Fibrillar Collagen Type Research Articles

Abstract A035: 3D High-Density Collagen I Improves the Modeling of Aggressive Pancreatic Cancer

Abstract Background: Fibrillar collagen type I constitutes the majority of the extracellular matrix in pancreatic ductal adenocarcinoma (PDAC), yet most in vivo murine models involve the use of basement membrane extracts, which instead contain collagen type IV, as matrices to embed PDAC cells. Type I collagen in the PDAC tumor microenvironment has been associated with worse prognosis. Our hypothesis is that PDAC cells grown in 3D high-density collagen I (HDC) gels behave more aggressively compared to the same cells grown using conventional basement membrane matrix (BMM). Methods: The KPC1199 2D cell line was previously generated from a tumor-bearing male KPC mouse. KPC1199 cells were cultured in vitro in HDC or BMM for one week. Orthotopic tumors were established by embedding KPC1199 cells in HDC (N=10) or BMM (N=10) in the pancreatic tail of syngeneic C57BL/6J mice via laparotomy. Half of the tumors were collected five weeks after tumor implantation, and the other half utilized for a survival study. Collagen was imaged by second harmonic generation (SHG) microscopy. Results: In vitro imaging of single KPC1199 cells grown in HDC revealed an aggressive phenotype with clonal network-like structures compared to a spheroidal, non-aggressive phenotype for cells grown in BMM over the course of seven days. SHG imaging confirmed consistent formation and organization of collagen in HDC tumors but minimal to faint collagen formation in BMM tumors. Gross examination of HDC mice showed 60% with visible liver metastasis compared with none in the BMM mice. In the survival study, the entire HDC group died before the BMM group (p-value < 0.01). Conclusions: Our pilot study is the first, to our knowledge, to successfully generate orthotopic PDAC tumors using 3D high-density collagen I in a mouse model. KPC1199 cells embedded in HDC showed an aggressive phenotype in vitro, and resultant tumors demonstrated more robust collagen organization compared to the same cells grown in the BMM substrate. This aggressive phenotype translated to visible liver metastasis within five weeks in the HDC mice and prior to death from primary tumor burden. This is not typical for most KPC-derived cell lines but is typical for human PDAC patients. Our preliminary findings support the use of HDC as an alternative substrate for better modeling of this aggressive cancer. Citation Format: Kim Nguyen-Ta, Sural Ranamukhaarachchi, Jeffrey Turner, Utsav Joshi, Himangshu Sonowal, Jorge de la Torre Medina, Herve Tiriac, Stephanie Fraley, Rebekah White. 3D High-Density Collagen I Improves the Modeling of Aggressive Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A035.

Acupotomy ameliorates knee osteoarthritis-related collagen deposition and fibrosis in rabbit skeletal muscle through the TGF-β/Smad pathway

ObjectiveTo investigate the effects of acupotomy on skeletal muscle fibrosis and collagen deposition in a rabbit knee osteoarthritis (KOA) model. MethodsRabbits (n = 18) were randomly divided into control, KOA, and KOA + acupotomy (Apo) groups (n = 6). The rabbits in the KOA and Apo groups were modeled using the modified Videman's method for 6 weeks. After modeling, the Apo group was subjected to acupotomy once a week for 3 weeks on the vastus medialis, vastus lateralis, rectus femoris, biceps femoris, and anserine bursa tendons around the knee. The behavior of all animals was recorded, rectus femoris tissue was obtained, and histomorphological changes were observed using Masson staining and transmission electron microscopy. The expression of transforming growth factor-β1 (TGF-β1), Smad 3, Smad 7, fibrillar collagen types I (Col-I) and III (Col-III) was detected using Western blot and real-time polymerase chain reaction (RT-PCR). ResultsHistological analysis revealed that acupotomy improved the microstructure and reduced the collagen volume fraction of rectus femoris, compared with the KOA group (P = .034). Acupotomy inhibited abnormal collagen deposition by modulating the expression of fibrosis-related proteins and mRNA, thus preventing skeletal muscle fibrosis. Western blot and RT-PCR analysis revealed that in the Apo group, Col-I, and Col-III protein levels were significantly lower than those in the KOA group (both P < .01), same as Col-I and Col-III mRNA levels (P = .0031; P = .0046). Compared with the KOA group, the protein levels of TGF-β1 and Smad 3 were significantly reduced (both P < .01), as were the mRNA levels of TGF-β1 and Smad 3 (P = .0007; P = .0011). Conversely, the levels of protein and mRNA of Smad 7 were significantly higher than that in the KOA group (P < .01; P = .0271). ConclusionAcupotomy could alleviate skeletal muscle fibrosis and delay KOA progress by inhibiting collagen deposition through the TGF-β/Smad pathway in the skeletal muscle of KOA rabbits.

Spectroscopic analysis of the sum-frequency response of the carbon-hydrogen stretching modes in collagen type I.

We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon-hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements Xijk2 of collagen's nonlinear susceptibility. Using the three-dimensional atomic positions derived from published crystallographic data of collagen type I, we simulated its Xijk2 elements for the methylene stretching vibration and compared the predicted response with the experimental results. Our analysis revealed that the carbon-hydrogen stretching range of the SFG spectrum is dominated by symmetric stretching modes of methylene bridge groups on the pyrrolidine rings of the proline and hydroxyproline residues, giving rise to a dominant peak near 2942cm-1 and a shoulder at 2917cm-1. Weak asymmetric stretches of the methylene bridge group of glycine are observed in the region near 2870cm-1, whereas asymmetric CH2-stretching modes on the pyrrolidine rings are found in the 2980 to 3030cm-1 range. These findings help predict the protein's nonlinear optical properties from its crystal structure, thus establishing a connection between the protein structure and SFG spectroscopic measurements.

IMPACT OF BMP-1 MODULATION IN EARLY FRACTURE REPAIR

BMP-1 is the major procollagen-C-peptidase activating, besides fibrillar collagen types I-III, several enzymes and growth factors involved in the generation of extracellular matrix. This study investigated the effect of adding and inhibiting BMP-1 directly post fracture.Standardised femoral fractures were stabilized by an intramedullary nail in 12 week-old female C57Bl/6J mice. We injected either 20 µL recombinant active BMP-1, activity buffer or the BMP-1 specific inhibitor “sizzled”. After 7, 14 and 28 days, mice were sacrificed. Femurs were dissected and paraffin slides were prepared. Callus composition was divided into soft tissue, mineralized and cartilaginous callus. Murine MC3T3 pre-osteoblastic cells were kept in culture adding BMP-1 and sizzled during osteoblastic differentiation. Putative cytotoxicity was determined using MTT-vitality assay. Cell calcification, collagen deposition, and BMP-2 and myostatin protein quantity were characterized.Adding BMP-1 displayed a weak positive effect on the outcome. After 7 days, more mineralised callus was present, meanwhile the cartilaginous callus was apparently remodelled at higher rate. In the case of BMP-1 inhibition, we observed more cartilaginous callus, which may indicate reduced stability. In cell culture, we could observe a high interference with mineralisation capabilities depending on the stage of osteoblastic development when adding BMP-1 or inhibiting it. Addition and inhibition impaired myostatin (anti-osteogen) and BMP-2 (pro-osteogen) expression.Interfering with BMP-1 homeostasis in this early stage of fracture repair seems to have rather negative effects. Inhibition apparently yields lower callus quality while the addition of BMP-1 does not significantly accelerate the healing outcome. Cell culture experiments show that BMP-1 application after 7 days of healing leads to higher collagen output but has no effect on mineralisation. This may suggest that BMP-1 application at a later time-point may lead to more pronounced beneficial effects on fracture repair.

The Structural Interactions of Molecular and Fibrillar Collagen Type I with Fibronectin and Its Role in the Regulation of Mesenchymal Stem Cell Morphology and Functional Activity.

The observed differences in the structure of native tissue and tissue formed in vitro cause the loss of functional activity of cells cultured in vitro. The lack of fundamental knowledge about the protein mechanism interactions limits the ability to effectively create in vitro native tissue. Collagen is able to spontaneously assemble into fibrils in vitro, but in vivo, other proteins, for example fibronectin, have a noticeable effect on this process. The molecular or fibrillar structure of collagen plays an equally important role. Therefore, we studied the interaction of the molecular and fibrillar structure of collagen with fibronectin. Atomic force and transmission electron microscopy showed that the presence of fibronectin does not affect the native structure and diameter of collagen fibrils. Confocal microscopy demonstrated that the collagen structure affects the cell morphology. Cells are better spread on molecular collagen compared with cells cultured on fibrillar collagen. Fibronectin promotes the formation of a large number of focal contacts, while in combination with collagen of both forms, its effect is leveled. Thus, understanding the mechanisms of the relationship between the protein structure and composition will effectively manage the creation in vitro of a new tissue with native properties.

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Pharmacologic inhibition of discoidin domain Receptor 2 (DDR2) sensitizes homologous recombination proficient ovarian cancer models to treatment with olaparib (245)

Extracellular Matrix in Heart Disease: Focus on Circulating Collagen Type I and III Derived Peptides as Biomarkers of Myocardial Fibrosis and Their Potential in the Prognosis of Heart Failure: A Concise Review.

Accumulating evidence indicates that two major proteins are responsible for the structural coherence of bounding cardiomyocytes. These biomolecules are known as myocardial fibrillar collagen type I (COL1) and type III (COL3). In addition, fibronectin, laminin, fibrillin, elastin, glycoproteins, and proteoglycans take part in the formation of cardiac extracellular matrix (ECM). In physiological conditions, collagen synthesis and degradation in human cardiac ECM are well-regulated processes, but they can be impaired in certain cardiovascular diseases, such as heart failure (HF). Myocardial remodeling is part of the central mechanism of HF and involves cardiomyocyte injury and cardiac fibrosis due to increased fibrillar collagen accumulation. COL1 and COL3 are predominantly involved in this process. Specific products identified as collagen-derived peptides are released in the circulation as a result of abnormal COL1 and COL3 turnover and myocardial remodeling in HF and can be detected in patients’ sera. The role of these products in the pathogenesis of cardiac fibrosis and the possible clinical implications are the focus of numerous investigations. This paper reviews recent studies on COL1- and COL3-derived peptides in patients with HF. Their potential application as indicators of myocardial fibrosis and prognostic markers of HF is also highlighted.

A Comprehensive Analysis of Fibrillar Collagens in Lamprey Suggests a Conserved Role in Vertebrate Musculoskeletal Evolution.

Vertebrates have distinct tissues which are not present in invertebrate chordates nor other metazoans. The rise of these tissues also coincided with at least one round of whole-genome duplication as well as a suite of lineage-specific segmental duplications. Understanding whether novel genes lead to the origin and diversification of novel cell types, therefore, is of great importance in vertebrate evolution. Here we were particularly interested in the evolution of the vertebrate musculoskeletal system, the muscles and connective tissues that support a diversity of body plans. A major component of the musculoskeletal extracellular matrix (ECM) is fibrillar collagens, a gene family which has been greatly expanded upon in vertebrates. We thus asked whether the repertoire of fibrillar collagens in vertebrates reflects differences in the musculoskeletal system. To test this, we explored the diversity of fibrillar collagens in lamprey, a jawless vertebrate which diverged from jawed vertebrates (gnathostomes) more than five hundred million years ago and has undergone its own gene duplications. Some of the principal components of vertebrate hyaline cartilage are the fibrillar collagens type II and XI, but their presence in cartilage development across all vertebrate taxa has been disputed. We particularly emphasized the characterization of genes in the lamprey hyaline cartilage, testing if its collagen repertoire was similar to that in gnathostomes. Overall, we discovered thirteen fibrillar collagens from all known gene subfamilies in lamprey and were able to identify several lineage-specific duplications. We found that, while the collagen loci have undergone rearrangement, the Clade A genes have remained linked with the hox clusters, a phenomenon also seen in gnathostomes. While the lamprey muscular tissue was largely similar to that seen in gnathostomes, we saw considerable differences in the larval lamprey skeletal tissue, with distinct collagen combinations pertaining to different cartilage types. Our gene expression analyses were unable to identify type II collagen in the sea lamprey hyaline cartilage nor any other fibrillar collagen during chondrogenesis at the stages observed, meaning that sea lamprey likely no longer require these genes during early cartilage development. Our findings suggest that fibrillar collagens were multifunctional across the musculoskeletal system in the last common ancestor of vertebrates and have been largely conserved, but these genes alone cannot explain the origin of novel cell types.

Ex vivo observation of granulocyte activity during thrombus formation

BackgroundThe process of thrombus formation is thought to involve interactions between platelets and leukocytes. Leukocyte incorporation into growing thrombi has been well established in vivo, and a number of properties of platelet-leukocyte interactions critical for thrombus formation have been characterized in vitro in thromboinflammatory settings and have clinical relevance. Leukocyte activity can be impaired in distinct hereditary and acquired disorders of immunological nature, among which is Wiskott-Aldrich Syndrome (WAS). However, a more quantitative characterization of leukocyte behavior in thromboinflammatory conditions has been hampered by lack of approaches for its study ex vivo. Here, we aimed to develop an ex vivo model of thromboinflammation, and compared granulocyte behavior of WAS patients and healthy donors.ResultsThrombus formation in anticoagulated whole blood from healthy volunteers and patients was visualized by fluorescent microscopy in parallel-plate flow chambers with fibrillar collagen type I coverslips. Moving granulocytes were observed in hirudinated or sodium citrate-recalcified blood under low wall shear rate conditions (100 s−1). These cells crawled around thrombi in a step-wise manner with an average velocity of 90–120 nm/s. Pre-incubation of blood with granulocyte priming agents lead to a significant decrease in mean-velocity of the cells and increase in the number of adherent cells. The leukocytes from patients with WAS demonstrated a 1.5-fold lower mean velocity, in line with their impaired actin polymerization. It is noteworthy that in an experimental setting where patients’ platelets were replaced with healthy donor’s platelets the granulocytes’ crawling velocity did not change, thus proving that WASP (WAS protein) deficiency causes disruption of granulocytes’ behavior. Thereby, the observed features of granulocytes crawling are consistent with the neutrophil chemotaxis phenomenon. As most of the crawling granulocytes carried procoagulant platelets teared from thrombi, we propose that the role of granulocytes in thrombus formation is that of platelet scavengers.ConclusionsWe have developed an ex vivo experimental model applicable for observation of granulocyte activity in thrombus formation. Using the proposed setting, we observed a reduction of motility of granulocytes of patients with WAS. We suggest that our ex vivo approach should be useful both for basic and for clinical research.

The Ehlers-Danlos Syndromes against the Backdrop of Inborn Errors of Metabolism.

The Ehlers–Danlos syndromes are a group of multisystemic heritable connective tissue disorders with clinical presentations that range from multiple congenital malformations, over adolescent-onset debilitating or even life-threatening complications of connective tissue fragility, to mild conditions that remain undiagnosed in adulthood. To date, thirteen different EDS types have been recognized, stemming from genetic defects in 20 different genes. While initial biochemical and molecular analyses mainly discovered defects in genes coding for the fibrillar collagens type I, III and V or their modifying enzymes, recent discoveries have linked EDS to defects in non-collagenous matrix glycoproteins, in proteoglycan biosynthesis and in the complement pathway. This genetic heterogeneity explains the important clinical heterogeneity among and within the different EDS types. Generalized joint hypermobility and skin hyperextensibility with cutaneous fragility, atrophic scarring and easy bruising are defining manifestations of EDS; however, other signs and symptoms of connective tissue fragility, such as complications of vascular and internal organ fragility, orocraniofacial abnormalities, neuromuscular involvement and ophthalmological complications are variably present in the different types of EDS. These features may help to differentiate between the different EDS types but also evoke a wide differential diagnosis, including different inborn errors of metabolism. In this narrative review, we will discuss the clinical presentation of EDS within the context of inborn errors of metabolism, give a brief overview of their underlying genetic defects and pathophysiological mechanisms and provide a guide for the diagnostic approach.

Progressive alcohol-related liver fibrosis is characterised by imbalanced collagen formation and degradation.

SummaryBackgroundLiver fibrosis accumulation is considered a turnover disease, with formation exceeding degradation, although this hypothesis has never been tested in humans.AimsTo investigate extracellular matrix (ECM) remodelling in a biopsy‐controlled study of alcohol‐related liver disease (ALD) patients.MethodsWe evaluated the relationship between formation and degradation of four collagens as a function of histological fibrosis, inflammation and steatosis in 281 patients with ALD and 50 matched healthy controls. Post hoc, we tested the findings in a cohort of patients with alcohol‐related cirrhosis and assessed the collagens' prognostic accuracy. We assessed the fibrillar collagens type III (PRO‐C3/C3M) and V (PRO‐C5/C5M), the basement membrane collagen IV (PRO‐C4/C4M), and the microfilament interface collagen VI (PRO‐C6/C6M).ResultsMean age was 54 ± 6 years, 74% male, fibrosis stage F0/1/2/3/4 = 33/98/84/18/48. Compared to controls, patients with ALD had higher levels of type III collagen formation and degradation, with the highest concentrations in those with cirrhosis (PRO‐C3 = 8.2 ± 1.7 ng/mL in controls, 14.6 ± 13.5 in ALD, 34.8 ± 23.1 in cirrhosis; C3M 7.4 ± 1.9 in controls, 9.3 ± 4.4 in ALD, 14.0 ± 5 in cirrhosis). ECM remodelling became increasingly imbalanced in higher stages of liver fibrosis, with formation progressively superseding degradation. This was particularly pronounced for type III collagen. We observed similar imbalance for inflammatory severity, but not steatosis.ConclusionsALD is characterised by both elevated collagen formation and degradation, which becomes increasingly imbalanced with more severe disease. Net increase in fibrillar collagens contributes to fibrosis progression. This has important implications for monitoring and very early identification of patients at highest risk of progressing to cirrhosis.

Preventing ovarian cancer cell metastasis through inhibition of discoidin domain receptor 2 (DDR2) in the tumor microenvironment: a metabolomic analysis

Objectives: Discoidin domain receptor 2 (DDR2) is a tyrosine kinase activated by fibrillar collagen type 1. DDR2 expression in both cancer cells and stromal cells is critical for metastases in breast cancer. In ovarian cancer, DDR2 stabilizes Snail, a critical factor in the epithelial to mesenchymal transition, which facilitates cancer cell invasion and metastases. Snail has also been shown to reprogram glucose metabolism in breast cancer cells, thereby allowing cancer cell survival under metabolic stress. This study aimed to investigate how stromal DDR2 expression in normal omental and ovarian cancer associated fibroblasts (NOF and CAF) regulates tumor cell invasion by analyzing changes in stromal cell metabolomics. Methods: Matrigel invasion assays were performed using immortalized normal omental fibroblasts (NOF206) and ES2 ovarian cancer cells. NOF206s were transfected with two different siRNAs to produce transient siDDR2 knockdown. Lysates and conditioned media were collected of siControl (DDR2-expressing), Dharmacon siDDR2 and Sigma siDDR2 (DDR2 non-expressing) cells. Mass spectrometry was performed and metabolites were identified. Quantitative RT-PCR was performed to evaluate changes in the expression of mesenchymal markers based on DDR2 expression. Results: DDR2 expressing tumor cells invaded less when co-cultured with DDR2-non expressing NOFs compared to DDR2 expressing NOFs (51 vs 155 cells per hpf, p Conclusions: Genetic inactivation of DDR2 results in marked changes in the metabolism of normal omental fibroblasts resulting in decreased tumor cell invasion. These findings suggest a novel role of DDR2 in metabolism and stromal cell energy generation. The significance of these findings requires further study on the complex relationship between ovarian cancer and the stromal environment.

Discoidin Domain Receptor 2 (DDR2) expression in combination with mutation status is predictive of survival in patients with ovarian cancer

<h3>Objectives:</h3> Dicoidin Domain Receptor 2 (DDR2) is a tyrosine kinase receptor which binds the most common extracellular matrix protein, fibrillar collagen type 1. Expression of DDR2 is critical for invasion and migration of ovarian cancer tumor cells. Clinically, high DDR2 expression has been shown to be associated with advanced stage disease and decreased survival. It is not known how <i>BRCA</i> mutation status, a known marker for treatment sensitivity, impacts this relationship between DDR2 expression and survival. This study aimed to determine whether DDR2 expression was correlated with <i>BRCA</i> mutation status and to quantify the effects of this relationship on survival. <h3>Methods:</h3> Immunohistochemical (IHC) analysis of DDR2 expression in two ovarian cancer tissue microarrays (TMA) was performed. DDR2 staining on the membrane of tumor cells was scored microscopically according to the intensity and percentage of cells positive for DDR2 expression compared to controls. High DDR2 staining was defined as >70% and low staining <69%. Clinical information, including germline mutational status and survival data, was abstracted from the electronic medical record. Statistical analysis was performed using descriptive statistics and unpaired t-tests. Survival analysis was performed using the Kaplan-Meier method with statistical significance determined using the log-rank test. <h3>Results:</h3> The cohort included 164 patients diagnosed between 2/1994 and 1/2017. The majority had advanced stage disease (79%) and high-grade serous histology (76%). Germline mutation status was known in 93 patients (57%). <i>BRCA1</i> was the most common mutation in 38 patients (64%) followed by <i>BRCA2</i> in 20 patients (34%). 34 patients had negative germline testing. DDR2 expression was significantly associated with mutational status. Patients without a germline mutation had significantly higher DDR2 expression (64%) compared to <i>BRCA1</i> (37%) (p=0.002) and <i>BRCA2</i> had significantly higher DDR2 expression (55%) compared to <i>BRCA1</i> (37%) (p=0.03). DDR2 expression did not vary by stage, grade or platinum sensitive status. In survival analysis, patients with high DDR2 expression had significantly lower overall survival (33mo vs 83mo, HR 1.94, p=0.002) and progression free survival (17mo vs 34mo, HR 1.668, p=0.04) compared to patients with low DDR2 expression. When survival analysis was stratified by mutation status and DDR2 expression, survival differences were seen. In patients without a germline mutation, high DDR2 expression was associated with worse overall survival (42mo vs 147mo, HR=2.818, p=0.04) compared to low DDR2 expression. <h3>Conclusions:</h3> Tumor DDR2 expression is correlated with mutation status and when combined, provides useful prognostic information. Studies are ongoing to identify additional <i>BRCA</i> deficient tumors for further survival analysis.

Increasing sensitivity to olaparib through inhibition of discoidin domain receptor 2 (DDR2) in homologous-recombination proficient ovarian cancer models

<h3>Objectives:</h3> Discoidin Domain Receptor 2 (DDR2) is a tyrosine kinase receptor which binds the most common extracellular matrix protein, fibrillar collagen type 1. DDR2 expression is critical for invasion and migration of ovarian cancer tumor cells, making DDR2 a potential target for new treatments. This study aimed to determine whether genetic inactivation of DDR2 in ovarian cancer cells would increase sensitivity to treatment with PARP inhibitor. <h3>Methods:</h3> Two ovarian cancer cells lines, ES2 and COV362, were used for <i>in vitro</i> assays. Stable hairpin control and DDR2 knockdowns (shSCRM and shDDR2) were used for ES2 and transient small interfering RNA knockdown of DDR2 (siControl and siDDR2) were used for COV362. Homologous recombination (HR) status was determined by quantification of RAD-51 foci relative to controls using immunofluorescence after irradiation. Cell viability and survival after treatment with olaparib was quantified using MTS assays and clonogenics. The DNA damage response after treatment with olaparib was analyzed using immunofluorescence. <i>In vivo</i> studies to evaluate the sensitivity to olaparib in the presence or absence of DDR2 expression were performed in nude mice after intraperitoneal injection with ES2shSCRM or shDDR2 cells. <h3>Results:</h3> ES2 and COV362 cells were found to have a 2-fold increase in RAD51 foci after irradiation indicating HR proficiency. DDR2 genetically inactivated cells (ES2shDDR2) were found to be more HR deficient than DDR2 expressing cells (ES2shSCRM) which remained HR proficient (0.80 vs 4.78 relative RAD51 foci per cell). We found that genetic inactivation of DDR2 in the ES2s and COV362s led to increased sensitivity to olaparib with an 80% (15uM vs 73uM) and 68% (62uM vs 195uM) reduction in IC50s, respectively, compared to DDR2 expressing cells. Additionally, ES2shDDR2 cells had decreased cell survival by clonogenic assay after treatment with olaparib compared to ES2shSCRM cells as measured by absorbance (0.64 vs 0.80, p=0.005). We found that DNA damage and the repair response was altered in DDR2 genetically inactivated cells treated with olaparib with an increase in gamma-H2AX (8 vs 6 foci per cell, p<0.0001) and 53BP1 foci (3 vs 1 foci per cell, p<0.0001) when compared to DDR2 expressing cells. In an intraperitoneal metastatic mouse model with the HR-proficient cell line ES2, we found that genetic inactivation of DDR2 led to decreased tumor burden when treated with olaparib compared to DDR2-expressing cells treated with olaparib (1499mm³ vs 3236mm³, p=0.04). <h3>Conclusions:</h3> DDR2 knockdown sensitizes HR proficient ovarian cancer cells to treatment with PARP inhibitor in both <i>in vitro</i> and <i>in vivo</i> models through a mechanism of induced HR deficiency and increased DNA damage. Future experiments will explore whether a DDR2 inhibitor can improve sensitivity to PARP inhibitor in HR proficient ovarian cancer models.

Abstract 388: Non-invasive biomarker potential of the fibroblast-derived collagen type III and collagen type XI for predicting outcome in metastatic melanoma patients treated with anti-PD-1 therapy

Abstract Background: Biomarkers for predicting immune checkpoint inhibitor (ICI) efficacy are essential for patient selection for current ICI regimens and new combination therapies. Patients with T-cell excluded tumors have poor effect on ICI therapy. T-cell exclusion is driven by upregulated TGF-β-signaling and activated fibroblasts, which produce increased levels of fibrillar collagens (desmoplasia). In this study, we investigated if non-invasive biomarkers reflecting deposition of the fibrillar collagen type III (PRO-C3) and collagen type XI (PRO-C11) associated with response and survival outcomes in patients treated with ICI therapy. Methods: N-terminal pro-peptides of collagen type III (PRO-C3) and collagen type XI collagen (PRO-C11) were measured with ELISAs in pre-treatment serum from metastatic melanoma patients treated with anti-PD-1 therapy (pembrolizumab) (n=35). The association between PRO-C3 and PRO-C11 levels and progression-free survival (PFS) and overall survival (OS) were assessed by Cox regression analyses, alone and after adjusting for tumor PDL1 expression and BRAF mutational status. Results: Univariate Cox regression identified high (&amp;lt;75th percentile) baseline PRO-C3 and PRO-C11 as predictors of poor PFS (PRO-C3: HR=2.77, 95%CI=1.01-7.55, p=0.047, and PRO-C11: HR=2.87, 95%CI=1.05-7.87, p=0.040) and OS (PRO-C3: HR=6.54, 95%CI=1.56-27.53, p=0.010, and PRO-C11: HR=4.58, 95%CI=1.13-18.65, p=0.034). Moreover, when PRO-C3 and PRO-C11 were adjusted for PDL1 expression (≥1%) and BRAF mutations, the biomarkers remained independently predictive of poor PFS (PRO-C3: HR=3.00, 95%CI=1.07-8.42, p=0.038, and PRO-C11: HR=3.66, 95%CI=1.22-10.98, p=0.021) and OS (PRO-C3: HR=6.20, 95%CI=1.47-26.12, p=0.013, and PRO-C11: HR=7.85, 95%CI=1.32-46.78, p=0.024). PRO-C3 was furthermore significantly elevated in patients with progressive disease compared to patients with complete response, partial response and stable disease (p=0.007). Conclusions: Non-invasive biomarkers of the fibroblast-derived collagen type III (PRO-C3) and collagen type XI (PRO-C11) reflecting fibroblast activity predict survival outcomes in metastatic melanoma patients treated with anti-PD-1 therapy independent of PD-L1 expression and BRAF mutational status. These findings support the link between desmoplasia (tumor fibrosis) and poor response to ICIs and indicate a promise of non-invasive collagen-based biomarkers for selecting metastatic melanoma patients for anti-PD-1 therapy at baseline and hereby improve the response rate. Citation Format: Christina Jensen, Anders Kverneland, Marco Donia, Neel I. Nissen, Morten A. Karsdal, Inge Marie Svane, Nicholas Willumsen. Non-invasive biomarker potential of the fibroblast-derived collagen type III and collagen type XI for predicting outcome in metastatic melanoma patients treated with anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 388.

USF1 Suppresses Expression of Fibrillar Type I, II, and III Collagen and pNP Adamts-3 in Osteosarcoma Cells

Collagens are the main components of human tissues. Various regulatory factors and cytokines may influence expression levels for collagen-encoding genes, and, therefore, contrubite to some collagen-associated pathologies. In this study, we demonstrate regulatory effects of USF1 on expression of genes encoding fibrillar collagen types I, II, and III in osteoblastic Saos-2 and MG-63 cells. An ectopic expression of the human USF1 led to a decrease in both mRNA and protein expression levels of the collagen-encoding genes mentioned above. ADAMTS-3 is a proteinase primarily responsible for the amino-terminal cleavage of type I and type II collagen precursors. The ADAMTS-3 promoter region contains potential binding sites for USF1. Here we show that an overexpression of USF1 lead to a decrease in ADAMTS-3 mRNA and protein expression levels. In co-transfection studies, USF1 negatively regulated ADAMTS-3 promoter activity. Further, in EMSA studies, we showed that USF1 binds to the ADAMTS-3 promoter region. In conclusion, it seems that ADAMTS-3 and USF1 contribute to the regulation of collagen encoding genes in osteosarcoma.

Fibronectin-functionalization of 3D collagen networks supports immune tolerance and inflammation suppression in human monocyte-derived macrophages

The extracellular matrix (ECM) is dynamically reorganized during wound healing. Concomitantly, recruited monocytes differentiate into macrophages. However, the role of the wound's ECM during this transition remain to be fully understood. Fibronectin is a multifunctional glycoprotein present in early wound ECM with a potential immunomodulatory role during monocyte-to-macrophage differentiation. Hence, to investigate the impact of fibronectin during this differentiation step, 3D fibrillar collagen type I networks with or without fibronectin-functionalization were engineered with defined topology (fibril and pore diameter: 0.8 μm; 7 μm) and amount of adsorbed fibronectin (0.15 μg per μg collagen). Primary, human monocytes were then differentiated into macrophages inside these networks. The immunological imprinting of the resulting macrophages was monitored by means of the expression of FABP4, CLEC4E, SLC2A6, and SOD2 which discriminate naïve and tolerized macrophages, as well pro-inflammatory (M1) and anti-inflammatory (M2) macrophage polarization. The analyses indicate that fibronectin-functionalization of collagen I networks induces macrophage tolerance rather than M1 or M2 macrophage phenotypes. This finding was confirmed by release profiles of pro- and anti-inflammatory cytokines such as IL6, IL8, CXCL10, and IL10. Nevertheless, upon LPS challenge, immune suppression by fibronectin was overridden since these macrophages could then deploy an efficient immune response. Our results therefore provide new perspectives in biomaterial science of wound healing scaffolds and the design of instructive materials for human monocyte-derived cells.

Collagenases MMP-1, MMP-13, and Tissue Inhibitors TIMP-1, TIMP-2: Their Role in Healthy and Complicated Pregnancy and Potential as Preeclampsia Biomarkers—A Brief Review

Extracellular matrix (ECM) turnover is characterized by a unique balance between matrix metalloproteinases’ degradation activity and their natural inhibition by collagen specific tissue inhibitors. Human uterine ECM is a complex structure, majorly consisting of proteins as fibrillar collagen types I and III, fibronectin, and laminin. Collagenases are enzymes from the matrix metalloproteinases’ family, which are predominantly involved in fibrillar collagen types I and III degradation. They are mainly represented by matrix metalloproteinase-1, -13 (MMP-1, -13), naturally inhibited by tissue inhibitors (TIMP-1, -2). The collagen structure of the uterus has been shown to be impaired in women with preeclampsia. This is a result of MMPs/TIMPs dysregulation interplay. This review article summarizes the actual available research data in the literature about the role of MMP-1, MMP-13 and TIMP-1, and TIMP-2 in collagen types I and III turnover in healthy and complicated pregnancy. Their potential use as circulating markers for diagnosis, prognosis, and monitoring of the development of preeclampsia is discussed as well.

Distinct Expression Patterns of Fibrillar Collagen Types I, III, and V in Association with Mammary Gland Remodeling during Pregnancy, Lactation and Weaning.

The mammary gland structurally and functionally remodels during pregnancy, during lactation and after weaning. There are three types of fibrillar collagens, types I, III, and V, in mammary stromal tissue. While the importance of the fibrillar structure of collagens for mammary morphogenesis has been suggested, the expression patterns of each type of fibrillar collagen in conjunction with mammary remodeling remain unclear. In this study, we investigated their expression patterns during pregnancy, parturition, lactation and involution. Type I collagen showed a well-developed fibril structure during pregnancy, but the fibrillar structure of type I collagen then became sparse at parturition and during lactation, which was concurrent with the downregulation of its mRNA and protein levels. The well-developed fibrillar structure of type I collagen reappeared after weaning. On the other hand, type V collagen showed a well-developed fibrillar structure and upregulation in the lactation period but not in the periods of pregnancy and involution. Type III collagen transiently developed a dense fibrillar network at the time of parturition and exhibited drastic increases in mRNA expression. These results indicate that each type of fibrillar collagen is distinctly involved in structural and functional remodeling in mammary glands during pregnancy, parturition, lactation, and involution after weaning. Furthermore, in vitro studies of mammary epithelial cells showed regulatory effects of type I collagen on cell adhesion, cell proliferation, ductal branching, and β-casein secretion. Each type of fibrillar collagen may have different roles in defining the cellular microenvironment in conjunction with structural and functional mammary gland remodeling.

The Ehlers-Danlos syndromes.

The Ehlers-Danlos syndromes (EDS) are a heterogeneous group of hereditary disorders of connective tissue, with common features including joint hypermobility, soft and hyperextensible skin, abnormal wound healing and easy bruising. Fourteen different types of EDS are recognized, of which the molecular cause is known for 13 types. These types are caused by variants in 20 different genes, the majority of which encode the fibrillar collagen types I, III and V, modifying or processing enzymes for those proteins, and enzymes that can modify glycosaminoglycan chains of proteoglycans. For the hypermobile type of EDS, the molecular underpinnings remain unknown. As connective tissue is ubiquitously distributed throughout the body, manifestations of the different types of EDS are present, to varying degrees, in virtually every organ system. This can make these disorders particularly challenging to diagnose and manage. Management consists of a care team responsible for surveillance of major and organ-specific complications (for example, arterial aneurysm and dissection), integrated physical medicine and rehabilitation. No specific medical or genetic therapies are available for any type of EDS.