- Email: [email protected]
Ultrastructural investigation of the time-dependent relationship between breast cancer cells and thrombosis induction
Micron 90 (2016) 59–63
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Ultrastructural investigation of the time-dependent relationship between breast cancer cells and thrombosis induction Wendy J. van der Spuy ∗ , Tanya N. Augustine School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
a r t i c l e
i n f o
Article history: Received 1 June 2016 Received in revised form 9 August 2016 Accepted 16 August 2016 Available online 27 August 2016 Keywords: Platelet Fibrin network Microparticle SEM Ultrastructure
a b s t r a c t Thromboembolic complications are a common cause of death in breast cancer patients. The in vivo relationship between coagulation factors and circulating tumours is a multifaceted one, with tumour cells implicated in thrombocytosis and platelets associated with coagulation-mediated metastasis. Platelets and fibrin networks are known to be morphologically altered in patients with cancer, and their relationship with breast cancer cells themselves may increase thrombosis susceptibility. This was investigated in vitro, assessing such morphological alterations through the establishment of a MCF-7 breast cancer cell co-culture system with blood plasma. Co-culture duration ranged from zero to thirty minutes, with five-minute intervals, in order to assess the time-dependent ultrastructural conformations of platelet and fibrin networks, using scanning electron microscopy. It was found that enhanced coagulability was related to co-culture duration. Changes in platelet and fibrin network morphology from normal were visible as early as five minutes in co-culture with MCF-7 cells. With longer co-culture duration thrombosis-linked variation in structural configuration was intensified, including advanced platelet aggregation and adherence characteristics, compression of fibrin networks into plaques of increased density, and upsurge of microparticulate extrusion implicated in amplifying procoagulant events during the metastatic process. These results confirm that cancer cells are stimulators of coagulation even in an in vitro system and breast cancer patients may become increasingly susceptible to thrombotic-related consequences with increased exposure to tumour cells, especially during metastasis. © 2016 Published by Elsevier Ltd.
1. Introduction Thromboembolic complications are the second most common cause of mortality in breast cancer patients (Falanga and Donati, 2000; Nash et al., 2002). The in vivo interaction between circulating tumours and coagulation factors is complex and reciprocal – with platelet activation and aggregation implicated in facilitating coagulation-mediated metastasis, and tumour-derived cytokines and growth factors implicated in thrombocytosis (Bambace and Holmes, 2011). Specifically, following intravasation into the vascular system, tumour cell-secreted factors induce platelet activation and aggregation, thereby protecting tumour cells from high velocity shear forces and immunosurveillance (Bambace and Holmes, 2011). Thus malignancy itself is associated with increased thromboembolic risk (Falanga and Donati, 2000; Nash et al., 2002).
∗ Corresponding author at: School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193, South Africa. E-mail addresses: [email protected], [email protected] (W.J. van der Spuy). http://dx.doi.org/10.1016/j.micron.2016.08.006 0968-4328/© 2016 Published by Elsevier Ltd.
Furthermore, as depicted in Fig. 1, inflammation (leukocyte involvement) and platelet activation are closely interlinked (van der Spuy and Pretorius, 2013), and though not in the scope of this paper, inflammation is imperative in malignant processes (Jurasz et al., 2004). Morphological alterations in platelet and fibrin network morphology have long been associated with thrombotic risk and such architectural shifts are evident in disease conditions such as diabetes, stroke and cancer (Pretorius et al., 2009, 2011a,b). It is thus understood that the in vivo relationship between breast cancer cells and coagulation factors may lead to changes in platelet and fibrin network morphology as well as function, increasing susceptibility to thrombosis. It is known that cancer cells are able to produce platelet agonists including thrombin, which not only induce platelet activation (Picker, 2011) but are a requirement for fibrin network formation. During normal haemostasis, the generation of a fibrin clot and subsequent fibrinolysis forms part of the coagulation process. However, in disease states including thromboembolic ischemic stroke, fibrin clot structure and fibrinolysis can be dysregulated (Pretorius et al., 2011b). The higher the amount of coagulation agonists produced and the longer the exposure dura-
60
W.J. van der Spuy, T.N. Augustine / Micron 90 (2016) 59–63
Fig. 1. Platelet activation is central to cancer progression. Platelet activation amplifies the inflammatory response, stimulates thrombosis processes, assists in the extravasation of circulating cancer cells, and has a role in the establishment of new tumour growth.
tion to these agonists, the further and more rapidly are platelets expected to advance through phases of activation, aggregation and full adherence. This study was designed to investigate the in vitro environment in order to ascertain how soon tumour cells affect coagulation ability or propensity for thrombosis, as a starting point for further studies into their relationship.
2. Materials and methods A co-culture system was established, culturing MCF-7 luminal phenotype breast cancer cells with the blood plasma of seemingly healthy female individuals. Applicable exclusion criteria included smoking, contraceptive use or pregnancy, and a history of autoimmune disease or cancer. The experiment was repeated in triplicate (conducted separately) with the blood of two different volunteers per experiment (n = 6). Ethical clearance was obtained from the University of the Witwatersrand’s Human Ethics Committee (M081036 and M140155). MCF-7 cells were plated at a concentration of 1 × 105 cells per coverslip in a 24-well plate and incubated for 24 h at 37 ◦ C and 5% CO2 in normal DMEM media (Dulbecco’s Modified Eagles Medium, Lonza, Verviers, Belgium; BE12-604F), with 10% heat-inactivated FBS (Foetal Bovine Serum, Biowest, South America; S1810-500) and 0.1% P/S (Penicillin/Streptomycin, Highveld Biological, RSA; 214). Normal media was replaced with Phenol Red Free (PRF)-DMEM media (Lonza, Verviers, Belgium; BE12-115F) with 10% Dextran Coated Charcoal (DCC)-stripped FBS, 0.1% P/S and l-glutamine, and incubated for 48 h at 37 ◦ C and 5% CO2 for cell cycle synchronization (Gil et al., 2013). Peripheral blood was obtained via venepuncture into citratecontaining vacutainers (Lasec, South Africa). Diagnostic data suggests that citrated platelets – in plasma which is separated from the whole blood whence it is obtained by centrifugation – do not activate upon standing even after 1500g centrifugation for up to 15 min, and such separated plasma remains stable for diagnostic haemostasis testing up to 24 h in an unrefrigerated state (Favaloro et al., 2012). However, we have also shown that centrifugation at 400×g for the generation of PRP, may induce early activation as evidenced by the loss of CD62p (p-selectin) containing-microparticles (Augustine et al., 2016). As such in this study, platelet-rich plasma (PRP) was obtained by centrifuging whole blood for 5 min at 1000 rpm (200×g). Co-cultures were thus implemented by expos-
ing PRP to cells for periods of zero (control – no exposure of plasma to cells) up to 30 min with intervals of 5 min, after which platelet and fibrin network coagula were prepared on glass coverslips for scanning electron microscopy (SEM). MCF-7 cells were not prepared for scanning electron microscopy in this experiment, as it was designed specifically to determine co-culture duration for further experiments. Platelet coagula were prepared using only 20 l of platelet rich plasma, whereas fibrin coagula were prepared through activation of 15 l platelet rich plasma with 5 l of 20 U/ml human thrombin (SANBS, South Africa (van der Spuy and Pretorius, 2013)). Prepared coverslips were incubated on 0.1 M phosphate buffered saline (PBS)-dampened filter paper for 2 min to ensure adherence, after which a washing process followed where the coverslips were submerged in PBS on a shaker for 20 min to ensure removal of trapped proteins. This was followed by primary fixation in 2.5% formaldehyde/glutaraldehyde solution for 15 min, three rinses in PBS, secondary fixation in 1% osmium tetroxide (OsO4 ) for 15 min, three rinses in PBS and then serial dehydration in 30%, 50%, 70%, 90% and three times absolute ethanol. SEM procedures were completed by drying the samples with hexamethyldisilazane (HMDS), mounting on aluminium stubs and coating by carbon evaporation. The ultrastructural alterations in platelet and fibrin network morphology were then assessed with a ZEISS ULTRA plus FEG Scanning Electron Microscope at the University of Pretoria’s Microscopy and Microanalysis Unit in South Africa. Accelerating voltage was set at 1 kV for high quality surface analysis and samples imaged with the In-lens secondary electron detector. For each of the coagulum coverslips prepared, ten low magnification images were acquired to give an overall perspective of the level of coagulation, and ten high magnification images were acquired for clear visualization of morphological characteristics. Therefore, for each time point, 60 high magnification images were taken of platelets and fibrin networks, respectively, and a representative image was selected for depiction in this report.
3. Results Control preparations of PRP not exposed to tumour cells showed typical morphology for platelets and fibrin networks. Platelets in the zero co-culture control groups displayed discoid to slightly rounded platelets with smooth membrane surfaces and little to no pseudopodia. No aggregates were present in these groups. Fibrin
W.J. van der Spuy, T.N. Augustine / Micron 90 (2016) 59–63
61
Fig. 2. Control platelet and fibrin network ultrastructure. Ultrastructure was studied preparing coagula from plasma without (plasma) or with (fibrin) the addition of thrombin. (A) Control platelet, unexposed to tumour cells, showing typically discoid-shaped cell body and smooth membrane surfaces. (B) Control fibrin networks, unexposed to tumour cells, showing typical mesh formation with major (thick) and minor (thin) fibres interspersed sparingly between them (Scale bar = 1 m). Key: Star, platelet central body; thin dotted arrow, major fibrin fibre; thick dotted arrow, minor fibrin fibre.
networks were smooth in appearance showing typical major and minor fibre lattices. Fig. 2 shows a typical control platelet (Fig. 2A) and normal fibrin network formation (Fig. 2B). Fig. 3 shows experimental platelet coagula alongside corresponding fibrin networks as per co-culture duration. After 5 min of co-culture with MCF-7 cells, platelets (Fig. 3A) were found to be ball- to hemisphere-shaped indicating activation and firm but reversible adhesion. Some aggregation of platelets in small groups of two to four was visible. Membrane surfaces were still smooth but with the appearance of folds and pseudopodia. The spread of hyalomeres was evident in approximately 20% of platelets at this time. Fibrin networks (Fig. 3B) displayed thickening and clumping of primary fibres and net formation adjacent to and over primary fibre masses at this time. After 10 and 15 min of co-culture, platelet (Fig. 3C) hyalomere spread had increased and was evident in approximately 40–50% of platelets, indicating a shift towards full and irreversible adhesion. Pseudopodia were less prominent at this time and in some samples markedly shortened when compared to the 5-min point. Most platelets were visible in aggregates of two or more and their membrane surfaces appeared roughened. Fibrin networks (Fig. 3D) showed further thickening of primary fibres as they clumped together further, indicating increased stickiness of the fibres. Nets became increasingly dense and in some instances formed plaques. Additionally, intertwined platelets became conspicuous. After 20 and 25 min of co-culture, larger aggregated groups of platelets were seen (Fig. 3E). Pseudopodia were short and thickened and hyalomere spreads tended to be conjoined. Membrane surfaces still displayed rough folds and microparticular debris was visible. In fibrin (Fig. 3F), the dense nets completely masked primary fibres which were sometimes visible beneath the net through larger holes. Visible platelets were found to be completely trapped within the dense nets and more plaque formation was also evident. After 30 min of co-culture, separate platelet (Fig. 3G) cell bodies were not clearly distinguishable but pseudopodia and some hyalomere spread were discernible. Apart from the rough folds visible on the platelet surfaces, microparticular debris was abundant on and surrounding platelets and small scale fibrin networks appeared to be forming among the platelet aggregates. In fibrin network preparations at this time (Fig. 3H), heavy plaques had formed and though nets were still visible, primary fibrin networks could no longer be seen beneath them due to the structural density. The changes in platelet ultrastructure seen here with increased co-culture duration indicates increased activation, aggregation and adherence, increasing the risk of thrombosis. The increased density of fibrin network ultrastructure with increased co-culture duration indicates heightened inflammation, with characteristics not only expressing an increased risk for thrombosis but also increased clot lysing difficulty.
4. Discussion MCF-7 cells represent a luminal phenotype breast adenocarcinoma, presenting with both oestrogen and progesterone receptors (Augustine et al., 2015). Tumour cells, including the MCF-7 cell line have been shown to activate platelet function and induce aggregation via the release of ADP (Oleksowicz et al., 1995; Jurasz et al., 2004) and potent generation of thrombin (Marchetti et al., 2012). Our results show definitive ultrastructural alterations indicative of platelet activation that supplement these findings. With activation, platelets lose their resting discoid shape as pseudopodia appear and the central body tends to bulge to a more rounded shape allowing the platelet to roll in the vascular system. This is followed by a spreading phase in which the central body flattens out to a hemisphere shape for firmer adherence after which further extensive spreading of the hyalomere takes place allowing for irreversible adhesion (Hughes et al., 2000; Kuwahara et al., 2002). It is suggested that the presence of microparticles surrounding platelets is increased under inflammatory circumstances and is more evident in environments where thrombin is present in higher concentration (Hughes et al., 2000). Fibrin networks typical in non-inflammatory circumstances contain mostly major or thick fibres with minor or thin fibres distributed sparsely between them. Inflammation has been shown to not only tighten the fibre arrangement but also increase the density of fibrin networks, particularly increasing minor fibre content which can be seen as dense matted deposits (Pretorius et al., 2010; Lipinski et al., 2012). Higher thrombin concentrations are thought to play a role in the formation of denser fibrin networks, also increasing lysing duration. Changes in morphology from normal (zero minute controls) were visible as early as 5 min in co-culture with MCF-7 cells. From our results it would seem that increased activation signals affect both platelets and fibrin networks concurrently (Bastyr et al., 1990; Franchini, 2006). At 5 min, platelets displayed activation characteristics with extension of pseudopodia and less smooth membrane surfaces as well as early stage aggregation; while fibrin preparations showed increased clumping of major fibres associated with stickiness and an increase in density of minor fibres developing nets which covered the primary fibres. At 10 and 15 min, platelet pseudopodia appeared to shorten and thicken, and spreading of hyalomeres was most pronounced; while fibrin displayed heavier net density in areas and plaque formation became evident, indicative of a highly inflammatory state (Pretorius et al., 2011a,b; Undas and Ariëns, 2011). Where platelets were visible within the network, their pseudopodia were integrated with fibrin fibres. By 20 and 25 min, the central bodies of platelets seemed to spread into their hyalomeres as full adhesion was attained and aggregates were more closely associated than at earlier time points. At this time, fib-
62
W.J. van der Spuy, T.N. Augustine / Micron 90 (2016) 59–63
Fig. 3. Experimental platelet and fibrin network ultrastructure. Coagula were prepared from PRP co-cultured with MCF-7 cells for 5 up to 30 min. At 5 min (A) platelets showed extension of pseudopodia and increased surface membrane folds as well as early evidence of aggregation; and (B) fibrin showed network tightening, with minor fibres forming a net over major fibres in places. 10 and 15 min co-culture duration resulted in (C) thickening of platelet pseudopodia and initiation of hyalomere spread, with rough surface membranes, indicating a high level of inflammation and displaying full activation/adhesion characteristics; and (D) further tightening and clumping of fibrin fibres into plaques as well as platelet integration into the network in places. At 20 and 25 min co-culture duration, (E) expansion of central platelet bodies into the conjoined hyalomere spreads and rough surface membrane folds were visible, indicating further flattening of central bodies required for irreversible adhesion; and (F) dense fibrin net and plaque formation completely integrated platelets (one pictured) into the coagulum. At 30 min (G) single platelet morphology was indistinguishable, with bulbous membranes and microparticle debris surrounding the aggregate, including net formation places; and (H) fibrin network compacting advanced further to major plaque formation (Scale bar = 1 m). Key: Star, platelet central body; solid arrow, platelet pseudopod; hash/pound, platelet hyalomere spread; thin dotted arrow, major fibrin fibre; thick dotted arrow, minor fibrin fibre/net/plaque.
rin networks displayed dense net and plaque formation and where platelets were visible within the network, they were completely trapped within the network. At 30 min, platelet aggregates were large and showed evidence of fibrin nets forming between them; where fibrin networks advanced by tightening further into dense plaques.
Furthermore, microparticles, the generation of which are associated with increased thrombin levels, were present (Zwicker et al., 2007). Microparticle extrusion is associated primarily with the final stages of platelet activation and is implicated in facilitating interaction between platelets and other cell types (Burnouf et al., 2014). The presence of microparticular debris in our study
W.J. van der Spuy, T.N. Augustine / Micron 90 (2016) 59–63
was noted to rise with increasing duration of exposure to MCF7 cells. Microparticles amplify procoagulation events during the metastatic process (Zwicker et al., 2007) in which vascular components have heightened exposure to cancer cells. This postulation is substantiated by the ultrastructural alterations noted in the platelets themselves and also in matched fibrin network samples. In this study, these microparticles are possibly integrated into aggregates, acting as further scaffolding as fibrin networks become compacted into dense plaques. This induced progression of cellular and fibre characteristics resembles that of previously studied blood plasma preparations of severe inflammation- and thrombosis-prone patients (Pretorius et al., 2009, 2011a,b). As heightened activation characteristics are found to perpetuate in this experiment, it seems to confirm that cancer cells are stimulators of coagulation even in an in vitro co-culture system; which increasingly affects platelet and fibrin ultrastructure with longer duration co-culture. 4.1. Limitations and future direction In this experiment, which was used as a foundation for the development of protocols, cancer cells were synchronized to improve repeatability. The use of unsynchronized cells is proposed to more closely mimic the in vivo environment; and the preparation of non-control blood samples, from patients with diagnosed cancer, are applicable in clinical practice period. Protocols are being designed to follow patients from diagnosis, through chemotherapy, and surgical alternatives. Though protocols for platelet and fibrin network preparation are well established, preparation of samples for scanning electron microscopy is lengthy and costly, notwithstanding that electron microscopy knowledge is required to identify and interpret changes in morphology. Desktop scanning electron microscopes are available for diagnostic uses, but research publication warrants the use of high-resolution microscopes. For ongoing studies in this lab, once 5-min co-culture duration was determined to produce early ultrastructural changes, further protocols were developed, which showed that flow cytometry results correspond with scanning electron microscopy in platelet activation studies (Augustine et al., 2016). The added benefit of using flow cytometry, besides time to result yield, is that results can be readily quantified. Of further benefit to platelet activation studies would be the inclusion of inhibitors of stimulatory cytokine release from cancer cells as well as inhibitors of platelet activation in investigations, to further advance understanding of the relationship between cancer cells and thrombosis. 5. Conclusion In conclusion, our results seem to indicate that even in the in vitro environment, cancer cells are able to stimulate thrombosisrelated ultrastructural changes in platelets and fibrin networks, evidencing that the reciprocal interaction between tumour cells and the coagulation system itself induces thrombosis, over and above the influence of other systemic factors. The more rigid the construction of platelet and fibrin networks, the higher the propensity for clotting and the more impaired fibrinolysis would be. These results offer picturesque confirmation that breast cancer patients would be increasingly susceptible to thrombotic-related consequences, largely dependent on duration of exposure to circulating tumour cells. Acknowledgements This research was funded by the National Research Foundation of South Africa grant to WJS (NRF 87935) and the Carnegie Large
63
Research Grant to TNA (001.408.8421101). The authors declare no conflict of interests and are alone responsible for the content and writing of the paper. The authors extend gratitude to the phlebotomists and volunteers who collected and donated blood respectively, as well as their postgraduate student Lindsay Kaberry for laboratory assistance. Lastly we wish to express immense gratitude to the Unit for Microscopy and Microanalysis at the University of Pretoria. References Augustine, T.N., Dix-Peek, T., Duarte, R., Candy, G.P., 2015. Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer. J. Immunol. Methods 426, 1–13. Augustine, T.N., van der Spuy, W.J., Kaberry, L.L., Shayi, M., 2016. Thrombin-mediated platelet activation of lysed whole blood and platelet rich plasma: a comparison between platelet activation markers and ultrastructural alterations. Microsc. Microanal. (in press). Bambace, N.M., Holmes, C.E., 2011. The platelet contribution to cancer progression. J. Thromb. Haemost. 9 (2), 237–249. Bastyr 3rd, E.J., Kadrofske, M.M., Vinik, A.I., 1990. Platelet activity and phosphoinositide turnover increase with advancing age. Am. J. Med. 88, 601–606. Burnouf, T., Goubran, H.A., Chou, M.L., Devos, D., Radosevic, M., 2014. Platelet microparticles: detection and assessment of their paradoxical functional roles in disease and regenerative medicine. Blood Rev. 28, 155–166. Falanga, A., Donati, M.B., 2000. Pathogenesis of thrombosis in patients with malignancy. Int. J. Haematol. 73, 137–144. Favaloro, E.J., Funk, D.M., Lippi, G., 2012. Pre-analytical variables in coagulation testing associated with diagnostic errors in hemostasis. Lab. Med. 43 (2), 1–10. Franchini, M., 2006. Hemostasis and aging. Crit. Rev. Oncol. Hematol. 60, 144–151. Gil, J.F., Augustine, T.N., Hosie, M.J., 2013. Anastrozole and RU486: effects on estrogen receptor ␣ and Mucin 1 expression and correlation in the MCF-7 breast cancer cell line. Acta Histochem. 115, 851–857. Hughes, M., Hayward, C.P.M., Warkentin, T.E., Horsewood, P., Chorneyko, K.A., Kelton, J.G., 2000. Morphological analysis of microparticulate generation in heparin-induced thrombocytopenia. Blood 96 (1), 188–194. Jurasz, P., Alonso-Escolano, D., Radomski, M.W., 2004. Platelet-cancer interactions: mechanisms and pharmacology of tumour cell-induced platelet aggregation. Br. J. Pharmacol. 143 (7), 819–826. Kuwahara, M., Sugimoto, M., Tsuji, S., Matsui, H., Mizuno, T., Miyata, S., Yashioka, A., 2002. Platelet shape changes and adhesion under high shear flow. Arterioscler. Thromb. Vasc. Biol. 22, 329–334. Lipinski, B., Pretorius, E., Oberholzer, H.M., van der Spuy, W.J., 2012. Interaction of fibrin with red blood cells: the role of iron. Ultrastruct. Pathol. 36, 79–84. Marchetti, M., Diani, E., ten Cate, H., Falanga, A., 2012. Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography. Haematologica 97 (8), 1173–1180. Nash, G.F., Turner, L.F., Scully, M.F., Kakkar, A.K., 2002. Platelets and cancer. Lancet Oncol. 3 (7), 425–430. Oleksowicz, L., Mrowiec, Z., Schwartz, E., Khorshidi, M., Dutcher, J.P., Puszkin, E., 1995. Characterization of tumor-induced platelet aggregation: the role of immunorelated GPIb and GPIIb/IIIa expression by MCF-7 breast cancer cells. Thromb. Res. 79 (3), 261–274. Picker, S.M., 2011. In-vitro assessment of platelet function. Transfus. Apher. Sci. 44 (3), 305–319. Pretorius, E., Bornman, M.S., Reif, S., Oberholzer, H.M., Franz, R.C., 2009. Ultrastructural changes of platelet aggregates and fibrin networks in a patient with renal clear cell adenocarcinoma: a scanning electron microscopy study. Microsc. Res. Tech. 72 (9), 679–683. Pretorius, E., Oberholzer, H.M., van der Spuy, W.J., Meiring, J.H., 2010. Age-related changes in fibrin networks and platelets of individuals over 75: a scanning electron microscopy study showing “thrombotic preparedness”. J. Thromb. Thrombolysis 29, 271–275. Pretorius, E., Oberholzer, H.M., van der Spuy, W.J., Swanepoel, A.C., Soma, P., 2011a. Qualitative scanning electron microscopy analysis of fibrin networks and platelet abnormalities in diabetes. Blood Coagul. Fibrinolysis 22, 463–467. Pretorius, E., Swanepoel, A.C., Oberholzer, H.M., van der Spuy, W.J., Duim, W., Wessels, P.F., 2011b. A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic sstroke. J. Thromb. Thrombolysis 31 (4), 507–513. Undas, A., Ariëns, R.A., 2011. Fibrin clot structure and function: a role in the pathophysiology of arterial and venous thromboembolic diseases. Arterioscler. Thromb. Vasc. Biol. 31, e88–e99. van der Spuy, W.J., Pretorius, E., 2013. A place for ultrastructural analysis of platelets in cerebral ischemic research. Microsc. Res. Tech. 76 (8), 795–802. Zwicker, J.I., Furie, B.C., Furie, B., 2007. Cancer-associated thrombosis. Crit. Rev. Oncol. Hematol. 62 (2), 126–136.
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Ultrastructural investigation of the time-dependent relationship between breast cancer cells and thrombosis induction Wendy J. van der Spuy ∗ , Tanya N. Augustine School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
a r t i c l e
i n f o
Article history: Received 1 June 2016 Received in revised form 9 August 2016 Accepted 16 August 2016 Available online 27 August 2016 Keywords: Platelet Fibrin network Microparticle SEM Ultrastructure
a b s t r a c t Thromboembolic complications are a common cause of death in breast cancer patients. The in vivo relationship between coagulation factors and circulating tumours is a multifaceted one, with tumour cells implicated in thrombocytosis and platelets associated with coagulation-mediated metastasis. Platelets and fibrin networks are known to be morphologically altered in patients with cancer, and their relationship with breast cancer cells themselves may increase thrombosis susceptibility. This was investigated in vitro, assessing such morphological alterations through the establishment of a MCF-7 breast cancer cell co-culture system with blood plasma. Co-culture duration ranged from zero to thirty minutes, with five-minute intervals, in order to assess the time-dependent ultrastructural conformations of platelet and fibrin networks, using scanning electron microscopy. It was found that enhanced coagulability was related to co-culture duration. Changes in platelet and fibrin network morphology from normal were visible as early as five minutes in co-culture with MCF-7 cells. With longer co-culture duration thrombosis-linked variation in structural configuration was intensified, including advanced platelet aggregation and adherence characteristics, compression of fibrin networks into plaques of increased density, and upsurge of microparticulate extrusion implicated in amplifying procoagulant events during the metastatic process. These results confirm that cancer cells are stimulators of coagulation even in an in vitro system and breast cancer patients may become increasingly susceptible to thrombotic-related consequences with increased exposure to tumour cells, especially during metastasis. © 2016 Published by Elsevier Ltd.
1. Introduction Thromboembolic complications are the second most common cause of mortality in breast cancer patients (Falanga and Donati, 2000; Nash et al., 2002). The in vivo interaction between circulating tumours and coagulation factors is complex and reciprocal – with platelet activation and aggregation implicated in facilitating coagulation-mediated metastasis, and tumour-derived cytokines and growth factors implicated in thrombocytosis (Bambace and Holmes, 2011). Specifically, following intravasation into the vascular system, tumour cell-secreted factors induce platelet activation and aggregation, thereby protecting tumour cells from high velocity shear forces and immunosurveillance (Bambace and Holmes, 2011). Thus malignancy itself is associated with increased thromboembolic risk (Falanga and Donati, 2000; Nash et al., 2002).
∗ Corresponding author at: School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193, South Africa. E-mail addresses: [email protected], [email protected] (W.J. van der Spuy). http://dx.doi.org/10.1016/j.micron.2016.08.006 0968-4328/© 2016 Published by Elsevier Ltd.
Furthermore, as depicted in Fig. 1, inflammation (leukocyte involvement) and platelet activation are closely interlinked (van der Spuy and Pretorius, 2013), and though not in the scope of this paper, inflammation is imperative in malignant processes (Jurasz et al., 2004). Morphological alterations in platelet and fibrin network morphology have long been associated with thrombotic risk and such architectural shifts are evident in disease conditions such as diabetes, stroke and cancer (Pretorius et al., 2009, 2011a,b). It is thus understood that the in vivo relationship between breast cancer cells and coagulation factors may lead to changes in platelet and fibrin network morphology as well as function, increasing susceptibility to thrombosis. It is known that cancer cells are able to produce platelet agonists including thrombin, which not only induce platelet activation (Picker, 2011) but are a requirement for fibrin network formation. During normal haemostasis, the generation of a fibrin clot and subsequent fibrinolysis forms part of the coagulation process. However, in disease states including thromboembolic ischemic stroke, fibrin clot structure and fibrinolysis can be dysregulated (Pretorius et al., 2011b). The higher the amount of coagulation agonists produced and the longer the exposure dura-
60
W.J. van der Spuy, T.N. Augustine / Micron 90 (2016) 59–63
Fig. 1. Platelet activation is central to cancer progression. Platelet activation amplifies the inflammatory response, stimulates thrombosis processes, assists in the extravasation of circulating cancer cells, and has a role in the establishment of new tumour growth.
tion to these agonists, the further and more rapidly are platelets expected to advance through phases of activation, aggregation and full adherence. This study was designed to investigate the in vitro environment in order to ascertain how soon tumour cells affect coagulation ability or propensity for thrombosis, as a starting point for further studies into their relationship.
2. Materials and methods A co-culture system was established, culturing MCF-7 luminal phenotype breast cancer cells with the blood plasma of seemingly healthy female individuals. Applicable exclusion criteria included smoking, contraceptive use or pregnancy, and a history of autoimmune disease or cancer. The experiment was repeated in triplicate (conducted separately) with the blood of two different volunteers per experiment (n = 6). Ethical clearance was obtained from the University of the Witwatersrand’s Human Ethics Committee (M081036 and M140155). MCF-7 cells were plated at a concentration of 1 × 105 cells per coverslip in a 24-well plate and incubated for 24 h at 37 ◦ C and 5% CO2 in normal DMEM media (Dulbecco’s Modified Eagles Medium, Lonza, Verviers, Belgium; BE12-604F), with 10% heat-inactivated FBS (Foetal Bovine Serum, Biowest, South America; S1810-500) and 0.1% P/S (Penicillin/Streptomycin, Highveld Biological, RSA; 214). Normal media was replaced with Phenol Red Free (PRF)-DMEM media (Lonza, Verviers, Belgium; BE12-115F) with 10% Dextran Coated Charcoal (DCC)-stripped FBS, 0.1% P/S and l-glutamine, and incubated for 48 h at 37 ◦ C and 5% CO2 for cell cycle synchronization (Gil et al., 2013). Peripheral blood was obtained via venepuncture into citratecontaining vacutainers (Lasec, South Africa). Diagnostic data suggests that citrated platelets – in plasma which is separated from the whole blood whence it is obtained by centrifugation – do not activate upon standing even after 1500g centrifugation for up to 15 min, and such separated plasma remains stable for diagnostic haemostasis testing up to 24 h in an unrefrigerated state (Favaloro et al., 2012). However, we have also shown that centrifugation at 400×g for the generation of PRP, may induce early activation as evidenced by the loss of CD62p (p-selectin) containing-microparticles (Augustine et al., 2016). As such in this study, platelet-rich plasma (PRP) was obtained by centrifuging whole blood for 5 min at 1000 rpm (200×g). Co-cultures were thus implemented by expos-
ing PRP to cells for periods of zero (control – no exposure of plasma to cells) up to 30 min with intervals of 5 min, after which platelet and fibrin network coagula were prepared on glass coverslips for scanning electron microscopy (SEM). MCF-7 cells were not prepared for scanning electron microscopy in this experiment, as it was designed specifically to determine co-culture duration for further experiments. Platelet coagula were prepared using only 20 l of platelet rich plasma, whereas fibrin coagula were prepared through activation of 15 l platelet rich plasma with 5 l of 20 U/ml human thrombin (SANBS, South Africa (van der Spuy and Pretorius, 2013)). Prepared coverslips were incubated on 0.1 M phosphate buffered saline (PBS)-dampened filter paper for 2 min to ensure adherence, after which a washing process followed where the coverslips were submerged in PBS on a shaker for 20 min to ensure removal of trapped proteins. This was followed by primary fixation in 2.5% formaldehyde/glutaraldehyde solution for 15 min, three rinses in PBS, secondary fixation in 1% osmium tetroxide (OsO4 ) for 15 min, three rinses in PBS and then serial dehydration in 30%, 50%, 70%, 90% and three times absolute ethanol. SEM procedures were completed by drying the samples with hexamethyldisilazane (HMDS), mounting on aluminium stubs and coating by carbon evaporation. The ultrastructural alterations in platelet and fibrin network morphology were then assessed with a ZEISS ULTRA plus FEG Scanning Electron Microscope at the University of Pretoria’s Microscopy and Microanalysis Unit in South Africa. Accelerating voltage was set at 1 kV for high quality surface analysis and samples imaged with the In-lens secondary electron detector. For each of the coagulum coverslips prepared, ten low magnification images were acquired to give an overall perspective of the level of coagulation, and ten high magnification images were acquired for clear visualization of morphological characteristics. Therefore, for each time point, 60 high magnification images were taken of platelets and fibrin networks, respectively, and a representative image was selected for depiction in this report.
3. Results Control preparations of PRP not exposed to tumour cells showed typical morphology for platelets and fibrin networks. Platelets in the zero co-culture control groups displayed discoid to slightly rounded platelets with smooth membrane surfaces and little to no pseudopodia. No aggregates were present in these groups. Fibrin
W.J. van der Spuy, T.N. Augustine / Micron 90 (2016) 59–63
61
Fig. 2. Control platelet and fibrin network ultrastructure. Ultrastructure was studied preparing coagula from plasma without (plasma) or with (fibrin) the addition of thrombin. (A) Control platelet, unexposed to tumour cells, showing typically discoid-shaped cell body and smooth membrane surfaces. (B) Control fibrin networks, unexposed to tumour cells, showing typical mesh formation with major (thick) and minor (thin) fibres interspersed sparingly between them (Scale bar = 1 m). Key: Star, platelet central body; thin dotted arrow, major fibrin fibre; thick dotted arrow, minor fibrin fibre.
networks were smooth in appearance showing typical major and minor fibre lattices. Fig. 2 shows a typical control platelet (Fig. 2A) and normal fibrin network formation (Fig. 2B). Fig. 3 shows experimental platelet coagula alongside corresponding fibrin networks as per co-culture duration. After 5 min of co-culture with MCF-7 cells, platelets (Fig. 3A) were found to be ball- to hemisphere-shaped indicating activation and firm but reversible adhesion. Some aggregation of platelets in small groups of two to four was visible. Membrane surfaces were still smooth but with the appearance of folds and pseudopodia. The spread of hyalomeres was evident in approximately 20% of platelets at this time. Fibrin networks (Fig. 3B) displayed thickening and clumping of primary fibres and net formation adjacent to and over primary fibre masses at this time. After 10 and 15 min of co-culture, platelet (Fig. 3C) hyalomere spread had increased and was evident in approximately 40–50% of platelets, indicating a shift towards full and irreversible adhesion. Pseudopodia were less prominent at this time and in some samples markedly shortened when compared to the 5-min point. Most platelets were visible in aggregates of two or more and their membrane surfaces appeared roughened. Fibrin networks (Fig. 3D) showed further thickening of primary fibres as they clumped together further, indicating increased stickiness of the fibres. Nets became increasingly dense and in some instances formed plaques. Additionally, intertwined platelets became conspicuous. After 20 and 25 min of co-culture, larger aggregated groups of platelets were seen (Fig. 3E). Pseudopodia were short and thickened and hyalomere spreads tended to be conjoined. Membrane surfaces still displayed rough folds and microparticular debris was visible. In fibrin (Fig. 3F), the dense nets completely masked primary fibres which were sometimes visible beneath the net through larger holes. Visible platelets were found to be completely trapped within the dense nets and more plaque formation was also evident. After 30 min of co-culture, separate platelet (Fig. 3G) cell bodies were not clearly distinguishable but pseudopodia and some hyalomere spread were discernible. Apart from the rough folds visible on the platelet surfaces, microparticular debris was abundant on and surrounding platelets and small scale fibrin networks appeared to be forming among the platelet aggregates. In fibrin network preparations at this time (Fig. 3H), heavy plaques had formed and though nets were still visible, primary fibrin networks could no longer be seen beneath them due to the structural density. The changes in platelet ultrastructure seen here with increased co-culture duration indicates increased activation, aggregation and adherence, increasing the risk of thrombosis. The increased density of fibrin network ultrastructure with increased co-culture duration indicates heightened inflammation, with characteristics not only expressing an increased risk for thrombosis but also increased clot lysing difficulty.
4. Discussion MCF-7 cells represent a luminal phenotype breast adenocarcinoma, presenting with both oestrogen and progesterone receptors (Augustine et al., 2015). Tumour cells, including the MCF-7 cell line have been shown to activate platelet function and induce aggregation via the release of ADP (Oleksowicz et al., 1995; Jurasz et al., 2004) and potent generation of thrombin (Marchetti et al., 2012). Our results show definitive ultrastructural alterations indicative of platelet activation that supplement these findings. With activation, platelets lose their resting discoid shape as pseudopodia appear and the central body tends to bulge to a more rounded shape allowing the platelet to roll in the vascular system. This is followed by a spreading phase in which the central body flattens out to a hemisphere shape for firmer adherence after which further extensive spreading of the hyalomere takes place allowing for irreversible adhesion (Hughes et al., 2000; Kuwahara et al., 2002). It is suggested that the presence of microparticles surrounding platelets is increased under inflammatory circumstances and is more evident in environments where thrombin is present in higher concentration (Hughes et al., 2000). Fibrin networks typical in non-inflammatory circumstances contain mostly major or thick fibres with minor or thin fibres distributed sparsely between them. Inflammation has been shown to not only tighten the fibre arrangement but also increase the density of fibrin networks, particularly increasing minor fibre content which can be seen as dense matted deposits (Pretorius et al., 2010; Lipinski et al., 2012). Higher thrombin concentrations are thought to play a role in the formation of denser fibrin networks, also increasing lysing duration. Changes in morphology from normal (zero minute controls) were visible as early as 5 min in co-culture with MCF-7 cells. From our results it would seem that increased activation signals affect both platelets and fibrin networks concurrently (Bastyr et al., 1990; Franchini, 2006). At 5 min, platelets displayed activation characteristics with extension of pseudopodia and less smooth membrane surfaces as well as early stage aggregation; while fibrin preparations showed increased clumping of major fibres associated with stickiness and an increase in density of minor fibres developing nets which covered the primary fibres. At 10 and 15 min, platelet pseudopodia appeared to shorten and thicken, and spreading of hyalomeres was most pronounced; while fibrin displayed heavier net density in areas and plaque formation became evident, indicative of a highly inflammatory state (Pretorius et al., 2011a,b; Undas and Ariëns, 2011). Where platelets were visible within the network, their pseudopodia were integrated with fibrin fibres. By 20 and 25 min, the central bodies of platelets seemed to spread into their hyalomeres as full adhesion was attained and aggregates were more closely associated than at earlier time points. At this time, fib-
62
W.J. van der Spuy, T.N. Augustine / Micron 90 (2016) 59–63
Fig. 3. Experimental platelet and fibrin network ultrastructure. Coagula were prepared from PRP co-cultured with MCF-7 cells for 5 up to 30 min. At 5 min (A) platelets showed extension of pseudopodia and increased surface membrane folds as well as early evidence of aggregation; and (B) fibrin showed network tightening, with minor fibres forming a net over major fibres in places. 10 and 15 min co-culture duration resulted in (C) thickening of platelet pseudopodia and initiation of hyalomere spread, with rough surface membranes, indicating a high level of inflammation and displaying full activation/adhesion characteristics; and (D) further tightening and clumping of fibrin fibres into plaques as well as platelet integration into the network in places. At 20 and 25 min co-culture duration, (E) expansion of central platelet bodies into the conjoined hyalomere spreads and rough surface membrane folds were visible, indicating further flattening of central bodies required for irreversible adhesion; and (F) dense fibrin net and plaque formation completely integrated platelets (one pictured) into the coagulum. At 30 min (G) single platelet morphology was indistinguishable, with bulbous membranes and microparticle debris surrounding the aggregate, including net formation places; and (H) fibrin network compacting advanced further to major plaque formation (Scale bar = 1 m). Key: Star, platelet central body; solid arrow, platelet pseudopod; hash/pound, platelet hyalomere spread; thin dotted arrow, major fibrin fibre; thick dotted arrow, minor fibrin fibre/net/plaque.
rin networks displayed dense net and plaque formation and where platelets were visible within the network, they were completely trapped within the network. At 30 min, platelet aggregates were large and showed evidence of fibrin nets forming between them; where fibrin networks advanced by tightening further into dense plaques.
Furthermore, microparticles, the generation of which are associated with increased thrombin levels, were present (Zwicker et al., 2007). Microparticle extrusion is associated primarily with the final stages of platelet activation and is implicated in facilitating interaction between platelets and other cell types (Burnouf et al., 2014). The presence of microparticular debris in our study
W.J. van der Spuy, T.N. Augustine / Micron 90 (2016) 59–63
was noted to rise with increasing duration of exposure to MCF7 cells. Microparticles amplify procoagulation events during the metastatic process (Zwicker et al., 2007) in which vascular components have heightened exposure to cancer cells. This postulation is substantiated by the ultrastructural alterations noted in the platelets themselves and also in matched fibrin network samples. In this study, these microparticles are possibly integrated into aggregates, acting as further scaffolding as fibrin networks become compacted into dense plaques. This induced progression of cellular and fibre characteristics resembles that of previously studied blood plasma preparations of severe inflammation- and thrombosis-prone patients (Pretorius et al., 2009, 2011a,b). As heightened activation characteristics are found to perpetuate in this experiment, it seems to confirm that cancer cells are stimulators of coagulation even in an in vitro co-culture system; which increasingly affects platelet and fibrin ultrastructure with longer duration co-culture. 4.1. Limitations and future direction In this experiment, which was used as a foundation for the development of protocols, cancer cells were synchronized to improve repeatability. The use of unsynchronized cells is proposed to more closely mimic the in vivo environment; and the preparation of non-control blood samples, from patients with diagnosed cancer, are applicable in clinical practice period. Protocols are being designed to follow patients from diagnosis, through chemotherapy, and surgical alternatives. Though protocols for platelet and fibrin network preparation are well established, preparation of samples for scanning electron microscopy is lengthy and costly, notwithstanding that electron microscopy knowledge is required to identify and interpret changes in morphology. Desktop scanning electron microscopes are available for diagnostic uses, but research publication warrants the use of high-resolution microscopes. For ongoing studies in this lab, once 5-min co-culture duration was determined to produce early ultrastructural changes, further protocols were developed, which showed that flow cytometry results correspond with scanning electron microscopy in platelet activation studies (Augustine et al., 2016). The added benefit of using flow cytometry, besides time to result yield, is that results can be readily quantified. Of further benefit to platelet activation studies would be the inclusion of inhibitors of stimulatory cytokine release from cancer cells as well as inhibitors of platelet activation in investigations, to further advance understanding of the relationship between cancer cells and thrombosis. 5. Conclusion In conclusion, our results seem to indicate that even in the in vitro environment, cancer cells are able to stimulate thrombosisrelated ultrastructural changes in platelets and fibrin networks, evidencing that the reciprocal interaction between tumour cells and the coagulation system itself induces thrombosis, over and above the influence of other systemic factors. The more rigid the construction of platelet and fibrin networks, the higher the propensity for clotting and the more impaired fibrinolysis would be. These results offer picturesque confirmation that breast cancer patients would be increasingly susceptible to thrombotic-related consequences, largely dependent on duration of exposure to circulating tumour cells. Acknowledgements This research was funded by the National Research Foundation of South Africa grant to WJS (NRF 87935) and the Carnegie Large
63
Research Grant to TNA (001.408.8421101). The authors declare no conflict of interests and are alone responsible for the content and writing of the paper. The authors extend gratitude to the phlebotomists and volunteers who collected and donated blood respectively, as well as their postgraduate student Lindsay Kaberry for laboratory assistance. Lastly we wish to express immense gratitude to the Unit for Microscopy and Microanalysis at the University of Pretoria. References Augustine, T.N., Dix-Peek, T., Duarte, R., Candy, G.P., 2015. Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer. J. Immunol. Methods 426, 1–13. Augustine, T.N., van der Spuy, W.J., Kaberry, L.L., Shayi, M., 2016. Thrombin-mediated platelet activation of lysed whole blood and platelet rich plasma: a comparison between platelet activation markers and ultrastructural alterations. Microsc. Microanal. (in press). Bambace, N.M., Holmes, C.E., 2011. The platelet contribution to cancer progression. J. Thromb. Haemost. 9 (2), 237–249. Bastyr 3rd, E.J., Kadrofske, M.M., Vinik, A.I., 1990. Platelet activity and phosphoinositide turnover increase with advancing age. Am. J. Med. 88, 601–606. Burnouf, T., Goubran, H.A., Chou, M.L., Devos, D., Radosevic, M., 2014. Platelet microparticles: detection and assessment of their paradoxical functional roles in disease and regenerative medicine. Blood Rev. 28, 155–166. Falanga, A., Donati, M.B., 2000. Pathogenesis of thrombosis in patients with malignancy. Int. J. Haematol. 73, 137–144. Favaloro, E.J., Funk, D.M., Lippi, G., 2012. Pre-analytical variables in coagulation testing associated with diagnostic errors in hemostasis. Lab. Med. 43 (2), 1–10. Franchini, M., 2006. Hemostasis and aging. Crit. Rev. Oncol. Hematol. 60, 144–151. Gil, J.F., Augustine, T.N., Hosie, M.J., 2013. Anastrozole and RU486: effects on estrogen receptor ␣ and Mucin 1 expression and correlation in the MCF-7 breast cancer cell line. Acta Histochem. 115, 851–857. Hughes, M., Hayward, C.P.M., Warkentin, T.E., Horsewood, P., Chorneyko, K.A., Kelton, J.G., 2000. Morphological analysis of microparticulate generation in heparin-induced thrombocytopenia. Blood 96 (1), 188–194. Jurasz, P., Alonso-Escolano, D., Radomski, M.W., 2004. Platelet-cancer interactions: mechanisms and pharmacology of tumour cell-induced platelet aggregation. Br. J. Pharmacol. 143 (7), 819–826. Kuwahara, M., Sugimoto, M., Tsuji, S., Matsui, H., Mizuno, T., Miyata, S., Yashioka, A., 2002. Platelet shape changes and adhesion under high shear flow. Arterioscler. Thromb. Vasc. Biol. 22, 329–334. Lipinski, B., Pretorius, E., Oberholzer, H.M., van der Spuy, W.J., 2012. Interaction of fibrin with red blood cells: the role of iron. Ultrastruct. Pathol. 36, 79–84. Marchetti, M., Diani, E., ten Cate, H., Falanga, A., 2012. Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography. Haematologica 97 (8), 1173–1180. Nash, G.F., Turner, L.F., Scully, M.F., Kakkar, A.K., 2002. Platelets and cancer. Lancet Oncol. 3 (7), 425–430. Oleksowicz, L., Mrowiec, Z., Schwartz, E., Khorshidi, M., Dutcher, J.P., Puszkin, E., 1995. Characterization of tumor-induced platelet aggregation: the role of immunorelated GPIb and GPIIb/IIIa expression by MCF-7 breast cancer cells. Thromb. Res. 79 (3), 261–274. Picker, S.M., 2011. In-vitro assessment of platelet function. Transfus. Apher. Sci. 44 (3), 305–319. Pretorius, E., Bornman, M.S., Reif, S., Oberholzer, H.M., Franz, R.C., 2009. Ultrastructural changes of platelet aggregates and fibrin networks in a patient with renal clear cell adenocarcinoma: a scanning electron microscopy study. Microsc. Res. Tech. 72 (9), 679–683. Pretorius, E., Oberholzer, H.M., van der Spuy, W.J., Meiring, J.H., 2010. Age-related changes in fibrin networks and platelets of individuals over 75: a scanning electron microscopy study showing “thrombotic preparedness”. J. Thromb. Thrombolysis 29, 271–275. Pretorius, E., Oberholzer, H.M., van der Spuy, W.J., Swanepoel, A.C., Soma, P., 2011a. Qualitative scanning electron microscopy analysis of fibrin networks and platelet abnormalities in diabetes. Blood Coagul. Fibrinolysis 22, 463–467. Pretorius, E., Swanepoel, A.C., Oberholzer, H.M., van der Spuy, W.J., Duim, W., Wessels, P.F., 2011b. A descriptive investigation of the ultrastructure of fibrin networks in thrombo-embolic ischemic sstroke. J. Thromb. Thrombolysis 31 (4), 507–513. Undas, A., Ariëns, R.A., 2011. Fibrin clot structure and function: a role in the pathophysiology of arterial and venous thromboembolic diseases. Arterioscler. Thromb. Vasc. Biol. 31, e88–e99. van der Spuy, W.J., Pretorius, E., 2013. A place for ultrastructural analysis of platelets in cerebral ischemic research. Microsc. Res. Tech. 76 (8), 795–802. Zwicker, J.I., Furie, B.C., Furie, B., 2007. Cancer-associated thrombosis. Crit. Rev. Oncol. Hematol. 62 (2), 126–136.