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The matricellular protein SPARC (secreted proteome acidic and rich in cysteine) is known to bind collagens and regulate fibrillogenesis. Cleavage of SPARC at a single peptide bond, increases the affinity for collagens up to 20-fold. To investigate if this specific cleavage has pathological relevance in fibrotic disorders, we developed a competitive ELISA targeting the generated neo-epitope on the released fragment and quantified it in serum from patients with lung cancer, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and healthy subjects. Furthermore, the ability of SPARC to protect fibrillar collagens from proteolytic degradation was investigated in vitro, potentially adding a new collagen chaperone function to SPARC. The fragment was significantly elevated in lung cancer patients when compared to healthy subjects measured in a discovery cohort (p = 0.0005) and a validation cohort (p < 0.0001). No significant difference was observed for IPF and COPD patients compared to healthy subjects. When recombinant SPARC was incubated with type I or type III collagen and matrix metalloproteinase-9, collagen degradation was completely inhibited. Together, these data suggest that cleavage of SPARC at a specific site, which modulates collagen binding, is a physiological mechanism increased during pathogenesis of lung cancer. Furthermore, inhibition of fibrillar collagen degradation by SPARC adds a new chaperone function to SPARC which may play additional roles in the contribution to increased collagen deposition leading to a pro-fibrotic and tumorigenic environment.

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BACKGROUND: Decorin is one of the most abundant proteoglycans of the extracellular matrix and is mainly secreted and deposited in the interstitial matrix by fibroblasts where it plays an important role in collagen turnover and tissue homeostasis. Degradation of decorin might disturb normal tissue homeostasis contributing to extracellular matrix remodeling diseases. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific fragment of degraded decorin, which has potential as a novel non-invasive serum biomarker for fibrotic lung disorders. METHODS: A fragment of decorin cleaved in vitro using human articular cartilage was identified by mass-spectrometry (MS/MS). Monoclonal antibodies were raised against the neo-epitope of the cleaved decorin fragment and a competitive ELISA assay (DCN-CS) was developed. The assay was evaluated by determining the inter- and intra-assay precision, dilution recovery, accuracy, analyte stability and interference. Serum levels were assessed in lung cancer patients, patients with idiopathic pulmonary fibrosis (IPF), patients with chronic obstructive pulmonary disease (COPD) and healthy controls. RESULTS: The DCN-CS ELISA was technically robust and was specific for decorin cleaved by cathepsin-S. DCN-CS was elevated in lung cancer patients (p < 0.0001) and IPF patients (p < 0.001) when compared to healthy controls. The diagnostic power for differentiating lung cancer patients and IPF patients from healthy controls was 0.96 and 0.77, respectively. CONCLUSION: Cathepsin-S degraded decorin could be quantified in serum using the DCN-CS competitive ELISA. The clinical data indicated that degradation of decorin by cathepsin-S is an important part of the pathology of lung cancer and IPF.

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During cancer progression, the homeostasis of the extracellular matrix becomes imbalanced with an excessive collagen remodeling by matrix metalloproteinases. As a consequence, small protein fragments of degraded collagens are released into the circulation. We have investigated the potential of protein fragments of collagen type I, III and IV as novel biomarkers for colorectal cancer. Specific fragments of degraded type I, III and IV collagen (C1M, C3M, C4M) and type III collagen formation (Pro-C3) were assessed in serum from colorectal cancer patients, subjects with adenomas and matched healthy controls using well-characterized and validated ELISAs. Serum levels of the biomarkers were significantly elevated in colorectal cancer patients compared to subjects with adenomas (C1M, Pro-C3, C3M) and controls (C1M, Pro-C3). When patients were stratified according to their tumour stage, all four biomarkers were able to differentiate stage IV metastatic patients from all other stages. Combination of all markers with age and gender in a logistic regression model discriminated between metastatic and non-metastatic patients with an AUROC of 0.80. The data suggest that the levels of these collagen remodeling biomarkers may be a measure of tumour activity and invasiveness and may provide new clinical tools for monitoring of patients with advanced stage colorectal cancer.

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Chronic fibro-proliferative diseases are associated with nearly 45% of all deaths in the developed world. Matrix metalloproteinase (MMP) mediated remodeling of the extracellular matrix (ECM) plays an important role in disease development. Degradation of type I collagen is considered having a major role in this matter. C1M is a biomarker measuring type I collagen degradation fragments in blood. The aim of the current study was to investigate whether MMP mediated type I collagen degradation (C1M) was predictive of mortality in a large prospective cohort of Danish women aged 48-89 (n = 5855). Subjects with high serum C1M showed significant increased mortality. The adjusted three year HR was 2.02 [95% CI: 1.48-2.76] for all-cause mortality, 2.32 [95% CI: 1.51-3.56] for cancer and 1.77 [95% CI: 0.98-3.17] for cardiovascular diseases. The adjusted nine year HR was 1.50 [95% CI: 1.28-1.75] for all-cause mortality, 1.49 [95% CI: 1.16-1.90] for cancer and 1.69 [95% CI: 1.27-2.24] for cardiovascular diseases. High MMP-mediated type I collagen degradation was associated with increased mortality. Subjects with high C1M had a 2-fold increase in mortality compared to subjects with low levels of this collagen degradation product.

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