Abstract Type XIII collagen is an integral membrane protein in type II orientation. In cells and tissues type XIII collagen has been located in various adhesive structures, like focal adhesions. Due to this, its biological role has been implicated in cell adhesion. This collagen also exists as a soluble protein due to the release of the ectodomain from the plasma membrane. In this thesis, ectodomain shedding, i.e. enzymatic release of the extracellular domain, was studied in detail, focusing on the phenomenon as it occurs in mammalian cells. It was found that the ectodomain is released by members of the mammalian proprotein convertase family, e.g. furin. Shedding was shown to take place at the cell surface, but based on additional observations, this cleavage may also take place intracellularly in the Golgi apparatus. Various intracellular mechanisms, depending on cell type, were found to be involved in the regulation of ectodomain shedding. Apparently, due to the liberation of the ectodomain, the level of type XIII collagen on the plasma membrane is maintained at a relatively even amount. The released ectodomain was shown to retain biological activity. It showed distinct matrix-specificity so that on vitronectin its influence on cell functions was anti-adhesive, anti-migratory, anti-proliferative and non-supportive of cell spreading. It was also demonstrated to affect the fibronectin matrix assembly in a manner that resulted in reduced amounts of the fibrillar fibronectin matrix. A large collection of human epithelial and mesenchymal cancer samples were screened for type XIII collagen mRNA expression and compared to the expression levels of pre-malignant and normal samples. It was discovered that malignant transformation upregulates the expression of type XIII collagen in mesenchymal cancers and particularly in the stroma of epithelial cancers, more so than in cancer epithelia. TGF-β1 was demonstrated as one factor contributing to the stimulation of expression. Based on cell culture experiments in this study, it was also deduced that the upregulated expression of type XIII collagen and the concomitant shedding of the ectodomain can remodel the tumour stroma, making it inauspicious for adhesion-dependent cell functions, particularly in vitronectin-rich milieu.

Abstract Adhesive interactions between cells and extracellular matrix proteins play a vital role in biological processes such as cell proliferation, differentiation and survival. Integrins comprise a major family of cell surface receptors that mediate these interactions. Integrin engagement triggers adhesion-dependent intracellular signalling cascades that include the phosphorylation of tyrosines in intracellular signalling proteins. Integrin-dependent signals act in concert with signals from growth factors and other signalling receptors. The objective of this thesis was to study how cell adhesion and growth factors interact with intracellular components to regulate cell behavior in normal and transformed cells. One of the main proteins phosphorylated following integrin ligation in several different cell types is the docking protein p130Cas (Cas), which is tyrosine phosphorylated after stimulation of cells with low concentrations of epidermal growth factor (EGF). Tyrosine-phosphorylated Cas associates with an adapter protein c-Crk, the main binding protein for Cas, suggesting a novel role for EGF in Cas signalling. The interaction of cells with a variety of agonists such as growth factors and integrin ligation results in stimulation of mitogen-activated protein kinases (MAPKs), which control the expression of genes important for many cell functions. Expression of Cas and Crk induces activation of C-Jun N-terminal kinases (JNKs), which are members of MAPK family. JNK activation induced by integrin ligand binding is blocked by the expression of a dominant-negative mutant of Cas or Crk demonstrating an important role for the Cas-Crk complex in integrin-mediated JNK activation. The proto-oncogene product p120Cbl (Cbl) was identified as the main tyrosine-phosphorylated protein following integrin ligation in hematopoietic cells of myeloid lineage. Tyrosine-phosphorylated Cbl interacts with and activates other signalling proteins, such as Src tyrosine kinase and phosphatidylinositol 3"-kinase (PI 3-kinase), thereby mediating adhesion-dependent signals in hematopoietic cells. Unlike the cellular Cbl, the transforming mutants of Cbl were tyrosine-phosphorylated in an adhesion-independent manner and interacted with and activated signalling molecules both in suspended and in adherent cells. Further, the oncogenic forms of Cbl induced anchorage-independent but serum-dependent proliferation of cells. These results support the view that transformation by Cbl results from constitutive activation of integrin-dependent rather than growth factor-dependent signalling events.

No description available.

Glaucoma is the leading cause of irreversible blindness worldwide, and is a major cause of blindness in the United States. It affects approximately 5% of Caucasians and 10% of African- Americans over the age of 60 years. Elevated intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma. Impaired outflow of aqueous humor from the eye is thought to be the cause of pathologically elevated IOP. However, the etiology of outflow impairment is unknown. Anatomically, the aqueous humor drains into the iridocorneal angle of the eye, where the iris inserts at the transition between the cornea and sclera. In humans, approximately 80-90% of the aqueous traverses through the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), Schlemm’s canal, collector channels and empties into episcleral veins. Abnormalities at these sites are thought to cause impaired outflow. Abnormal accumulation of extracellular matrix (ECM) in the TM or JCT, abnormal endothelial function in Schlemm’s canal, or a combination of these components have been strongly implicated. Our laboratory has focused on the role of Secreted Protein Acidic and Rich in Cysteine (SPARC) in regulating outflow. SPARC is the prototypical matricellular protein that mediates ECM organization and turnover in numerous human tissues. Our lab was first to demonstrate that SPARC is highly expressed in the TM and JCT regions of the eye, and that the SPARC knockout (KO) mouse has a significant decrease in IOP of 15-20%. SPARC may affect the degree of segmental flow, a theory that states that variable aqueous outflow occurs around the circumference of the eye; only certain portions of the TM are thought to display active outflow at any particular moment. The cytokine transforming growth factor-ß2 (TGFß2) has been shown to modulate multiple ECM proteins, including SPARC. TGFß2 is significantly upregulated by 2 to 3-fold in the aqueous humor of glaucoma patients compared to controls. In addition, when TGFß2 is overexpressed in rodent eyes, increased ECM deposition is observed within the trabecular meshwork leading to IOP elevation. SPARC is one of the most highly upregulated proteins by TGFß2, and is downstream of TGFß2. We hypothesized that wild-type (WT) mice would demonstrate segmental flow, while SPARC KO mice would display a more continuous pattern of outflow around the eye. We also believed that IOP would be inversely correlated with outflow area. We also hypothesized that SPARC is essential to the process of TGFß2-mediated ocular hypertension, and that the lack of SPARC would impair IOP elevation. We conducted a tracer study utilizing fluorescent microbeads to determine the location of outflow circumferentially around the mouse TM. Microbeads were injected intracamerally into the eyes of WT and KO mice. After a 45-minute incubation period, the mice were euthanized and eyes were processed for confocal, light, and electron microscopy. During the second group of experiments, empty or TGFß2-containing adenovirus was injected intravitreally into WT and SPARC KO mice and IOP was measured for 2 weeks. Immunohistochemistry was completed on all tissues to assess for changes in major ECM proteins. Percentage effective filtration length (PEFL), or area of the TM labeled by tracer, was significantly increased in SPARC KO mice (70.61% ± 11.36%, p<0.005; N=11) compared to WT mice (54.68% ± 9.95%; N=11). In addition, the pressures between the two sets of eyes were significantly different with mean pressures of 16.3 mm Hg in WT mice and 12.6 mm Hg in KO mice (p<0.005, N=11 pairs). In addition, PEFL and IOP were inversely correlated with R2 = 0.72 (N=10 pairs); in eyes with higher IOP, PEFL was reduced. Electron microscopy demonstrated that high-tracer TM areas had a greater separation between trabecular beams. Collagen fibril diameter was found to be smaller in the KO (28.272 nm) compared to WT (34.961 nm; p<0.0005, N=3 pairs). These data provided structural correlations to the functional data regarding segmental flow. In the second set of experiments, IOP was found to be significantly elevated in TGFß2- injected WT mice compared to empty vector-injected WT mice during days 4-11 (p<0.05, N=8). However, IOP was not significantly elevated in TGFß2-injected KO mice compared to controls. Immunohistochemistry demonstrated that TGFß2 increased expression of collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC within the TM of WT mice, but only PAI-1 and CTGF in KO mice (p<0.05, N=3 pairs). These data support our hypotheses, indicating that SPARC plays an integral role in the modulation of aqueous humor outflow. In addition, it appears as though SPARC is essential to the regulation of TGFß2-mediated ocular hypertension. Aside from providing further evidence of the importance of ECM in IOP regulation, our work presents the novel discovery of segmental flow in the mouse. Given the potential role of SPARC in TGFß2-mediated ocular hypertension, SPARC may not only play an integral role in ECM homeostasis within the trabecular meshwork, but may be a valuable target for pharmacologic therapy in treating primary open-angle glaucoma.

Individuals with diabetes have an increased mortality due to the macro- and microvascular complications, which are commonly preceded by endothelial dysfunction. We have shown that endothelial microparticles (eMPs) are markers and mediators of vascular injury and pathology. However, their utility as a biomarker of hyperglycemia-induced endothelial damage and their influence on the vasculature remains unclear. We hypothesized that high glucose (HG) exposure alters eMPs protein composition, making them reflective of active signalling processes characteristic of a hyperglycemic environment. In addition, HG alters eMPs bioactivity, making them more potent inducers of oxidative stress, thrombosis and endothelial damage. Therefore, we assessed the exclusive effects of HG on eMPs formation, composition, and signalling. Results: Exposure of endothelial cells to high glucose for 24 hours caused a 3-fold increase in eMPs formation, increased mean vesicle size and their absolute electronegativity. Proteomic analysis of eMPs identified 1,212 independent proteins, with 68 exclusive to HG and associated with signalling processes related to metabolic processes, oxidation-reduction reactions, hemostasis and thrombosis and cellular interactions at the vascular wall. Compared to eMPs formed under normal conditions, eMPs formed in response to HG possess a ~3-fold greater procoagulant activity, induced a greater production of cellular ROS and were more potent inhibitors of endothelial-dependent relaxation. Conclusions/Interpretation: Taken together our results indicate HG alters the composition of eMPs, making them more potent mediators of endothelial damage. With similar changes in bioactivity being evident in the protein composition and the associated enriched biological processes, eMPs protein content may provide insight into the pathophysiological status of the cells in a hyperglycemic environment and provide use clinically, to identify dysregulated pathways for therapeutic targeting.

The unique pluripotent character of human embryonic stem cells (hESCs) places them in the forefront of scientific research, especially as they hold great promise for application in regenerative medicine, as well as drug discovery and toxicity analyses. Conventionally hESCs are cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) that are derived from E13.5 mouse embryos. One of the biggest challenges in the hESC field is the development of a reproducible and defined hESC culture system that would eliminate batch-to-batch variability of the MEFs as well as exposure to feeder cells that makes hESCs less applicable for clinical use. Previous studies have shown that maintenance of pluripotency can be achieved using Matrigel, a mixture of ECM components, or ECM derived from MEFs or human fibroblasts (Xu, et al., 2001, Klimanskaya, et al., 2005). Other groups have succeeded in culturing feeder-free hESCs by using extracellular matrix (ECM) proteins, such as fibronectin, vitronectin or laminin, as substrates for hESC culture in the absence of feeders, confirming that ECM plays a key role in maintaining hESC growth (Amit, et al., 2004, Braam, et al., 2008, Baxter, et al., 2009, Rodin, et al., 2010).The aim of this work was to investigate the ECM deposited by MEF feeder cells and to isolate and identify proteins in the ECM that support undifferentiated growth of hESCs in the absence of feeders. We have investigated whether matrices derived from different passage feeders differ in their ability to support pluripotency. I also assessed the integrin receptor profile of hESCs in order to define the mechanisms of ECM engagement. ECM was extracted from two strains of feeder cells, CD1 x CD1 and MF1 x CD1, at passages 4 (early passage), 9 and 14 (late passage), and assessed for its ability to support hESC self-renewal over at least 3 passages. Tandem mass spectrometry was used to analyse the ECM composition of each MEF line, thereby allowing a comparison between different passages and different cell lines. More than 100 proteins were identified for each sample, the majority of which were ECM proteins and shared between different passage feeders. As predicted, fibronectin, which is known to support hESC self-renewal was the most prevalent species in all MEF-derived matrices. Furthermore a proteomic analysis of matrix derived from hESCs cultured in feeder-free conditions on fibronectin coating substratum revealed a number of proteins shared between supportive MEF populations and hESC, suggesting other potential candidates that may either assist or interfere with the maintenance of pluripotent hESCs. Of the proteins identified fibrillin-1, perlecan, fibulin-2 were tested as substrates for culturing hESCs in the absence of feeders, with the prospect of developing an optimised feeder-free culturing system that uses a combination defined animal-free substrates. Finally this study sought to dissect the interaction between ECM and growth factors and how these extrinsic factors may affect self-renewal and maintenance of pluripotency-associated gene expression. Interruption of hESC attachment, as well as removal of growth factors appeared to affect transcript levels of pluripotency genes, OCT4 and NANOG, suggesting that the microenvironment can influence hESC fate.

Sepsis is among the most prevalent diagnosed critical illnesses in the United States today. Although advances have reduced the overall morbidity and mortality associated with this illness, the enormous number of deaths associated with it shows a need for improved diagnostic and therapeutic optionsgent. Our laboratory has utilized RNA based technologies to aid in the treatment of histone induced multiple organ dysfunction syndrome seen in sepsis. Histones are proteins found in the nucleus of every cell in our body and have been shown to be released during sepsis. Such release induces damage to other cells, causing a feed forward cycle that results in organ failure and death. Several therapeutics have been utilized to neutralize histones but have shown considerable toxicity. This thesis describes the generation of single stranded RNA aptamers to bind and neutralize histone mediate damage without unwanted toxicity. We demonstrate that our aptamers selectively bind to histones but not serum proteins. In addition, we establish that our aptamers can neutralize all histone mediated cellular response in vitro and in vivo. Finally, we determined that our aptamers are able inhibit the histone feed forward cycle in a temporal fashion in our murine model of multiple organ dysfunction. This novel therapeutic demonstrates the selectivity and effectiveness needed to inhibit histones in several critical illnesses.

Amyotrophic lateral sclerosis is a relentlessly progressive and fatal neuromuscular disease with no effective biomarkers, treatments or cure. In the early stages of ALS, it can be difficult to provide a diagnosis as patients do not meet diagnostic criteria until they become symptomatic, a sign of neuron degeneration. Early detection is therefore crucial to provide access to therapeutics prior to significant neuron loss. Extracellular vesicles are an ideal source of biomarkers as they contain a mix of proteins and nucleic acids reflective of the physiological state and are released from all cell types. We identified valosin-containing protein, integrin-beta 1 and gelsolin as potential biomarkers for ALS14 through proteomic analysis of EVs isolated from cell lines carrying the ALS-associated VCP-R155H mutation. My results indicate that EVs may serve as a valuable source of protein biomarkers in diagnostic, prognostic and predictive applications.

The ability to differentiate into mesenchymal lineages, as well as immunomodulatory, anti-inflammatory, anti-apoptotic, and angiogenic properties give ASCs great therapeutic potential. Through their culture as multicellular, three-dimensional spheroids this potential can even be enhanced. Accordingly, 3D spheroids are not only promising candidates for the application in regenerative medicine and inflammatory disease therapy, but also for the use as building blocks in tissue engineering approaches. Due to the resemblance to physiological cell-cell and cell-matrix interactions, 3D spheroids gain higher similarity to real tissues, what makes them a valuable tool in the development of bioactive constructs equivalent to native tissues in terms of its cellular and extracellular structure. Especially, to overcome the still tremendous clinical need for adequate implants to repair soft tissue defects, 3D spheroids consisting of ASCs are a promising approach in adipose tissue engineering. Nevertheless, studies on the use of ASC-based spheroids as building blocks for fat tissue reconstruction have so far been very rare. In order to optimally exploit their therapeutic potential to further their use in regenerative medicine, including adipose tissue engineering approaches, a 3D spheroid model consisting of ASCs was characterized extensively in this work. This included not only the elucidation of the structural features, but also the differentiation capacity, gene expression, and secretory properties. In addition, the elucidation of underlying mechanisms contributing to the improved therapeutic efficiency was addressed. / Humane mesenchymale Stammzellen aus dem Fettgewebe (ASCs) verfügen über ein großes therapeutisches Potenzial. Dieses reicht von ihrer Fähigkeit zur Differenzierung entlang der mesenchymalen Linien bis hin zu entzündungshemmenden und immunmodulatorischen Eigenschaften, was sie zu vielversprechenden Kandidaten im Einsatz für die regenerative Zelltherapie macht. Die Routinekultur dieser Zellen unter herkömmlichen 2D-Kulturbedingungen führt durch den Verlust der Umgebung in einem dreidimensionalen Gewebeverbund zur Beeinträchtigung ihrer regenerativen Fähigkeiten. Die Kultivierung in dreidimensionalen Zellaggregaten (Sphäroiden), in denen die Zellen in einem mehr physiologischen 3D Verbund innerhalb ihrer sekretierten Matrix interagieren können, erscheint somit als geeignete Möglichkeit das therapeutische Potential von ASCs zu steigern. Dies macht ASC-basierte Sphäroide nicht nur zu einem vielversprechenden Ansatz in der regenerativen Medizin im Allgemeinen, sondern auch zu einem innovativen Tool in ihrer Verwendung als „Bausteine“ für das Konzept des Tissue Engineerings. Insbesondere im Bereich des Fettgewebe-Engineerings besteht ein immenser klinischer Bedarf an der Entwicklung geeigneter Implantate für die Rekonstruktion von Weichteildefekten. Hierfür erscheinen Sphäroide aus den leicht und in ausreichender Menge zu isolierenden ASCs besonders attraktiv. Der effektive Einsatz solcher Sphäroide in der regenerativen Medizin, sowie im Fettgewebe-Engineering, erfordert jedoch zunächst ihre umfassende Charakterisierung in Bezug auf Strukturmerkmale, Differenzierungsfähigkeit und sekretorische Eigenschaften. Dazu wurden im ersten Teil dieser Arbeit der Prozess der Sphäroidbildung sowie die daran beteiligten Zell-Zell- und Zell-ECM-Interaktionen untersucht. Mit Hilfe der sogenannten Liquid-Overlay Technik gelang die reproduzierbare und kontrollierte Zusammenlagerung der ASCs in dreidimensionale Sphäroide innerhalb von 24 h. Hinsichtlich der Entwicklung von Extrazellulärmatrix-Komponenten während der Sphäroidbildung zeigte sich Fibronektin als die Matrix-Komponente, welche als Erste während des Prozesses zu detektieren war. Durch das Blockieren des Fibronektin-spezifischen β1-Integrins mittels eines spezifischen Antikörpers konnte ein Einfluss dieser frühen Fibronektin-Expression auf den Verlauf der Zellaggregation gezeigt werden. Bei dem hier blockierten β1-Integrin handelt es sich um einen zellulären Rezeptor, welcher maßgeblich an der Fibronektinbindung beteiligt ist. Eine Abhängigkeit der Sphäroidbildung von der Zell-Zell Interaktion mittels Cadherine konnte hingegen für die ASCs nicht nachgewiesen werden. Somit konnten im ersten Abschnitt neben einer detaillierten Beschreibung der ASC-Sphäroidbildung zusätzlich erste Erkenntnisse über die dem Prozess zugrundeliegenden Mechanismen gewonnen werden. Der effektive Einsatz von ASC-basierten Sphäroiden als Bausteine für das Fettgewebe-Engineering erfordert Kenntnisse sowohl über die Entwicklung der ECM als wichtigen gewebe-inhärenten Faktor, als auch über eine effiziente adipogene Induktion. Somit lag der Fokus im zweiten Teil dieser Arbeit auf der Charakterisierung der Extrazellulärmatrix in den ASC-Sphäroiden, sowie auf deren Fähigkeit zur adipogenen Differenzierung im Vergleich zur konventionellen 2D-Kultur unter der Verwendung verschiedener Induktionsprotokolle. Differenzierte Sphäroide weisen mit Laminin, Kollagen Typ I, IV und VI als nachgewiesenen Hauptbestandteilen eine Fettgewebs-spezifische Matrixzusammensetzung auf, die eine ausgeprägte Ähnlichkeit zu der des nativen Fettgewebes zeigt. Darüber hinaus konnte eine verbesserte Differenzierungsfähigkeit der ASC-Sphäroide nach einem kurzen induktiven Stimulus gegenüber der 2D kultivierten Zellen gezeigt werden. Dies wurde sowohl hinsichtlich des Triglyceridgehalts, sowie der Expression adipogener Markergene nachgewiesen. Diese verbesserte Differenzierbarkeit und die Möglichkeit mit den Sphäroiden fettgewebeähnliche Mikrogewebe herzustellen, machen diese Zellaggregate zu vielversprechenden Bausteinen für Ansätze im Fettgewebe-Engineering. Inzwischen ist bekannt, dass die Extrazellulärmatrix nicht nur als inertes, unterstützendes Gerüst wirkt, sondern auch zelluläre Prozesse, einschließlich der Differenzierung, durch die Interaktion mit zellulären Rezeptoren beeinflusst. Laminin als wichtige ECM Komponente der Basalmembran reifer Adipozyten konnte in der ECM der adipogen differenzierenden Sphäroide im Gegensatz zu 2D Kulturen zu einem sehr frühen Zeitpunkt der Adipogenese nachgewiesen werden. Um einen positiven Einfluss des Laminins auf den Prozess der adipogenen Differenzierung weitergehend zu untersuchen, wurde ein shRNA-vermittelter Knockdown eines spezifischen Laminin-Gens, welches für die Lamininkette α4 des adipospezifischen Laminin-8 Heterotrimers kodiert, durchgeführt. Obwohl ein stabiler Knockdown des LAMA4 Gens in den ASCs erreicht wurde, so konnte dennoch keine direkte Korrelation zur adipogenen Differenzierungsfähigkeit der Zellen festgestellt werden, da die reduzierte Expression dieses einzelnen Laminin-Gens sich nicht konsequent in der Expression des Gesamtlaminins auf Proteinebene durchsetzte. Das verbesserte therapeutische Potenzial mesenchymaler Stammzellen in einer dreidimensionalen Umgebung beschränkt sich nicht nur auf ihre verbesserte Differenzierungsfähigkeit, sondern umfasst auch die parakrine Sekretion der Zellen, die angiogene, anti-apoptotische, entzündungshemmende und immunmodulatorische Eigenschaften vermittelt. Um dies auch für ASCs zu bestätigen, wurden im letzten Teil dieser Arbeit die Genexpression von ASC-basierten Sphäroiden im Vergleich zu 2D kultivierten Zellen untersucht. Durch mRNA Genexpressionsanalysen konnte für ausgewählte entzündungshemmende, anti-apoptotische und anti-tumor wirksame Zytokine eine signifikant höhere Expression in den Sphäroiden gegenüber der 2D Kultur gezeigt werden. Für einen der wichtigsten entzündungshemmenden Faktoren, Prostaglandin E2, konnte eine erhöhte Sekretion von ASCs, kultiviert als 3D Sphäroide, auch auf Sekretionsebene bestätigt werden. Die Identifizierung intrinsischer Faktoren, welche zu dem verbesserten therapeutischen Potenzial der Zellen in einer dreidimensionalen Umgebung beitragen, ist noch ausstehend. Zur Untersuchung des Einflusses von Zell-Zellkontakten diente ein PCR-Array zum Screening hierfür relevanter Gene. Durch den Vergleich der beiden Kulturbedingungen, 2D Monolayer und 3D Sphäroide, sollten mögliche Unterschiede in den ASCs festgestellt werden können, welche eventuell für das verbesserte therapeutische Potenzial der Sphäroide mitunter verantwortlich sind. Dabei konnten eindeutige Unterschiede zwischen ASCs aus der 3D bzw. 2D-Kultur festgestellt werden. Diese Ergebnisse bilden die Grundlage für weitere Untersuchungen auf diesem Gebiet, um einem umfassenden Verständnis der Rolle von Zell-Zell- und Zell-ECM-Interaktionen in einem 3D-Sphäroid-Kultursystem näherzukommen. Zusammengefasst tragen die Ergebnisse dieser Arbeit zu einer umfangreichen Charakterisierung der ASC Sphäroide hinsichtlich struktureller Merkmale, Differenzierungsfähigkeit und sekretorischer Eigenschaften bei. Es wurde gezeigt, dass ASCs der 3D Sphäroide nicht nur eine verbesserte Differenzierungsfähigkeit, sowie eine erhöhte Expression von Genen aufweisen, welche für verschiedene Zytokine codieren, sondern ebenso in der Lage sind Fettgewebs-ähnliche Mikrogewebe zu bilden. Diese Erkenntnisse tragen dadurch insgesamt zur Förderung der ASC Sphäroide in ihrer effektiven Anwendung in der regenerativen Medizin und im Bereich des Fettgewebe-Engineerings bei.

Breast cancer (BC) is the second leading cause of death in Canadian women. Detection of the disease at an early stage greatly increases the average 5-year survival rate, however non-invasive early detection methods are not available to-date. Cells release various types of extracellular vesicles (EVs) to mediate intercellular communication by transferring signals in the form of bioactive molecules such as proteins, metabolites, and nucleic acids. Understanding the composition of these biomolecules may shed light on the physiological state of the cell of origin. Therefore, EVs are a promising source of biomarkers for non-invasive detection of BC. However, the surface proteome of EVs is not yet understood well enough to propose BC biomarkers that could be detected directly from biofluids. In this study, small EVs (sEVs) and medium EVs (mEVs) were isolated by differential ultracentrifugation from breast cancer MDA-MB-231 and MCF7, and non-cancerous breast epithelial MCF10A cell lines and analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. EV proteins were analyzed by two approaches: (1) global proteomic analysis and (2) enrichment of EV surface proteins by labelling surface-accessible proteins with a Sulfo-NHS-SS-Biotin reagent. Potential BC biomarkers were obtained from the first approach (1) by identifying the presence of cell line specific sEV proteins, filtering for membrane/surface proteins using UniProt annotations, and predicting the co-localization of proteins on sEVs with known EV marker proteins (CD63, CD9, CD81) by correlation analysis. This resulted in 11 potential BC sEV biomarkers (C8A, AXL, ST14, FAM20B, PROM2, CLDN3, ITGA7, MEGF10, SHISA2, GJC1, IFNGR1); the presence of ST14, CLDN3 and ITGA7 was validated by Western blot analysis. The surface labelling approach (2) enriched proteins previously not identified using the first approach (1). Potential general BC biomarkers were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. Annotation with known BC disease associations from DisGeNET yielded 9 and 2 potential surface proteins on sEVs and mEVs, respectively. This study demonstrates the emerging role of EVs as a rich source of known and novel biomarkers which may be used for non-invasive detection of BC.