowel disease (IBD) is not well understood. In this study, we analyzed the expression of TNC in the inflamed mucosa of patients with ulcerative colitis (UC) and Crohn's disease (CD). Serum TNC levels were determined by the enzyme-linked immunosorbent assay (ELISA), and the levels of TNC in patients with different disease activities were compared. The expression of TNC was derived from a GEO dataset. THP-1 cells were stimulated with TNC to evaluate the proinflammatory role of TNC. We found higher TNC expression in the inflamed mucosa of patients with UC and CD compared with normal controls (NCs). TNC was mainly expressed in the stromal area of the intestinal mucosa. The median serum levels of TNC were significantly higher in UC (median 74.1 ng/ml, range 42.6-102.1 ng/ml) and CD (median 59.2 ng/ml, range 44.0-80.9 ng/ml). We also found that serum TNC levels were correlated with Mayo scores in UC and Crohn's disease activity index (CDAI) in CD. Through GSE14580, we demonstrated that patients who were nonresponsive to infliximab treatment had higher mucosal TNC mRNA expression. High TNC mRNA expression in the inflamed intestinal mucosa was associated with poor response to infliximab therapy in patients with UC. Furthermore, THP-1 cells stimulated with TNC showed increased expression of IL-6, but not TNF-α, IL-8, MCP-1, or IL-1ß. Thus, increased TNC levels may participate in the pathogenesis of IBD and may serve as a biomarker for disease activity and response to treatment with infliximab.

Serum Levels and Urinary Excretion of Tenascin-C and TIMP-1 in Acute Kidney Injury.

BACKGROUND: Both the extracellular matrix molecule tenascin-C (Tn-C) and tissue inhibitors of metalloproteinases (TIMPs) have a role in tissue injury, inflammation, and remodeling. In this pilot study, we tried to evaluate the role of these markers in acute kidney injury (AKI). METHODS: A total of 52 subjects were enrolled in this study. Group 1 consisted of 27 patients with AKI (stage 1, 2, and 3), and Group 2 consisted of 25 age- and gender-matched healthy subjects. Serum and urine samples (to determine Tn-C and TIMP-1) were obtained from the participants at the beginning of the study. Second samples were obtained from Group 1 patients when renal function improved (at discharge). RESULTS: Serum TIMP-1 concentrations (admission and discharge) were higher in Group 1 than Group 2 (p = 0.0001 for both comparisons). Tn-C excretion in spot urine was significantly higher in healthy controls than at the admission levels of the patient group (p = 0.036). However, TIMP-1 excretion in spot urine was lower in healthy controls than in admission and discharge levels of the patient group (p = 0.0001 for both comparisons). CONCLUSIONS: Our results show that these biomarkers (especially TIMP-1) may have a role in the pathophysiology of AKI. Further studies are needed in this field.

Dynamics of extracellular matrix proteins in cerebrospinal fluid and serum and their relation to clinical outcome in human traumatic brain injury.

Background Brevican, neurocan, tenascin-C and tenascin-R are extracellular matrix proteins present in brain that show increased expression in experimental animal models of brain injury. However, little is known about the dynamics of these proteins in human body fluids, such as cerebrospinal fluid (CSF) and serum, after traumatic brain injury (TBI). The aims of this study were to investigate if matrix proteins in CSF and serum are associated with functional outcome following traumatic brain injury, if their concentrations change over time and to compare their levels between brain injured patients to controls. Methods In total, 42 traumatic brain injury patients, nine healthy controls and a contrast group consisting of 38 idiopathic normal pressure hydrocephalus patients were included. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the concentrations of proteins. Results Increased concentrations of brevican, tenascin-C and tenascin-R in CSF correlated with unfavourable outcome, with stronger outcome prediction ability compared to other biomarkers of brain tissue injury. CSF brevican, tenascin-R and serum neurocan gradually decreased with time (p = 0.04, p = 0.008, p = 0.005, respectively), while serum tenascin-C (p = 0.01) increased. CSF concentrations of brevican, neurocan and tenascin-R (only in time point 3) after TBI were lower than in the idiopathic normal pressure hydrocephalus group (p < 0.0001, p < 0.0001, and p = 0.0008, respectively). In serum, tenascin-C concentration was higher and neurocan lower compared to healthy controls (p = 0.02 and p = 0.0009). Conclusions These findings indicate that levels of extracellular matrix proteins are associated with clinical outcome following TBI and may act as markers for different pathophysiology than currently used protein biomarkers.

Expression of tenascin C in cardiovascular lesions of Kawasaki disease.

BACKGROUND/OBJECTIVE: To examine tenascin C (TN-C) expression in coronary artery lesions (CALs) and myocardial lesions (MLs) in Kawasaki disease (KD). METHODS AND RESULTS: Twenty-five KD autopsy cases (post-KD-onset range of 6 days to 17 years) were examined in this study. Time-course analysis based on the disease day was performed of the histological findings for the CALs and MLs, as well as the localization and intensity of expression of TN-C. TN-C expression was observed to coincide with the areas where inflammatory cell infiltration was present in both coronary arteries and myocardium during the acute stage of KD, and the intensity of its expression correlated with the degree of inflammation. Obvious TN-C expression persisted in the thickened intima and media of CALs even after Disease Day 27. However, in spite of the presence of inflammatory cell infiltration, TN-C expression became weaker in the adventitia and surrounding connective tissue. After 8 months or more, TN-C was not expressed in the vasculitis scars of most cases, but expression was observed around newly formed vessels in the thickened intima and around recanalized vessels after thrombotic occlusion. CONCLUSIONS: The findings suggest a correlation between the degree of inflammation and TN-C expression in the cardiovascular lesions of acute-stage Kawasaki disease.

Tenascin-C as a prognostic determinant of colorectal cancer through induction of epithelial-to-mesenchymal transition and proliferation.

Although Tenascin-C (TNC) as an extracellular matrix protein involved in various cancers, the mechanisms by which TNC leads to decreased survival time remain to be clarified in CRC. We assessed the expression of TNC and its relationship with cancer associated fibroblasts (CAFs) markers, epithelial-to-mesenchymal transition (EMT) and cell cycle markers in 100 paraffin-embedded CRC tissue samples using immunohistochemistry. TNC expression was higher in CRC tissue samples than in adjacent non-tumor-tissues (P < .001). In addition, TNC was involved in clinical stage (P = .030), pT stage (P = .049), distant metastasis (P = .004), tumor recurrence (P = .007), and tumor budding (P < .001). TNC play crucial roles in regulating the poor 5-year CRC survival rate by Kaplan-Meier analysis, and was an independent predictor of poor overall survival (P = .007) and disease-free survival (P = .004) in CRC. Moreover, it was postively correlated with CAF (SMA (P < .001) and FSP1 (P = .005)) and cell cycle marker p27 (P = .013) along with EMT (E-cadherin, P = .599; Snail, P < .001; vimentin, P = .012). TNC may promote EMT-like change and proliferation, which lead to poor prognosis for patients with CRC.

Targeted matrisome analysis identifies thrombospondin-2 and tenascin-C in aligned collagen stroma from invasive breast carcinoma.

Increasing evidence demonstrates an important role for the extracellular matrix (ECM) in breast cancer progression. Collagen type I, a core constituent of the fibrous ECM, undergoes a significant set of changes that accompany tumor progression, termed Tumor Associated Collagen Signatures (TACS). Late stages of this progression are characterized by the presence of bundled, straight collagen (TACS-2) that become oriented perpendicular to the tumor-stromal boundary (TACS-3). Importantly, the presence of TACS-3 collagen is an independent predictor of poor patient outcome. At present, it remains unclear whether reorganization of the collagen matrix is the consequence of mechanical or compositional tissue remodeling. Here, we identify compositional changes in ECM correlating to collagen fiber reorganization from nineteen normal and invasive ductal carcinoma (IDC) patient biopsies using matrisome-targeted proteomics. Twenty-seven ECM proteins were significantly altered in IDC samples compared to normal tissue. Further, a set of nineteen matrisome proteins positively correlate and five proteins inversely correlate with IDC tissues containing straightened collagen fibers. Tenascin-C and thrombospondin-2 significantly co-localized with aligned collagen fibers in IDC tissues. This study highlights the compositional change in matrisome proteins accompanying collagen re-organization during breast cancer progression and provides candidate proteins for investigation into cellular and structural influences on collagen alignment.

A biosensor based on a 3D-DNA walking machine network and distance-controlled electrochemiluminescence energy transfer for ultrasensitive detection of tenascin C and lead ions.

An electrochemiluminescence biosensor was proposed based on distance-controlled energy transfer and a 3D-DNA walking machine network to significantly improve mass transport and offer an ultrasensitive and controllable strategy for the detection of different types of target.

Internal Affairs: Tenascin-C as a Clinically Relevant, Endogenous Driver of Innate Immunity.

To protect against danger, the innate immune system must promptly and accurately sense alarm signals, and mount an appropriate response to restore homeostasis. One endogenous trigger of immunity is tenascin-C, a large hexameric protein of the extracellular matrix. Upregulated upon tissue injury and cellular stress, tenascin-C is expressed during inflammation and tissue remodeling, where it influences cellular behavior by interacting with a multitude of molecular targets, including other matrix components, cell surface proteins, and growth factors. Here, we discuss how these interactions confer upon tenascin-C distinct immunomodulatory capabilities that make this matrix molecule necessary for efficient tissue repair. We also highlight in vivo studies that provide insight into the consequences of misregulated tenascin-C expression on inflammation and fibrosis during a wide range of inflammatory diseases. Finally, we examine how its unique expression pattern and inflammatory actions make tenascin-C a viable target for clinical exploitation in both diagnostic and therapeutic arenas.

How to detect and purify tenascin-C.

The extracellular matrix molecule tenascin-C (TNC) was discovered over 30 years ago, and its tightly regulated pattern of expression since sparked keen interest in the scientific community. In adult tissues, TNC expression is restricted to specific niches and areas of active remodeling or high mechanical strain. However, while most healthy tissues contain little TNC, its transient expression upon cellular stress or tissue injury helps to mediate repair and restore homeostasis. Persistent expression of TNC is associated with chronic inflammation, fibrosis, and cancer, where methods for its detection are emerging as a reliable means to predict disease onset, prognosis, and response to treatment. Because studying the expression of this large matrix molecule is not always straightforward, here we describe basic techniques to examine tissue levels of TNC mRNA and protein. We also describe methods for purifying recombinant TNC, knocking down its expression, and creating cell-derived matrices with or without TNC within.

Investigating cell-type specific functions of tenascin-C.

The extracellular matrix molecule tenascin-C (TNC) has received a lot of attention since its discovery 30 years ago because of its multiple roles in tissue repair, and in pathologies such as chronic inflammation, fibrosis, and cancer. Mouse models with high or no TNC expression have enabled the validation of key roles for TNC in immunity and angiogenesis. In parallel, many approaches including primary cell or organ cultures have shed light on the cellular and molecular mechanisms by which TNC exerts its multiple actions in vivo. Here, we will describe assays that investigate its antiadhesive properties and that measure the effect of TNC on the actin cytoskeleton, cell survival, proliferation, and migration. We will also describe assays to assess the impact of TNC on endothelial and immune cells in cell and organ culture, and to compare the responses of fibroblasts from normal and diseased tissues.

Tenascin C is a prognostic determinant and potential cancer-associated fibroblasts marker for breast ductal carcinoma.

Tenascin C (TNC) is a key of extracellular matrix glycoprotein and highly express in numerous human malignancies. Herein, we attempted to clarify the clinicopathological significance of TNC as a prognostic determinant of breast ductal carcinoma. Then, we investigated TNC immunohistochemical expression in 150 breast ductal carcinomas and 27 normal breast tissue samples. Clinical relevance of TNC expression and the association TNC expression with other factors related to cancer-associated fibroblasts were also examined. In results, TNC expression was significantly higher in breast ductal carcinoma (56.0%) than normal breast tissues (25.9%). The upregulation TNC in cancer stromal were associated with pT stage (P=0.003), lymph node metastasis (P=0.002) and tumor node metastasis stage (P=0.001), also was correlated with an increase in tumor-associated macrophage population (P<0.001). The microvessel density (MVD) was significantly higher in TNC positive group than in negative group (P<0.001). In both univariate and multivariate Cox regression analyses, TNC was an independent poor prognostic factor for overall survival (OS) in breast ductal carcinoma patients. Importantly, over-expression TNC (P<0.001), FSP1 (P<0.001), SMA (P=0.002) and Vimentin (P=0.049) were significantly correlation with the lower OS (P<0.005). In addition, TNC expression in breast ductal carcinoma stromal was positively correlated with FSP1 (P<0.001), SMA (P=0.001) and Vimentin (P<0.001). In conclusion, the high expression of TNC could be a useful cancer-associated fibroblasts marker for the prediction of prognosis of breast ductal carcinoma patients.

Overexpression of teneurin transmembrane protein 1 is a potential marker of disease progression in papillary thyroid carcinoma.

Although papillary thyroid cancer is a relatively indolent malignancy, its progression may be associated with dedifferentiation and resistance to radioactive iodine treatment. In this study, patterns of differentially expressed genes in association with disease progression were systemically evaluated. We firstly performed transcriptome analyses for four matched cancerous and noncancerous tissue pairs of the classical subtype of papillary thyroid cancer. Among the upregulated and downregulated genes, the expression of 164 and 183 genes increased and decreased, respectively, from stage I to stage IV. Functional enrichment and pathway analysis showed that angiogenesis pathway was upregulated, whereas oxidation-reduction and metabolism of reactive oxygen species were downregulated. Teneurin transmembrane protein 1 (TENM1) expression was highly upregulated in cancerous tissues and negative in benign thyroid tissues. By immunohistochemistry, TENM1 expression in papillary thyroid cancer was associated with the classical subtype (p = 0.018), extrathyroidal invasion (p = 0.001), BRAF V600E mutation (p < 0.001), and an advanced stage (p = 0.019). Taken together, our results indicate that distinct pathways are involved in papillary thyroid cancer progression, and TENM1 is a potential marker of cancer progression.

Tenascin-C Is a Major Component of the Fibrogenic Niche in Kidney Fibrosis.

Kidney fibrosis initiates at certain focal sites in which the fibrogenic niche provides a specialized microenvironment that facilitates fibroblast activation and proliferation. However, the molecular identity of these fibrogenic niches is poorly characterized. Here, we determined whether tenascin-C (TNC), an extracellular matrix glycoprotein, is a component of the fibrogenic niche in kidney fibrosis. In vivo, TNC expression increased rapidly in kidneys subjected to unilateral ureteral obstruction or ischemia/reperfusion injury and predominantly localized at the foci rich in fibroblasts in renal interstitium. In vitro, TNC selectively promoted renal interstitial fibroblast proliferation, bromodeoxyuridine incorporation, and the expression of proliferation-related genes. The mitogenic activity of TNC required the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling cascade. Using decellularized extracellular matrix scaffolds, we found that TNC-enriched scaffolds facilitated fibroblast proliferation, whereas TNC-deprived scaffolds inhibited proliferation. Matrix scaffold prepared from fibrotic kidney also promoted greater ex vivo fibroblast proliferation than did scaffolds prepared from healthy kidney. Conversely, small interfering RNA-mediated knockdown of TNC in vivo repressed injury-induced fibroblast expansion and renal fibrosis. These studies identify TNC as a major constituent of the fibrogenic niche that promotes fibroblast proliferation, and illustrate a pivotal role for the TNC-enriched microenvironment in kidney fibrogenesis.

High expression of intratumoral stromal proteins is associated with chemotherapy resistance in breast cancer.

We studied the changes of intratumoral stromal proteins including THBS1, TNC, FN, SPARC and α-SMA, following neoadjuvant chemotherapy. The underlying mechanisms by which THBS1 and TNC regulated resistance to docetaxel were further studied using functional studies. 100 patients with newly diagnosed breast cancer were treated with alternating sequential doxorubicin and docetaxel. Immunohistochemistry (IHC) staining for stromal proteins was performed on pre- and post-treatment core biopsies respectively. THBS1 and TNC were further validated with IHC in an independent cohort of 31 patients. A high baseline combined expression score of the 5 stromal proteins predicted independently for poor progression-free (HRadjusted 2.22, 95% CI 1.06-4.64) and overall survival (HRadjusted 5.94, 95% CI 2.25-15.71). After 1-2 cycles of chemotherapy, increased expression of THBS1, TNC, FN, SPARC and α-SMA was seen in patients with subsequent pathological lymph node involvement at surgery. Increased expression of THBS1 and TNC compared to baseline was also seen in intrinsically resistant tumors, but not in sensitive ones. Both THBS1 and TNC-associated chemoresistance were confirmed in an independent validation cohort. Exogenous THBS1 and TNC protected MCF-7 cells against proliferation inhibition induced by docetaxel through activating integrin ß1/mTOR pathway. Thus, up-regulation of THBS1, TNC, FN, SPARC and α-SMA following neoadjuvant chemotherapy was associated with chemotherapy resistance in breast cancer patients. Functional studies showed THBS1 and TNC to mediate chemoresistance through the integrin ß1/mTOR pathway, suggesting that therapies targeting integrin ß1/mTOR pathway may be a promising strategy to overcome chemotherapy resistance.

Tenascin-C Is Associated with Cored Amyloid-ß Plaques in Alzheimer Disease and Pathology Burdened Cognitively Normal Elderly.

Tenascin-C (TN-C) is an extracellular matrix glycoprotein linked to inflammatory processes in pathological conditions including Alzheimer disease (AD). We examined the distribution of TN-C immunoreactivity (ir) in relation to amyloid-ß (Aß) plaques and vascular Aß deposits in autopsy brain tissues from 14 patients with clinical and neuropathological AD and 10 aged-matched controls with no cognitive impairment; 5 of the controls had Aß plaques and 5 did not. TN-C ir was abundant in cortical white matter and subpial cerebral gray matter in all cases, whereas TN-C ir was weak in blood vessels. In all cases with Aß plaques but not in plaque-free controls, TN-C ir was detected as large (>100 µm in diameter) diffuse extracellular deposits in cortical grey matter. TN-C plaques completely overlapped and surrounded cored Aß plaques labeled with X-34, a fluorescent derivative of Congo red, and they were associated with reactive astrocytes astrocytes, microglia and phosphorylated tau-containing dystrophic neurites. Diffuse Aß plaques lacking amyloid cores, reactive glia or dystrophic neurites showed no TN-C ir. In cases with cerebral amyloid angiopathy, TN-C ir in vessel walls did not spread into the surrounding neuropil. These results suggest a role for TN-C in Aß plaque pathogenesis and its potential as a biomarker and therapy target.

Tenascin-C, a Prognostic Determinant of Esophageal Squamous Cell Carcinoma.

BACKGROUND: Tenascin-C, an adhesion modulatory extracellular matrix molecule, is highly expressed in numerous human malignancies; thus, it may contribute to carcinogenesis and tumor progression. We explored the clinicopathological significance of Tenascin-C as a prognostic determinant of esophageal squamous cell carcinoma (ESCC). METHODS: In ESCC patient tissues and cell lines, the presence of isoforms were examined using western blotting. We then investigated Tenascin-C immunohistochemical expression in 136 ESCC tissue samples. The clinical relevance of Tenascin-C expression and the correlation between Tenascin-C expression and expression of other factors related to cancer-associated fibroblasts (CAFs) were also determined. RESULTS: Both 250 and 350 kDa sized isoforms of Tenascin-C were expressed only in esophageal cancer tissue not in normal tissue. Furthermore, both isoforms were also identified in all of four CAFs derived from esophageal cancer tissues. Tenascin-C expression was remarkably higher in ESCC than in adjacent non-tumor esophageal epithelium (p < 0.001). Tenascin-C expression in ESCC stromal fibroblasts was associated with patient's age, tumor (pT) stage, lymph node metastasis, clinical stage, and cancer recurrence. Tenascin-C expression in cancer cells was correlated with an increase in tumor-associated macrophage (TAM) population, cancer recurrence, and hypoxia inducible factor1α (HIF1α) expression. Moreover, Tenascin-C overexpression in cancer cells and stromal fibroblasts was an independent poor prognostic factor for overall survival (OS) and disease-free survival (DFS). In the Cox proportional hazard regression model, Tenascin-C overexpression in cancer cells and stromal fibroblasts was a significant independent hazard factor for OS and DFS in ESCC patients in both univariate and multivariate analyses. Furthermore, Tenascin-C expression in stromal fibroblasts of the ESCC patients was positively correlated with platelet-derived growth factor α (PDGFRα), PDGFRß, and smooth muscle actin (SMA) expression. The 5-year OS and DFS rates were remarkably lower in patients with positive expressions of both Tenascin-C and PDGFRα (p < 0.001), Tenascin-C and PDGFRß (p < 0.001), Tenascin-C and SMA (p < 0.001), Tenascin-C and fibroblast activation protein (FAP) (p < 0.001), and Tenascin-C and fibroblast-stimulating protein-1 (FSP1) (p < 0.001) in ESCC stromal fibroblasts than in patients with negative expressions of both Tenascin-C and one of the abovementioned CAF markers. CONCLUSION: Our results show that Tenascin-C is a reliable and significant prognostic factor in ESCC. Tenascin-C may thus be a potent ESCC therapeutic target.

Expression analysis of α-smooth muscle actin and tenascin-C in the periodontal ligament under orthodontic loading or in vitro culture.

α-smooth muscle actin (α-SMA) and tenascin-C are stress-induced phenotypic features of myofibroblasts. The expression levels of these two proteins closely correlate with the extracellular mechanical microenvironment. We investigated how the expression of α-SMA and tenascin-C was altered in the periodontal ligament (PDL) under orthodontic loading to indirectly reveal the intrinsic mechanical microenvironment in the PDL. In this study, we demonstrated the synergistic effects of transforming growth factor-ß1 (TGF-ß1) and mechanical tensile or compressive stress on myofibroblast differentiation from human periodontal ligament cells (hPDLCs). The hPDLCs under higher tensile or compressive stress significantly increased their levels of α-SMA and tenascin-C compared with those under lower tensile or compressive stress. A similar trend was observed in the tension and compression areas of the PDL under continuous light or heavy orthodontic load in rats. During the time-course analysis of expression, we observed that an increase in α-SMA levels was matched by an increase in tenascin-C levels in the PDL under orthodontic load in vivo. The time-dependent variation of α-SMA and tenascin-C expression in the PDL may indicate the time-dependent variation of intrinsic stress under constant extrinsic loading.