Collagen biology making inroads into prognosis and treatment of cancer progression and metastasis.

Progression through dissemination to tumor-surrounding tissues and metastasis development is a hallmark of cancer that requires continuous cell-to-cell interactions and tissue remodeling. In fact, metastization can be regarded as a tissue disease orchestrated by cancer cells, leading to neoplastic colonization of new organs. Collagen is a major component of the extracellular matrix (ECM), and increasing evidence suggests that it has an important role in cancer progression and metastasis. Desmoplasia and collagen biomarkers have been associated with relapse and death in cancer patients. Despite the increasing interest in ECM and in the desmoplastic process in tumor microenvironment as prognostic factors and therapeutic targets in cancer, further research is required for a better understanding of these aspects of cancer biology. In this review, published evidence correlating collagen with cancer prognosis is retrieved and analyzed, and the role of collagen and its fragments in cancer pathophysiology is discussed.

Signals of the Neuropilin-1-MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells.

The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1-MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the C-type lectin-related protein rhodocetin-αß (RCαß) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCαß reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin-mediated transmission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-κB. Simultaneously, RCαß-triggered signaling via the NRP1-MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCαß-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This was due to RCαß-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCαß induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Additionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1-MET signaling and influenced by intercellular forces.

Standardization and Validation of Fluorescence-Based Quantitative Assay to Study Human Platelet Adhesion to Extracellular-Matrix in a 384-Well Plate.

Platelets play a crucial role in the immunological response and are involved in the pathological settings of vascular diseases, and their adhesion to the extracellular matrix is important to bring leukocytes close to the endothelial cells and to form and stabilize the thrombus. Currently there are several methods to study platelet adhesion; however, the optimal parameters to perform the assay vary among studies, which hinders their comparison and reproducibility. Here, a standardization and validation of a fluorescence-based quantitative adhesion assay to study platelet-ECM interaction in a high-throughput screening format is proposed. Our study confirms that fluorescence-based quantitative assays can be effectively used to detect platelet adhesion, in which BCECF-AM presents the highest sensitivity in comparison to other dyes.

Extracellular Vesicle Transfer from Endothelial Cells Drives VE-Cadherin Expression in Breast Cancer Cells, Thereby Causing Heterotypic Cell Contacts.

Cadherins mediate cohesive contacts between isotypic cells by homophilic interaction and prevent contact between heterotypic cells. Breast cancer cells neighboring endothelial cells (ECs) atypically express vascular endothelial (VE)-cadherin. To understand this EC-induced VE-cadherin expression in breast cancer cells, MCF7 and MDA-MB-231 cells expressing different endogenous cadherins were co-cultured with ECs and analyzed for VE-cadherin at the transcriptional level and by confocal microscopy, flow cytometry, and immunoblotting. After losing their endogenous cadherins and neo-expression of VE-cadherin, these cells integrated into an EC monolayer without compromising the barrier function instantly. However, they induced the death of nearby ECs. EC-derived extracellular vesicles (EVs) contained soluble and membrane-anchored forms of VE-cadherin. Only the latter was re-utilized by the cancer cells. In a reporter gene assay, EC-adjacent cancer cells also showed a juxtacrine but no paracrine activation of the endogenous VE-cadherin gene. This cadherin switch enabled intimate contact between cancer and endothelial cells in a chicken chorioallantoic membrane tumor model showing vasculogenic mimicry (VM). This EV-mediated, EC-induced cadherin switch in breast cancer cells and the neo-expression of VE-cadherin mechanistically explain the mutual communication in the tumor microenvironment. Hence, it may be a target to tackle VM, which is often found in breast cancers of poor prognosis.

From Patients to Platelets and Back Again: Pharmacological Approaches to Glycoprotein VI, a Thrilling Antithrombotic Target with Minor Bleeding Risks.

Despite significant advances in the treatment of thrombogenic diseases, antiplatelet therapies are still associated with a high bleeding risk. Consequently, potential benefits of preventing thromboembolic events by pharmacological agents need to be balanced with the potential harm of inducing hemorrhage. Glycoprotein VI (GPVI) is a platelet-specific receptor, which plays a crucial role in thrombus formation. GPVI deficiency has been identified in patients who suffer from significant reduction of collagen-induced thrombus formation, with a slight tendency for mild bleeding. However, an isolated GPVI deficiency can reduce thrombus formation while not resulting in severe bleeding. Together, these observations strongly suggest that physiological hemostasis does not require GPVI, but pharmacological GPVI modulation may provide novel "bleeding-free" antithrombotic therapies. In this review, we discuss recent findings regarding the biological role of GPVI in platelet-related disorders and highlight the efforts to develop potential therapeutic strategies based on its structure, signaling pathways, and biological effects.

The spatial molecular pattern of integrin recognition sites and their immobilization to colloidal nanobeads determine α2ß1 integrin-dependent platelet activation.

Collagen, a strong platelet activator, is recognized by integrin α2ß1 and GPVI. It induces aggregation, if added to suspended platelets, or platelet adhesion if immobilized to a surface. The recombinant non-prolylhydroxylated mini-collagen FC3 triple helix containing one α2ß1 integrin binding site is a tool to specifically study how α2ß1 integrin activates platelet. Whereas soluble FC3 monomers antagonistically block collagen-induced platelet activation, immobilization of several FC3 molecules to an interface or to colloidal nanobeads determines the agonistic action of FC3. Nanopatterning of FC3 reveals that intermolecular distances below 64 nm between α2ß1 integrin binding sites trigger signaling through dot-like clusters of α2ß1 integrin, which are visible in high resolution microscopy with dSTORM. Upon signaling, these integrin clusters increase in numbers per platelet, but retain their individual size. Immobilization of several FC3 to 100 nm-sized nanobeads identifies α2ß1 integrin-triggered signaling in platelets to occur at a twentyfold slower rate than collagen, which activates platelet in a fast integrative signaling via different platelet receptors. As compared to collagen stimulation, FC3-nanobead-triggered signaling cause a significant stronger activation of the protein kinase BTK, a weak and dispensable activation of PDK1, as well as a distinct phosphorylation pattern of PDB/Akt.