# Neutrophils, Cancer and Thrombosis: The New Bermuda Triangle in Cancer Research

Mélanie Langiu, Ana-Luisa Palacios-Acedo, Lydie Crescence, Diane Mege, Christophe Dubois, Laurence Panicot-Dubois

► To cite this version:

Mélanie Langiu, Ana-Luisa Palacios-Acedo, Lydie Crescence, Diane Mege, Christophe Dubois, et al. Neutrophils, Cancer and Thrombosis: The New Bermuda Triangle in Cancer Research. International Journal of Molecular Sciences, 2022, 23 (3), pp.1257. 10.3390/ijms23031257. hal-03685893

HAL Id: hal-03685893

https://hal.science/hal-03685893

Submitted on 26 Jun 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Review

# Neutrophils, Cancer and Thrombosis: The New Bermuda Triangle in Cancer Research

Mélanie Langiu <fnref n="1" />,†, Ana-Luisa Palacios-Acedo <fnref n="1" />,†, Lydie Crescence <fnref n="1" />, Diane Mege <fnref n="1" />/<fnref n="2" />, Christophe Dubois <fnref n="1" />,*, and Laurence Panicot-Dubois <fnref n="1" /> ID

**Abstract:** Spontaneous venous thrombosis is often the first clinical sign of cancer, and it is linked to a worsened survival rate. Traditionally, tumor-cell induced platelet activation has been the main actor studied in cancer-associated-thrombosis. However, platelet involvement alone does not seem to be sufficient to explain this heightened pro-thrombotic state. Neutrophils are emerging as key players in both thrombus generation and cancer progression. Neutrophils can impact thrombosis through the release of pro-inflammatory cytokines and expression of molecules like P-selectin and Tissue Factor (TF) on their membrane and on neutrophil-derived microvesicles. Their role in cancer progression is evidenced by the fact that patients with high blood-neutrophil counts have a worsened prognosis. Tumors can attract neutrophils to the cancer site via pro-inflammatory cytokine secretions and induce a switch to pro-tumoral (or N2) neutrophils, which support metastatic spread and have an immunosuppressive role. They can also expel their nuclear contents to entrap pathogens forming Neutrophil Extracellular Traps (NETs) and can also capture coagulation factors, enhancing the thrombus formation. These NETs are also known to have pro-tumoral effects by supporting the metastatic process. Here, we strived to do a comprehensive literature review of the role of neutrophils as drivers of both cancer-associated thrombosis (CAT) and cancer progression.

**Keywords:** cancer; thrombosis; neutrophil; NET

<figure><img src="image_3.png" /></figure>

check for
updates

**Citation:** Langiu, M.; Palacios-Acedo, A.-L.; Crescence, L.; Mege, D.; Dubois, C.; Panicot-Dubois, L. Neutrophils, Cancer and Thrombosis: The New Bermuda Triangle in Cancer Research. *Int. J. Mol. Sci.* **2022**, *23*, 1257. https://doi.org/10.3390/ijms23031257

Academic Editor: Isabella Russo

Received: 24 November 2021

Accepted: 21 January 2022

Published: 23 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_4.png" /></figure>

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

The connection between cancer and thrombosis has been described since the XIX century by Dr Trousseau who described the presence of spontaneous coagulation in oncological patients [1,2]. Cancer patients have an increased incidence of both venous thromboembolism (VTE) (4% to 20%) and arterial thrombosis (2% to 5%) [3,4]. In fact, between 20 to 30% of all first venous thrombotic events are cancer related, and their presence portends poor prognosis and a significant decrease in patient survival [5,6]. The incidence of cancer-associated-thrombosis (CAT) is also associated with the tumor type, stage and treatment administered. CAT is the second most common cause of death in cancer patients (after cancer evolution itself) and thrombi can be found in half of deceased cancer patients during autopsy [1,7–9].

Platelets are at the crossroads of hemostasis, thrombosis, and inflammation. As such, their involvement in CAT has been largely investigated. Cancer cells can activate platelets through tumor cell induced platelet activation (TCIPA) [2]. In a direct manner, platelet receptors like αIIbβ3 and αVIβ1 can bind to tumor αVβ3 and ADAM9 respectively, as well as binding with P-selectins through PSGL-1 interaction, platelet toll-like receptor 4 and facilitating CLEC2-podoplanin interactions [10–14]. In an indirect manner, tumor cells secrete platelet agonists (like thromboxane A2 and ADP) into the tumor microenvironment,

<footnote n="1">¹ Aix Marseille Univ INSERM, INRAE, C2VN, 13005 Marseille, France; melanie.langiu@univ-amu.fr (M.L.); ana-luisa.palacios-acedo@univ-amu.fr (A.-L.P.-A.); Lydie.crescence@univ-amu.fr (L.C.); diane.mege@univ-amu.fr (D.M.); laurence.panicot-dubois@univ-amu.fr (L.P.-D.)</footnote>

<footnote n="2">² Department of Digestive Surgery, La Timone University Hospital, 13005 Marseille, France</footnote>

<footnote>* Correspondence: christophe.dubois@univ-amu.fr</footnote>

<footnote>† These authors contributed equally to this work.</footnote>
activating platelets which then release more platelet agonists and create a potent activation loop [11–13,15]. Tumor-activated platelets can also secrete growth factors into the tumor microenvironment like transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF) [2,13,16]. This platelet degranulation can also support local angiogenesis and increase the endothelial expression of adhesion molecules [13,17,18]. This overall increased platelet activity is related to a heightened risk of venous and arterial thrombosis in cancer patients [2,19–21].

However, platelet involvement alone does not seem to be sufficient to explain the heightened pro-thrombotic state in cancer patients. Recently, neutrophils have been beginning to enter the spotlight for their role in both tumor progression and CAT. Indeed, recent studies described that an increase in circulating neutrophil numbers was indicative of a worsened prognosis in gliomas, lung, and esophageal cancer [22–24]. To date, the relevant molecular mechanisms are not entirely clear into the literature. In this review will strive to compile the current state of the art of neutrophil involvement in cancer progression and in cancer-associated thrombosis.

## 2. Neutrophils and Thrombosis

Neutrophils are the most abundant leucocytes in humans and constitute an important pillar of the innate immunity [25,26]. They are the first immune cells to be recruited to inflammatory sites [25,26]. Neutrophils are also key players in intravascular immunity; their microbicidal activity prevents the spread of circulating pathogens [25]. Neutrophils have the capacity to mediate phagocytosis and intracellular killing of different pathogens as well as eliminate cellular debris in their phagolysosomal granules [25,27,28].

Circulating neutrophils are recruited to extra-vascular inflammatory sites via a chemokine gradient [29]. Studies have shown that neutrophils react preferentially to a hierarchy of chemokines, allowing them to reach the desired endothelial placement and then transigrate to the surrounding tissue [29]. For example, bacterial-derived N-formyl-Methionyl-leucyl-phenylalanine (fMLP) and complement C5a override CXCL8/IL-8 and LTB4 chemotactic stimuli [29,30]. The actual recruiting cascade involves several well characterized steps: rolling-adhesion-tethering-crawling and transmigration. The endothelium is stimulated by inflammatory mediators like histamine and cytokines or pattern-recognition-receptors that will enhance P-selectin and E-selectin expression on the endothelial-cell surface to maximize neutrophil recruitment [29]. Neutrophils will begin "rolling" on the endothelial surface before being primed and activated by molecules like tumor-necrosis-factor-α (TNFα) as well as by direct contact with activated endothelial cells [29]. Priming of the neutrophils is necessary to achieve activation of the NADPH oxidase pathway used to destroy pathogens [29–31]. As the neutrophils slow down, they "crawl" between endothelial cells to reach the cell-cell junctions via ICAM1-MAC1 signaling [29,30]. Eventually neutrophils tether to the endothelium before transmigrating, a complex process that requires multiple integrin interactions (ICAM1 and 2, VCAM1, PECAM1 and EPCAM to name a few) [29]. Transmigration can occur between endothelial cells or transcellularly, and once neutrophils are in the tissue, they can zoom-in on the inflammatory site [26,29,30].

Neutrophils can eliminate pathogens in different ways; in both an intra and extracellular manner [27,31]. They can internalize opsonized pathogens or cellular debris into their phagolysosomal granules for destruction with reactive oxygen species (NADPH oxidase pathway) [29]. They also secrete their granule content to combat extracellular pathogens [29,31,32]. Neutrophils have three distinct granule types: **(1) Azurophilic or primary** containing myeloperoxidase (MPO), cathepsin G, neutrophil elastase (NE) and bactericidal/permeability increasing protein (BPI) [31–33]. **(2) Specific or secondary granules** with alkaline phosphatase, NADPH oxidase, collagenase, lactoferrin and histaminase and **(3) the tertiary granules** with collagenase, cathepsin and gelatinase [29,31–33].

The role of neutrophils in the innate immunity is well characterized; however, their importance in other phenomena like thrombosis and hemostasis has only recently begun to be determined. It is also clear that in an altered pathological state like cancer, most
of the known mechanisms can be heightened, creating an amplification loop. The role of neutrophils in thrombosis began to be elucidated in 2005 when it was reported that leukocytes were rolling onto the growing thrombus site within the first two minutes post laser injury [34]. However, it was not until 2012 that the importance of neutrophils in thrombus formation was cemented [35]. Indeed, it was shown by Darbousset et al. that mice neutrophils were the first cells to accumulate at the site of arterial injury, even before platelets were detected [35]. Neutrophils accumulate through the interaction of intracellular-adhesion-molecule-1 (ICAM1) with leukocyte-function-associated-antigen-1 (LFA1) and express TF [35]. Moreover, neutrophil depletion has been shown to have a significant attenuation effect on thrombus formation in mice in vivo [36]. Interestingly, soluble fibrinogen found in prothrombotic conditions can also activate neutrophils in a CD11-b dependent mechanism which contributes to an increase in proinflammatory signals [37].

Neutrophils are a crucial leukocyte subset recruited at the site of injury, in fact, they release neutrophil elastase and augment their intracellular calcium mobilization, evidencing their activated state [38–40]. Additionally, ATP secreted during the thrombo-inflammatory process can directly activate neutrophils through the P2X1 membrane receptor [38]. It has been shown that P2X1 present on PMNs is involved in thrombin generation, and that its expression at the surface of platelets is needed for thrombus formation [38]. Several researchers, like Darbousset, R. et al. have shown the effect of ATP on thrombosis; and how it can activate human neutrophils in vitro [38]. On the other hand, in in vivo studies, P2X1-deficient mice had less neutrophil-accumulation and decreased thrombus formation at a laser-induced-injury site [38]. P2X1 activation leads to both platelet and neutrophil activation and activated platelets will then secrete their granules containing ADP, which enhances the platelet activation at the thrombus site [2,38,41]. These platelets will undergo intracellular calcium mobilization and adhere to the forming thrombus [42].

Another possible route in which neutrophils can impact thrombus formation is through the direct expression of TF on their membrane; this TF can be “obtained” after the neutrophil interacts with monocytes and/or macrophages [28,43]. Neutrophils that are first to arrive to the site of a laser injury can also express TF on their surface after interaction with endothelial cells [35]. More specifically, human neutrophils can express TF after stimulation with P-selectins and fMLP [44]. In inflammatory conditions, IgG triggers complement activation and C5a generation, which can induce TF gene transcription in activated human neutrophils [45].

A proposed molecular mechanism through which neutrophils impact thrombosis is through the generation of microvesicles (MVs). Microvesicles are small membrane vesicles released during cellular activation that contain the same membrane markers and proteins as the cell of origin [46–48]. MVs are known to have important contributions in inter-cellular communications and in pathological conditions like cancer [48–50]. MVs express phosphatidyl-serine on their outer membrane, which not only facilitates formation of coagulation complexes, but also promotes the ability of tissue factor (TF) to initiate coagulation [51]. Moreover, neutrophil-derived MVs may contain MPO, which has been shown to play a role in thrombus propagation by causing endothelial damage [28–48,52]. Human Neutrophil-MVs obtained after a PMA stimulation contain functionally active MAC-1 integrins that interact with GPIb on resting platelets. This interaction can activate platelets and induce the expression of the adhesion molecule P-selectin [53]. Please find a schematic revision of the role of neutrophils in thrombosis in Figure 1.
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Schematic representation of neutrophil interactions and implications in thrombosis. Following endothelial injury, activated neutrophils express TF, which initiates the coagulation cascade resulting in thrombin generation and platelet activation. Activated platelets create a positive feedback loop to recruit more circulating platelets. Figure created using Servier Medical Art available at http://smart.servier.com/ (accessed on 15 September 2021).</figcaption></figure>

**3. Neutrophil Extracellular Traps or Activated Neutrophils in Venous Thrombosis**

The role of neutrophil extracellular traps (NETs) in thrombosis has been recently described (Figure 2). NETs are part of the innate immunity's defense against pathogens in which the neutrophil sacrifices itself by expulsing its genetic material, thus forming «neutrophil extracellular traps» (NETs) [54].
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Schematic representation of NET interactions and implications in thrombosis. NETs are capable of trapping platelets and red blood cells, forming a red thrombus. In addition, they express pro-coagulant molecules such as tissue factor, fibrinogen, fibronectin or Von Willebrand factor, which initiate the coagulation cascade and participated to the thrombus formation. Figure created using Servier Medical Art available at http://smart.servier.com/ (accessed on 15 September 2021).</figcaption></figure>

NETs are fibers of decondensed chromatin (DNA and histones) coated with antimicrobial proteins (myeloperoxidase (MPO), cathepsin G and neutrophil elastase (NE)) that are released by the neutrophil when it detects pathogens (bacteria, protozoa, fungi) in the extracellular environment [54]. These chromatin fibers create a network that entraps pathogens, preventing their dissemination in the host organism and eliminating them due to their anti-microbial properties [54,55].

The reaction of NET formation is catalyzed by peptidyl-arginine deiminase 4 (PAD4); which enables the chromatin de-condensation; causing Histone 3 to be citrullinated (CitH3) [56]. Therefore, by detecting either CitH3 or extracellular DNA, researchers can identify NETs. Indeed, the involvement of NETs in thrombosis was demonstrated when CitH3 was found in the thrombi of patients with VTE, and it was shown that the dissociation of NETs could promote the lysis of the thrombus [57].

Interestingly, studies have also highlighted the role of the enzyme PAD4 in the involvement of NETs in thrombosis [58]. Indeed, Martinod and colleagues demonstrated that after 48 h of inferior vena cava ligation only 10% of PAD4-deficient mice developed thrombi versus 90% in wild-type mice [58]. So PAD4 appears to be crucial in pathological venous thrombosis formation. Furthermore, no NETs were found in the thrombi from the PAD4-deficient mice, further highlighting their role in thrombus formation [55].

The network formed by NETs to trap pathogens can also trap platelets, an essential player in thrombosis [59]. In fact, Fuchs and colleagues have shown that NETs provide a scaffold for the activation and aggregation of platelets, as well as for red blood cells which form the red portion of the thrombus [59]. These data are supported by the fact that when mice were injected with DNase-I (a DNA-cleaving nuclease), NETs were degraded and no platelet aggregates were formed [59]. This suggests that NETs form an essential pro-thrombotic substrate for thrombosis.

The interaction of platelets and molecules that are entrapped in the NETs like von Willebrand factor (vWF), fibronectin and fibrinogen can also induce platelet aggregation [59]. The presence of fibrinogen causes the clots to consolidate through its transformation into fibrin in a thrombin-dependent manner [59]. In fact, Longstaff and colleagues have shown that contact between NETs (DNA + histones) and fibrin induces thicker fibers with improved stability and stiffness, and that the combination of histones and DNA significantly prolongs clot lysis time [60].

Additionally, Stakos and colleagues demonstrated that NETs were capable of secreting functional tissue factor (TF) [61]. TF is the initiating molecule of the extrinsic coagulation pathway and ultimately leads to thrombus formation [62]. The chromatin fibers can
also inhibit the inhibitors of the extrinsic pathway, resulting in an over-activity of this coagulation pathway [63].

Nevertheless, Carminita et al. have demonstrated that following laser-induced injury, neutrophils -but not NETs- are involved in thrombus formation [41]. In fact, activated neutrophils already express CitH3 and PAD4, confirming that they are markers of neutrophil activation rather than NET formation [41]. Moreover, they showed that the inhibition of thrombus formation by DNase-I could be independent of NET formation [41]. This shows the current need for more research on the real role of NETs in thrombus formation.

### 4. Neutrophils in Cancer Development

Neutrophils can play a role in the development of cancer. This is highlighted in the relationship between neutrophilia and worsened prognosis in oncological patients [64]. Cancer cells can secrete chemokines, like IL-8, as well as GRO chemokines (CXCL1/2/3) and TGF-β to induce neutrophil migration to the primary tumor [64,65]. This chemokine explains why tumor-associated neutrophils (TANs) are localized at the margins of the tumor site in the early stages of cancer and may then massively infiltrate the tumor center in advanced stages [64].

Neutrophil secretion of chemokines is also enhanced during cancer development [66,67]. Cytokine IL17 can impact the tumoral microenvironment and cause the tumoral stroma to develop pro-tumorigenic functions, indeed Hayata et al. showed that in a mouse model inhibition of IL17a actually increased the cytotoxicity of tumor-infiltrating lymphocytes [66,68]. They also participate in immune cell recruitment to the tumor and enhancing the cancer-associated inflammation and promoting pathogenic T-cells [67].

In 2019, Wisdom and colleagues demonstrated that neutrophils could promote tumor resistance to radiation therapy in a genetically modified mouse model of sarcoma [69]. They do so by increasing a MAPK-regulated transcriptional program downstream of Kras and upregulating the expression of the AP-1 family transcription factors Fos and Jun, to promote cell proliferation [69]. Moreover, TANs are divided into two subpopulations: the N1, which have an anti-tumor behavior, and the N2, which have a pro-tumor one [70] (Figure 3). This pro-tumor profile of neutrophils can be favored by the cancer cells themselves [71,72]. It is described in the literature that tumor cells can differentially secrete cytokines such as IL-35, IL-10 or TGF-β to induce a switch from N1 to N2 neutrophil phenotype in the early stages of cancer [71,72]. Indirectly, tumor-derived exosomes can also polarize neutrophils into a N2 phenotype via HMGB1/TLR4/NF-κB signaling [73–75]. Therefore, tumor cells themselves can tip the N1-N2 balance to favor a pro-tumoral phenotype of neutrophils.

Pro-tumoral N2 neutrophils can produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) to create DNA damage and genetic instability which can potentially initiate the tumor process [74,76]. They can also secrete mediators such as Oncostatin M (OSM), Matrix Metalloproteinase 9 (MMP9), IL-17 and VEGF into the tumoral microenvironment to induce angiogenesis and support tumor growth [72,77,78].

Pro-tumoral N2 neutrophils play a key role in the formation of metastases by secreting MMP9 and neutrophil elastase (NE) to favor the remodeling of the extracellular matrix necessary for tumor progression [77]. In addition, Li, S. et al. have shown that neutrophils can secrete IL-17A to activate the JAK2/STAT3 pathway to induce the epithelial-mesenchymal transition (EMT) [78]. Moreover, N2 neutrophils secrete inflammatory cytokines such as IL-1B and OSM to promote cancer cell migration and invasion [79]. Pro-tumoral N2 neutrophils are important allies that contribute to the tumor growth and spread through different mechanisms.
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Summary of the role of N2 neutrophils during tumor development. Tumor cells can recruit circulating neutrophils and induce a switch from N1 to N2 neutrophils. Indeed, N2 neutrophils play an important role in several stages of cancer development, like tumor initiation and growth or metastasis formation by secreting several key molecules. Figure created using Servier Medical Art available at http://smart.servier.com/ (accessed on 15 September 2021).</figcaption></figure>

Interestingly, N2 neutrophils also have an immunosuppressive role [72,80,81]. They can eliminate tumor-infiltrating T lymphocytes (TIL) by activating TGF-β via MMP9 [80,81]. They also secrete high levels of arginase-1 and can activate the STAT3 and ERK pathways that lead to iNOS production and suppression of T-lymphocyte activation [70,72,81]. Arginase-1 and iNOS allow the transformation of L-arginine either into urea and L-Ornithine or Nitric Oxide (NO) and citrulline. Resulting in a decrease in L-Arginine levels, a molecule that is essential for the generation of CD3ζ75 [70,81,82]. CD3ζ is a chain of the CD3 complex that associates with the T cell receptor (TCR) and is essential for TCR signaling via the ITAM (immunoreceptor tyrosine-based activation motif) [82]. Without ITAM, the formation of a functional TCR and the proliferation of T lymphocytes are impacted [83]. In addition, extracellular vesicles derived from gastric cancer cells have been shown to induce PD-L1 expression on neutrophils to inactivate TLs [80,83].

Tumors are known to recruit macrophages and platelets to their microenvironment [14,84,85]. Indeed, high-infiltration of both macrophages and platelets can be correlated to a worsened prognosis [2]. Tumor-associated-neutrophils can influence macrophage
polarization to tumor-associated-macrophages (TAM) by secreting Il-8, TNF-α and MPO [84,86–88]. These TAMs have T-cell immunosuppressive properties through PD-PD-L1 signaling and can upregulate Treg functions, thus contributing to the local immuno-suppression that favors tumor growth [83,87,88].

Tumoral platelet infiltration is known to give tumors a survival advantage by degran-ulation, supporting both angiogenesis and tumor-growth [2]. It is logical to assume that neutrophils also interact with these cell types. Activated platelets express P-selectin, which can bind to neutrophils through the PSGL-1 receptor to create platelet-neutrophil aggregates that support metastasis by hiding cancer cells from shear forces in circulation [14,89,90]. Activated platelets can also secrete transforming growth factor beta (TGF-β1) to recruit more neutrophils to the tumoral site and increase T-cell immunosuppression [2,65,70,80].

**5. NETs in Cancer Development**

NETs can support a pro-tumoral role as they are believed to be involved in tumor growth, metastasis, and the awakening of dormant cancer cells [91] (Figure 4). This could explain why NETs are a marker of poor prognosis in cancer patients, especially in terminal cancer patients [92]. In 2016, Demers et colleagues demonstrated with PAD4-deficient mice that NETs are essential for promoting tumor growth [93]. However, the exact mechanisms involved remain unknown to date. In 2019, Yazdani et al. proposed that neutrophil elastase (NE) released by NETs activates TLR4 in cancer cells which results in upregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1A), a transcription coactivator that leads to increased mitochondrial biogenesis to provide additional energy for the tumor to accelerate its growth [94].

Interestingly, Zhang et al. found that the number of circulating NETs in peripheral blood correlated with disease stage in gastric cancer [95]. Indeed, Zhang et al. considered nucleosome-bound NE to indicate NET formation in serum and plasma of peripheral blood samples; these complexes were identified using capture ELISA [95].

In 2017, Park and colleagues demonstrated that blocking NET formation with DNase-I treatment reduced cancer invasion and prevented lung metastasis in mice [96]. Similarly, in colorectal cancer (CRC), it has been shown that increased NETs contribute to the devel-opment of CRC liver metastasis and that their digestion with DNase-I limits the increase in liver metastasis associated with NETs [97].

The exact mechanisms by which NETs are involved in metastasis remain somewhat unclear and controversial. Indeed, two initial hypotheses as to their involvement have been put forward: (1) that the DNA network formed by NETs may trap circulating cancer cells at the site of dissemination or (2) that they may increase local vascular permeability that facilitates the extravasation of cancer cells into the surrounding tissues [96]. Several studies support the first hypothesis that NETs facilitate the adhesion of circulating tumor cells to form metastases [98–100]. In 2020, Yang and colleagues demonstrated that NETs trap cancer cells but do not exert cytotoxicity on them [97]. NETs can increase the cancer cell's proliferative and invasive capacity by triggering tumor IL-8 expression [97]. The over-expression of IL-8 can in turn activate neutrophils and generate NETs that promote metastasis [97].

A third hypothesis (3) regarding the role of NETs in metastasis formation has recently been put forward [101]. The extracellular DNA represented by NETs can act as a chemo-tactic factor to attract cancer cells, instead of simply entrapping them [101]. This has been shown in several mouse models and would occur via the CCDC25 receptor present on the surface of cancer cells [101]. In fact, the CCDC25 receptor then interacts with NETs to recruit ILK to initiate the β-parvin-RAC1-CDC42 cascade, which induces cytoskeletal rearrangement and tumor cell migration [101].
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 4.** Schematic representation of the 3 main hypotheses of NETs in cancer (1) DNA network formed by NETs may trap circulating cancer cells at the site of dissemination. (2) DNA network formed by NETs may increase local vascular permeability that facilitates the extravasation of cancer cells into the surrounding tissues. (3) The extracellular DNA represented by NETs can act as a chemotactic factor to attract cancer cells. Figure created using Servier Medical Art available at http://smart.servier.com/ (accessed on 15 September 2021).</figcaption></figure>

Last but not least, it has been shown in the literature that NETs are also involved in the
awakening of so-called “dormant” or senescent cancer cells [102]. Tumor cells originating
from the primary site can be disseminated in other tissues and remain dormant [102]. The
stimuli that induce them to awaken are not well known. It has been described in mouse
models that NE and MMP9 released by NETs can cleave laminin and induce the prolifera-
tion of dormant cancer cells by activating integrin α3β1 to mediate cell migration [102,103].
Thus, NETs seem to have many pro-tumor effects, but the exact molecular mechanisms remain elusive.

However, the role of NETs in cancer progression remains controversial as anti-tumor effects have also been described [104]. For example, Arelaki and colleagues demonstrated in 2016 that NETs generated in vitro prevent the growth of colorectal cancer cells and primary myeloid leukemia cells, by inducing their apoptosis and/or inhibiting their proliferation [104]. These anti-tumor effects remain to date mostly unknown.

## 6. NETs and Cancer-Associated Thrombosis

Recently, researchers began to study the role of NETs in CAT, especially since CitH3 was shown to be present in the thrombi of cancer patients [105,106]. Numerous studies have corroborated the link between NETs and CAT, including a 2-year prospective study of 946 patients that shows that patients with elevated blood CitH3 levels had a higher cumulative incidence of VTE [107,108].

As we have previously stated, NETs can induce a pro-thrombotic and pro-coagulant state via multiple mechanisms. This pro-coagulant state can be amplified by the cancer cells themselves since they can secrete G-CSF (Granulocyte-Colony Stimulating Factor) in very large quantities [93,109,110]. G-CSF release induces a higher production of neutrophils and, as Demers and colleagues have shown, it also allows neutrophils to spontaneously create more NETs [109]. In addition, cancer cells themselves are also able to release PAD4 into their microenvironment, which promotes citrullination of histone 3 and chromatin de-condensation, leading to NET formation [95].

On the other hand, the tumor microenvironment can also increase the pro-thrombotic state. Indeed, tumors often grow faster than their blood supply, thus provoking hypoxia (which is characteristic of most solid tumors) [110]. McInturff et al. have shown that hypoxia favors the formation of NETs via the mammalian target rapamycin (mTOR) which regulates NET formation by post-transcriptional control of the expression of hypoxia-inducible factor 1 α (HIF-1α) [111].

Thus, NETs may be an attractive target for reducing CAT. Indeed, Boone and colleagues have shown that the use of chloroquine reduces the hypercoagulability observed in pancreatic cancer by inhibiting NETs [112]. Furthermore, it has been shown in an orthotopic mouse model of breast cancer that the use of dunnione, a potent substrate of NAD(P)H quinone oxidoreductase 1, attenuates the pro-thrombotic state by inhibiting TF and NETs formation [113].

Elaskani et al. studied the NET-induced platelet aggregation and found that targeting the NET scaffold was not an effective strategy to reduce platelet activation [114]. More traditionally used anticoagulant and anti-platelet drugs like low molecular weight or unfractioned heparin or direct-acting oral anticoagulants (Apixaban, dabigatran, rivaroxaban or endoxaban) continue to be the gold standard for thrombosis treatment in CAT [115,116]. Recently, the use of platelet P2RY12 inhibitors has been proposed to both prevent and treat TCIPA and CAT, but this application has not yet been validated in clinical trials [15,117].

## 7. Conclusions

We have endeavored to describe the current state of the literature on the relationship between neutrophils, thrombosis, and cancer. Neutrophilia is associated with worse prognosis in cancer patients and this increased neutrophil count has a direct impact on the development of cancer and CAT. Cancer cells themselves can participate in this relationship, creating a vicious circle that enhances both tumor growth and CAT. An important contributor to this relationship appears to be NET formation by neutrophils. NETS potentially sustain not only cancer growth, but also the development of cancer-associated thrombosis; yet the exact molecular mechanisms remain to be elucidated. We have shown that neutrophils play a key role in thrombus and CAT development; highlighting the necessity for further research to harness the power of neutrophils as new potential therapeutic or diagnostic targets in cancer.
**Author Contributions:** Conceptualization, M.L., A.-L.P.-A., L.C., C.D. and L.P.-D.; investigation, M.L. and A.-L.P.-A.; data curation, M.L. and A.-L.P.-A.; writing—original draft preparation, M.L. and A.-L.P.-A.; writing—review and editing M.L., A.-L.P.-A., L.C., D.M., C.D. and L.P.-D.; supervision, L.C., C.D. and L.P.-D. All authors have read and agreed to the published version of the manuscript.

**Funding:** Ana-Luisa Palacios-Acedo was supported by the PAUSE program (College de France-PARIS).

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Abbreviations**

<table><tr><td>ATP</td><td>Adenosine triphosphate</td></tr><tr><td>BPI</td><td>Bactericidal/permeability increasing protein</td></tr><tr><td>CAT</td><td>Cancer associated thrombosis</td></tr><tr><td>CCDC25</td><td>Coiled-Coil Domain Containing 25</td></tr><tr><td>CitH3</td><td>Citrullinated histone H3</td></tr><tr><td>CRC</td><td>Colorectal cancer</td></tr><tr><td>CXCL8/IL-8</td><td>Interleukin 8</td></tr><tr><td>EMT</td><td>Epithelial-mesenchymal transition</td></tr><tr><td>EPCAM</td><td>Epithelial cell adhesion molecule</td></tr><tr><td>ERK</td><td>Extracellular signal-regulated kinases</td></tr><tr><td>fMLP</td><td>N-formyl-Methionyl-leucyl-phenylalanine</td></tr><tr><td>G-CSF</td><td>Granulocyte-Colony Stimulating Factor</td></tr><tr><td>HIF-1α</td><td>Hypoxia-inducible factor 1 α</td></tr><tr><td>HMGB1</td><td>High-mobility group box 1</td></tr><tr><td>ICAM1/CD54</td><td>Intercellular Adhesion Molecule-1</td></tr><tr><td>ILK</td><td>Integrin-linked kinase</td></tr><tr><td>iNOS</td><td>Inducible nitric oxide synthase</td></tr><tr><td>ITAM</td><td>Immunoreceptor tyrosine-based activation motif</td></tr><tr><td>JAK2</td><td>Janus kinase 2</td></tr><tr><td>LFA1</td><td>Leukocyte-function-associated-antigen-1</td></tr><tr><td>LTB4</td><td>Leukotriene B4</td></tr><tr><td>MAC1</td><td>Macrophage-1 Antigen</td></tr><tr><td>MAPK</td><td>Mitogen-activated protein kinases</td></tr><tr><td>MMP9</td><td>Matrix Metalloproteinase 9</td></tr><tr><td>MPO</td><td>Myeloperoxidase</td></tr><tr><td>mTOR</td><td>Mammalian target rapamycin</td></tr><tr><td>TNFα</td><td>Tumor-necrosis-factor-α</td></tr><tr><td>VCAM1</td><td>Vascular Cell Adhesion Molecule-1</td></tr><tr><td>VEGF</td><td>Vascular Endothelial Growth Factor</td></tr><tr><td>MV</td><td>Microvesicles</td></tr><tr><td>NE</td><td>Neutrophil elastase</td></tr><tr><td>NET</td><td>Neutrophil extracellular traps</td></tr><tr><td>NF-κB</td><td>Nuclear factor-kappa B</td></tr><tr><td>nMV</td><td>Neutrophil derived MVs</td></tr><tr><td>NO</td><td>Nitric Oxide</td></tr><tr><td>OSM</td><td>Oncostatin M</td></tr><tr><td>P2RX1/P2X1</td><td>P2X purinoceptor 1</td></tr><tr><td>PAD4</td><td>Peptidylarginine deiminase 4</td></tr></table>
<table><tr><td>PDGF</td><td>Platelet derived growth factor</td></tr><tr><td>PD-L1</td><td>Programmed death-ligand 1</td></tr><tr><td>PECAM1</td><td>Platelet endothelial cell adhesion molecule-1</td></tr><tr><td>PGC1A</td><td>Peroxisome proliferator-activated receptor gamma coactivator 1-alpha</td></tr><tr><td>PMN</td><td>Polymorphonuclear cells</td></tr><tr><td>RNS</td><td>Reactive nitrogen species</td></tr><tr><td>ROS</td><td>Reactive oxygen species</td></tr><tr><td>STAT3</td><td>Signal transducer and activator of transcription 3</td></tr><tr><td>TAM</td><td>Tumor-associated macrophage</td></tr><tr><td>TAN</td><td>Tumor-associated neutrophils</td></tr><tr><td>TCIPA</td><td>Tumor cell induced platelet activation</td></tr><tr><td>TCR</td><td>T cell receptor</td></tr><tr><td>TF</td><td>Tissue Factor</td></tr><tr><td>TGF-β</td><td>Transforming growth factor beta</td></tr><tr><td>TIL</td><td>Tumor-infiltrating T lymphocytes</td></tr><tr><td>TLR4</td><td>Toll Like Receptor 4</td></tr><tr><td>TLs</td><td>T Lymphocytes</td></tr><tr><td>VEGF</td><td>Vascular endothelial growth factor</td></tr><tr><td>VTE</td><td>Venous thromboembolism</td></tr><tr><td>vWF</td><td>von Willebrand factor</td></tr><tr><td>MV</td><td>Microvesicles</td></tr><tr><td>NE</td><td>Neutrophil elastase</td></tr></table>

## References

1. Ikushima, S.; Ono, R.; Fukuda, K.; Sakayori, M.; Awano, N.; Kondo, K. Trousseau's syndrome: Cancer-associated thrombosis. *Jpn. J. Clin. Oncol.* **2015**, *46*, 204–208. [CrossRef] [PubMed]

2. Palacios-Acedo, A.L.; Mège, D.; Crescence, L.; Dignat-George, F.; Dubois, C.; Panicot-Dubois, L. Platelets, Thrombo-Inflammation, and Cancer: Collaborating with the Enemy. *Front. Immunol.* **2019**, *10*, 1805. [CrossRef] [PubMed]

3. Falanga, A.; Marchetti, M.; Vignoli, A. Coagulation and cancer: Biological and clinical aspects. *J. Thromb. Haemost.* **2012**, *11*, 223–233. [CrossRef] [PubMed]

4. Hisada, Y.; Mackman, N. Cancer-associated pathways and biomarkers of venous thrombosis. *Blood* **2017**, *130*, 1499–1506. [CrossRef] [PubMed]

5. Timp, J.F.; Braekkan, S.K.; Versteeg, H.; Cannegieter, S.C. Epidemiology of cancer-associated venous thrombosis. *Blood* **2013**, *122*, 1712–1723. [CrossRef]

6. Plantureux, L.; Crescence, L.; Dignat-George, F.; Panicot-Dubois, L.; Dubois, C. Effects of platelets on cancer progression. *Thromb. Res.* **2018**, *164*, S40–S47. [CrossRef]

7. Varki, A. Trousseau's syndrome: Multiple definitions and multiple mechanisms. *Blood* **2007**, *110*, 1723–1729. [CrossRef]

8. Croninfenton, D.P.; Søndergaard, F.; Pedersen, L.; Fryzek, J.P.; Cetin, K.; Acquavella, J.F.; Baron, J.A.; Sørensen, H.T. Hospitalisation for venous thromboembolism in cancer patients and the general population: A population-based cohort study in Denmark, 1997–2006. *Br. J. Cancer* **2010**, *103*, 947–953. [CrossRef]

9. Khorana, A.A.; Francis, C.W.; Culakova, E.; Kuderer, N.M.; Lyman, G.H. Thromboembolism is a leading cause of death in cancer patients receiving outpatient chemotherapy. *J. Thromb. Haemost.* **2007**, *5*, 632–634. [CrossRef]

10. Mammadova-Bach, E.; Zigrino, P.; Brucker, C.; Bourdon, C.; Freund, M.; De Arcangelis, A.; Abrams, S.I.; Orend, G.; Gachet, C.; Mangin, P.H. Platelet integrin α6β1 controls lung metastasis through direct binding to cancer cell-derived ADAM9. *JCI Insight* **2016**, *1*, e88245. [CrossRef]

11. Boukerche, H.; Berthier-Vergnes, O.; Penin, F.; Tabone, E.; Lizard, G.; Bailly, M.; McGregor, J.L. Human melanoma cell lines differ in their capacity to release ADP and aggregate platelets. *Br. J. Haematol.* **1994**, *87*, 763–772. [CrossRef] [PubMed]

12. Kloss, L.; Dollt, C.; Schledzewski, K.; Krewer, A.; Melchers, S.; Manta, C.; Sticht, C.; De La Torre, C.; Utikal, J.; Umansky, V.; et al. ADP secreted by dying melanoma cells mediates chemotaxis and chemokine secretion of macrophages via the purinergic receptor P2Y12. *Cell Death Dis.* **2019**, *10*, 760. [CrossRef] [PubMed]

13. Xu, X.R.; Yousef, G.M.; Ni, H. Cancer and platelet crosstalk: Opportunities and challenges for aspirin and other antiplatelet agents. *Blood* **2018**, *131*, 1777–1789. [CrossRef] [PubMed]

14. Schlesinger, M. Role of platelets and platelet receptors in cancer metastasis. *J. Hematol. Oncol.* **2018**, *11*, 125. [CrossRef] [PubMed]

15. Palacios-Acedo, A.L.; Mezouar, S.; Mège, D.; Crescence, L.; Dubois, C.; Panicot-Dubois, L. P2RY12-Inhibitors Reduce Cancer-Associated Thrombosis and Tumor Growth in Pancreatic Cancers. *Front. Oncol.* **2021**, *11*, 704945. [CrossRef]

16. Holmes, C.E.; Levis, J.E.; Schneider, D.J.; Bambace, N.M.; Sharma, D.; Lal, I.; Wood, M.E.; Muss, H.B. Platelet phenotype changes associated with breast cancer and its treatment. *Platelets* **2016**, *27*, 703–711. [CrossRef]
17. Plantureux, L.; Mège, D.; Crescence, L.; Carminita, E.; Robert, S.; Cointe, S.; Brouilly, N.; Ezzedine, W.; Dignat-George, F.; Dubois, C.; et al. The Interaction of Platelets with Colorectal Cancer Cells Inhibits Tumor Growth but Promotes Metastasis. *Cancer Res.* **2020**, *80*, 291–303. [CrossRef]

18. Mezouar, S.; Darbousset, R.; Dignat-George, F.; Panicot-Dubois, L.; Dubois, C. Inhibition of platelet activation prevents the P-selectin and integrin-dependent accumulation of cancer cell microparticles and reduces tumor growth and metastasis in vivo. *Int. J. Cancer* **2014**, *136*, 462–475. [CrossRef]

19. Mitrugno, A.; Yunga, S.T.; Sylman, J.L.; Zilberman-Rudenko, J.; Shirai, T.; Hebert, J.F.; Kayton, R.; Zhang, Y.; Nan, X.; Shatzel, J.J.; et al. The role of coagulation and platelets in colon cancer-associated thrombosis. *Am. J. Physiol. Physiol.* **2019**, *316*, C264–C273. [CrossRef]

20. Mezouar, S.; Frère, C.; Darbousset, R.; Mege, D.; Crescence, L.; Dignat-George, F.; Panicot-Dubois, L.; Dubois, C. Role of platelets in cancer and cancer-associated thrombosis: Experimental and clinical evidences. *Thromb. Res.* **2016**, *139*, 65–76. [CrossRef]

21. Franco, A.T.; Corken, A.; Ware, J. Platelets at the interface of thrombosis, inflammation, and cancer. *Blood* **2015**, *126*, 582–588. [CrossRef] [PubMed]

22. Schernberg, A.; Moureau-Zabotto, L.; Del Campo, E.R.; Escande, A.; Ducreux, M.; Nguyen, F.; Goere, D.; Chargari, C.; Deutsch, E. Leukocytosis and neutrophilia predict outcome in locally advanced esophageal cancer treated with definitive chemoradiation. *Oncotarget* **2017**, *8*, 11579–11588. [CrossRef] [PubMed]

23. Schernberg, A.; Nivet, A.; Dhermain, F.; Ammari, S.; Escande, A.; Pallud, J.; Louvel, G.; Deutsch, E. Neutrophilia as a biomarker for overall survival in newly diagnosed high-grade glioma patients undergoing chemoradiation. *Clin. Transl. Radiat. Oncol.* **2018**, *10*, 47–52. [CrossRef] [PubMed]

24. Schernberg, A.; Mezquita, L.; Boros, A.; Botticella, A.; Caramella, C.; Besse, B.; Escande, A.; Planchard, D.; Le Péchoux, C.; Deutsch, E. Neutrophilia as prognostic biomarker in locally advanced stage III lung cancer. *PLoS ONE* **2018**, *13*, e0204490. [CrossRef]

25. Pfeiler, S.; Stark, K.; Massberg, S.; Engelmann, B. Propagation of thrombosis by neutrophils and extracellular nucleosome networks. *Haematologica* **2016**, *102*, 206–213. [CrossRef]

26. Castanheira, F.V.S.; Kubes, P. Neutrophils and NETs in modulating acute and chronic inflammation. *Blood* **2019**, *133*, 2178–2185. [CrossRef]

27. Noubouossie, D.F.; Reeves, B.N.; Strahl, B.D.; Key, N.S. Neutrophils: Back in the thrombosis spotlight. *Blood* **2019**, *133*, 2186–2197. [CrossRef]

28. Kapoor, S.; Opneja, A.; Nayak, L. The role of neutrophils in thrombosis. *Thromb. Res.* **2018**, *170*, 87–96. [CrossRef]

29. Kolaczkowska, E.; Kubes, P. Neutrophil recruitment and function in health and inflammation. *Nat. Rev. Immunol.* **2013**, *13*, 159–175. [CrossRef]

30. Phillipson, M.; Kubes, P. The neutrophil in vascular inflammation. *Nat. Med.* **2011**, *17*, 1381–1390. [CrossRef]

31. Cassatella, M.A.; Östberg, N.; Tamassia, N.; Soehnlein, O. Biological Roles of Neutrophil-Derived Granule Proteins and Cytokines. *Trends Immunol.* **2019**, *40*, 648–664. [CrossRef] [PubMed]

32. Cowland, J.B.; Borregaard, N. Granulopoiesis and granules of human neutrophils. *Immunol. Rev.* **2016**, *273*, 11–28. [CrossRef] [PubMed]

33. Kobayashi, S.D.; Malachowa, N.; DeLeo, F.R. Influence of Microbes on Neutrophil Life and Death. *Front. Cell. Infect. Microbiol.* **2017**, *7*, 159. [CrossRef] [PubMed]

34. Gross, P.L.; Furie, B.C.; Merrill-Skoloff, G.; Chou, J.; Furie, B. Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development. *J. Leukoc. Biol.* **2005**, *78*, 1318–1326. [CrossRef] [PubMed]

35. Darbousset, R.; Thomas, G.; Mezouar, S.; Frère, C.; Bonier, R.; Mackman, N.; Renné, T.; Dignat-George, F.; Dubois, C.; Panicot-Dubois, L. Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation. *Blood* **2012**, *120*, 2133–2143. [CrossRef] [PubMed]

36. Von Brühl, M.L.; Stark, K.; Steinhart, A.; Chandraratne, S.; Konrad, I.; Lorenz, M.; Khandoga, A.; Tirniceriu, A.; Coletti, R.; Köllnberger, M.; et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. *J. Exp. Med.* **2012**, *209*, 819–835. [CrossRef]

37. Rubel, C.; Fernández, G.C.; Dran, G.; Bompadre, M.B.; Isturiz, M.A.; Palermo, M.S. Fibrinogen Promotes Neutrophil Activation and Delays Apoptosis. *J. Immunol.* **2001**, *166*, 2002–2010. [CrossRef]

38. Darbousset, R.; Delierneux, C.; Mezouar, S.; Hego, A.; Lecut, C.; Guillaumat, I.; Riederer, M.A.; Evans, R.J.; Dignat-George, F.; Panicot-Dubois, L.; et al. P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice. *Blood* **2014**, *124*, 2575–2585. [CrossRef]

39. Schaff, U.Y.; Dixit, N.; Procyk, E.; Yamayoshi, I.; Tse, T.; Simon, S.I. Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear flow. *Blood* **2010**, *115*, 657–666. [CrossRef]

40. Dubois, C.; Panicot-Dubois, L.; Gainor, J.F.; Furie, B.C.; Furie, B. Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model. *J. Clin. Investig.* **2007**, *117*, 953–960. [CrossRef]

41. Carminita, E.; Crescence, L.; Brouilly, N.; Altié, A.; Panicot-Dubois, L.; Dubois, C. DNAse-dependent, NET-independent pathway of thrombus formation in vivo. *Proc. Natl. Acad. Sci. USA* **2021**, *118*, e2100561118. [CrossRef] [PubMed]

42. Dubois, C.; Panicot-Dubois, L.; Furie, B.C.; Furie, B. Dynamics of Calcium Mobilization in Platelets during Thrombus Formation in a Living Mouse. *Blood* **2005**, *106*, 649. [CrossRef]
43. Egorina, E.M.; Sovershaev, M.A.; Olsen, J.O.; Østerud, B. Granulocytes do not express but acquire monocyte-derived tissue factor in whole blood: Evidence for a direct transfer. *Blood* **2008**, *111*, 1208–1216. [CrossRef] [PubMed]

44. Maugeri, N.; Brambilla, M.; Camera, M.; Carbone, A.; Tremoli, E.; Donati, M.B.; DE Gaetano, G.; Cerletti, C. Human polymor-
phonuclear leukocytes produce and express functional tissue factor upon stimulation1. *J. Thromb. Haemost.* **2006**, *4*, 1323–1330.
[CrossRef]

45. Ritis, K.; Doumas, M.; Mastellos, D.; Micheli, A.; Giaglis, S.; Magotti, P.; Rafail, S.; Kartalis, G.; Sideras, P.; Lambris, J. A Novel C5a Receptor-Tissue Factor Cross-Talk in Neutrophils Links Innate Immunity to Coagulation Pathways. *J. Immunol.* **2006**, *177*, 4794–4802. [CrossRef]

46. Mege, D.; Crescence, L.; Ouaissi, M.; Sielezneff, I.; Guieu, R.; Dignat-George, F.; Dubois, C.; Panicot-Dubois, L. Fibrin-bearing microparticles: Marker of thrombo-embolic events in pancreatic and colorectal cancers. *Oncotarget* **2017**, *8*, 97394–97406. [CrossRef]

47. Willms, A.; Müller, C.; Julich, H.; Klein, N.; Schwab, R.; Güsgen, C.; Richardsen, I.; Schaaf, S.; Krawczyk, M.; Krawczyk, M.; et al. Tumour-associated circulating microparticles: A novel liquid biopsy tool for screening and therapy monitoring of colorectal carcinoma and other epithelial neoplasia. *Oncotarget* **2016**, *7*, 30867–30875. [CrossRef]

48. Mege, D.; Panicot-Dubois, L.; Dubois, C. Tumor-Derived Microparticles to Monitor Colorectal Cancer Evolution. In *Advanced Structural Safety Studies*; Springer: Berlin/Heidelberg, Germany, 2018; Volume 1765, pp. 271–277.

49. Gong, J.; Jaiswal, R.; Dalla, P.; Luk, F.; Bebawy, M. Microparticles in cancer: A review of recent developments and the potential for clinical application. *Semin. Cell Dev. Biol.* **2015**, *40*, 35–40. [CrossRef]

50. Mezouar, S.; Mege, D.; Darbousset, R.; Farge, D.; Debourdeau, P.; Dignat-George, F.; Panicot-Dubois, L.; Dubois, C. Involvement of Platelet-Derived Microparticles in Tumor Progression and Thrombosis. *Semin. Oncol.* **2014**, *41*, 346–358. [CrossRef]

51. Nomura, S.; Shimizu, M. Clinical significance of procoagulant microparticles. *J. Intensiv. Care* **2015**, *3*, 2–11. [CrossRef]

52. Pitanga, T.N.; França, L.D.A.; Rocha, V.C.J.; Meirelles, T.; Borges, V.M.; Gonçalves, M.S.; Pontes-De-Carvalho, L.C.;oronha-Dutra A.A.; Dos-Santos, W.L.C. Neutrophil-derived microparticles induce myeloperoxidase-mediated damage of vascular endothelial cells. *BMC Cell Biol.* **2014**, *15*, 21. [CrossRef]

53. Pluskota, E.; Woody, N.M.; Szpak, D.; Ballantyne, C.M.; Soloviev, D.A.; Simon, D.I.; Plow, E.F. Expression, activation, and function of integrin αMβ2 (Mac-1) on neutrophil-derived microparticles. *Blood* **2008**, *112*, 2327–2335. [CrossRef] [PubMed]

54. Brinkmann, V.; Reichard, U.; Goosmann, C.; Fauler, B.; Uhlemann, Y.; Weiss, D.S.; Weinrauch, Y.; Zychlinsky, A. Neutrophil extracellular traps kill bacteria. *Science* **2004**, *303*, 1532–1535. [CrossRef] [PubMed]

55. Kumar, S.; Gupta, E.; Kaushik, S.; Jyoti, A. Neutrophil Extracellular Traps: Formation and Involvement in Disease Progression. *Iran. J. Allergy Asthma Immunol.* **2018**, *17*, 208–220.

56. Wang, Y.; Li, M.; Stadler, S.C.; Correll, S.; Li, P.; Wang, D.; Hayama, R.; Leonelli, L.; Han, H.; Grigoryev, S.A.; et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. *J. Cell Biol.* **2009**, *184*, 205–213. [CrossRef] [PubMed]

57. Savchenko, A.S.; Martinod, K.; Seidman, M.; Wong, S.L.; Borisoff, J.I.; Piazza, G.; Libby, P.; Goldhaber, S.Z.; Mitchell, R.N.; Wagner, D.D. Neutrophil extracellular traps form predominantly during the organizing stage of human venous thromboembolism development. *J. Thromb. Haemost.* **2014**, *12*, 860–870. [CrossRef] [PubMed]

58. Martinod, K.; Demers, M.; Fuchs, T.A.; Wong, S.L.; Brill, A.; Gallant, M.; Hu, J.; Wang, Y.; Wagner, D.D. Neutrophil histone modi fi cation by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice. *Proc. Natl. Acad. Sci. USA* **2013**, *110*, 8674–8679. [CrossRef]

59. Fuchs, T.A.; Brill, A.; Duerschmied, D.; Schatzberg, D.; Monestier, M.; Myers, D.D., Jr.; Wrobleski, S.K.; Wakefield, T.W.; Hartwig, J.H.; Wagner, D.D. Extracellular DNA traps promote thrombosis. *Proc. Natl. Acad. Sci. USA* **2010**, *107*, 15880–15885. [CrossRef]

60. Longstaff, C.; Varjú, I.; Sótonyi, P.; Szabó, L.; Krumrey, M.; Hoell, A.; Bóta, A.; Varga, Z.; Komorowicz, E.; Kolev, K. Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones. *J. Biol. Chem.* **2013**, *288*, 6946–6956. [CrossRef]

61. Stakos, D.A.; Kambas, K.; Konstantinidis, T.; Mitroulis, I.; Apostolidou, E.; Arelaki, S.; Tsironidou, V.; Giatromanolaki, A.; Skendros, P.; Konstantinides, S.; et al. Expression of functional tissue factor by neutrophil extracellular traps in culprit artery of acute myocardial infarction. *Eur. Heart J.* **2015**, *36*, 1405–1414. [CrossRef]

62. Butenas, S.; Orfeo, T.; Mann, K.G. Tissue Factor in Coagulation. *Arter. Thromb. Vasc. Biol.* **2009**, *29*, 1989–1996. [CrossRef]
[PubMed]

63. Massberg, S.; Grahl, L.; von Bruehl, M.-L.; Manukyan, D.; Pfeiler, S.; Goosmann, C.; Brinkmann, V.; Lorenz, M.; Bidzhekov, K.; Khandagale, A.B.; et al. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. *Nat. Med.* **2010**, *16*, 887–896. [CrossRef] [PubMed]

64. Mishalian, I.; Bayuh, R.; Levy, L.; Zolotarov, L.; Michaeli, J.; Fridlender, Z.G. Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression. *Cancer Immunol. Immunother.* **2013**, *62*, 1745–1756. [CrossRef] [PubMed]

65. SenGupta, S.; Hein, L.E.; Xu, Y.; Zhang, J.; Konwerski, J.R.; Li, Y.; Johnson, C.; Cai, D.; Smith, J.L.; Parent, C.A. Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-β and CXCR2 Ligands. *Front. Immunol.* **2021**, *12*, 973. [CrossRef]

66. Mucciolo, G.; Curcio, C.; Roux, C.; Li, W.Y.; Capello, M.; Curto, R.; Chiarle, R.; Giordano, D.; Satolli, M.A.; Lawlor, R.; et al. IL17A critically shapes the transcriptional program of fibroblasts in pancreatic cancer and switches on their protumorigenic functions. *Proc. Natl. Acad. Sci. USA* **2021**, *118*, e2020395118. [CrossRef]
67. McGinley, A.M.; Sutton, C.E.; Edwards, S.C.; Leane, C.M.; Decoursey, J.; Teijeiro, A.; Hamilton, J.A.; Boon, L.; Djouder, N.; Mills, K.H. Interleukin-17A Serves a Priming Role in Autoimmunity by Recruiting IL-1β-Producing Myeloid Cells that Promote Pathogenic T Cells. *Immunity* **2020**, *52*, 342–356.e6. [CrossRef]

68. Hayata, K.; Iwahashi, M.; Ojima, T.; Katsuda, M.; Iida, T.; Nakamori, M.; Ueda, K.; Nakamura, M.; Miyazawa, M.; Tsuji, T.; et al. Inhibition of IL-17A in Tumor Microenvironment Augments Cytotoxicity of Tumor-Infiltrating Lymphocytes in Tumor-Bearing Mice. *PLoS ONE* **2013**, *8*, e53131. [CrossRef]

69. Wisdom, A.J.; Hong, C.S.; Lin, A.J.; Xiang, Y.; Cooper, D.E.; Zhang, J.; Xu, E.S.; Kuo, H.-C.; Mowery, Y.M.; Carpenter, D.; et al. Neutrophils promote tumor resistance to radiation therapy. *Proc. Natl. Acad. Sci. USA* **2019**, *116*, 18584–18589. [CrossRef]

70. Fridlender, Z.G.; Sun, J.; Kim, S.; Kapoor, V.; Cheng, G.; Ling, L.; Scott Worthen, G.; Albelda, S.M. Polarization of tumor-associated neutrophil pheno-type by TGF-beta: "N1" versus "N2" TAN. *Cancer Cell* **2009**, *16*, 183–194. [CrossRef]

71. Sagiv, J.Y.; Michaeli, J.; Assi, S.; Mishalian, I.; Kisos, H.; Levy, L.; Damti, P.; Lumbroso, D.; Polyansky, L.; Sionov, R.V.; et al. Phenotypic diversity and plasticity in circulating neutrophil subpopulations in cancer. *Cell Rep.* **2015**, *10*, 562–573. [CrossRef]

72. Zou, J.-M.; Qin, J.; Li, Y.-C.; Wang, Y.; Li, D.; Shu, Y.; Luo, C.; Wang, S.-S.; Chi, G.; Guo, F.; et al. IL-35 induces N2 phenotype of neutrophils to promote tumor growth. *Oncotarget* **2017**, *8*, 33501–33514. [CrossRef] [PubMed]

73. Scalerandi, M.V.; Peinetti, N.; Leimgruber, C.; Rubio, M.M.C.; Nicola, J.P.; Menezes, G.B.; Maldonado, C.A.; Quintar, A. Inefficient N2-Like Neutrophils Are Promoted by Androgens During Infection. *Front. Immunol.* **2018**, *9*, 1980. [CrossRef] [PubMed]

74. Powell, D.R.; Huttenlocher, A. Neutrophils in the Tumor Microenvironment. *Trends Immunol.* **2015**, *37*, 41–52. [CrossRef] [PubMed]

75. Zhang, X.; Shi, H.; Yuan, X.; Jiang, P.; Qian, H.; Xu, W. Tumor-derived exosomes induce N2 polarization of neutrophils to promote gastric cancer cell migration. *Mol. Cancer* **2018**, *17*, 146. [CrossRef]

76. Furumaya, C.; Martinez-Sanz, P.; Bouti, P.; Kuijpers, T.W.; Matlung, H.L. Plasticity in Pro- and Anti-tumor Activity of Neutrophils: Shifting the Balance. *Front. Immunol.* **2020**, *11*, e2100. [CrossRef]

77. Wang, X.; Qiu, L.; Li, Z.; Wang, X.-Y.; Yi, H. Understanding the Multifaceted Role of Neutrophils in Cancer and Autoimmune Diseases. *Front. Immunol.* **2018**, *9*, 2456. [CrossRef]

78. Li, T.-J.; Jiang, Y.-M.; Hu, Y.-F.; Huang, L.; Yu, J.; Zhao, L.-Y.; Deng, H.-J.; Mou, T.-Y.; Liu, H.; Yang, Y.; et al. Interleukin-17-Producing Neutrophils Link Inflammatory Stimuli to Disease Progression by Promoting Angiogenesis in Gastric Cancer. *Clin. Cancer Res.* **2016**, *23*, 1575–1585. [CrossRef]

79. Bruchard, M.; Ghiringhelli, F. Microenvironnement tumoral. *Med./Sci. Paris* **2014**, *30*, 429–435. [CrossRef]

80. Germann, M.; Zangger, N.; Sauvain, M.O.; Sempoux, C.; Bowler, A.D.; Wirapati, P.; Kandalaft, L.E.; Delorenzi, M.; Tejpar, S.; Coukos, G.; et al. Neutrophils suppress tumor-infiltrating T cells in colon cancer via matrix metalloproteinase-mediated activation of TGF β. *EMBO Mol. Med.* **2020**, *12*, e10681. [CrossRef]

81. Li, S.; Cong, X.; Gao, H.; Lan, X.; Li, Z.; Wang, W.; Song, S.; Wang, Y.; Li, C.; Zhang, H.; et al. Tumor-associated neutrophils induce EMT by IL-17a to promote migration and invasion in gastric cancer cells. *J. Exp. Clin. Cancer Res.* **2019**, *38*, 6. [CrossRef]

82. Bald, T.; Quast, T.; Landsberg, J.; Rogava, M.; Glodde, N.; Lopez-Ramos, D.; Kohlmeyer, J.; Riesenberg, S.; van den Boorn-Konijnenberg, D.; Hömig-Hölzel, C.; et al. Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma. *Nature* **2014**, *507*, 109–113. [CrossRef] [PubMed]

83. Shi, Y.; Zhang, J.; Mao, Z.; Jiang, H.; Liu, W.; Shi, H.; Ji, R.; Xu, W.; Qian, H.; Zhang, X. Extracellular Vesicles from Gastric Cancer Cells Induce PD-L1 Expression on Neutrophils to Suppress T-Cell Immunity. *Front. Oncol.* **2020**, *10*, 629. [CrossRef] [PubMed]

84. Kim, J.; Bae, J.-S. Tumor-Associated Macrophages and Neutrophils in Tumor Microenvironment. *Mediat. Inflamm.* **2016**, *2016*, 6058147. [CrossRef] [PubMed]

85. Lin, Y.; Xu, J.; Lan, H. Tumor-associated macrophages in tumor metastasis: Biological roles and clinical therapeutic applications. *J. Hematol. Oncol.* **2019**, *12*, 76. [CrossRef]

86. Casazza, A.; Laoui, D.; Wenes, M.; Rizzolio, S.; Bassani, N.; Mambretti, M.; Deschoemaeker, S.; Van Ginderachter, J.A.; Tamagnone, L.; Mazzone, M. Impeding Macrophage Entry into Hypoxic Tumor Areas by Sema3A/Nrp1 Signaling Blockade Inhibits Angiogenesis and Restores Antitumor Immunity. *Cancer Cell* **2013**, *24*, 695–709. [CrossRef]

87. Laoui, D.; Keirsse, J.; Morias, Y.; Van Overmeire, E.; Geeraerts, X.; Elkrim, Y.; Kiss, M.; Bolli, E.; Lahmar, Q.; Sichien, D.; et al. The tumour microenvironment harbours ontogenically distinct dendritic cell populations with opposing effects on tumour immunity. *Nat. Commun.* **2016**, *7*, 13720. [CrossRef]

88. Savage, N.D.L.; De Boer, T.; Walburg, K.V.; Joosten, S.A.; Van Meijgaarden, K.; Geluk, A.; Ottenhoff, T.H.M. Human Anti-Inflammatory Macrophages Induce Foxp3+GITR+CD25+ Regulatory T Cells, Which Suppress via Membrane-Bound TGFβ-1. *J. Immunol.* **2008**, *181*, 2220–2226. [CrossRef]

89. Templeton, A.J.; Mcnamara, M.G.; Šeruga, B.; Vera-Badillo, F.E.; Aneja, P.; Ocaña, A.; Leibowitz-Amit, R.; Sonpavde, G.; Knox, J.J.; Tran, B.; et al. Prognostic Role of Neutrophil-to-Lymphocyte Ratio in Solid Tumors: A Systematic Review and Meta-Analysis. *J. Natl. Cancer Inst.* **2014**, *106*, dju124. [CrossRef]

90. Lecot, P.; Sarabi, M.; Abrantes, M.P.; Mussard, J.; Koenderman, L.; Caux, C.; Bendriss-Vermare, N.; Michallet, M.-C. Neutrophil Heterogeneity in Cancer: From Biology to Therapies. *Front. Immunol.* **2019**, *10*, 2155. [CrossRef]

91. Nolan, E.; Malanchi, I. Neutrophil 'safety net' causes cancer cells to metastasize and proliferate. *Nature* **2020**, *583*, 32–33. [CrossRef]
92. Rosell, A.; Aguilera, K.; Hisada, Y.; Schmedes, C.; Mackman, N.; Wallén, H.; Lundström, S.; Thålin, C. Prognostic value of circulating markers of neutrophil activation, neutrophil extracellular traps, coagulation and fibrinolysis in patients with terminal cancer. *Sci. Rep.* **2021**, *11*, 5074. [CrossRef] [PubMed]

93. Demers, M.; Wong, S.L.; Martinod, K.; Gallant, M.; Cabral, J.E.; Wang, Y.; Wagner, D.D. Priming of neutrophils toward NETosis promotes tumor growth. *OncoImmunology* **2016**, *5*, e1134073. [CrossRef] [PubMed]

94. Yazdani, H.O.; Roy, E.; Comerci, A.J.; van der Windt, D.J.; Zhang, H.; Huang, H.; Loughran, P.; Shiva, S.; Geller, D.A.; Bartlett, D.L.; et al. Neutrophil Extracellular Traps Drive Mitochondrial Homeostasis in Tumors to Augment Growth. *Cancer Res.* **2019**, *79*, 5626–5639. [CrossRef] [PubMed]

95. Zhang, Y.; Hu, Y.; Ma, C.; Sun, H.; Wei, X.; Li, M.; Wei, W.; Zhang, F.; Yang, F.; Wang, H.; et al. Diagnostic, Therapeutic Predictive, and Prognostic Value of Neutrophil Extracellular Traps in Patients with Gastric Adenocarcinoma. *Front. Oncol.* **2020**, *10*, 1036. [CrossRef] [PubMed]

96. Park, J.; Wysocki, R.W.; Amoozgar, Z.; Maiorino, L.; Fein, M.R.; Jorns, J.; Schott, A.F.; Kinugasa-Katayama, Y.; Lee, Y.; Won, N.H.; et al. Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps. *Sci. Transl. Med.* **2016**, *8*, 361ra138. [CrossRef] [PubMed]

97. Yang, L.; Liu, L.; Zhang, R.; Hong, J.; Wang, Y.; Wang, J.; Zuo, J.; Zhang, J.; Chen, J.; Hao, H. IL-8 mediates a positive loop connecting increased neutrophil extracellular traps (NETs) and colorectal cancer liver metastasis. *J. Cancer* **2020**, *11*, 4384–4396. [CrossRef]

98. Ferrucci, P.F.; Ascierto, P.A.; Pigozzo, J.; Del Vecchio, M.; Maio, M.; Cappellini, G.C.A.; Guidoboni, M.; Queirolo, P.; Savoia, P.; Mandalà, M.; et al. Baseline neutrophils and derived neutrophil-to-lymphocyte ratio: Prognostic relevance in metastatic melanoma patients receiving ipilimumab. *Ann. Oncol.* **2016**, *27*, 732–738. [CrossRef]

99. Tohme, S.; Yazdani, H.O.; Al-Khafaji, A.B.; Chidi, A.P.; Loughran, P.; Mowen, A.K.; Wang, Y.; Simmons, R.L.; Huang, H.; Tsung, A. Neutrophil extracellular traps promote the development and progression of liver metastases after surgical stress. *Cancer Res.* **2016**, *76*, 1367–1380. [CrossRef]

100. Cools-Lartigue, J.; Spicer, J.; Najmeh, S.; Ferri, L. Neutrophil extracellular traps in cancer progression. *Cell. Mol. Life Sci.* **2014**, *71*, 4179–4194. [CrossRef]

101. Yang, L.; Liu, Q.; Zhang, X.; Liu, X.; Zhou, B.; Chen, J.; Huang, D.; Li, J.; Li, H.; Chen, F.; et al. DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25. *Nature* **2020**, *583*, 133–138. [CrossRef]

102. Albrengues, J.; Shields, M.A.; Ng, D.; Park, C.G.; Ambrico, A.; Poindexter, M.E.; Upadhyay, P.; Uyeminami, D.L.; Pommier, A.; Küttner, V.; et al. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. *Science* **2018**, *361*, eaao4227. [CrossRef] [PubMed]

103. Kreidberg, J.A. Functions of α3β1 integrin. *Curr. Opin. Cell Biol.* **2000**, *12*, 548–553. [CrossRef]

104. Arelaki, S.; Arampatzioglou, A.; Kambas, K.; Papagoras, C.; Miltiades, P.; Angelidou, I.; Mitsios, A.; Kotsianidis, I.; Skendros, P.; Sivridis, E.; et al. Gradient Infiltration of Neutrophil Extracellular Traps in Colon Cancer and Evidence for Their Involvement in Tumour Growth. *PLoS ONE* **2016**, *11*, e0154484. [CrossRef] [PubMed]

105. Thålin, C.; Hisada, Y.; Lundström, S.; Mackman, N.; Wallén, H. Neutrophil Extracellular Traps: Villains and Targets in Arterial, Venous, and Cancer-Associated Thrombosis. *Arterioscler. Thromb. Vasc. Biol.* **2019**, *39*, 1724–1738. [CrossRef] [PubMed]

106. Thålin, C.; Demers, M.; Blomgren, B.; Wong, S.L.; von Arbin, M.; von Heijne, A.; Laska, A.C.; Wallén, H.; Wagner, D.D.; Aspberg, S. NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation. *Thromb. Res.* **2016**, *139*, 56–64. [CrossRef]

107. Mauracher, L.-M.; Posch, F.; Martinod, K.; Grilz, E.; Däullary, T.; Hell, L.; Brostjan, C.; Zielinski, C.; Ay, C.; Wagner, D.D.; et al. Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients. *J. Thromb. Haemost.* **2018**, *16*, 508–518. [CrossRef]

108. Hisada, Y.; Grover, S.P.; Maqsood, A.; Houston, R.; Ay, C.; Noubouossie, D.F.; Cooley, B.C.; Wallén, H.; Key, N.S.; Thålin, C.; et al. Neutrophils and neutrophil extracellular traps enhance venous thrombosis in mice bearing human pancreatic tumors. *Haematologica* **2019**, *105*, 218–225. [CrossRef]

109. Demers, M.; Krause, D.S.; Schatzberg, D.; Martinod, K.; Voorhees, J.R.; Fuchs, T.A.; Scadden, D.T.; Wagner, D.D. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. *Proc. Natl. Acad. Sci. USA* **2012**, *109*, 13076–13081. [CrossRef]

110. Bertout, J.A.; Patel, S.A.; Simon, M.C. The impact of O₂ availability on human cancer. *Nat. Cancer* **2008**, *8*, 967–975. [CrossRef]

111. McInturff, A.M.; Cody, M.J.; Elliott, E.A.; Glenn, J.W.; Rowley, J.W.; Rondina, M.T.; Yost, C.C. Mammalian target of rapamycin regulates neutrophil extracellular trap formation via induction of hypoxia-inducible factor 1α. *Blood* **2012**, *120*, 3118–3125. [CrossRef]

112. Boone, B.A.; Murthy, P.; Miller-Ocuin, J.; Doerfler, W.R.; Ellis, J.T.; Liang, X.; Ross, M.A.; Wallace, C.T.; Sperry, J.L.; Lotze, M.T.; et al. Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps. *BMC Cancer* **2018**, *18*, 678. [CrossRef] [PubMed]

113. Cao, W.; Zhu, M.-Y.; Lee, S.-H.; Lee, S.-B.; Kim, H.-J.; Park, B.-O.; Yoon, C.-H.; Khadka, D.; Oh, G.-S.; Shim, H.; et al. Modulation of Cellular NAD⁺ Attenuates Cancer-Associated Hypercoagulability and Thrombosis via the Inhibition of Tissue Factor and Formation of Neutrophil Extracellular Traps. *Int. J. Mol. Sci.* **2021**, *22*, 12085. [CrossRef] [PubMed]
114. Elaskalani, O.; Razak, N.B.A.; Metharom, P. Neutrophil extracellular traps induce aggregation of washed human platelets independently of extracellular DNA and histones. *Cell Commun. Signal.* **2018**, *16*, 24. [CrossRef] [PubMed]

115. Streiff, M.B.; Abutalib, S.A.; Farge, D.; Murphy, M.; Connors, J.M.; Piazza, G. Update on Guidelines for the Management of Cancer-Associated Thrombosis. *Oncology* **2020**, *26*, e24-e40. [CrossRef]

116. Sikorska, J.; Uprichard, J. Direct Oral Anticoagulants: A Quick Guide. *Eur. Cardiol. Rev.* **2017**, *12*, 40-45. [CrossRef]

117. Elaskalani, O.; Domenichini, A.; Razak, N.B.A.; Dye, D.E.; Falasca, M.; Metharom, P. Antiplatelet Drug Ticagrelor Enhances Chemotherapeutic Efficacy by Targeting the Novel P2Y12-AKT Pathway in Pancreatic Cancer Cells. *Cancers* **2020**, *12*, 250. [CrossRef]