ATHANASIOS ARAMPATZIOGLOU

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1 DEMOCRITUS UNIVERSITY OF THRACE SCHOOL OF HEALTH SCIENCIES DEPARTMENT OF MOLECULAR BIOLOGY & GENETICS Master s Programme of Studies «Translational Research in Molecular Biology and Genetics» Title Neutrophil extracellular traps impede cancer cell growth in vitro by inducing apoptosis and reducing the proliferation by ATHANASIOS ARAMPATZIOGLOU MASTER THESIS March 2016

2 THESIS COMMITTEE 1. IOANNA MAROULAKOU (Prof. of Genetics Supervisor) 2. KONSTANTINOS RITIS (Prof. of Internal Medicine co-supervisor) 3. PANAGIOTIS SKENDROS (Ass. Prof. of Internal Medicine) 2

3 TABLE OF CONTENTS Page Ευχαριστίες....5 Abbreviations...7 Abstract Introduction Key facts about cancer To date progress in cancer knowledge and treatment Cancer and immune response Neutrophils, a key player in the immune response Neutrophils in cancer NETs, a key mechanism of neutrophils The key role of NETs in non-infectious diseases NETs in cancer and cancer-associated thrombosis TF, the main in vivo initiator of coagulation and its non-thrombotic manifestations Rationale of the study Materials and Methods Cell isolation NET structure generation and isolation Verification and quantification of NET structures Cell culture..38 3

4 2.5. Stimulation and inhibition studies Surface area covered by cells Cell proliferation and apoptosis Staining Statistical analysis Results NETs inhibit in vitro growth and induce apoptosis of colon cancer cells NETs inhibit acute myeloid leukemia cells growth in vitro Discussion 49 References 52 4

5 ΕΥΧΑΡΙΣΤΙΕΣ Η παρούσα μεταπτυχιακή εργασία εκπονήθηκε από τον φοιτητή Αραμπατζιόγλου Αθανάσιο, στα πλαίσια παρακολούθησης του Μεταπτυχιακού Προγράμματος Σπουδών με τίτλο «Μεταφραστική Έρευνα στη Μοριακή Βιολογία και Γενετική», του τμήματος Μοριακής Βιολογίας και Γενετικής του Δημοκρίτειου Πανεπιστημίου Θράκης (ΔΠΘ). Το πειραματικό μέρος της μελέτης πραγματοποιήθηκε στο εργαστήριο Μοριακής Αιματολογίας που ανήκει στον τομέα Γενικής Παθολογίας του Τμήματος Ιατρικής του ΔΠΘ, υπό την από κοινού επίβλεψη της Καθηγήτριας Γενετικής στο τμήμα Μοριακής Βιολογίας και Γενετικής κας Ιωάννας Μαρουλάκου και του Καθηγητή Παθολογίας στην Α Παθολογική Κλινική του Πανεπιστημιακού Γενικού Νοσοκομείου Έβρου (ΠΓΝΕ) και Διευθυντή του Εργαστηρίου Μοριακής Αιματολογίας κ. Κωσταντίνου Ρίτη. Θα ήθελα να ευχαριστήσω θερμά τον κ. Κωσταντίνο Καμπά, Μοριακό Βιολόγο και Διδάκτορα Ανοσολογίας, την κα Ακριβή Χρυσανθοπούλου, Βιολόγο και Διδάκτορα Ανοσολογίας, την κα Βικτώρια Τσιρωνίδου, Βιολόγο Τεχνικό Εργαστηρίου και Υποψήφια Διδάκτορα Ανοσολογίας, καθώς και τον κ. Παναγιώτη Σκένδρο, Επίκουρο Καθηγητή Παθολογίας, για τη στήριξη, τη βοήθεια που μου παρείχαν και τις πολύτιμες συμβουλές τους. Ιδιαίτερες ευχαριστίες θα ήθελα να εκφράσω στην κα. Στέλλα Αρελάκη, Ειδικευόμενη Ιατρό και Υποψήφια Διδάκτορα της Παθολογικής Ανατομικής, για την άριστη συνεργασία που είχαμε καθ όλη τη διάρκεια διεξαγωγής της συγκεκριμένης μελέτης. Επίσης, θα ήθελα να ευχαριστήσω θερμά τον κ. Θεοχάρη Κωνσταντινίδη, Ιατρό Βιοπαθολόγο και Υποψήφιο Διδάκτορα Ανοσολογίας, την κα. Ειρήνη Αποστολίδου, Ειδικευόμενη Ιατρό της Παθολογίας, την κα. Ηλιάνα Αγγελίδου Βιολόγο και Υποψήφια Διδάκτορα Ανοσολογίας, καθώς και τον κ. Αλέξανδρο Μήτσιο, Μοριακό Βιολόγο και μεταπτυχιακό φοιτητή του Μεταπτυχιακού Προγράμματος Σπουδών με τίτλο «Μεταφραστική Έρευνα στη Μοριακή Βιολογία και Γενετική», για τις εποικοδομητικές μας συζητήσεις και τη βοήθεια που μου προσέφεραν. 5

6 Επιπλέον, θέλω να ευχαριστήσω βαθύτατα τους γονείς μου, Αντώνιο Αραμπατζιόγλου και Ιωάννα Κιοσέ, καθώς και τον αδερφό μου, Δημήτριο Αραμπατζιόγλου, για την αμέριστη ηθική υποστήριξη, τη συνεχή συμπαράσταση και την κατανόηση που έδειξαν προς το πρόσωπό μου όλον αυτόν τον καιρό. Τέλος, θα ήθελα να τονίσω πως αυτή η διπλωματική εργασία δεν θα ήταν δυνατό να πραγματοποιηθεί χωρίς την αμέριστη στήριξη και αρωγή της κας Ιωάννας Μαρουλάκου, Καθηγήτριας Γενετικής του τμήματος Μοριακής Βιολογίας και Γενετικής, καθώς και του κ. Κωσταντίνου Ρίτη, Καθηγητή Παθολογίας στην Α Παθολογική Κλινική του ΠΓΝΕ και Διευθυντή του Εργαστηρίου Μοριακής Αιματολογίας του ΔΠΘ. Οφείλω και στους δύο εξίσου τις θερμές και ειλικρινείς μου ευχαριστίες για την ευκαιρία που μου έδωσαν να συνεργαστώ μαζί τους, την επιστημονική καθοδήγηση, την υποστήριξη και τις γνώσεις που μου προσέφεραν καθ όλη τη διάρκεια διεκπεραίωσης της παρούσας διπλωματικής εργασίας, καθώς και για την προσπάθειά τους να μου παρέχουν κάθε ευκαιρία για τη μελλοντική μου εξέλιξη. Οι υποδείξεις και οι συμβουλές τους με κατεύθυναν σε έναν σωστό τρόπο σκέψης και μου προσέφεραν σημαντικά εφόδια για τη μετέπειτα ακαδημαϊκή μου πορεία. 6

7 ABBREVIATIONS AAV: ANCA associated vasculitis AML: acute myeloid leukemia ANCA: anti-neutrophil cytoplasmic antibody APS: antiphospholipid syndrome C5a: complement component 5 alpha CFSE: 5(6)-carboxyfluorescein diacetate N-succinimidyl ester CG: cathepsin G CTCs: circulating tumour cells DCs: dendritic cells DNase I: Deoxyribonuclease I ECM: extracellular matrix EMEM: Eagle's Minimum Essential Medium ERK: extracellular signal-regulated kinase ES: Ewing sarcoma FBS: fetal bovine serum FMF: familial Mediterranean fever IL: interleukin MAPK: mitogen-activated protein kinase MF: myofibroblasts MMP: matrix metalloproteinase 7

8 MPO: myeloperoxidase MPs: microparticles NE: neutrophil elastase NETs: neutrophil extracellular traps NK: natural killer cells PAD4: peptidylarginine deiminase 4 PARs: protease activated receptors PBS: phosphate-buffered saline solution PenStrep: penicillin and streptomycin PI: propidium iodide PI3K: phosphoinositide3-kinase PMA: phorbol 12-myristate 13-acetate PMNs: polymorphonuclear cells ROS: reactive oxygen species SLE: systemic lupus erythematosus STEMI: ST-segment elevation acute myocardial infarction TANs: tumour-associated neutrophils TF: tissue factor TFPI: Tissue Factor Pathway Inhibitor WHO: World Health Organization 8

9 ABSTRACT Background The role of neutrophils in tumour biology is largely unresolved. Nowadays it is well known that, under certain inflammatory stimuli, neutrophils produce neutrophil extracellular traps (NETs), fibrous structures composed of nuclear DNA and granular cytoplasmic and nuclear proteins. Recently, independent studies indicated NETs or Tissue Factor (TF) involvement in cancer biology and associated thrombosis. However, the mechanisms by which neutrophils, NETs and TF are implicated in cancer progression are still unidentified. Methods Both TF-bearing and TF-negative NETs were generated in vitro, following treatment of neutrophils derived from healthy donors with the appropriate agents. NET release was verified and quantified using MPO/DNA complex ELISA. Subsequently, these in vitro generated NETs were co-cultured with Caco-2 cell line and acute myeloid leukemia (AML) primary cells, in order to examine the effect of NETs on cancer cell growth. Finally, proliferation and apoptosis/necrosis of cancer cells were analyzed by flow cytometry, after 4 days of co-culture with NETs. Results Irrespectively of the NET-generating stimulus, NETs acted as potent in vitro inhibitors of colon cancer cells growth by inducing apoptosis in these cells. Moreover, NETs impeded growth of acute myeloid leukemia primary cell cultures by inducing apoptosis and inhibiting proliferation. Conclusions These data support the role of neutrophils and NETs in cancer biology, suggesting that NETs, independently of their triggering stimulus and the presence or absence of TF, act as generic potent inhibitors of cancer cells by inducing apoptosis and reducing the proliferation rate. 9

10 1. INTRODUCTION 1.1. Key facts about cancer Cancer is a leading cause of morbidity and mortality worldwide. According to the World Health Organization (WHO), 8.2 millions of people die each year from cancer, with metastasis and cancer-associated thrombosis being the main causes of death. Estimated incidence, mortality and prevalence for all cancers (excluding nonmelanoma skin cancer), worldwide in Nowadays, it is known that more than 100 different cancer types exist, each requiring unique diagnosis and treatment [1]. Among men, the 5 most common sites of cancer are lung, prostate, colorectum, stomach, and liver, while breast, colorectal, lung, uterine cervix, and stomach cancer are the most common types of cancer among women [1]. More than 30% of cancer deaths could be prevented by modifying or avoiding key risk factors, including tobacco use, being overweight or obese, unhealthy diet with low fruit and vegetable intake, lack of physical activity, alcohol use, sexually transmitted HPV-infection, infection by HBV, ionizing and non-ionizing radiation, urban air pollution, and indoor smoke from household use of solid fuels [1]. Early detection and accurate diagnosis are essential for adequate and effective treatment, as every cancer type requires a specific treatment regimen which encompasses one or more therapeutic approaches such as surgery, and/or radiotherapy, and/or chemotherapy [1]. The primary target is to cure cancer or to considerably prolong life. Improving the patient's quality of life is also an important goal. 10

11 Estimated age-standardized incidence and mortality rates for men, worldwide. Estimated age-standardized incidence and mortality rates for women, worldwide. 11

12 1.2. To date progress in cancer knowledge and treatment Over the last 60 years, cancer research and clinical practice have led to significant progress in the knowledge of cancer biology and treatment of the disease [2]. Many causes and factors have been proposed to account for cancer. Namely, cancer has been attributed to multiple factors such as weakness of the anti-tumour activity of the immune system, expression of genes involved in the positive (proto-oncogenes) or negative (suppressor-genes) regulation of cell growth, imbalance of anti-apoptotic and pro-apoptotic pathways in favour of the former and disruption of cell differentiation [2]. However, the ultimate secret leading to the cure of malignant tumours has not been found yet. Nowadays, the attention of the biomedical world has been focused mainly on the harnessing of patient s own immune system in order to fight cancer [2]. The existence of an immune response to the emergence of cancerous cells is generally accepted, and there has been made great progress on elucidating such an anti-tumour response and the way to activate it, but the results obtained in clinical practice were until now rather discouraging [2,3]. The strong relationship between cancer development and the immune response demonstrates that the progress in cancer knowledge and treatment is probably indebted to the progress in cancer immunity [2,3] Cancer and immune response It is well known that the relationship between cancer and the immune system is not limited only to surveillance against rising monoclonal disorders, but also against established malignant tumours. The interplay between the immune system and cancer determines many aspects of the clinical presentation and the progression of the disease. Inflammation is one of the commonly recognized hallmark features of cancer. Cancer-related inflammation pertains mainly to the local immune response found at the site of the tumour, which frequently precedes and contributes to its development [3]. In established cancers, there is increasing evidence that both local and systemic inflammation are implicated in progression of the disease and survival of cancer 12

13 patients [3]. Local inflammation encompasses both tumour-derived and host-derived cytokines, inflammatory protein mediators and infiltrating immune cells, which act in the local tumour microenvironment and can mediate initiation and promotion of carcinogenesis [3]. On the other hand, systemic inflammation involves cytokines, inflammatory proteins and immune cells, which are present and detectable in the systemic circulation and can give rise to the paraneoplastic symptoms observed in patients with cancer [3]. There is substantial cross-talk between the mediators and the cytokines of the local tumour microenvironment and those of the systemic circulation, so it is commonly believed that the targeting of cancer-related inflammation has the potential to favourably affect both compartments, and thus benefit the patient [3]. Local and systemic inflammatory response (Diakos et al, Lancet Oncol, 2014). 13

14 1.4. Neutrophils, a key player in the immune response Human neutrophils are an essential part of the innate immune system, as they constitute the majority of the white blood cells, representing 50 to 70% of the total circulating leukocytes [4,5]. They are also known as granulocytes due to the presence of granules in their cytoplasm, or as polymorphonuclear cells (PMNs) due to their distinctive lobed nuclei (2 to 5 lobes are present per nucleus) [4,5]. Neutrophil granules contain a variety of toxic substances that kill or inhibit a variety of pathogens. Neutrophil granules contain myeloperoxidase (MPO), neutrophil elastase (NE), cathepsin G, lysozyme and difensins [4,5]. A simplified schematic representation of the neutrophil morphology. Neutrophils are produced through the process of hematopoiesis in the bone marrow. They have a very short lifespan, so neutrophil homeostasis is maintained by continuous release of many neutrophils from the bone marrow [4-6]. The bone marrow of a normal healthy adult produces more than 100 billion neutrophils per day. The average lifespan of non-activated circulating neutrophils is 5 to 90 hours, but when they are activated they live for another 2 days in the migration tissue [6]. Accelerated neutrophil death decreases neutrophil counts (a situation known as neutropenia) and increases susceptibility to infection. In turn, delayed neutrophil death increases neutrophil counts (a situation known as neutrophilia) and intensifies innate defenses, possibly promoting chronic inflammation [6]. 14

15 Blood smear showing a neutrophil circulating within the blood stream ( Under normal circumstances, neutrophils circulate within the blood stream waiting to be called into action [4-7]. During inflammation, a variety of inflammatory mediators are produced that act as chemotactic factors, attracting neutrophils to the site of inflammation. These mediators may be components of the complement, components of the blood coagulation system, or even cytokines produced by the T- helper cells and the macrophages [7]. Cell surface receptors allow neutrophils to detect these molecules (e.g. interleukin-8, interferon gamma, C3a, C5a) and as a result, neutrophils leave the blood stream and are rapidly recruited to the site of infection, where they constitute the first line of defense [7]. Under inflammatory conditions, neutrophils are attracted to the site of inflammation (iahealth.net/inflammation). 15

16 In order to contain and clear an infection, neutrophils employ three strategies: Phagocytosis, release of soluble anti-microbials (commonly known as degranulation), and generation of neutrophil extracellular traps (also known as NETs) [7,8]. A simplified schematic representation of the three anti-microbial strategies of neutrophils (Papayannopoulos et al, Trends in Immunology, 2009) Neutrophils in cancer Although neutrophils generally constitute the most prominent inflammatory cell population, relatively little is known about their implication in human cancers. Nevertheless, recent studies demonstrated that neutrophils mediate the crosstalk between cancer and immune system [9,10]. Various immune and non-immune cells are present in the tumour microenvironment, in addition to tumour cells themselves. Prior studies have demonstrated important roles for many of these cells, including lymphocytes, natural killer (NK) cells, macrophages, fibroblasts, endothelial cells, and pericytes [11]. As regards neutrophils, until recently they were commonly thought to be just a casual observer in the tumour microenvironment, and not a disease modifying entity. This view was mainly based on the belief that such a short-lived cell could not impact a chronic, progressive disease. However, recent studies demonstrated that tumour-associated neutrophils (also known as TANs) are fully capable of modifying tumour growth and invasiveness [9,10,12-17]. 16

17 In untreated tumours, TANs appear to develop a pro-tumourigenic phenotype, termed N2 TAN in analogy to the M2 macrophage phenotype, which seems to contribute to tumour growth and suppression of the antitumour immune response [9,10]. Nowadays it is well known that many cell types within the tumour microenvironment are capable of secreting neutrophil chemotactic substances. Interestingly, the tumour cells themselves often secrete CXC chemokines, such as IL-8, mediating neutrophil recruitment to sites of tumourigenesis [9,10]. This finding strongly suggests that TANs are not a means of host defense. Interestingly, neutrophil depletion experiments on murine models have led to inhibition of tumour growth [15], limitation of metastases number [16], and reduction of endothelial cell recruitment to tumours [17]. Furthermore, neutrophilia has been associated with a poorer prognosis in cancer patients [18-20], while the neutrophil to lymphocyte ratio has been proposed as a prognostic factor in several types of cancer, such as colon cancer [21]. Namely, according to a relatively recent study, the presence of intratumoural neutrophils in patients with localized renal cell carcinoma is related to increased mortality [22]. In addition, according to a prior study, increased levels of tumour infiltrating PMNs in patients with bronchioalveolar carcinoma are significantly associated with poor outcomes [23]. Moreover, TANs have been identified as the main component and driver of metastatic establishment within the pre-metastatic lung microenvironment, in mouse breast cancer models [13]. In this direction, tumour-infiltrating neutrophils have been reported to facilitate metastasis, mainly by altering the microenvironment of the metastatic lesion [12-14]. And last but not least, several recent studies highlight the importance of TANs by using neutrophil-derived agents, such as chemokines and/or cytokines, reactive oxygen species (ROS), and matrix-degrading proteinases, in order to impact tumour immune surveillance, metastasis, angiogenesis, and cellular proliferation [9]. 17

18 A scheme of the cells and factors mediating intratumoural recruitment of neutrophils (Fridlender et al, Carcinogenesis, 2012). Effects of neutrophil-derived products on the tumour microenvironment (Gregory et al, Cancer Res, 2011). 18

19 In contrast to the above mentioned studies reporting several pro-tumourigenic effects of TANs, there are also some recent studies, mainly based on engineered tumour cell lines or specific therapies, which report anti-tumourigenic roles for these cells [9]. Such studies demonstrate that neutrophils can assume a more tumourcytotoxic phenotype, termed N1 TAN, having the potential to kill tumour cells and inhibit tumour growth [24-27]. Thus, depletion of these N1 TANs either augments tumour growth and/or blunts the anti-tumourigenic effects of immunologic treatments [24,28-30]. Additionally, there are studies reporting that TANs can be modified such that they become more cytotoxic to tumour cells [24-26,31]. However, it is not clarified whether manipulation of neutrophils into this N1 form is dangerous for the host tissue. Hence, inhibiting neutrophil recruitment or neutrophil-derived substances with known tumour-promoting properties might prove to be more efficacious and with fewer concerns for toxicity. Concluding, TANs are a distinct population of neutrophils, which in their basic unmanipulated state are induced by the tumour microenvironment in order to elicit pro-tumourigenic responses (N2 phenotype). However, there is increasing evidence that these cells are also capable of assuming anti-tumourigenic roles (N1 phenotype). Thus, it seems that neutrophils are an important, underappreciated cell population in cancer biology, and their functions need to be better characterized. Deciphering the way that TANs support or fight cancer is crucial to develop strategies directing the immune system against tumours. The pro- and anti-tumourigenic effects that have been described in neutrophils (Fridlender et al, Carcinogenesis, 2012). 19

20 1.6. NETs, a key mechanism of neutrophils As mentioned above, generation and release of neutrophil extracellular traps, a process commonly termed NETosis, is one of the main strategies that neutrophils employ in order to contain and kill pathogens [4-8]. NETs are extracellular chromatin structures, formed under certain inflammatory stimuli and composed of cytoplasmic, granular and nuclear components of neutrophils [4-8,32]. The impact of NETs as an antimicrobial response derives from the combined antimicrobial activities of both cytoplasmic and granular proteins, as well as histones [4-8]. As regards NETs morphology, their ultrastructure is unusual. They consist of smooth filaments with a diameter of about 17 nm, composed of stacked and probably modified nucleosomes [33]. This backbone is studded with globular domains with a diameter of about 50 nm, made of granular proteins. In high-resolution scanning electron microscopy, this morphology easily differentiates NETs from other fibrous structures, such as fibrin [33]. Interestingly, unfixed, fully hydrated NETs have a cloud-like appearance and occupy a space that is 10 to 15-fold bigger than the volume of the cells they originate from [33]. This reflects what they may look like in vivo, when space is available. Scanning electron microscopy of bacteria caught in NETs (Brinkmann et al, J Cell Biol, 2012). 20

21 Neutrophil extracellular traps are the result of a unique form of cell death, termed NETosis, during which activated neutrophils undergo dramatic morphological changes [4-8,34]. Intracellular membranes disintegrate and elastase enters the nucleus, followed by hypercitrullination of histones, chromatin decondensation and extrusion of nuclear material from the cell. Extracellular DNA complexes with histones and granular enzymes to form a sticky network of NETs that can entrap endogenous (e.g., platelets) and exogenous (e.g., bacteria) particles and molecules [4-8,34]. Namely, minutes after activation, neutrophils flatten and firmly attach to the substratum. During the next hour, the nucleus loses its lobules, the chromatin decondenses, and the inner and outer nuclear membranes progressively detach from each other. Concomitantly, the granules disintegrate. After 1 h, the nuclear envelope disaggregates into vesicles and the nucleoplasm and cytoplasm form a homogenous mass. Finally, the cells round up and seem to contract until the cell membrane ruptures and the interior of the cell is ejected into the extracellular space, forming NETs [4-8,34]. Notably, despite the intermixing of cellular compartments, during the last phase of NETosis, less than 30 proteins are present on NETs. Most of them originate from granules, few are from the nucleus, while cytoplasmic NET components are rare [4-8,34]. In conclusion, NETosis is morphologically quite different from apoptosis and other forms of cell death [6]. A schematic representation of the mechanism of NETosis (Brinkmann et al, J Cell Biol, 2012). To date, there have been reported various physiological NET inducers, such as infections with bacteria, fungi, and HIV parasites [4-8,34]. In suspension, NET formation is poor, probably in order to prevent excessive NETosis in circulation and thus avoid thrombus formation, which is mentioned in details below. 21

22 At molecular level, there are some events that have been reported to be required for NETosis. These events are, sequentially, the production of ROS, the migration of NE and later MPO from the granules to the nucleus, the processing of histones, and eventually the rupture of the cell [6,34]. As regards the production of ROS, the NADPH oxidase enzyme complex (also called phagocytic oxidase; PHOX) assembles at the cell and phagosomal membrane and reduces molecular oxygen into superoxide anions by transferring electrons from NADPH. Superoxide dismutates into hydrogen peroxide, which in turn acts as substrate for one of the most abundant enzymes in the neutrophil s granules, the MPO. MPO reacts with hydrogen peroxide to generate hypohalous acids, such as hypochlorous acid (HOCl). ROS oxidize various types of molecules including nucleic acids, lipids, and proteins [6,34]. During NETosis, the segregation between eu- and heterochromatin is lost, and the nucleoplasm appears homogenous. This depends on the activity of NE and MPO, which are stored in azurophilic granules. NE is released, through an unknown mechanism, from granules and enters the nucleus, where it degrades the linker histone H1 and processes core histones [6,34,35]. NE activity is essential for NET formation because NE-deficient mice do not make NETs, which contributes to their immune deficiency. MPO also migrates to the nucleus later than NE, where it enhances chromatin decondensation [6,34,35]. In agreement with this requirement, patients without MPO activity cannot produce NETs, and hypochlorous acid, the product of MPO, is sufficient for NET release. In addition to partial degradation by NE, histones undergo further modifications to decondense the chromatin structure [6,34,35]. Upon neutrophil activation, the enzyme peptidylarginine deiminase 4 (PAD4) catalyzes the conversion of arginine residues to citrulline in three of the four core histones. In NETs and decondensed nuclei, but not in the nucleus of unstimulated neutrophils, histones are citrullinated [6]. The relevance of PAD4 was tested pharmacologically in cell lines, which make few NETs, if any, but not in neutrophils. In PAD4-null mice, hypercitrullination of H3 was not detectable, and the strain failed to produce NETs [36]. Interestingly, in a S. pyogenes infection model, PAD4-null mice developed larger lesions than their PAD4-expressing siblings, but NET formation remains to be quantified in this model [36]. 22

23 A schematic representation of the proposed model for the regulation of NETosis (Remijsen et al, Cell Death and Differentiation, 2011). Eventually, NETs are removed during the resolution of inflammation. NETs are susceptible to Deoxyribonuclease I (DNase I), an enzyme produced by the pancreas [37]. It is not known what happens to the debris left by DNase I, but perhaps phagocytes, macrophages, and neutrophils newly recruited to the inflammatory site clean up the mess. 23

24 NETs are rather fragile structures, and much effort is required to unambiguously detect and quantify them. NET quantification should rely on their unique composition: chromatin tightly linked to neutrophil proteins such as NE, MPO, or calgranulin. This definition excludes chromatin released by other forms of cell death. Published methods of NET quantification include microscopy and DNA detection either with membrane-impermeable DNA dyes or by staining the DNA in the supernatant after release of the NETs with a mild nuclease treatment. Immunostaining is an obvious way to detect NETs, but it is prone to biases introduced by the observer [4,7]. Moreover, automatic microscopy is an objective and quantitative method to measure NET formation [38]. Changes in nuclear morphology (loss of lobules and expansion of the nucleus) and composition (migration of NE and MPO to the nucleus) are specific and quantitative markers of the progress of NETosis [4,7]. Anti-chromatin antibodies stain the compact nuclei of unstimulated neutrophils weakly, but the signal increases as the chromatin relaxes [4,7]. In tissue sections and in secretions, NETs have been identified using the same markers. Computer-assisted analysis of the overlap between chromatin and neutrophil markers can quantify NETs in tissue sections. Although more technically challenging, NETs can also be identified in vitro and in vivo by measuring their size and detecting their antigens by scanning or transmission electron microscopy. NET visualization as chromatin and DNA filamens, decorated with NE (Brinkmann et al, J Cell Biol, 2012). 24

25 1.7. The key role of NETs in non-infectious diseases Besides the role of NETs against infections, recent studies demonstrate that neutrophil extracellular traps are also strongly implicated in non-infectious diseases, such as thrombosis [39,40], autoimmune diseases [41], autoinflammatory disorders [42], cardiovascular diseases [40], fibrosis [43], and cancer [44]. Coagulation is a way to reduce blood loss after injury, but it also represents a primitive innate immune response that limits microbial spreading [39-41]. Coagulation is an example of how the amount of NET formation can determine a good or bad outcome [39-41]. NETs participate in timely clot formation, but if present in excess they induce massive coagulation that can stop the blood supply of organs, causing severe ischemia. Arterial blood clots are often induced by damage to the endothelium. In contrast, venous thrombi mainly develop when the blood flow is reduced for several hours. In both situations, neutrophils accumulate and adhere tightly to the endothelium [39-41]. There, neutrophils produce NETs that serve as a scaffold for the stimulation of thrombus formation [40]. Both NE and cathepsin G, two serine proteases that are on the NETs, degrade inhibitors of coagulation. In mice deficient in both enzymes, during arterial thrombosis, fibrin deposition and clot formation are reduced, as is the case when the mice are treated with an anti-net antibody [46,47]. Interestingly, in experimental Escherichia coli sepsis, the proportion of bacteria sequestered in the microvasculature of the liver was higher in animals with functional NETs than in animals treated with an anti-chromatin antibody that blocks NET function, underlining the fact that coagulation also reduces bacterial spread to other organs [45]. Together, these data indicate that clotting is enhanced by NETs, promoting defense against pathogens. Although the vessel is not damaged at the onset of venous thrombogenesis, numerous neutrophils and macrophages are recruited and play a major role during formation of the thrombus. There, activated platelets stimulate neutrophils to form NETs [45], which serve as a prothrombotic scaffold and bind and activate FXII [46]. Consequently, NETs can be detected in venous thrombi [47]. 25

26 As regards the implication of NETs in autoimmune diseases, a recent study described a novel mechanism for the hypercoagulability and infliction of tissue injury in anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) [41]. Neutrophil activation by inflammatory mediators and ANCA induces expression of tissue factor (TF) and release of TF expressing NETs and microparticles (MPs). It is proposed that the subsequent activation of the extrinsic coagulation cascade may have a significant pathogenic role in hypercoagulability that characterizes active AAV [41]. Additionally, signaling through protease activated receptors (PARs) and resulting activation of endothelial, epithelial and/or mesangial cells could be an alternative pathway for the involvement of neutrophil derived TF in the pathogenesis of AAV [41]. Recently has been demonstrated that the inflammatory attack of familial Mediterranean fever (FMF), a classical autoinfammatory disorder, is characterized by the production of bioactive interleukin-1β (IL-1β) by PMNs and its release through NETs [42]. These findings propose a two-hit model for the trigger of acute inflammatory response in FMF that possibly characterizes various neutrophilassociated disorders [42]. Furthermore, regarding the implication of NETs in cardiovascular diseases, a recent study demonstrated for the first time the interaction between activated platelets and TF-loaded neutrophils at sites of atherosclerotic plaque rupture for the release of active TF-bearing NETs in humans with ST-segment elevation acute myocardial infarction (STEMI) [40]. A variety of inflammatory stimuli in STEMI promote the de novo expression of TF in neutrophils. In parallel, locally activated platelets interact with neutrophils for the release of TF-bearing NETs inside the culprit artery [40]. The functionality of TF depends on the integrity of the NET structure and is able to induce thrombin generation and platelet activation, creating a possible vicious cycle that leads to thrombus propagation and stability [40]. 26

27 A schematic representation of the two hit TF/NET model in STEMI (Stakos et al, European Heart Journal, 2015). Recently, the implication of NETs in fibrosis has also been demonstrated. It is suggested that neutrophils migrate into inflamed tissue in response to inflammatory stimuli to promote fibrosis through NET release [43]. NET components, such as chromatin, histones, and MPO, are involved in the differentiation of fibroblasts, whereas other NET molecules (e.g. cytokines, such as interleukin-17) further promote the fibrotic potential of myofibroblast (MF) phenotype cells [43]. It is proposed that neutrophil infiltration in tissues affected by chronic inflammation or recurrent inflammatory bouts, caused by either pathogens or environmental agents, may perpetuate tissue injury through NET release [43]. NET components, including histones, antimicrobial peptides, and cytokines, in conjunction with a possible defect in NET clearance by either DNase or macrophages may impact on fibroblast activation, contributing to disease progression towards fibrosis [43]. 27

28 1.8. NETs in cancer and cancer-associated thrombosis Recently, NETs have redefined the role of neutrophils in tumour biology [12-14,44,48-50]. To date, it has been suggested that NETs may act within the primary tumour promoting tumour progression [12,44,49], while at remote sites they might sequester circulating cancer cells favoring metastasis [13,14,50]. Additionally, NETs have been implicated in cancer-associated thrombosis [44,48]. The role that NETs play in tumour progression remains poorly understood, as it has only recently started to be characterized. To date only few studies have been performed, but there is increasing evidence suggesting a potential association between intra-tumoural NET deposition and tumour progression, in both experimental models and cancer patients [49-51]. A finding that supports the important role of NETs in tumour biology is the ability of tumour cells to predispose neutrophils to undergo NETosis. Namely, a recent study reported that in Ewing sarcoma (ES), the second most common primary bone cancer afflicting adolescents and young adults, ES cells can stimulate TANs to release NETs, which may serve as a marker of poor prognosis [49]. Moreover, another recent study demonstrated that multiple tumour types, including hematologic, mammary and lung neoplasms, are able to predispose circulating neutrophils to produce NETs [48]. The data presented thus far suggest that NET formation is induced within the primary tumour by a number of neoplasms. Based on the redundancy of this finding across a number of tumour types, it is feasible that intra-tumoural NET deposition confers some type of advantage to the neoplasm in question [51]. Indeed, NETs appear to facilitate primary tumour development through the inhibition of apoptosis and also via a direct proliferative effect [48,49,51-53]. Given the above mentioned findings, arises the possibility that NETs promote tumour progression, ultimately leading to metastasis. Collectively, the data presented thus far suggest that NETs may act within the primary tumour, promoting tumour progression. However, until today, no studies have demonstrated the mechanism(s) underlying these observations. As previously mentioned, NETs are composed of neutrophil-derived chromatin decorated with antimicrobial proteins and peptides, such as matrix metalloproteinase 9 (MMP-9), cathepsin G (CG) and NE. The role of these NET components in tumour progression has been examined, but no specific reference to NETs has been made [12]. As stated 28

29 before, NETs provide a microenvironment that traps pathogens and brings them into close proximity with antimicrobial peptides, favoring their elimination. Thus, it is possible that in the context of malignancy NETs play an analogous role, whereby tumour cells are exposed to a high local concentration of biologically active proteins, favoring interactions that may act to promote proliferation, inhibit apoptosis and support egress from the primary tumour [12]. As mentioned before, the role of neutrophils in metastasis remains unclear. However, recent studies have shown that neutrophils directly interact with cancer cells and favor their migration [54]. Moreover, the presence of neutrophils was shown to establish a seeding bed for metastatic cancer cells [55]. On the other hand, a toxic effect of tumour-activated neutrophils on cancer cells has also been described [56]. Some recent studies demonstrate increasing evidence that the aforementioned phenomena may also implicate NETs. Namely, in proximity of the vessel wall, NETs may favor the attachment of the cancer cell to the vessel and support extravasation [57]. Moreover, through their proteases and the binding of adhesion molecules, such as fibronectin, NETs may generate a seeding soil and promote tumour cell migration [57]. Finally, NETs could also cover circulating cancer cells with platelets and enhance immune escape. The inappropriate or excessive NET deposition has been associated with ongoing inflammation and tissue damage [8,58-60]. In cancer, this persistent inflammatory state may result in ongoing expression of adhesion molecules, facilitating tumour cell entrapment and interaction with the extracellular matrix (ECM) [61-64]. Adhesive events between disseminated tumour cells and end organ vasculature have been shown to support the development of gross metastasis. Moreover, both in vitro and in vivo studies have revealed that neutrophils play a central role in arresting circulating tumour cells (also known as CTCs) [50,54,62,64,65]. Under infectious/inflammatory conditions, when NET formation is supported, such interactions appear to be enhanced [62,64,66]. Thus, given that one of the primary roles attributed to NETs is the sequestration of intravascular bacteria, probably NETs act in an analogous manner to capture CTCs. Accordingly, by sequestering tumour cells and bringing them into close proximity with a number of neutrophil-derived factors, NETs may generate a microenvironment for the trapped tumour cells rich in proteins and enzymes that 29

30 facilitate their progression. Following dissemination and adhesion, CTCs must be able to proliferate in order to form stable metastatic foci. A recent study suggests that, under certain conditions, NETs may play a direct proliferative role and may also inhibit apoptosis, promoting metastasis formation [51]. Another study demonstrates that, following sequestration within NETs, CTCs are able to form stable micro metastatic foci and ultimately go on to form macro metastases. This implies that trapped cells are able to survive interactions with NETs, while they are also able to grow [13,14,50]. Taken together, these data support a potential pro-metastatic role for NETs, involved in every step of the metastatic cascade, from early adhesion, proliferation and invasion to angiogenesis. A schematic representation of the postulated pro-tumorigenic roles of NETs (Cools-Lartigue et al, Cell Mol Life Sci, 2014). 30

31 NETs could be implicated in many steps of tumor progression (Demers et al, OncoImmunology, 2013). NETs have also been implicated in cancer-associated thrombosis, the second most common cause of death in cancer patients. Even in the absence of obvious thrombosis, cancer patients represent a hypercoagulable condition without a clear etiology. A recent study demonstrated that, through the generation of NETs, neutrophils provide a scaffold and a stimulus for platelet adhesion and thrombus formation [57]. NETs were shown to promote coagulation as well [57,67]. Since an increased risk of thrombosis is associated with cancer, it was hypothesized that tumour-induced neutrophils might have a role in cancer-associated thrombosis. Thus, based on a mammary cancer model, another recent study demonstrated that as cancer progresses, NETs are spontaneously formed in the blood and their presence correlates with signs of thrombosis [48]. Moreover, further studies based on murine models reported that both leukemia and solid tumours produce a factor that primes neutrophils to undergo NETosis and predisposes the host to thrombosis. In this direction, G-CSF, which induces neutrophilia and neutrophil activation and is produced by many tumours, was reported to be such a priming factor [48]. Concluding, NETs have been identified as a key player in cancer-associated thrombosis and, thus, as a new potential target in the effort to minimize the incidence of thrombotic events in cancer patients [48]. 31

32 1.9. TF, the main in vivo initiator of coagulation and its non-thrombotic manifestations TF is a 47 kda transmembrane glycoprotein that shares high homology in secondary and tertiary structure with interferon γ receptors, and constitutes a member of the human class II cytokine receptor family. Nowadays, TF is considered as the main in vivo initiator of coagulation [40,68-70]. The presence of multiple binding sites in the promoter region of the gene indicates multipotent expression in a large variety of cells and under a vast array of stimuli [68]. Under normal conditions, TF is not expressed in endothelial cells, but only in sub-endothelial tissue, thus creating a protecting envelope between blood and sites of expression. However, under specific inflammatory conditions, TF is expressed in endothelial cells and myeloid leukocytes [40,68-70]. There is increasing evidence indicating the presence of circulating TF in blood, commonly known as blood-borne TF. The peripheral blood cells are a potential source of blood-borne TF. Although monocytes have been reported to constitutively express TF, there is emerging evidence indicating that other cell populations may also be implicated in the generation of blood-borne TF [40,68-70]. Apart from the role of the extrinsic coagulation system (also known as the TFthrombin axis) in thrombosis, this system has also been associated with several nonthrombotic models, such as angiogenesis, tumor growth and metastasis, inflammation, and fibrosis [70,71]. The serine proteases of this pathway, namely TF/FVIIa, Xa, and thrombin, are able to signal through the protease activated receptor (PAR) receptor family to produce intracellular signals via the phosphoinositide3-kinase (PI3K) pathway, the Src tyrosine kinase pathway, the extracellular signal-regulated kinase (ERK) pathway, and the mitogen-activated protein kinase (MAPK) pathway [70,71]. The activation of these pathways results in the secretion of cytokines and chemokines implicated in several biological functions. Increased prevalence of venous thrombotic events is a long standing observation in patients suffering from infectious and sterile inflammatory disorders. Venous thrombosis constitutes a major morbidity and mortality factor in inflammatory diseases, including sepsis, systemic lupus erythematosus (SLE), inflammatory bowel disease, and vasculitis [68-71]. Additionally, recent clinical data derived from patients with rheumatoid arthritis and SLE support the critical role of inflammation 32

33 in accelerated atherothrombosis. Experimental evidence links the observed thrombogenicity with TF-dependent activation of extrinsic coagulation cascade. Increased TF expression by endothelial and blood cells exposed to inflammatory mediators is proposed as an essential part of the pathogenic mechanism for arterial and venous thromboembolism that characterizes inflammatory disorders [68-71]. These observations indicate a potential triggering role of inflammation in thrombosis. However, the relationship between inflammation and thrombosis is bidirectional, since thrombosis can reignite inflammation creating a persistent or recurrent inflammatory environment [68-70]. TF-thrombin axis enhances the inflammatory response in several clinical models such as arthritis, antiphospholipid syndrome (APS), ischemia/reperfusion injury, and sepsis. Signaling through PARs plays a critical role for this reciprocal process. TF/FVIIa complex has been implicated in the induction of inflammation in the aforementioned clinical models. In an endotoxemic animal model, both TF deficiency and combined inhibition of thrombin and deficiency in PAR2 reduced inflammation. Further studies in animal models of sepsis demonstrated that extrinsic coagulation cascade inhibition with a varying range of anticoagulants, such as natural anticoagulants, Tissue Factor Pathway Inhibitor (TFPI), Protein C, and Antithrombin III, attenuated the persisting inflammation. Moreover, it has been recently shown that thrombin is able to generate biologically active C5a from C5 in the absence of C3, indicating a significant role in the reignition of inflammation. However, the physiological contribution of this pathway has to be further investigated. This data establish the reciprocal and close relationship between the thrombosis and inflammation [68-70]. A schematic representation of the TF - thrombin axis ( 33

34 In addition, TF has been demonstrated to play a key role in both cancer-associated thrombosis and metastasis, although up to date the main focus was TF originating from cancer cells [72,73]. As already mentioned, thrombosis is a major cause of morbidity and mortality in cancer patients. The pathogenesis of the cancer-associated hypercoagulability is determined by the cancer cell-specific prothrombotic properties, together with the host cell inflammatory response [72,73]. TF is known to be the most important procoagulant protein expressed by cancer cells, as it highly contributes to the procoagulant phenotype of malignant cells [72,73]. Recent studies indicate that, in cancer tissues, oncogenes determine the expression of the procoagulant proteins, including TF. Moreover, it is suggested that TF is also overexpressed by host normal blood cells, triggered by cancer-derived inflammatory stimuli [72,73]. Accordingly, as demonstrated by aberrations of circulating thrombotic biomarkers, a subclinical activation of blood coagulation is typically present in cancer patients. The importance of measuring these biomarkers, in order to determine the patient thrombotic risk level, is under investigation. The ultimate goal is to identify the high-risk subgroups, so as to establish more accurate and targeted anticoagulation strategies in order to prevent thrombosis in cancer patients. Finally, the clarification of the particular molecular mechanisms triggering blood coagulation in specific cancer types may also reveal alternative ways to inhibit clotting activation in such patients [72]. However, in this direction, the combined role of TF and NETs in cancer biology has never been addressed. 34

35 1.10. Rationale of the study Recent studies demonstrated that neutrophils participate in the crosstalk between cancer environment and immune system, however their biological significance and functional role in human cancers remains largely elusive. A great body of evidence indicates NET formation as a key effector and regulatory mechanism of neutrophils in infection, inflammation and thrombosis. Furthermore, the role of NETs in tumour biology represents an emerging research field today. However, most of the data derive from animal experimental studies, while the pathophysiological significance of NETs in human cancer has not been investigated yet. Taken together, the elucidation of the mechanisms through which NETs are involved in cancer and cancer-associated thrombosis, would provide insights triggering further research for novel diagnostic and/or therapeutic targets. Thus, we investigated for the first time the presence of NETs in primary tumours and metastatic lymph nodes of colon adenocarcinoma patients, and their in vitro effects in cultures of colon cancer cells and primary leukemic cells. 35

36 2. MATERIALS AND METHODS 2.1. Cell isolation Peripheral blood neutrophils were isolated from heparinized blood samples, derived from healthy donors: 3 ml of blood were dissolved in 3 ml of 0.9% sodium chloride. Diluted blood was stowed in 3 ml Ficoll-histopaque 1119 (Polysucrose, 6.0 g/dl and sodium diatrizoate, 16.7 g/l; Sigma-Aldrich, St Louis, MO, USA) and in 3 ml Ficoll-Histopaque 1077 (Polysucrose 57 g/l, and sodium diatrizoate, 90 g/l; Stem Cell Technologies, Vancouver, Canada). A centrifugation was performed at 2000 rpm for 30 min, at room temperature. Two distinct opaque layers were formatted. Each layer was washed with 10 ml of phosphate-buffered saline solution (PBS; Gibco BRL, New York, USA) 1x. A centrifugation was performed at 1400 rpm for 10 min, at room temperature. Supernatant was discarded and cells were measured and assessed for viability. The preparation of the gradient must be done immediately before use. As blood ages the cell recoveries will drop, so the procedure has to be as fast as possible. Primary human acute myeloid leukemia cells were isolated from peripheral blood samples, derived from patients at the University Hospital of Alexandroupolis, Greece. Mononuclear cells were isolated using Biocoll Separating Solution according to the manufacturer's instructions (Biochrom, Berlin, DE), frozen in liquid nitrogen, and processed within 4 months after cryopreservation. The AML samples consisted of more than 95% of CD34+ cells, therefore, no further purification was performed. After thawing, the cells were resuspended in Myeloid Long-Term Culture Medium (MyeloCult H5100; Stem Cell Technologies). Peripheral blood samples of patients with acute myeloid leukemia were obtained after informed consent. AML diagnosis was made in accordance with the World Health Organization criteria NET structure generation and isolation To generate NETs, neutrophils, derived from healthy donors, were seeded in each well of a six-well culture plate (Corning Incorporated, New York, USA), in low-serum RPMI medium (Gibco BRL). After 20 min, neutrophils were exposed to sepsis serum [39], isolated from blood samples derived from septic 36

37 patients at the Academic Hospital of Alexandroupolis, Greece, or to phorbol 12- myristate 13-acetate (PMA) (40 ng/ml; Sigma-Aldrich), a generic inducer of NET release, for 210 min. These concentrations and time points were optimal for neutrophil stimulation according to optimizing experiments. Similarly, untreated neutrophils were used as control. Cells in each well of the culture plate were washed with 1 ml of RPMI medium, pre-warmed in room temperature for 10 min, in order to get the stimuli removed. Vigorous shaking of the culture plate was performed for 2 min, in order to get the NETs detached from the bottom and released into the medium. 750 μl of the supernatant were collected from each well of the plate, in a 15 ml centrifuge tube. A centrifugation was performed at 500 rpm for 5 min, at 4 o C. 700 μl of the supernatant fluid (in which NETs float at this point) were collected in a new falcon. NETs containing solution was stored at -20 o C. MPO/DNA complex ELISA was used to quantify NET release and MPO/DNA complex was measured in NET structures isolated from neutrophils Verification and quantification of NET structures To quantify NET release, MPO/DNA complex ELISA (R&D Systems, Minneapolis, USA) was used in NET structures isolated from neutrophils. NET release was depicted as % increase compared with controls. Each well of the plate with high binding F-bottom was covered with 50 μl anti-mpo (5 μg/ml) antibodies, diluted in PBS 1x (1/500). The plate was next covered with parafilm and stored at 4 o C overnight. At the next day we removed the solution (hit and wash 3 times with 200 μl PBS 1x). In each well we transferred 20 μl from the sample for assessment and 80 μl incubation buffer containing anti-dna Mab (1:25 dilution). We incubated the plate for 2 h at room temperature, covered with parafilm and shaking. Next, we removed the solution and washed 3 times with incubation buffer. We added ABTS solution (poroxidase substrate) and incubated the plate for 20 min at room temperature, shaking, resulting in the production of green colour. The intensity of the green colour was proportional to the quantity of MPO/DNA complex. Thereafter, the reaction was terminated by adding acid ABTS (Stop Solution). Each sample was measured in 405 nm. 37

38 2.4. Cell culture Caco-2 cells were cultured in 5% CO2, at 37 C, in L-glutamine Eagle's Minimum Essential Medium (EMEM; Gibco BRL, New York, USA) containing 10% of fetal bovine serum (FBS; Gibco BRL) and 1% of penicillin and streptomycin (PenStrep; Gibco BRL). Cells were subcultured using trypsin-edta (Gibco BRL) at a subcultivation ratio of 1:4 to 1:6, as soon as they reached about 80% confluent. Medium was renewed 1 to 2 times per week. Frozen cells were stored at -80 o C. AML cells were cultured in 5% CO2, at 37 C, in Myeloid Long-Term Culture Medium (MyeloCult H5100; Stem Cell Technologies) containing 10% of FBS and 1% of PenStrep. Fresh medium was added every 2 days of culture. Frozen cells were cryopreserved in liquid nitrogen vapor Stimulation and inhibition studies Both Caco-2 and AML cells were stimulated with 500 ng of NET structures isolated from neutrophils treated with PMA or sepsis serum, or whole PMNs pretreated with these agents, for 4 days. For NET scaffold inhibition, isolated NET structures were pre-incubated with DNase I (10 U/ml; Fermentas, Vilnius, Lithuania) or heparin (heparin sodium, 100 μg/ml; LEO Pharma A/S, Ballerup, Denmark) for 60 min. For TF inhibition an IgG1 mouse anti-human TF mab (10 μg/ml; Sekisui Diagnostics) was used Surface area covered by cells Four images randomly taken from different regions of each well with 100x magnification per experiment were analysed. Average surface area covered by cells was calculated with Fiji/ImageJ [74] Cell proliferation and apoptosis For the analysis of cell proliferation, CFSE proliferation assay was used. After washing with PBS 1x, AML cells were resuspended at 10 x 10 6 cells/ml in PBS 1x 38

39 containing 6% of FBS and labeled with a final concentration of 3 μm 5(6)- carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Sigma-Aldrich) for 8 minutes, at room temperature. The reaction was stopped by the addition of an equal volume of 100% FBS, followed by incubation for 1 min, at room temperature. Cells were washed once with 1 ml of PBS 1x containing 50% of FBS and once with PBS 1x. The CFSE-labeled mononuclear cells were resuspended in L-glutamine Eagle's Minimum Essential Medium containing 10% of FBS and 1% of PenStrep, and cultured for 4 days in 5% CO2, at 37 C. On day 4, cells were collected, washed and stained extracellularly with the following monoclonal antibodies: CD34 APC (clone 8G12; BD Biosciences, New Jersey, USA) and CD45 PerCp (clone 2D1; BD Biosciences). For the analysis of apoptosis/necrosis, Phosphatidyl Serine Detection Kit (IQ Products, Groningen, Netherlands) was used. After washing with 500 μl of calciumbuffer, Caco-2 and AML cells were resuspended in 100 μl of calciumbuffer and stained with 5 μl of FITC-annexin V (BD Biosciences) for 30 minutes at room temperature. AML cells were also labeled with 3 μl of CD34 APC (clone 8G12) during this incubation. After washing with 500 μl of calciumbuffer, cells were resuspended in 200 μl of calciumbuffer and stained with 5 μl of propidium iodide (PI; Sigma-Aldrich) for 30 minutes at room temperature. Proliferation and apoptosis analysis were performed after 4 days of co-culture with NETs in a FACScalibur flow cytometer (BD Biosciences). All data were analyzed with Flowjo V Staining To assess the effect of PMA and sepsis serum-induced NETs on culture growth, Caco-2 and AML cells were stained with May-Grünwald (Merck KGaA, Darmstadt, Germany) and Giemsa (Merck KGaA). After washing three times with 2 ml of PBS 1x, cells were stained with 2 ml of May-Grünwald for 5 min, at room temperature. Cells were washed three times with 2 ml of destil H2O. Caco-2 and AML cells were stained with 2 ml of Giemsa for 15 min, at room temperature. Cells were washed three times with 2 ml of destil H2O. Visualization was performed by using light microscopy (Leica DM2000). 39

40 2.9. Statistical analysis Statistical analyses were performed using one-way analysis of variance (ANOVA) with Scheffé test for post hoc comparisons. P values less than 0.05 were considered significant. All statistical analyses were performed with OriginPro 8. 40

41 3. RESULTS 3.1. NETs inhibit in vitro growth and induce apoptosis of colon cancer cells Since it has been recently suggested that NETs are implicated in cancer progression and metastasis in murine lung and mammary tumour models [12,44,50], we investigated whether NETs are present in human colon adenocarcinoma. Interestingly, by examining tumour specimens of colectomy for adenocarcinoma from ten patients, we observed that both neutrophils and NETs are present in the vicinity of cancer cells, while the intensity of neutrophil infiltration and NET generation is proportional to the proximity to the tumour (not shown data, submitted for publication). Moreover, we observed that both neutrophils and NETs are important sources of TF in tumour microenvironment (not shown data, submitted for publication). Based on the above findings and bearing in mind that the biological significance of neutrophil extracellular traps in cancer progression remains unclear, we investigated the possible role of NETs in solid tumour progression in in vitro co-cultures of Caco-2 cells, a colon cancer cell line. In addition, to further examine the role of TF, we used both PMA-induced generic NETs that do not contain TF (i.e. TF-negative) and sepsis serum-induced NETs which are able to express TF (i.e. TF-bearing) [39]. In vitro generation and isolation of PMA-induced and sepsis serum-induced NETs were performed as mentioned thoroughly in the materials and methods section. Isolated NET structures were stored at -20 o C and they were thawed only once, within one month after generation, in order to be used in co-cultures. Verification and quantification of in vitro generated NETs were performed by using MPO/DNA complex ELISA (for more details see the materials and methods section). NET release was depicted as % formation of MPO/DNA complex (Figure 1). 41

42 Figure 1. Verification and quantification of in vitro generated NETs. NET release was depicted as % formation of MPO/DNA complex. Data from four independent experiments presented as mean ± SD. Both PMA and sepsis serum-induced NETs inhibited in vitro Caco-2 growth compared to controls (Figure 2), which also demonstrated apoptotic-like morphology. Dismantling of NETs with DNase I, a NET chromatin scaffold inhibitor, abolished their inhibitory effect (Figure 2). Figure 2. NETs inhibit growth in Caco-2 cultures. May-Grünwald-Giemsa staining of Caco-2 cells co-cultured with PMA or sepsis serum-induced NETs in the presence or absence of DNase I. One representative out of four independent experiments is shown. Original Magnification 100x. 42

43 Similar results were also obtained when either PMA or sepsis serum-pretreated neutrophils were used in Caco-2 cultures (Figure 3). Figure 3. PMA or sepsis serum-pretreated neutrophils inhibit growth in Caco-2 cultures. May-Grünwald-Giemsa staining of Caco-2 cells co-cultured with PMNs pretreated with PMA or sepsis serum. One representative out of four independent experiments is shown. Original Magnification 100x. The measurement of the surface area covered by Caco-2 cells in each case demonstrated that the presence of PMA or sepsis serum-induced NETs led to a remarkable reduction of in vitro Caco-2 growth, compared to untreated cells (Figure 4). Figure 4. NETs remarkably reduce in vitro Caco-2 growth. Percentage of surface area covered by cells. Data from four independent experiments presented as mean ± SD. n.s. - not significant compared to control, *p <

44 We next investigated whether NETs inhibit Caco-2 growth via apoptosis. Thus, Caco-2 cells were treated with the aforementioned agents and demonstrated increased levels of apoptotic and late apoptotic cells, as assessed by Annexin V/PI flow cytometry (Figures 5A-B). DNase I attenuated this effect on apoptosis (Figures 5A- B). Moreover, apoptosis induction by NETs was concentration-dependent (data not shown). Figure 5. NETs induce apoptosis in Caco-2 cultures. Annexin V/PI flow cytometry of Caco-2 cells co-cultured with PMA or sepsis serum-induced NETs in the presence or absence of DNase I. (A) Representative scatter plots. (B) Data from four independent experiments presented as mean ± SD. n.s. - not significant compared to control, *p <

45 These data indicate that NETs, irrespectively of the NET-generating stimulus, act as potent inhibitors of colon cancer cells growth in vitro by inducing apoptosis in these cells NETs inhibit acute myeloid leukemia cells growth in vitro To further clarify whether this inhibitory role of NETs in cancer cell growth is specific only to colon adenocarcinoma or occurs with other types of malignancy as well, we investigated their effects in human hematopoietic cancer. Thus, we conducted in vitro co-culture experiments of primary AML cells in the presence of NETs. Similarly to Caco-2 cells, PMA and sepsis serum-induced NETs significantly suppressed the growth of AML cells in vitro (Figure 6), compared to controls. DNase I abolished this inhibitory effect of NETs (Figure 6). Figure 6. NETs inhibit growth of AML cells in vitro. May-Grünwald-Giemsa staining of AML cells co-cultured with PMA or sepsis serum-induced NETs in the presence or absence of DNase I. One representative out of four independent experiments is shown. Original Magnification 100x. Similar results were also obtained when either PMA or sepsis serum-pretreated neutrophils were used in AML cultures (Figure 7). 45

46 Figure 7. PMA or sepsis serum-pretreated neutrophils inhibit growth of AML cells in vitro. May-Grünwald-Giemsa staining of AML cells co-cultured with PMNs pretreated with PMA or sepsis serum. One representative out of four independent experiments is shown. Original Magnification 100x. We investigated whether NETs suppress AML growth via affecting their proliferative activity. As assessed by flow cytometry, NET-treated AML cells demonstrated a remarkable reduction of their proliferation compared to untreated cells (Figures 8A-B). Figure 8. NETs reduce proliferation of AML cells in vitro. CFSE flow cytometry of AML cells co-cultured with either PMA or sepsis serum-induced NETs in the presence or absence of DNase I. (A) Representative scatter plots. (B) Data from four independent experiments presented as mean ± SD. n.s. - not significant compared to control, *p <