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Magalhaes MA, Glogauer JE, Glogauer M. Neutrophils and oral squamous cell carcinoma: lessons learned and future directions. J Leukoc Biol. 2014 Nov;96(5):695-702.

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Neutrophils and oral squamous cell carcinoma: lessons learned and future

directions

Marco A. O. Magalhaes DDS, PhD 1,2 , Judah E. Glogauer 1 and Michael Glogauer DDS,

PhD 1

1 Matrix Dynamics group, Faculty of Dentistry, University of Toronto. 241-150

College street, Toronto, Ontario, Canada, M5S3E2 2 Department of Oral Pathology and Oral Medicine, Faculty of Dentistry, University

of Toronto, 511-124 Edward Street, Toronto, Ontario, Canada

Corresponding author: marco.magalhaes@utoronto.ca

Summary sentence:

This review focuses on how neutrophils modulate the behavior of oral squamous

cell carcinoma and the possible use of neutrophils as biomarkers of OSCC

Abbreviations

OSCC - Oral Squamous Cell Carcinoma INFb - Interferon Beta TGFb - Transforming Growth Factor Beta MMP9 - Matrix metallopeptidase 9 AKT - Protein kinase B NET - Neutrophil extracellular traps PMN - Polymorphonuclear neutrophils NLR - Neutrophil to Lymphocyte Ratio HNSCC - Head and neck squamous cell carcinoma HNP - Human Defensins NGAL - Neutrophil Gelatinase Associated Lipocalin SCC - Squamous Cell Carcinoma HNSCC – Head and neck squamous cell carcinoma TNF - Tumor Necrosis Factor HSPG - Heparin sulfate side chains of proteoglycans TACI - Calcium modulator and cyclophilin ligand interactor TNFa - Tumor Necrosis Factor Alpha ROS - Reactive Oxygen Species TRAIL - TNF-related apoptosis-inducing ligand MAPK - Mitogen-activated protein kinases iNOS - Calcium insensitive Nitric Oxide Synthase NO - Nitric Oxide TAN - Tumor Associated Neutrophils MMP8 - Matrix Metalloproteinase 8 MT1MMP - Membrane type 1-matrix metalloproteinase 1 VEGF - Vascular endothelial growth factor TIMP-1 - TIMP metallopeptidase inhibitor 1 TIMP - Tissue inhibitors of metalloproteinases SN - Supernatant CREB - cAMP response element-binding protein MIF - Macrophage migration inhibitory factor

Abstract

The role of cells of the innate immune system in the pathogenesis of squamous cell

carcinoma has been the subject of intense research in recent years. In particular,

neutrophils have recently been shown to have either a pro-tumor or anti-tumor

phenotype in different cancers. Here we review the role of neutrophils as tumor

microenvironment and signaling modulators of oral squamous cell carcinoma

(OSCC) and their possible role as biomarkers of OSCC prognosis. Current evidence

supports a pro-tumorigenic role for neutrophils in OSCC but more research is

needed to clarify the precise mechanisms involved.

Introduction

Oral squamous cell carcinoma is the 5th leading cause of death in Canada [1]

and the overall high mortality rate (average 57% survival in 5 years) has changed

minimally over the previous 10 year period. A total of 42,440 new cases and 8,390

deaths are estimated in the United States in 2014 [2]. The high mortality rate of

patients with oral cancer is associated with its late detection and subsequent

increase in the occurrence metastatic disease. The 5-year survival rate for oral

cancer drops from approximately 60% to 30% for patients with metastatic disease

[1]. Recent advances have been made in understanding the genetic changes and

molecular mechanisms regulating OSCC progression, including the role of

neutrophils in this process.

Neutrophils are key mediators of the innate immune system. Neutrophil

activation is essential to protect the host system against infections and promote

normal healing [3]. For many decades, leukocytosis has been associated with a poor

prognosis in different types of malignancies [4]. Despite this, the specific role of

neutrophils and macrophages in the pathogenesis of cancer has only recently

become the subject of intense research [5, 6] with special focus on the association

between inflammation and cancer progression. This idea was initiated by Pekarek et

al. who showed that neutrophils can induce tumor angiogenesis [7], prompting a

significant number of research groups to investigate possible neutrophil pro-tumor

or anti-tumor roles as well as their potential uses as diagnostic and prognostic

markers. Recent work has shown that neutrophils are important players in cancer

biology [8, 9] and different neutrophil sub-populations may have opposing roles in

cancer progression where an N1 population was demonstrated to have anti-tumor

properties and an N2 population of neutrophils was shown to promote tumor

progression. The N1 phenotype is induced by INFb and is characterized by reduced

MMP9 expression, increased reactive oxygen species (ROS) formation, and

apoptosis [10]. The N2 neutrophils develop a pro-tumor phenotype after TGFb

stimulation, leading to increased Arginase, MMP9 and collagenase expression, AKT

activation, and promotion of leukocyte recruitment [9]. This N2 phenotype is

particularly relevant for OSCC patients, since OSCC show increased expression of IL-

1b, IL-6 and TGFb [11]. These proposed opposing roles for neutrophils suggest that

they may be important biomarkers for OSCC and perhaps targets to control cancer

progression [12].

During the past 4 years, an increasing number of publications have shown

that neutrophils are present in a variety of tumors, including renal cell carcinomas

and head and neck carcinomas [13] and that they may contribute to tumor

progression [14, 15], metastasis [16], and extracellular traps (NET)-dependent

tumor metastasis [17]. The general roles of neutrophils in cancer pathogenesis and

prognosis have been reviewed elsewhere [6, 12], but the particular roles of

neutrophils in the diagnosis, prognosis and progression of oral squamous cell

carcinomas have not yet been reviewed. Considering the constant presence of

neutrophils in the oral tissues due to the oral biofilms, there is an increased interest

in analyzing how the presence of neutrophils modulates OSCC behavior. The

literature on OSCC and neutrophils is limited compared to other cancers and we

added publications on head and neck squamous cell carcinomas (HNSCC) to this

discussion.

In the next sections, we will analyze our current knowledge about the roles of

neutrophils in the progression of oral squamous cell carcinoma in three main

categories: biomarkers, microenvironment changes, and intercellular signaling

(Figure 1).

Neutrophils as biomarkers of OSCC

Significant efforts are being made to identify new diagnostic and prognostic

markers to better manage OSCC. Neutrophils are highly proteolytic and motile cells,

allowing them to have direct contact with various cells of the tumor

microenvironment. It is possible that the proteins they release can either directly or

indirectly be used for early detection, staging and prognosis of OSCC lesions. Due to

their accessibility in peripheral blood and saliva, neutrophils are excellent

candidates to develop new biomarkers for oral cancer. Here we describe the role of

neutrophils and neutrophil derived proteins as biomarkers of OSCC.

Neutrophil infiltration

Similar to the literature on other tumors, recent reports have shown that

high neutrophil infiltration in OSCC is associated with poor clinical outcomes.

Trellakis et al. used a retrospective histological analysis of head and neck squamous

cell carcinomas to show an association between PMN infiltration and squamous cell

carcinomas prognosis. Increased neutrophil infiltration as detected by CD66b

immunostaining correlated with poor patient survival [13]. These findings were

consistent with a recent report by Wang et al. who showed that tongue squamous

cell carcinomas with high neutrophil infiltration displayed increased lymph node

metastasis, higher clinical stage and increased chances of tumor recurrence [18].

Neutrophil-to-lymphocyte ratio (NLR)

The neutrophil-to-lymphocyte ratio (NLR) is a well established marker of

poor prognosis in various conditions including cardiovascular diseases [19] and

cancers [20, 21] including head and neck carcinomas [22]. An increase in NLR is

indicative of an ongoing inflammatory state with decrease in regulatory pathways.

Millrud and coworkers have correlated the activation pattern of leukocytes and

survival of HNSCC patients [23] where the prognostic markers (CD71, CD98, CD4/8

ratio, CD16/14) and a high NLR correlated with poor prognosis. Perisanidis et al.

evaluated 97 patients with biopsy proven OSCC who received neoadjuvant

chemotherapy and showed that a high NLR (>1.9) is an independent marker for

poor prognosis in OSCC patients [24]. Although this finding suggests that the NLR

may be an important OSCC prognostic biomarker, larger prospective studies are

needed to further clarify the use of NLR, the mechanisms and relevance behind the

changes in lymphocyte and neutrophil counts in OSCC.

Neutrophil-secreted proteins

Numerous studies have used neutrophil-secreted products as diagnostic and

prognostic markers of cancer. Among these, human defensins (HNP), TNF family

proteins and neutrophil gelatinase associated lipocalin (NGAL) have been studied in

the OSCC patients.

Human defensins (HNP1, HNP2 and HNP3) are known to induce cytotoxic

effects in numerous target cells, including SCC cells [25]. In a search for a possible

role for HNPs in cancer progression, Lundy et al. investigated the presence of HNPs

in OSSC and reported a 2-12 fold increase in their presence in localized tumor areas.

This finding also correlated with an increase in neutrophil infiltrates [26]. HNPs

were also elevated in the saliva of OSCC patients but the clinical significance of this

finding has yet to be clarified [27-29].

Members of the TNF superfamily of secreted proteins, including APRIL [30],

TRAIL and DR5 [31] were also investigated as possible oral cancer biomarkers. The

proliferation-induced ligand APRIL has been shown to regulate tumor cell survival

and proliferation through binding to heparin sulfate side chains of proteoglycans

(HSPG) or to the calcium modulator and cyclophilin ligand interactor (TACI)[32].

Jablonska et al. analyzed the expression of APRIL in peripheral blood neutrophils of

patients with OSCC and found a correlation between high expression and poor

prognosis. TRAIL is a soluble TNFa family ligand produced by numerous cells and

may be a promising candidate to promote cancer suppression. TRAIL induces

apoptosis by activation of DR receptors, including DR1 and DR5, and its expression

and release are decreased in late stage squamous cell carcinoma [31]. Since many

cells in the tumor microenvironment may secrete TRAIL, further studies are needed

to better understand the role and the prognostic importance of neutrophil-derived

TRAIL in OSCC progression.

Neutrophil gelatinase associated lipocalin (NGAL) is a regulator of iron and

hydrophobic-compounds transport, and has an important role in protecting MMP9

from degradation. Recent studies have described a pro-tumor role for NGAL in

different tumors, including breast and esophageal cancers [33]. NGAL has also been

shown to be up-regulated in well differentiated OSCC while poorly differentiated

tumors showed a weak expression, suggesting a possible role for this protein in

tumor staging [34].

Reactive oxygen species

Reactive oxygen species have an increasing number of roles in different

cellular processes, including phagocyte killing, chemotaxis, apoptosis, and

intracellular signaling [35]. The formation of ROS by neutrophils is decreased in

most cancers [9]. Peripheral blood neutrophils from patients with head and neck

squamous cell carcinoma had lower ROS production [36] and reduced apoptosis

[13]. This mechanism is dependent on the p38 MAPK pathway. Similarly, Jablonska

and others showed that iNOS expression and NO production are significantly

reduced in peripheral blood neutrophils of OSCC patients [37]. Although ROS are not

considered biomarkers of cancer progression, future studies will clarify the

signaling changes in TANs and the reduction of ROS in these cells. This may

contribute to the understanding of the crosstalk between cancer cells and TANs.

Neutrophils remodeling the cancer microenvironment

One of the most interesting recent findings suggests that neutrophils are

recruited to the niches of distant cancer metastasis, and may be a key player in the

establishment of metastatic spread [16]. Similarly, Huh et al. found that neutrophils

were recruited and increased the metastatic spread of melanoma through an IL-8

dependent mechanism [38].

Matrix Metaloproteases

The cancer microenvironment is a complex niche that is constantly being

remodelled by fibroblasts, inflammatory and cancer cells. Neutrophils secrete

MMP8 (collagenase), MMP9 (gelatinase), elastase, cathepsin G, proteinase 3 and

other matrix proteases that contribute to the remodelling of the tumor

microenvironment. These proteases can degrade the extracellular matrix directly

and facilitate the invasion of cancer cells [39], or alternatively activate MT1MMP in

the tumor cells and indirectly facilitate cancer progression [40]. Moilanen et al.

showed that MMP8 is also secreted by head and neck squamous cell carcinomas,

including oral cancers [41]. MMP9 is also known to regulate tumor angiogenesis, a

key factor in tumour progression (see below). In tumors where macrophages are

the main source of MMP9, inhibition of macrophage recruitment induces a

compensatory response, increasing the recruitment of MMP9+ neutrophils which

have a pro tumor phenotype [42]. Further studies are needed to verify the

significance of this mechanism in OSCC.

Angiogenesis

Neutrophils are known to promote tumor angiogensis through upregulation

of MMP9 and VEGF [43]. Bausch and others have shown that MMP9 is a VEGF-

independent angiogenic factor with an additive effect to VEGF-induced

angionenenis in hepatocellular carcinoma. Complete inhibition of angiogenesis

requires both MMP9 and VEGF inhibition [44]

Unlike other cells, neutrophils secrete proMMP9 without the inhibitor TIMP1,

providing a readily active MMP9, which is critical for tumor angiogenesis and

intravasation.. Inhibition of IL-8 decreases neutrophil recruitment to the tumor,

angiogenesis and metastasis (Bekes et al 2011). The combination of TIMP (MMP9

inhibitor) and anti-IL-8 antibody induces a substantial decrease in local

angiogenesis and intravasation of tumor cells in a given area [45]. This is a

fundamental link between inflammation and cancer progression, highlighting the

importance of neutrophils in cancer spread. The role of MMP9 in the progression

and spread of oral squamous cell carcinoma is not yet completely understood.

Neutrophil extracellular traps (NET) and adhesion molecules

Recent evidence shows that neutrophils may also contribute to the

metastatic spread of cancers by facilitating the seeding of circulating cancer cells

[46]. Several mechanisms have been described to explain the neutrophil-mediated

metastatic spread, including NETs. NETs are composed of extruded neutrophilic

DNA that can be seen under normal systemic inflammatory/infectious responses.

Cools-Lartigue et al. have showed that NETs sequester circulating cancer cells and

increase the formation of liver micrometastasis [17]. There are no publications

describing the role of NETs in OSCC metastasis. Further studies are needed to

understand the clinical relevance of this mechanism in OSCC .

Modulation of cell function and signaling

Both neutrophils and cancer cells influence the behaviour of each other in the

cancer microenvironment [39, 47]. Recent key studies have identified potential

pathways involved in the crosstalk between cancer cells and neutrophils. We will

focus this discussion on the regulation of cell function and signaling between OSCC

and neutrophils.

Dumitru and co-workers recently showed that tumor associated neutrophils

increased cortactin phosphorylation in oropharyngeal squamous cell carcinomas,

promoting cancer migration, leading to a poor prognosis [48]. This is a promising

result that links the presence of TAN with cytoskeletal changes in the cancer cells.

More importantly, cortactin is an essential actin binding protein, regulating leading

edge formation and invadopodia formation in cancer cells [49].

Trellakis and coworkers performed a functional analysis of peripheral blood

neutrophils to show that the peripheral blood of HNSCC patients had an increase in

PMN, CXCL8, CCL4 and CCL5 [13]. In vitro analysis showed an increased migration

and increased survival of PMN exposed to FaDu cells or FaDu cells supernatant (SN).

This effect was significantly reduced by CXCL8 inhibition. Stimulation of PMN with

FaDu SN also increased the secretion of CCL4, MMP9 and lactoferrin. These results

show that head and neck squamous cell carcinomas establish a feedback loop with

neutrophils leading to increased inflammation.

Cancer cells can also change the activation state of neutrophils. Using a

protein phosphorylation array, Dumitru et al. showed that neutrophils challenged

with FaDu cancer cells showed a strong activation of p38/MAPK, CREB, and p27.

The activation of p38/MAPK was associated with an increase in chemotaxis, cell

survival and secretion of CCL4 and CXCL8. P27 and CREB also regulated the release

of MMP9 by neutrophils. The authors also demonstrated an increase in expression

of MMP9 and CCL4 by CD66b positive cells (neutrophils) in HNSCC [50].

Neutrophils show increased survival when exposed to supernatants from

different tumors, including squamous cell carcinoma, as shown by different groups

[51]. Several mechanisms are implicated in the prolonged survival of neutrophils,

including the release of cytokines and hyaluronan by tumor cells and other cells in

the microenvironment [52, 53]. The macrophage migration inhibitory factor (MIF)

was recently shown to modulate the activation and increase survival of neutrophils

[53]. MIF significantly increased neutrophil chemotaxis, reduced apoptosis, and

increased the expression of CCL4 and MMP9 through a CXCR2-dependent

mechanism. Samples from cancer patients were also used to show that MIF

expression in tumors correlate with neutrophil recruitment and poor survival. The

observation that neutrophils survive longer in the tumor microenvironment

challenges the initial understanding that neutrophils, as extremely short-lived cells,

could not participate effectively in tumor progression that occurs over an extended

period of time. Also, increased neutrophil survival translates into sustained

inflammation and secretion of neutrophil inflammatory mediators that may

contribute to tumor progression.

Concluding remarks

Increasing roles of innate immune cells in cancer biology

Considering the increased interest in the development of targeted therapies,

immune cells have become a primary target for prognosis and possible treatment of

cancers and this is valid for almost all malignancies, including OSCC. The dogma of

the short-lived, “non-specific” neutrophil in cancer is disappearing and is being

replaced by the concept of the neutrophil as a protagonist in cancer progression.

There are many reasons that support this hypothesis. First, the neutrophil

has extremely efficient motility machinery, allowing it to be recruited quickly to

areas of early cancer development and interact with tumor cells in the very early

steps of malignant transformation. Neutrophils can move in and out of the tumor

microenvironment, conveying important signaling cues and can also be detected in

the peripheral circulation. Second, contrary to initial beliefs, neutrophils in contact

with cancer cells have a prolonged life cycle and can be a resident of the tumor

microenvironment for extended periods of time. Third, neutrophils are equipped

with a variety of matrix remodeling proteases that can help shape the tumor

microenvironment.

Neutrophils, cancer and the oral microenvironment

Considering the oral microenvironment, the constant presence of neutrophils

in the saliva may be an important factor in early malignant transformation, signaling

and, most importantly, a marker for disease progression that is easily accessible

with a mouth rinse, without the need of blood collection [54, 55]. Also, there are

numerous conditions that are characterized by changes in the neutrophil population

in the mouth, including periodontal disease [55, 56]. These changes in neutrophils

may be involved in the observed increase in the rate of oral epithelial malignant

transformation in chronic inflammatory states [57, 58]. Certainly, more research is

needed to address these topics. Another important question is whether the

modulation of neutrophil activity can influence the development or prognosis of

oral cancers. There is evidence to suggest that maintaining good oral hygiene and

therefore low neutrophil counts in the saliva correlate with small prevalence of oral

cancer [59, 60]. Finally, the accessible oral microenvironment could also be a

potential candidate for local treatments for early OSCC.

Future directions

There are still many unanswered questions regarding the role of neutrophils

in cancer pathogenesis in general. The literature on oral squamous cell carcinoma

and neutrophils is limited compared to other models. New exciting results show tat

neutrophils inhibit seeding in the premetastastic lung in a breast cancer model [61]

and recruit T regulatory cells that may contribute to a decreased antitumor

response [62]. Further studies analyzing the role of these mechanisms in OSCC are

needed.

The most challenging topic to be addressed in the next few years is how to

modulate neutrophil activity to increase tumor surveillance and early suppression.

Identifying the signaling switches is essential to developing treatments to precisely

target this interaction. Also, considering the particularities of OSCC, how can we

develop localized treatment strategies? Since oral neutrophils are readily available,

research is needed to evaluate the genetic signature changes of neutrophils exposed

to OSCC.

The most promising area of research is the development of a sensitive and

specific diagnostic/prognostic marker for OSCC, which may be associated with

saliva tests. Analyzing neutrophils from OSCC patients’ saliva might prove to be a

good prognostic marker.

Authorship

MAOM designed and prepared the manuscript. JG contributed to manuscript

preparation. MG contributed to the design and the revision of the manuscript.

Acknowledgements

Thanks to Dr. Grace Bradley for the helpful discussion and support of this work. This

supported is supported by a Dental Research Institute grant. MG is supported by

CIHR.

Conflict of interest disclosure

The authors have no conflict of interest to disclose.

References

1. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Thun, M. J. (2009) Cancer statistics, 2009. CA Cancer J Clin 59, 225-49.

2. Society, A. C. (2014) Cancer Facts & Figures 2014. American Cancer Society, Atlanta.

3. Magalhaes, M. A. O. (2009) Effective neutrophil activation during innate immunity: Understanding the specific roles of Rac1 and Rac2. University of Toronto, 2009. 191 leaves.

4. Shoenfeld, Y., Tal, A., Berliner, S., Pinkhas, J. (1986) Leukocytosis in non hematological malignancies--a possible tumor-associated marker. J Cancer Res Clin Oncol 111, 54-8.

5. Bailey, S. C., Head, J. F., Greengard, O. (1989) Neutrophil maturation and hypersegmentation promoted in normal bone marrow by a carcinoma-elaborated protein factor. Am J Hematol 31, 159-65.

6. Mantovani, A. (2009) The yin-yang of tumor-associated neutrophils. Cancer Cell 16, 173-4.

7. Pekarek, L. A., Starr, B. A., Toledano, A. Y., Schreiber, H. (1995) Inhibition of tumor growth by elimination of granulocytes. J Exp Med 181, 435-40.

8. Welch, D. R., Schissel, D. J., Howrey, R. P., Aeed, P. A. (1989) Tumor-elicited polymorphonuclear cells, in contrast to "normal" circulating polymorphonuclear cells, stimulate invasive and metastatic potentials of rat mammary adenocarcinoma cells. Proc Natl Acad Sci U S A 86, 5859-63.

9. Fridlender, Z. G. and Albelda, S. M. (2012) Tumor-associated neutrophils: friend or foe? Carcinogenesis 33, 949-55.

10. Fridlender, Z. G., Sun, J., Kim, S., Kapoor, V., Cheng, G., Ling, L., Worthen, G. S., Albelda, S. M. (2009) Polarization of tumor-associated neutrophil phenotype by TGF-beta: "N1" versus "N2" TAN. Cancer Cell 16, 183-94.

11. Lee, J. J., Chang, Y. L., Lai, W. L., Ko, J. Y., Kuo, M. Y., Chiang, C. P., Azuma, M., Chen, C. W., Chia, J. S. (2011) Increased prevalence of interleukin-17-producing CD4(+) tumor infiltrating lymphocytes in human oral squamous cell carcinoma. Head Neck 33, 1301-8.

12. Gregory, A. D. and Houghton, A. M. (2011) Tumor-associated neutrophils: new targets for cancer therapy. Cancer Res 71, 2411-6.

13. Trellakis, S., Farjah, H., Bruderek, K., Dumitru, C. A., Hoffmann, T. K., Lang, S., Brandau, S. (2011) Peripheral blood neutrophil granulocytes from patients with head and neck squamous cell carcinoma functionally differ from their counterparts in healthy donors. Int J Immunopathol Pharmacol 24, 683-93.

14. Houghton, A. M., Rzymkiewicz, D. M., Ji, H., Gregory, A. D., Egea, E. E., Metz, H. E., Stolz, D. B., Land, S. R., Marconcini, L. A., Kliment, C. R., Jenkins, K. M., Beaulieu, K. A., Mouded, M., Frank, S. J., Wong, K. K., Shapiro, S. D. (2010) Neutrophil elastase-mediated degradation of IRS-1 accelerates lung tumor growth. Nat Med 16, 219-23.

15. Kuang, D. M., Zhao, Q., Wu, Y., Peng, C., Wang, J., Xu, Z., Yin, X. Y., Zheng, L. (2011) Peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma. J Hepatol 54, 948-55.

16. Spicer, J. D., McDonald, B., Cools-Lartigue, J. J., Chow, S. C., Giannias, B., Kubes, P., Ferri, L. E. (2012) Neutrophils promote liver metastasis via Mac-1-mediated interactions with circulating tumor cells. Cancer Res 72, 3919-27.

17. Cools-Lartigue, J., Spicer, J., McDonald, B., Gowing, S., Chow, S., Giannias, B., Bourdeau, F., Kubes, P., Ferri, L. (2013) Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest.

18. Wang, N., Feng, Y., Wang, Q., Liu, S., Xiang, L., Sun, M., Zhang, X., Liu, G., Qu, X., Wei, F. (2014) Neutrophils Infiltration in the Tongue Squamous Cell Carcinoma and Its Correlation with CEACAM1 Expression on Tumor Cells. PLoS One 9, e89991.

19. Bhat, T., Teli, S., Rijal, J., Bhat, H., Raza, M., Khoueiry, G., Meghani, M., Akhtar, M., Costantino, T. (2013) Neutrophil to lymphocyte ratio and cardiovascular diseases: a review. Expert Rev Cardiovasc Ther 11, 55-9.

20. Szkandera, J., Stotz, M., Eisner, F., Absenger, G., Stojakovic, T., Samonigg, H., Kornprat, P., Schaberl-Moser, R., Alzoughbi, W., Ress, A. L., Seggewies, F. S., Gerger, A., Hoefler, G., Pichler, M. (2013) External validation of the derived neutrophil to lymphocyte ratio as a prognostic marker on a large cohort of pancreatic cancer patients. PLoS One 8, e78225.

21. Sharaiha, R. Z., Halazun, K. J., Mirza, F., Port, J. L., Lee, P. C., Neugut, A. I., Altorki, N. K., Abrams, J. A. (2011) Elevated preoperative neutrophil:lymphocyte ratio as a predictor of postoperative disease recurrence in esophageal cancer. Ann Surg Oncol 18, 3362-9.

22. He, J. R., Shen, G. P., Ren, Z. F., Qin, H., Cui, C., Zhang, Y., Zeng, Y. X., Jia, W. H. (2012) Pretreatment levels of peripheral neutrophils and lymphocytes as independent prognostic factors in patients with nasopharyngeal carcinoma. Head Neck 34, 1769-76.

23. Millrud, C. R., Mansson Kvarnhammar, A., Uddman, R., Bjornsson, S., Riesbeck, K., Cardell, L. O. (2012) The activation pattern of blood leukocytes in head and neck squamous cell carcinoma is correlated to survival. PLoS One 7, e51120.

24. Perisanidis, C., Kornek, G., Pöschl, P. W., Holzinger, D., Pirklbauer, K., Schopper, C., Ewers, R. (2013) High neutrophil-to-lymphocyte ratio is an independent marker of poor disease-specific survival in patients with oral cancer. Med Oncol 30, 334.

25. McKeown, S. T., Lundy, F. T., Nelson, J., Lockhart, D., Irwin, C. R., Cowan, C. G., Marley, J. J. (2006) The cytotoxic effects of human neutrophil peptide-1 (HNP1) and lactoferrin on oral squamous cell carcinoma (OSCC) in vitro. Oral Oncol 42, 685-90.

26. Lundy, F. T., Orr, D. F., Gallagher, J. R., Maxwell, P., Shaw, C., Napier, S. S., Gerald Cowan, C., Lamey, P. J., Marley, J. J. (2004) Identification and overexpression of human neutrophil alpha-defensins (human neutrophil peptides 1, 2 and 3) in squamous cell carcinomas of the human tongue. Oral Oncol 40, 139-44.

27. Mizukawa, N., Sugiyama, K., Fukunaga, J., Ueno, T., Mishima, K., Takagi, S., Sugahara, T. (1998) Defensin-1, a peptide detected in the saliva of oral squamous cell carcinoma patients. Anticancer Res 18, 4645-9.

28. Sawaki, K., Mizukawa, N., Yamaai, T., Yoshimoto, T., Nakano, M., Sugahara, T. (2002) High concentration of beta-defensin-2 in oral squamous cell carcinoma. Anticancer Res 22, 2103-7.

29. Yoshimoto, T., Yamaai, T., Mizukawa, N., Sawaki, K., Nakano, M., Yamachika, E., Sugahara, T. (2003) Different expression patterns of beta-defensins in human squamous cell carcinomas. Anticancer Res 23, 4629-33.

30. Jabłońska, E., Wawrusiewicz-Kurylonek, N., Garley, M., Ratajczak-Wrona, W., Antonowicz, B., Dziemiańczyk-Pakieła, D., Jabłoński, J., Krętowski, A., Grabowska, S. Z. (2012) A proliferation-inducing ligand (APRIL) in neutrophils of patients with oral cavity squamous cell carcinoma. Eur Cytokine Netw 23, 93-100.

31. Jablonska, E., Jablonski, J., Marcinczyk, M., Grabowska, Z., Piotrowski, L. (2008) The release of soluble forms of TRAIL and DR5 by neutrophils of oral cavity cancer patients. Folia Histochem Cytobiol 46, 177-83.

32. Hahne, M., Kataoka, T., Schröter, M., Hofmann, K., Irmler, M., Bodmer, J. L., Schneider, P., Bornand, T., Holler, N., French, L. E., Sordat, B., Rimoldi, D., Tschopp, J. (1998) APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth. J Exp Med 188, 1185-90.

33. Bolignano, D., Donato, V., Lacquaniti, A., Fazio, M. R., Bono, C., Coppolino, G., Buemi, M. (2010) Neutrophil gelatinase-associated lipocalin (NGAL) in human neoplasias: a new protein enters the scene. Cancer Lett 288, 10-6.

34. Hiromoto, T., Noguchi, K., Yamamura, M., Zushi, Y., Segawa, E., Takaoka, K., Moridera, K., Kishimoto, H., Urade, M. (2011) Up-regulation of neutrophil gelatinase-associated lipocalin in oral squamous cell carcinoma: relation to cell differentiation. Oncol Rep 26, 1415-21.

35. Dupré-Crochet, S., Erard, M., Nüβe, O. (2013) ROS production in phagocytes: why, when, and where? J Leukoc Biol 94, 657-70.

36. Ratajczak-Wrona, W., Jablonska, E., Marcinczyk, M., Grabowska, Z., Piotrowski, L. (2009) Role of p38 MAPK pathway in induction of iNOS expression in neutrophils and peripheral blood mononuclear cells in patients with squamous cell carcinoma of the oral cavity. J Oral Maxillofac Surg 67, 2354-63.

37. Jabłońska, E., Puzewska, W., Marcińczyk, M., Grabowska, Z., Jabłoński, J. (2005) iNOS expression and NO production by neutrophils in cancer patients. Arch Immunol Ther Exp (Warsz) 53, 175-9.

38. Huh, S. J., Liang, S., Sharma, A., Dong, C., Robertson, G. P. (2010) Transiently entrapped circulating tumor cells interact with neutrophils to facilitate lung metastasis development. Cancer Res 70, 6071-82.

39. Dumitru, C. A., Lang, S., Brandau, S. (2013) Modulation of neutrophil granulocytes in the tumor microenvironment: mechanisms and consequences for tumor progression. Semin Cancer Biol 23, 141-8.

40. Shamamian, P., Schwartz, J. D., Pocock, B. J., Monea, S., Whiting, D., Marcus, S. G., Mignatti, P. (2001) Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis. J Cell Physiol 189, 197-206.

41. Moilanen, M., Pirilä, E., Grénman, R., Sorsa, T., Salo, T. (2002) Expression and regulation of collagenase-2 (MMP-8) in head and neck squamous cell carcinomas. J Pathol 197, 72-81.

42. Pahler, J. C., Tazzyman, S., Erez, N., Chen, Y. Y., Murdoch, C., Nozawa, H., Lewis, C. E., Hanahan, D. (2008) Plasticity in tumor-promoting inflammation: impairment of macrophage recruitment evokes a compensatory neutrophil response. Neoplasia 10, 329-40.

43. Jablonska, J., Leschner, S., Westphal, K., Lienenklaus, S., Weiss, S. (2010) Neutrophils responsive to endogenous IFN-beta regulate tumor angiogenesis and growth in a mouse tumor model. J Clin Invest 120, 1151-64.

44. Bausch, D., Pausch, T., Krauss, T., Hopt, U. T., Fernandez-del-Castillo, C., Warshaw, A. L., Thayer, S. P., Keck, T. (2011) Neutrophil granulocyte derived MMP-9 is a VEGF independent functional component of the angiogenic switch in pancreatic ductal adenocarcinoma. Angiogenesis 14, 235-43.

45. Bekes, E. M., Schweighofer, B., Kupriyanova, T. A., Zajac, E., Ardi, V. C., Quigley, J. P., Deryugina, E. I. (2011) Tumor-recruited neutrophils and neutrophil TIMP-free MMP-9 regulate coordinately the levels of tumor angiogenesis and efficiency of malignant cell intravasation. Am J Pathol 179, 1455-70.

46. McDonald, B., Spicer, J., Giannais, B., Fallavollita, L., Brodt, P., Ferri, L. E. (2009) Systemic inflammation increases cancer cell adhesion to hepatic sinusoids by neutrophil mediated mechanisms. Int J Cancer 125, 1298-305.

47. Amar, M., Pham Huu, T., Amit, N., Hakim, J. (1993) Molecular mechanisms involved in the inhibition of neutrophil locomotion by tumor cells. Blood Cells 19, 177-84; discussion 185-7.

48. Dumitru, C. A., Bankfalvi, A., Gu, X., Eberhardt, W. E., Zeidler, R., Lang, S., Brandau, S. (2013) Neutrophils Activate Tumoral CORTACTIN to Enhance Progression of Orohypopharynx Carcinoma. Front Immunol 4, 33.

49. Magalhaes, M. A., Larson, D. R., Mader, C. C., Bravo-Cordero, J. J., Gil-Henn, H., Oser, M., Chen, X., Koleske, A. J., Condeelis, J. (2011) Cortactin phosphorylation regulates cell invasion through a pH-dependent pathway. J Cell Biol 195, 903-20.

50. Dumitru, C. A., Fechner, M. K., Hoffmann, T. K., Lang, S., Brandau, S. (2012) A novel p38-MAPK signaling axis modulates neutrophil biology in head and neck cancer. J Leukoc Biol 91, 591-8.

51. Wu, Y., Zhao, Q., Peng, C., Sun, L., Li, X. F., Kuang, D. M. (2011) Neutrophils promote motility of cancer cells via a hyaluronan-mediated TLR4/PI3K activation loop. J Pathol 225, 438-47.

52. Glynn, P. C., Henney, E., Hall, I. P. (2002) The selective CXCR2 antagonist SB272844 blocks interleukin-8 and growth-related oncogene-alpha-mediated inhibition of spontaneous neutrophil apoptosis. Pulm Pharmacol Ther 15, 103-10.

53. Dumitru, C. A., Gholaman, H., Trellakis, S., Bruderek, K., Dominas, N., Gu, X., Bankfalvi, A., Whiteside, T. L., Lang, S., Brandau, S. (2011) Tumor-derived macrophage migration inhibitory factor modulates the biology of head and neck cancer cells via neutrophil activation. Int J Cancer 129, 859-69.

54. Forster, C., Aboodi, G., Lipton, J., Glogauer, M. (2012) A non-invasive oral rinse assay predicts bone marrow engraftment and 6 months prognosis following allogeneic hematopoietic stem cell transplantation. J Oral Pathol Med 41, 165-70.

55. Lakschevitz, F. S., Aboodi, G. M., Glogauer, M. (2013) Oral neutrophil transcriptome changes result in a pro-survival phenotype in periodontal diseases. PLoS One 8, e68983.

56. Lakschevitz, F. S., Aboodi, G. M., Glogauer, M. (2013) Oral neutrophils display a site-specific phenotype characterized by expression of T-cell receptors. J Periodontol 84, 1493-503.

57. Coussens, L. M. and Werb, Z. (2002) Inflammation and cancer. Nature 420, 860-7.

58. Piva, M. R., DE Souza, L. B., Martins-Filho, P. R., Nonaka, C. F., DE Santana Santos, T., DE Souza Andrade, E. S., Piva, D. (2013) Role of inflammation in oral carcinogenesis (Part II): CD8, FOXP3, TNF-α, TGF-β and NF-κB expression. Oncol Lett 5, 1909-1914.

59. Moergel, M., Kämmerer, P., Kasaj, A., Armouti, E., Alshihri, A., Weyer, V., Al-Nawas, B. (2013) Chronic periodontitis and its possible association with oral squamous cell carcinoma - a retrospective case control study. Head Face Med 9, 39.

60. Wen, B. W., Tsai, C. S., Lin, C. L., Chang, Y. J., Lee, C. F., Hsu, C. H., Kao, C. H. (2013) Cancer risk among gingivitis and periodontitis patients: A nationwide cohort study. QJM.

61. Granot, Z., Henke, E., Comen, E. A., King, T. A., Norton, L., Benezra, R. (2011) Tumor entrained neutrophils inhibit seeding in the premetastatic lung. Cancer Cell 20, 300-14.

62. Mishalian, I., Bayuh, R., Eruslanov, E., Michaeli, J., Levy, L., Zolotarov, L., Singhal, S., Albelda, S. M., Granot, Z., Fridlender, Z. G. (2014) Neutrophils recruit regulatory T-cells into tumors via secretion of CCL17-A new mechanism of impaired antitumor immunity. Int J Cancer.

Figure Legends

Figure 1. The role of neutrophils in the progression of OSCC. Neutrophils have

important roles in the progression of OSCC, including the remodeling of cancer

microenvironment, modulating cell function and signaling and a potential

biomarker. This figure summarizes the available information on each of these

categories.

Biomarkers

DefensinsTNF superfamilyNGAL

Neutrophil to lymphocyte ratioNeutrophil infiltrationNeutrophil secreted proteins

Modulation of cell function and signalling

Tumor microenvironment

Neutrophils

Cancer cells

Increased survival

Increased migrationCortactin phosphorylation

Increased CCL4, CXCL8, MMP9 and lactoferrin

AngiogenesisMatrix remodellingModulation of infammatory response