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Christina Jensen,1,2 Signe Holm Nielsen,1,3 Mohammed Eslam,4 Federica Genovese,1 Mette Juul Nielsen,1 Roslyn Vongsuvanh,4 Raj Uchila,4 David van der Poorten,4 Jacob George,4 Morten Asser Karsdal,1 Diana Julie Leeming,1 Nicholas Willumsen1 1Biomarkers & Research, Nordic Bioscience, Herlev, Denmark; 2Biotech Research & Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark; 3Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark; 4Storr Liver Centre, Westmead Institute for Medical Research, Westmead Hospital and University of Sydney, Sydney, NSW, AustraliaCorrespondence: Christina JensenNordic Bioscience, Herlev Hovedgade 205-207, Herlev 2730, DenmarkTel: +4544525252Fax: +4544525251Email chj@nordicbio.comPurpose: Non-invasive biomarkers for diagnosing and prognosing hepatocellular carcinoma (HCC) are urgently needed. Cirrhosis is present in 80– 90% of HCC patients. Cirrhosis is characterized by deposition and cross-linking of collagens that have crucial roles in HCC initiation and progression. We evaluated circulating cross-linked pro-peptides of type III collagen (PC3X) as a diagnostic and prognostic biomarker for HCC.Patients and Methods: PC3X was measured by ELISA in plasma from patients with HCC (n=79), cirrhosis (n=86), non-cirrhotic hepatitis-B infection (n=74) and from healthy controls (n=44). PC3X was compared to the liver fibrosis marker PRO-C3 and the HCC tumor-cell derived marker alpha-fetoprotein (AFP). Diagnostic and prognostic potential was evaluated by AUROC and by calculating hazard ratios (HR) for progression-free survival (PFS) and overall survival (OS).Results: PC3X, PRO-C3 and AFP were significantly elevated in patients with HCC compared to other liver diseases and healthy controls (p=0.0002, p< 0.0001). In patients with normal AFP (< 20 IU/mL), PC3X and PRO-C3 separated HCC from cirrhosis with an AUROC of 0.72 and 0.68, respectively. High PC3X and AFP predicted for poor PFS (HRPC3X=1.80, p=0.032; HRAFP=1.70, p=0.031) and OS (HRPC3X=2.12, p=0.024; HRAFP=2.55; p=0.003), whereas PRO-C3 did not (PFS: HR=1.19, p=0.059 and OS: HR=1.12, p=0.324). PC3X was independent of AFP (PFS: HR=1.74, p=0.045 and OS: HR=2.21, p=0.018) and combining the two improved prognostic value (PFS: HR=2.66, p=0.004 and OS: HR=5.86, p< 0.0001).Conclusion: PC3X is associated with HCC independent of AFP and provides diagnostic and prognostic value for HCC patients. If validated, this suggests that PC3X has biomarker potential for HCC.Keywords: tumor microenvironment, extracellular matrix, fibrosis, liver cancer

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BACKGROUND: During cancer the otherwise tightly controlled homeostasis of the extracellular matrix (ECM) is disturbed. The protein composition changes, the ECM stiffens and increased levels of proteases are secreted. The combination of these processes result in release of specific protein fragments (e.g. collagens) to the circulation, which when measured may reflect disease pathogenesis. OBJECTIVE: To investigate if biomarkers of protease-degraded collagen could differentiate ovarian and breast cancer patients from healthy controls when measured in serum. METHODS: The levels of markers reflecting MMP-degradation of type I (C1M), type III (C3M) and type IV (C4M, C4M12) collagen were assessed in serum from ovarian cancer patients (n = 10), breast cancer patients (n = 14) and healthy controls (n = 49) using validated ELISAs. The markers were compared using one way ANOVA and AUC was calculated. RESULTS: Allmarkers were significantly elevated in serum from ovarian cancer patients (p <0.0001) and breast cancer patients (p < 0.04-0.0001) compared to healthy controls. Furthermore, diagnostically the markers were able to differentiate ovarian (AUROC 90%-93%) and breast cancer patients (AUROC 76%-93%) from healthy controls, with C1M being the strongest differentiator of disease vs. controls. CONCLUSION: Four serum biomarkers reflecting altered MMP-mediated collagen turnover were able to differentiate ovarian and breast cancer patients from healthy controls. [ABSTRACT FROM AUTHOR]

A non-invasive method is applied for studying ion transport by single isolated epidermal mitochondria-rich (MR) cells. MR cells of toad skin (Bufo bufo) were prepared by trypsin (or pronase) treatment of the isolated epithelium bathed in Ca2+-free Ringer. Glass pipettes were pulled and heat- polished to obtain a tip of 2-4 mm with parallel walls and low tip resistances. The neck of an MR-cell was sucked into the tip of the pipette for being 'clamped mechanically' by the heat-polished glass wall. In this configuration the apical cell membrane faces the pipette solution while the major neck region and the cell body are in the electrically grounded bath. With Ringer in bath and pipette, transcellular voltage clamp currents were composed of an ohmic (Ileak) and a dynamic (Idynamic) component. The dynamic component was studied by stepping the transcellular potential (Vp) from a holding value of +50 mV to the hyperpolarizing region (50 > Vp ? ?100 mV). The steady state Idynamic?Vp relationship was strongly outward rectified with Idynamic being practically zero for Vp > 0 mV. At Vp = ?100 mV, MR cells isolated by trypsin or pronase generated a steady-state Idynamic of, ?2.72 ± 0.40 nA/cell (N = 21 MR cells). Continuous superfusion of the MR cell during recording increased the current to ?7.99 ± 1.48 nA/cell (N = 10 MR cells). The time course of the reversible activation of Gdynamic varied among cells, but was usually sigmoid with T1/2 decreasing with Vp (?25 ? Vp ? ?100 mV). T1/2 was in the order of 10 sec at Vp = ?100 mV. The single-MR-cell currents recorded in this study are fully compatible with Cl? currents estimated by relating density of MR cells to transepithelial ICl or by measurements with the self-referencing ('vibrating') probe technique. In the discussion, Ussing's work on epithelial shunt pathways is considered. His thinking and experiments leading to his theory of isotonic transport in leaky epithelia is emphasized. It is our thesis that the understanding of the physiology of epithelia owes as much to Ussing's studies of shunt pathways as to his studies of the active sodium pathway. [ABSTRACT FROM AUTHOR]

Altered architecture, composition and quality of the extracellular matrix (ECM) are pathological hallmarks of several inflammatory and fibro-proliferative pathological processes such as osteoarthritis (OA), rheumatoid arthritis (RA), fibrosis and cancer. One of the most important components of the ECM is fibronectin. Fibronectin serves as an adhesion molecule anchoring cells to the underlying basement membrane through direct interaction with integrin receptors. Fibronectin hereby modulates the properties of the ECM and affects cellular processes. Quantification of fibronectin remodeling could therefore be used to assess the changes in the ECM that occur during progression of fibro-proliferative pathologies. Ex vivo models are becoming state-of-the-art tools to study ECM remodeling as the cellular composition and the organization of the ECM are preserved. Ex vivo models may therefore be a valuable tool to study the ECM remodeling that occurs during progression of fibro-proliferative pathologies. The aim of this study was to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. A competitive The enzyme-linked immunosorbent assay (ELISA) against the C-terminus of fibronectin was developed (FBN-C). The assay was evaluated in relation to specificity, technical performance and as a marker for quantification of fibronectin in cartilage and cancer ex vivo models. The ELISA was specific and technically stable. Cleavage of tumor tissue with MMP-2 released significantly higher levels of FBN-C compared to tissue with buffer only and western blot analysis revealed that FBN-C recognizes both full length and degraded fibronectin. When ex vivo cartilage cultures were stimulated with the anabolic factor TGFβ and catabolic factors TNF-α and OSM, significantly higher levels of FBN-C were found in the conditioned media. Lastly, FBN-C was released from a cancer ex vivo model. In conclusion, we were able to quantify fibronectin remodeling in ex vivo models of cartilage and cancer. Quantification of fibronectin remodeling could be a valuable tool to understand ECM remodeling in ex vivo models of fibro-proliferative pathologies. [ABSTRACT FROM AUTHOR]

Purpose: There is an unmet need for new treatment options and biomarkers for patients with glioblastoma (GBM). Here we investigated three non-invasive biomarkers: type VI collagen degraded by granzyme B (C4G) and matrix metalloproteases (C4M), respectively, and ADAM10-degraded Tau (Tau-A)., Methods: Biomarker levels in pre- and on-treatment serum samples from patients with recurrent GBM (n = 39) treated with nivolumab and bevacizumab (NCT03890952) were compared to healthy levels (n = 22) and associated with overall survival (OS) outcome (median cutpoint). Longitudinal changes in biomarkers were investigated by a Mixed-effects analysis., Results: Tau-A (p < 0.0001) and C4G (p = 0.005), but not C4M (p = 0.106), were increased in patients. High Tau-A and C4G associated with improved OS (Tau-A: HR = 0.41, 95%CI = 0.20-0.86, C4G: HR = 0.47, 95%CI = 0.24-0.94). Only C4G increased with treatment (p = 0.024-0.005)., Conclusions: Tau-A and C4G are elevated in serum from patients with recurrent GBM and prognostic for OS. If validated, these biomarkers could be applied to clinical trials., (© 2024. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Introduction: Genetic studies have shown that variants in the microtubule-associated protein tau ( MAPT ) gene, which encodes tau protein, can increase the risk for Alzheimer's disease (AD). Additionally, two haplotypes of the MAPT gene (H1 and H2) are associated with various neurodegenerative disorders, including AD. This study aimed to test the association of MAPT haplotypes (H1 and H2) and MAPT haplotype-tagging polymorphisms (rs1467967, rs242557, rs3785883, rs2471738, del-In9, rs7521) with AD., Methods: The study included 964 individuals: 113 with AD, 53 with mild cognitive impairment (MCI), 54 with other dementias, and 744 healthy controls., Results: The results showed that individuals carrying the A allele in the MAPT rs1467967 polymorphism, the GG genotype in the MAPT rs7521 polymorphism, and the G allele in the MAPT rs242557 polymorphism had worse performance on various neuropsychological tests. Carriers of the C allele in MAPT rs2471738 polymorphism and CC homozygotes also showed worse performance on neuropsychological tests and pathological levels of several cerebrospinal fluid (CSF) biomarkers. However, T allele carriers in the MAPT rs2471738 polymorphism were more represented among patients with dementia and apolipoprotein E ( APOE ) ɛ4 carriers. Carriers of the H2 MAPT haplotype had worse performance on various neuropsychological tests, consistent with our previous study, which associated the H2 MAPT haplotype with pathological levels of CSF AD biomarkers. Regarding the MAPT rs3785883 polymorphism, further research is needed since both the AA and GG genotypes were associated with pathological levels of CSF and plasma AD biomarkers., Discussion: In conclusion, further genetic studies are needed to elucidate the role of MAPT haplotypes and MAPT haplotype-tagging polymorphisms in the development of AD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Babić Leko, Španić Popovački, Willumsen, Nikolac Perković, Pleić, Zubčić, Langer Horvat, Vogrinc, Boban, Borovečki, Zemunik, de Silva and Šimić.)

Understanding the tumor microenvironment (TME) and extracellular matrix (ECM) is crucial in cancer research due to their impact on tumor progression. Collagens, major ECM components, regulate cell signaling and behavior. Of the 28 reported collagens, type XII collagen is known to be vital for ECM organization. Over-produced by cancer-associated fibroblasts (CAFs), its upregulation correlates with poor survival in various cancers. This study aimed to develop an ELISA for quantifying circulating type XII collagen as a cancer biomarker. A specific ELISA targeting the C-terminal of type XII collagen was developed and used to analyze serum samples from cancer patients (n = 203) and healthy controls (n = 33). Additionally, type XII collagen expression was assessed in CAFs and normal fibroblasts (NFs) from different tissues, both under TGF-β stimulated and non-stimulated conditions. The nordicPRO-C12 ELISA demonstrated robustness and specificity for type XII collagen. PRO-C12 levels were significantly elevated in patients with various cancers compared to healthy controls and effectively distinguished between cancer patients and controls. Findings were validated using gene expression data. Furthermore, Western blot analysis revealed increased type XII collagen expression in both CAFs and NFs upon TGF-β1 stimulation, suggesting a potential role of TGF-β1 in modulating the expression of type XII collagen in cancerous and normal tissue microenvironments. This study unveils a promising avenue for harnessing PRO-C12 as a non-invasive serum biomarker, enabling the quantification of type XII collagen fragments in cancer patients. Further investigations are warranted to explore the potential of PRO-C12 across different cancer types and disease stages, shedding light on its multifaceted role in cancer development., (© 2024. The Author(s).)

Lipids play crucial roles in the susceptibility and brain cellular responses to Alzheimer's disease (AD) and are increasingly considered potential soluble biomarkers in cerebrospinal fluid (CSF) and plasma. To delineate the pathological correlations of distinct lipid species, we conducted a comprehensive characterization of both spatially localized and global differences in brain lipid composition in App NL-G-F mice with spatial and bulk mass spectrometry lipidomic profiling, using human amyloid-expressing (h-Aβ) and WT mouse brains controls. We observed age-dependent increases in lysophospholipids, bis(monoacylglycerol) phosphates, and phosphatidylglycerols around Aβ plaques in App NL-G-F mice. Immunohistology-based co-localization identified associations between focal pro-inflammatory lipids, glial activation, and autophagic flux disruption. Likewise, in human donors with varying Braak stages, similar studies of cortical sections revealed co-expression of lysophospholipids and ceramides around Aβ plaques in AD (Braak stage V/VI) but not in earlier Braak stage controls. Our findings in mice provide evidence of temporally and spatially heterogeneous differences in lipid composition as local and global Aβ-related pathologies evolve. Observing similar lipidomic changes associated with pathological Aβ plaques in human AD tissue provides a foundation for understanding differences in CSF lipids with reported clinical stage or disease severity., (© 2024 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

The fibrotic tumor microenvironment, characterized by its intricate extracellular matrix (ECM), consists of many collagens with diverse functions and unexplored biomarker potential. Type IX collagen is a member of the low-abundance collagen family known as the fibril-associated collagen with interrupted triple helices (FACITs) and is found mostly in cartilage. Its role in the tumor microenvironment remains unexplored. To investigate the biomarker potential of a type IX collagen in cancer, an immuno-assay was developed (PRO-C9) and technical assay performance was evaluated for the assessment of serum. PRO-C9 levels were measured in serum samples from 259 patients with various solid tumor types compared to serum levels from 73 healthy controls. PRO-C9 levels were significantly elevated in patients with solid tumors including bladder, breast, colorectal, gastric, head and neck, lung, melanoma, ovarian, pancreatic, and renal compared to levels in healthy controls ( p < 0.05- p < 0.0001). PRO-C9 could discriminate between patients with cancer and healthy controls, with the area under the receiver operating characteristic values ranging from 0.58 to 0.86 ( p < 0.3- p < 0.0001), indicating potential diagnostic utility. This study suggests that type IX collagen turnover is altered in patients with solid tumors and demonstrates the feasibility of using PRO-C9 as a non-invasive serum-based biomarker with relevance in multiple cancer types. Furthermore, these results underscore the potential utility of PRO-C9 to better elucidate the biology of FACITs in cancers.

Aging is associated with cell senescence and is the major risk factor for AD. We characterized premature cell senescence in postmortem brains from non-diseased controls (NDC) and donors with Alzheimer's disease (AD) using imaging mass cytometry (IMC) and single nuclear RNA (snRNA) sequencing (> 200,000 nuclei). We found increases in numbers of glia immunostaining for galactosidase beta (> fourfold) and p16 INK4A (up to twofold) with AD relative to NDC. Increased glial expression of genes related to senescence was associated with greater β-amyloid load. Prematurely senescent microglia downregulated phagocytic pathways suggesting reduced capacity for β-amyloid clearance. Gene set enrichment and pseudo-time trajectories described extensive DNA double-strand breaks (DSBs), mitochondrial dysfunction and ER stress associated with increased β-amyloid leading to premature senescence in microglia. We replicated these observations with independent AD snRNA-seq datasets. Our results describe a burden of senescent glia with AD that is sufficiently high to contribute to disease progression. These findings support the hypothesis that microglia are a primary target for senolytic treatments in AD., (© 2024. The Author(s).)

Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14., Competing Interests: Competing interests J.T.-U. and N.W. are employees of Nordic Bioscience A/S, and S.F.P. is cofounder of SOLID Therapeutics. All other authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Brain perfusion and blood-brain barrier (BBB) integrity are reduced early in Alzheimer's disease (AD). We performed single nucleus RNA sequencing of vascular cells isolated from AD and non-diseased control brains to characterise pathological transcriptional signatures responsible for this. We show that endothelial cells (EC) are enriched for expression of genes associated with susceptibility to AD. Increased β-amyloid is associated with BBB impairment and a dysfunctional angiogenic response related to a failure of increased pro-angiogenic HIF1A to increased VEGFA signalling to EC. This is associated with vascular inflammatory activation, EC senescence and apoptosis. Our genomic dissection of vascular cell risk gene enrichment provides evidence for a role of EC pathology in AD and suggests that reducing vascular inflammatory activation and restoring effective angiogenesis could reduce vascular dysfunction contributing to the genesis or progression of early AD., (© 2024. The Author(s).)

Fibroblast activation protein (FAP) is a known promoter of tumor development and is associated with poor clinical outcome for various cancer types. Being specifically expressed in pathological conditions including multiple types of fibrosis and cancers, FAP is an optimal target for diagnostics and treatment. Treatment strategies utilizing the unique proteolytic activity of FAP are emerging, thus emphasizing the importance of biomarkers to directly assess FAP activity. FAP is a type II transmembrane serine protease that has been shown to cleave collagens and other ECM components. In this study, we developed an ELISA assay (C3F) targeting a circulating type III collagen fragment derived from FAP cleavage to reflect FAP activity. We demonstrated that C3F was specific to the neoepitope of the cleavage site and that the fragment was generated through FAP cleavage of type III collagen. We measured C3F in serum from a cohort of patients with non-small cell lung cancer (NSCLC) ( n = 109) matched to healthy subjects ( n = 42) and a cohort of patients with spondyloarthritis (SpA) ( n = 17) matched to healthy subjects ( n = 19). We found that C3F was significantly elevated in patients with NSCLC and in patients with SpA compared to healthy controls ( p < 0.0001 and p = 0.0015, respectively). These findings suggest that C3F is a promising non-invasive biomarker reflecting FAP activity, which may aid in understanding tumor heterogeneity and potentially FAP-targeted therapies.

Cancer-associated fibroblasts (CAFs) deposit and remodel collagens in the tumor stroma, impacting cancer progression and efficacy of interventions. CAFs are the focus of new therapeutics with the aim of normalizing the tumor microenvironment. To do this, a better understanding of CAF heterogeneity and collagen composition in cancer is needed. In this study, we sought to profile the expression of collagens at multiple levels with the goal of identifying cancer biomarkers. We investigated the collagen expression pattern in various cell types and CAF subtypes in a publicly available single-cell RNA sequencing (RNA-seq) dataset of pancreatic ductal adenocarcinoma. Next, we investigated the collagen expression profile in tumor samples across cancer types from The Cancer Genome Atlas (TCGA) database and evaluated if specific patterns of collagen expression were associated with prognosis. Finally, we profiled circulating collagen peptides using a panel of immunoassays to measure collagen fragments in the serum of cancer patients. We found that pancreatic stellate cells and fibroblasts were the primary producers of collagens in the pancreas. COL1A1, COL3A1, COL5A1, COL6A1 were expressed in all CAF subtypes, whereas COL8A1, COL10A1, COL11A1, COL12A1 were specific to myofibroblast CAFs (myCAF) and COL14A1 specific to inflammatory CAFs (iCAF). In TCGA database, myCAF collagens COL10A1 and COL11A1 were elevated across solid tumor types, and multiple associations between high expression and worse survival were found. Finally, circulating collagen biomarkers were elevated in the serum of patients with cancer relative to healthy controls with COL11A1 (myCAF) having the best diagnostic accuracy of the markers measured. In conclusion, CAFs express a noncanonical collagen profile with specific collagen subtypes associated with iCAFs and myCAFs in PDAC. These collagens are deregulated at the cellular, tumor, and systemic levels across different solid tumors and associate with survival. These findings could lead to new discoveries such as novel biomarkers and therapeutic targets. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Collagens are the major components of the extracellular matrix (ECM) and are known to contribute to tumor progression and metastasis. There are 28 different types of collagens each with unique functions in maintaining tissue structure and function. Type XVII collagen (BP180) is a type II transmembrane protein that provides stable adhesion between epithelial cells and the underlying basement membrane. Aberrant expression and ectodomain shedding of type XVII collagen have been associated with epithelial damage, tumor invasiveness, and metastasis in multiple tumor types and may consequently be used as a potential (non-invasive) biomarker in cancer and treatment target., Method: An ELISA targeting the type XVII collagen ectodomain (PRO-C17) was developed for use in serum. PRO-C17 was measured in a cohort of patients with 11 different cancer types (n = 214) and compared to healthy controls (n = 23) (cohort 1). Based on the findings from cohort 1, PRO-C17 and its association with survival was explored in patients with metastatic colorectal cancer (mCRC) treated with bevacizumab in combination with chemotherapy (n = 212) (cohort 2)., Results: PRO-C17 was robust and specific towards the ectodomain of type XVII collagen. In cohort 1, PRO-C17 levels were elevated (p < 0.05) in serum from patients with CRC, kidney, ovarian, bladder, breast, and head and neck cancer compared to healthy controls. PRO-C17 was especially good at discriminating between CRC patients and healthy controls with an AUROC of 0.904. In cohort 2, patients with mCRC and high levels (tertile 3) of PRO-C17 had shorter overall survival (OS) with a median OS of 390 days compared to 539 days for patients with low levels of PRO-C17. When evaluated by multivariate Cox regression analysis, high PRO-C17 was predictive for poor OS independent of risk factors and the tumor fibrosis biomarker PRO-C3., Conclusion: PRO-C17 measures the ectodomain of type XVII collagen in serum and is a promising non-invasive biomarker that can aid in understanding tumor heterogeneity as well as elaborate on the role of collagen XVII in tumor progression. Moreover, the findings in the study proposes PRO-C17 as novel biomarker of epithelial damage in specific cancer types including CRC., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Small cell lung cancer (SCLC) is highly aggressive with limited therapeutic options and a poor prognosis. Moreover, noninvasive biomarker tools for detecting disease and monitoring treatment response are lacking. To address this, we evaluated serum biomarkers of extracellular matrix proteins not previously explored in SCLC., Methods: We measured biomarkers in the serum of 16 patients with SCLC before and after chemotherapy as well as in the serum of 11 healthy individuals., Results: Our findings demonstrated that SCLC serum had higher levels of collagen type I degradation, collagen type III formation, and collagen type XI formation than healthy controls. In addition, we observed higher levels of type XIX and XXII collagens, fibrinogen, and von Willebrand factor A formation in SCLC serum. The formation of type I collagen did not exhibit any discernible variation. However, we observed a decrease in the degradation of type I collagen following chemotherapy., Conclusion: Overall, our findings revealed elevated levels of collagen and blood-clotting markers in the serum of SCLC patients, indicating the potential of ECM proteins as noninvasive biomarkers for SCLC., (© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) arise due to acquired somatic driver mutations in stem cells and develop over 10-30 years from the earliest cancer stages (essential thrombocythemia, polycythemia vera) towards the advanced myelofibrosis stage with bone marrow failure. The JAK2V617F mutation is the most prevalent driver mutation. Chronic inflammation is considered to be a major pathogenetic player, both as a trigger of MPN development and as a driver of disease progression. Chronic inflammation in MPNs is characterized by persistent connective tissue remodeling, which leads to organ dysfunction and ultimately, organ failure, due to excessive accumulation of extracellular matrix (ECM). Considering that MPNs are acquired clonal stem cell diseases developing in an inflammatory microenvironment in which the hematopoietic cell populations are progressively replaced by stromal proliferation-"a wound that never heals"-we herein aim to provide a comprehensive review of previous promising research in the field of circulating ECM fragments in the diagnosis, treatment and monitoring of MPNs. We address the rationales and highlight new perspectives for the use of circulating ECM protein fragments as biologically plausible, noninvasive disease markers in the management of MPNs.

It has been known for decades that the tumor extracellular matrix (ECM) is dysfunctional leading to loss of tissue architecture and promotion of tumor growth. The altered ECM and tumor fibrogenesis leads to tissue stiffness that act as a physical barrier to immune cell infiltration into the tumor microenvironment (TME). It is becoming increasingly clear that the ECM plays important roles in tumor immune responses. A growing body of data now indicates that ECM components also play a more active role in immune regulation when dysregulated ECM components act as ligands to interact with receptors on immune cells to inhibit immune cell subpopulations in the TME. In addition, immunotherapies such as checkpoint inhibitors that are approved to treat cancer are often hindered by ECM changes. In this review we highlight the ways by which ECM alterations affect and regulate immunity in cancer. More specifically, how collagens and major ECM components, suppress immunity in the complex TME. Finally, we will review how our increased understanding of immune and immunotherapy regulation by the ECM is leading towards novel disruptive strategies to overcome immune suppression., Competing Interests: Authors DF and SL are employed by the company NextCure Inc. Authors MK, CJ and NW are employed by the company Nordic Bioscience., (Copyright © 2023 Flies, Langermann, Jensen, Karsdal and Willumsen.)

Autoantibodies have the potential as cancer biomarkers as they may associate with the outcome and immune-related adverse events (irAEs) following immunotherapy. Cancer and other fibroinflammatory diseases, such as rheumatoid arthritis (RA), are associated with excessive collagen turnover leading to collagen triple helix unfolding and denaturation with exposure of immunodominant epitopes. In this study, we aimed to investigate the role of autoreactivity against denatured collagen in cancer. A technically robust assay to quantify autoantibodies against denatured type III collagen products (anti-dCol3) was developed and then measured in pretreatment serum from 223 cancer patients and 33 age-matched controls. Moreover, the association between anti-dCol3 levels and type III collagen degradation (C3M) and formation (PRO-C3) was investigated. Anti-dCol3 levels were significantly lower in patients with bladder ( p = 0.0007), breast ( p = 0.0002), colorectal ( p < 0.0001), head and neck ( p = 0.0005), kidney ( p = 0.005), liver ( p = 0.030), lung ( p = 0.0004), melanoma ( p < 0.0001), ovarian ( p < 0.0001), pancreatic ( p < 0.0001), prostate ( p < 0.0001), and stomach cancers ( p < 0.0001) compared to controls. High anti-dCol3 levels were associated with type III collagen degradation (C3M, p = 0.0002) but not type III collagen formation (PRO-C3, p = 0.26). Cancer patients with different solid tumor types have downregulated levels of circulating autoantibodies against denatured type III collagen compared to controls, suggesting that autoreactivity against unhealthy type III collagen may be important for tumor control and eradication. This autoimmunity biomarker may have the potential for studying the close relationship between autoimmunity and cancer.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a pronounced fibrotic tumor microenvironment, which impairs treatment response. Type I and V collagens are responsible for the densely packed fibrils in the tumor fibrosis environment. While the role of the major type I collagen in cancer is well described, less is known about the minor type V collagen. Quantifying collagen propeptides in serum has been shown to have prognostic and predictive value. In this study, we evaluated the clinical utility of measuring the propeptide of type V collagen (PRO-C5) in serum from a discovery cohort and a validation cohort of patients with PDAC as well as in non-pancreatic solid tumor types to explore the relevance of the PRO-C5 biomarker in cancer. Methods: Serum PRO-C5 was measured in three cohorts: a discovery cohort (19 healthy controls, 12 patients with chronic pancreatitis and 33 patients with PDAC (stage I-IV)), a validation cohort (800 patients with PDAC (stage I-IV)), and a non-pancreatic solid tumor type cohort of 33 healthy controls and 200 patients with 10 different non-pancreatic solid tumor types. The levels of serum PRO-C5 in patients with cancer were compared to levels in healthy controls. The association between PRO-C5 levels and overall survival (OS) was evaluated in patients with PDAC after adjusting for established prognostic factors. Results: PRO-C5 was significantly increased in serum from patients with PDAC compared to healthy controls ( p < 0.001). High PRO-C5 levels were significantly associated with short OS in both the discovery- and the validation cohort, especially in early stages of PDAC (validation cohort stage II, HR = 2.0, 95%CI1.2-3.4). The association was independent of other prognostic parameters including stage, performance status and CA19-9. Furthermore, serum levels of PRO-C5 were significantly increased in serum from patients with other non-pancreatic solid tumor types compared to healthy controls. Conclusion: High levels of serum PRO-C5 is prognostic for short OS in patients with PDAC and may provide clinical value in many other tumor types beyond PDAC. This underlines the importance of type V collagen in tumor fibrosis. PRO-C5 could have the potential to be used in several aspects within drug discovery, patient stratification and drug efficacy., Competing Interests: NN, CJ, EM, JT-U, MK, and NW are all employed by Nordic Bioscience A/S. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nissen, Johansen, Chen, Jensen, Madsen, Hansen, Thorlacius-Ussing, Karsdal, Johansen, Diab, Jørgensen and Willumsen.)