1 1 the emerging role of cxc chemokines in epithelial ovarian

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The emerging role of CXC chemokines in epithelial ovarian cancer 2

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Prince Henry’s Institute, Monash Medical Centre, Clayton, Victoria 3168 4

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Adam Rainczuk*, Jyothsna Rao

*, Jessica Gathercole, Andrew N. Stephens 6

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Corresponding author: 9

[email protected] 10

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of 32 Reproduction Advance Publication first posted on 6 July 2012 as Manuscript REP-12-0153

Copyright © 2012 by the Society for Reproduction and Fertility.

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Abstract 48 In recent years, chemokines have generated intense investigations due to their 49

involvement in both physiological and pathological processes of inflammation, particularly in 50

ovarian biology. The physiological process of ovulation in the normal ovary involves various 51

chemokines that mediate the healing of the ruptured endometrium. It is now being reported 52

that many of these chemokines are also associated with the cancer of the ovary. Chronic 53

inflammation underlies the progression of ovarian cancer, therefore, it raises the possibility 54

that chemokines are involved in the inflammatory process and mediate immune responses 55

that may favour or inhibit tumour progression. 56

Ovarian cancer is a gynaecological cancer responsible for highest rate of mortality in 57

women. Although, there have been several investigations and advances in surgery and 58

chemotherapy, the survival rate for this disease remains low. This is mainly because of a lack 59

of specific symptoms and biomarkers for detection. In this review, we have discussed the 60

emerging role of the CXC chemokines in epithelial ovarian cancer. The CXC group of 61

chemokines are gaining importance in the field of ovarian cancer for being angiostatic and 62

angiogenic in function. While there have been several studies on the angiogenesis function, 63

emerging research shows that ELR- CXC chemokines, CXCL9 and CXCL10 are angiostatic. 64

Importantly, the angiostatic chemokines can inhibit the progression of epithelial ovarian 65

cancer. Given that there are currently no biomarkers or specific therapeutic targets for the 66

disease, these chemokines are emerging as promising targets for therapy. 67

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1. INTRODUCTION 71

1.1 An overview of the CXC chemokines and their functions 72

2. THE ROLE OF CXC CHEMOKINES IN NORMAL OVARIAN PHYSIOLOGY 73

3. EPITHELIAL OVARIAN CANCER 74

4. BALANCING INFLAMMATION, T-CELL RECRUITMENT, ANGIOGENESIS AND ANGIOSTASIS: 75 THE ROLE OF CHEMOKINES IN OVARIAN CANCER DEVELOPMENT AND PROGRESSION 76

4.1 Inflammation and the ovarian surface epithelium 77

4.2 Recruitment of T-cells to epithelial ovarian tumours 78

4.3 Angiostatic chemokine ligands: CXCL9 and CXCL10 expression can inhibit EOC progression 79

4.4 Angiogenic chemokine ligands can promote tumour development 80

5. DIRECT INVOLVEMENT THE ELR- CHEMOKINE CXCL12 IN METASTATIC SPREAD 81

6. CXC CHEMOKINES AS FUTURE THERAPEUTIC TARGETS FOR EOC 82

7. CONCLUSION 83

REFERENCES 84 85

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1. Introduction 87

As key mediators of the inflammatory response, chemokines represent an area of 88

intense interest and study. Their influence on the chemotactic recruitment of leukocytes 89

towards sites of inflammation is well established, and involves multiple cell and tissue types 90

(Charo & Ransohoff 2006). Apart from their established roles in chemotactic cell 91

recruitment, however, there is a growing body of evidence suggesting that interference with 92

their established functions may promote tumour development and metastasis (Mukaida & 93

Baba 2012). In particular, a complex chemokine signalling network may influence the 94

development and progression of epithelial ovarian cancers (EOC). The study of chemokines 95

is therefore becoming important in furthering our understanding of this disease. 96

1.1 An overview of the CXC chemokines and their functions. 97

Chemokines, comprising pairs of ligands and their associated receptors, are a 98

superfamily of heparin-binding cytokine molecules with molecular weights between 8-10 99

kDa (Mukaida & Baba 2012). They are important mediators of the formation of new blood 100

vessels by neovascularisation, involved in the angiogenic sprouting of vessels in both normal 101

(e.g., embryogenesis, menstruation) as well as pathological states (Keeley et al. 2008). This 102

role is achieved not solely through the promotion of angiogenesis, but via a complex 103

interplay between angiogenic and angiostatic signalling to influence the behaviour of the 104

vascular endothelium (Kiefer & Siekmann 2011). Chemokine ligands exert their effects 105

through their cognate G-protein-coupled receptors (GPCRs), a group of seven-106

transmembrane-spanning proteins expressed on the cell surface and belonging to the class A 107

rhodopsin-like family (Kiefer & Siekmann 2011) (Figure 1). 108

On the basis of their primary structure, chemokines are classified as either C, CC, 109

CXC or CX3C where ‘X’ represents a non-conserved amino acid substitution (Figure 1). 110

With the exception of the ‘C’ subgroup, all chemokines contain a common four cysteine 111

residue motif linked by disulfide bonds in conserved positions; one between the first and the 112

third, and one between the second and the fourth cysteine, to form triple stranded β-sheet 113

structures (Fernandez & Lolis 2002). The nomenclature for chemokines is extended by the 114

addition of receptor ‘R’ (e.g., CXCR1) and respective ligand ‘L’ (e.g., CXCL8). CXC 115

chemokines are further sub-grouped according to the presence or absence of a three amino 116

acid “ELR” Glu-Leu-Arg motif preceding the CXC sequence. Thus, the CXC chemokines are 117

often referred to as ELR+ or ELR

- (Strieter et al. 2004). 118

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Over the past two decades, many studies have examined the dual roles of CXC 119

chemokines in both the promotion and inhibition of angiogenesis. The angiogenic sprouting 120

of new, thin-walled capillary networks from pre-existing ones is a critical factor in tumour 121

growth and spread (Keeley et al. 2008, Kiefer & Siekmann 2011). As a general rule, ELR+ 122

CXC chemokines mediate angiogenesis, typically via interaction with the CXCR2 receptor 123

(see Table 1 for a listing of CXC chemokines and their cognate receptors) and are also 124

involved in other diverse functions such as stem cell migration, lymphocyte migration and 125

lymphoid tissue neogenesis (Romagnani et al. 2004, Kiefer & Siekmann 2011). CXCR2 is 126

typically expressed on endothelial cells, but can be constitutively expressed by specific 127

tissues and cells (Table 1) (Mukaida & Baba 2012). By contrast, the ELR- CXC chemokines 128

typically promote angiostasis (rather than angiogenesis), through their common receptor 129

CXCR3 (Mukaida & Baba 2012). Chemokine ligands lacking the ELR motif can promote 130

anti-tumour effects by recruiting both innate and adaptive immune effector cells (in the case 131

of CXCL9 and CXCL10 that can be secreted from tumour cells for example), and can also 132

inhibit endothelial cell migration and proliferation (Keeley et al. 2008, Kiefer & Siekmann 133

2011, Whitworth & Alvarez 2011). 134

There are, however, two exceptions to the rule. CXCL4 is an ELR+ chemokine, which 135

also binds to CXCR3. Unlike the other ELR+ members, CXCR4 promotes angiostasis similar 136

to the ELR- chemokines. Contrastingly, the ELR

- chemokine CXCL12 binds to the CXCR4 137

receptor to promote angiogenesis (Kiefer & Siekmann 2011). Therefore, both the ELR status 138

and the cognate receptor for each chemokine must be considered when evaluating the 139

function (either angiogenic or angiostatic) of the CXC chemokine family. The functions of 140

the CXC chemokines are also extensively regulated through post-translational modification. 141

This is a well-established mechanism for regulating their bioactivity and function, and has 142

been reviewed elsewhere (Mortier et al. 2011). 143

It is now well established that besides leukocytes, fibroblasts and endothelial cells, 144

tumour cells also express chemokines and their receptors (Balkwill 2004, Romagnani et al. 145

2004, Kiefer & Siekmann 2011, Mukaida & Baba 2012). Importantly, chemokines are 146

secreted by both the stromal and malignant part of the cancer tissue, and functionally the 147

chemokine ligand and receptor pairs can exert a direct effect on tumour proliferation and 148

survival (Erreni et al. 2009, Ha et al. 2011, Boimel et al. 2012). The chemokines from 149

stromal cells may influence the survival of malignant cells by binding to the functional 150

receptors acquired on the cancerous cells, enhancing metastasis in a chemokine rich 151

environment (Mantovani et al. 2010, Balkwill 2012). 152

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This review will discuss the emerging roles of the CXC chemokines in the 153

pathogenesis of epithelial ovarian cancer, in the context of their inflammatory, angiogenic 154

and metastatic properties. 155

2. The role of CXC chemokines in normal ovarian physiology 156

The surface epithelium of the ovary is a natural extension of the peritoneal lining, and 157

is directly exposed to any metabolic and environmental stress present in the peritoneal cavity 158

(Maccio & Madeddu 2012). Regular cycling and release of ova from the ovary causes the 159

ovarian epithelium to undergo regular cycles of wounding and repair throughout a female’s 160

reproductive life. This process is generally considered to induce an inflammatory response, 161

and triggers (Murdoch et al. 2010) the secretion of a large number of chemokines, cytokines, 162

enzymes and growth factors (Murdoch & McDonnel 2002). 163

A model of CXC chemokine-coordinated regulation of angiogenesis and 164

inflammation after wounding of a normal epithelial cell layer is summarised in Figure 2 165

(Griffioen & Molema 2000, Spinetti et al. 2001, Romagnani et al. 2004), and a more detailed 166

review of wound repair involving chemokines can be found in (Romagnani et al. 2004). At 167

the wound site, platelets release growth factors including vascular endothelial growth factor 168

(VEGF), platelet-derived growth factor (PDGF), and various cytokines including CXCL7, 169

CXCL1, CXCL5 (which initiate neutrophil recruitment) and CXCL4 (to assist in blood-clot 170

formation) (Griffioen & Molema 2000, Spinetti et al. 2001, Romagnani et al. 2004). 171

Wounded epithelial cells then express CXCL8, inducing an inflammatory response and 172

resulting in angiogenesis and the sprouting of new blood vessels with high CXCR2 173

expression (Romagnani et al. 2004). The epithelial layer also produces angiostatic CXCL9, 174

10, and 11, arresting the migration of proliferating CXCR3+ endothelial cells and leading to 175

lymphocyte recruitment during the period of healing (Romagnani et al. 2004). CXCL8 is also 176

involved in re-epithelialization of the wound site (not shown in Figure 2) (Romagnani et al. 177

2004). Other non-CXC chemokines are also involved in wound healing; for example, CCL2 178

expression during wound healing also recruits CCR2+ macrophages and monocytes to the 179

inflamed ovarian epithelium (Fader et al. 2010). The role of non-CXC chemokines in this 180

process is reviewed elsewhere (Charo & Ransohoff 2006). 181

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Chemokines and cytokines also modulate ovarian function via their involvement in 183

follicular development and ovulation, and the formation, function and death of the corpus 184

luteum (Auersperg et al. 1998). CXCL8, for example, is significantly elevated in normal 185

folliculogenesis suggesting a role in mediating neutrophil attraction (Ness & Modugno 2006). 186

Moreover, anti-CXCL8 antiserum can suppress hCG-induced ovulation and neutrophil 187

activity in women undergoing IVF (Ness & Modugno 2006). CXCL8 levels also correlate 188

with collagenases, which are crucial to the remodelling of the cervical matrix (Auersperg et 189

al. 1998). CXCL1, another neutrophil attractant, is present in periovulatory follicular fluid 190

and is expressed by ovarian stromal cells. On completion of ovulation, the ruptured 191

epithelium undergoes repair to form the corpus luteum, characterized by significantly 192

increased infiltration of leukocytes and modulated by pro-angiogenic factors in the follicular 193

fluid such as CXCL8 and CCL2 (Merritt & Cramer 2011). 194

Recent studies have shown that CXCL12 is expressed in the cells of the normal 195

ovarian surface epithelium, and the epithelium of the fallopian tube (Machelon et al. 2011). 196

Interestingly, CXCL12 is absent in the follicles and the oocytes (Machelon et al. 2011, 197

Merritt & Cramer 2011). Constitutive expression of CXCL10 has also been reported in 198

normal ovary, with cyclic increases observed during ovulation (Wong et al. 2002, Zhou et al. 199

2004). Whilst the precise roles played by these chemokines in the physiology of the ovary are 200

still unclear, their presence firmly establishes their importance in normal ovarian function. 201

3. Epithelial Ovarian Cancer 202

Epithelial ovarian cancers (EOC) account for between 80-95% of all ovarian tumours 203

diagnosed, and exhibit the highest mortality rate of any gynaecological tumour type (Roett & 204

Evans 2009b). EOCs are commonly classified according to histopathological appearance as 205

clear cell, endometrioid, mucinous or serous; amongst these, serous epithelial tumours are the 206

most commonly diagnosed tumour type (Kobel et al. 2008a). However, wide varieties of less 207

common of ovarian neoplasms exist and can include mixed, undifferentiated, or Brenner type 208

tumours (Kaku et al. 2003, Arnogiannaki et al. 2011). Whilst traditionally sub-grouped 209

according to FIGO stage and grade, a growing body of evidence now suggests that low- and 210

high- grade tumours have different etiologies arising from unrelated, underlying molecular 211

pathologies (Gilks et al. 2008, Kobel et al. 2008b). Low-grade tumours typically exhibit 212

micropapillary structures and originate from borderline tumours. These tumours are believed 213

to progress through benign and borderline stages to malignancy, and are generally associated 214

with good prognosis. They also have well-defined mutational pathways and often express 215

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mutated KRAS or BRAF (Cho 2009a). By contrast, a significantly greater number of TP53 216

gene mutations have been found in high grade serous tumours (Milner et al. 1993), these also 217

have a poorly defined mutational pathway with mutated TP53 expression found in up to 80% 218

of cases (Vang et al. 2009). They appear to arise spontaneously, metastasize early in their 219

progression and are associated with poor prognosis (Cho 2009b, Cho & Shih Ie 2009, Roett 220

& Evans 2009a). 221

4. Balancing inflammation, T-cell recruitment, angiogenesis and angiostasis: the role of 222

chemokines in ovarian cancer development and progression 223

4.1 Inflammation and the ovarian surface epithelium 224

225

Similar to other tumour types, chronic inflammation is an important condition 226

underlying the growth and progression of ovarian tumours (Ness & Cottreau 1999, Ness et al. 227

2000, Fleming et al. 2006, Son et al. 2007a, Coffelt & Scandurro 2008, Maccio & Madeddu 228

2012). As key mediators of inflammation, chemokines play two major roles; (i) they are 229

responsible for the recruitment and activation of immune cells into sites of malignancy (and 230

possibly areas of pre-neoplastic growth), and (ii) they mediate pro- and anti-angiogenic 231

effects, processes required for the vascularisation and progression of tumours. In addition, the 232

multiple biological effects exerted by CXC chemokines means that each of these processes is 233

inextricably linked. It is therefore of paramount importance to identify those molecules 234

involved, and their contribution towards anti-tumour activity and in preventing recurrence – 235

or, conversely, how their activity may promote tumour growth and metastasis. 236

Although an accepted view is that a majority of ovarian cancer cases originate from 237

the ovarian surface epithelium, emerging evidence suggests the involvement of the fimbriae 238

of fallopian tubes. Crum and colleagues showed that fallopian tubes and ovaries from women 239

with BRCA mutations often contained precursor lesions in the fimbriae of the tube (Medeiros 240

et al. 2006, Crum et al. 2007). By contrast, Clarke et al have demonstrated an association 241

between BRCA and inflammation in serous ovarian carcinomas. BRCA mutations (or 242

epigenetic loss) were associated with favourable prognosis via recruitment of intraepithelial 243

CD8+ (and to a lesser extent CD3

+) tumour infiltrating lymphocytes (TILs). This novel 244

association between intraepithelial T-cell infiltration and BRCA mutation in serous EOC has 245

also been observed with certain breast cancer types, and can lead to a favourable patient 246

prognosis (Clarke et al. 2009). Chronic inflammation from diseases such as hepatitis, human 247

papilloma virus in the cervix and ulcerative colitis are well known to cause cancer of the tube 248

(Demopoulos et al. 2001). Similarly, it has been suggested that proliferation and hyperplasia 249

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in the fallopian tube are associated with lesions that may be carcinogenic in nature (Moore & 250

Enterline 1975). Compelling evidence for this hypothesis was recently presented by Kim et 251

al. who demonstrated using a Dicer – PTEN knockout mouse that serous epithelial tumours 252

can arise in the fallopian tube and rapidly metastasize to the ovary. Mice in this study 253

exhibited a 100% mortality rate from tumours that bore a striking resemblance to clinical 254

disease (Kim et al. 2012). 255

Retrograde menstruation via the fallopian tube is a common condition, where the flow 256

of endometrial fluid bathes the tubes with inflammatory molecules such as CXCL8 (IL-8), 257

tumour necrosis factor alpha (TNF-α) and granulocyte macrophage-colony stimulating factor 258

(GM-CSF). Each of these are elevated in ovarian carcinomas (Strandell et al. 2004). It is well 259

established that a combination of tubal ligation and hysterectomy are protective against 260

ovarian cancer (Green et al. 1997, Seidman et al. 2002). Together these findings strongly 261

support the notion that inflammation in the fallopian tube, the ovarian epithelium, or both, are 262

contributing factors in the development ovarian cancer. 263

The inflammation-mediated release of chemokines, cytokines and growth factors can 264

also promote migration and proliferation of leukocytes, epithelial and endothelial cells 265

(Figure 2) (Mukaida & Baba 2012). At a molecular level, there is evidence that pro-266

inflammatory cytokines such as IL-1, IL-6 and TNF-α are linked to a poor prognosis in EOC 267

patients (Clendenen et al. 2011, Maccio & Madeddu 2012). Oxidative stress, cell necrosis, 268

and rapid cell division are hallmarks of chronic inflammation (Ness & Modugno 2006). 269

Rapid cell division gives rise to a possibility of replication error and the need for DNA repair, 270

such as at the key regulatory site TP53, increasing the risk of mutagenesis. This is further 271

supported by the high rate of TP53 mutation commonly observed in high-grade serous EOC 272

(Merritt & Cramer 2011). 273

Recently, inflammatory cytokine production in the human ovarian cancer 274

microenvironment has been described in terms of an autocrine cytokine network. The tumour 275

necrosis factor (TNF) network includes TNF-α, interleukin-6 (IL6) and CXCL12 (Kulbe et 276

al. 2012); in particular, the CXCL12/CXCR4 axis plays a major role in the proliferation and 277

metastasis of ovarian tumour cells (Barbieri et al. 2010). Enhanced TNF-network activity has 278

been associated with genes involving angiogenesis, inflammation, leukocyte infiltration. 279

Targeting members of the TNF network via siRNA or neutralizing antibodies reduced 280

experimental peritoneal ovarian tumour growth, as well as angiogenesis in human ovarian 281

tumour biopsies and mouse xenograft models (Kulbe et al. 2012). 282

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The NF-кB pathway is also important in modulating autocrine and paracrine 283

signalling between inflammatory markers in the cancer microenvironment (Son et al. 2007b). 284

Notably, NF-кB regulates the CXCL12/CXCR4 autocrine signalling in ovarian cancer cell 285

lines (Miyanishi et al. 2010). Over-expression of prostaglandins, leading to altered 286

expression of cytokines and chemokines, increases the invasiveness of ovarian tumour cells 287

(and other tumour types) (Aitokallio-Tallberg et al. 1988, Subbaramaiah et al. 1997, Ness & 288

Modugno 2006). The expression of cytokines and chemokines in inflamed malignancies of 289

the female genital tract may, therefore, lead to dysfunctional autocrine loops, resulting in 290

increased tumour progression and invasion. 291

292

4.2 Recruitment of T-cells to epithelial ovarian tumours 293

In the normal anti-tumour immune response, interactions between CXCL9 (MiG), 294

CXCL10 (IP-10) and their receptor CXCR3 influences the chemoattraction of Natural Killer 295

(NK) cells, activated Th1 cells, gamma-delta T-cells, macrophages and dendritic cells 296

towards tumours (Strieter et al. 2006, Whitworth & Alvarez 2011). The importance of 297

CXCL9 and CXCL10 to generate an anti-tumour response is supported by their roles as 298

mediators of interleukin-12 (IL-12), a potent immunoregulatory cytokine (also being trialled 299

as a therapeutic agent in ovarian cancer patients, although with modest results) (Whitworth & 300

Alvarez 2011). Additionally, in an investigation into links between coagulation and 301

inflammation, it was shown that culture of cells derived from ovarian cancer ascites and 302

peripheral blood mononuclear cells (PBMCs) from healthy donors also induced significant 303

upregulation of inflammatory cytokines (IL-1β & IL-6), along with CCL2 and CXCL8 304

(Naldini et al. 2011). This suggests that the ovarian cancer microenvironment is 305

inflammatory in nature, and can direct PBMCs to increase the release of inflammatory 306

chemokines and cytokines. 307

Recent gene expression studies identified CXCL10, CXCL11 and their receptor 308

CXCR3 as amplified in a subset of ovarian cancer patients from a sample of 489 high-grade, 309

serous epithelial tumours. These studies defined this group of tumours as “immunoreactive” 310

(CGARN 2011), suggesting an important role for these chemokines in the tumour 311

microenvironment. A primary function for these chemokine ligands is the trafficking of 312

leukocytes and the recruitment of activated CD4+ Th1 cells, CD8

+ T cells and NK cells 313

towards sites of inflammation (Clark-Lewis et al. 2003). High concentrations of CXCL9 and 314

CXCL10 are also associated with the presence of TILs, and this is a positive predictor of 315

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patient outcome with patients exhibiting lower recurrence, less dissemination and longer 316

survival (Zhang et al. 2003, Sato et al. 2005, Tomsova et al. 2008, Kryczek et al. 2009). By 317

contrast, negative patient outcomes are associated with tumour infiltration by regulatory T 318

cells (Treg) that mediate immune tolerance (Curiel et al. 2004, Sato et al. 2005, Kryczek et al. 319

2009). Sato and colleagues demonstrated that high intraepithelial CD8+/CD4

+ T-cell and 320

CD8+ T-cell/Treg ratios resulted in improved survival in EOC (Sato et al. 2005). Moreover, 321

neither intraepithelial CD4+ or other CD3

+ TILs alone were associated with survival in this 322

study (Sato et al. 2005). Others have more recently shown that longer survival is positively 323

correlated with both CD8+ and CD3

+ TILs (Hwang et al. 2012). However, CD8+ positive 324

TILs showed a more consistent association with patient survival in this meta-analysis (Hwang 325

et al. 2012). 326

Epithelial ovarian cancer tissue and ascites fluid contain high levels of CD4+ CD25

+ 327

FOXP3+ Treg cells, which are not present in normal ovarian tissues (Curiel et al. 2004). 328

FOXP3 is a marker of Treg cells, crucial for maintaining immunologic homeostasis by 329

preventing autoimmunity in a normal immune environment (Kelley & Parker 2010). In a 330

study of immune cell infiltration into tumours, a high level of the chemokine CCL22 331

(secreted by tumour-associated macrophages) was detected in tumour ascites, possibly 332

inducing Treg trafficking into tumours and suppressing the response of TILs (Figure 5) (Curiel 333

et al. 2004). In an apparent contradiction, however, under certain conditions in the tumour 334

microenvironment CXCL9, CXCL10, and the cytokine IL-17 can also be produced by 335

tumour-associated macrophages and contribute to positive outcomes in EOC (Kryczek et al. 336

2009). 337

An explanation as to why TILs are effective at improving EOC patient outcomes can 338

be partially attributed to the recent identification of T helper 17 (Th17) cells. These cells can 339

contribute to pathogenesis of autoimmune disease and initiate tumour growth in certain 340

models, but drive anti-tumour immunity in some epithelial malignancies (Zou & Restifo 341

2010, Wilke et al. 2011). Th17 cells are postulated not to be immunosuppressive, as they do 342

not express FOX3P (Wilke et al. 2011). An important study by Kryczek et al. showed that in 343

addition to NK cells and Th1 effector T cells, Th17 cells secreting IFN-G and IL-17 were 344

correlated with the elevation of Th1 type chemokines CXCL9 and CXCL10 in the ovarian 345

cancer microenvironment (Kryczek et al. 2009). CXCL9 and CXCL10 were produced by 346

primary ovarian cancer cells and macrophages, leading to the recruitment of CXCR3-347

expressing effector CD8+T cells and NK cells to the tumour. A significant association was 348

found with the presence of IL-17 in patient ascites, with high IL-17 levels (median patient 349

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survival 78 months) significantly improving survival time compared to low IL-17 levels 350

(median patient survival 27 months) (Kryczek et al. 2009). The levels of CXCL9 and 351

CXCL10 were directly correlated with tumour with tumour-infiltrating NK cells and CD8+T 352

cells, highlighting the importance of these ELR- chemokines in limiting the spread of EOC. 353

Figure 3 attempts to summarise the effect of increased CXCL9 and CXCL10 concentrations 354

in the EOC microenvironment, which are reported to lead to a positive survival outcome. 355

4.3 Angiostatic chemokine ligands: CXCL9 and CXCL10 expression can inhibit EOC 356

progression 357

The ELR- chemokines CXCL9, CXCL10 and CXCL11 can influence tumour 358

progression through their angiostatic effects (Strieter et al. 2006). Primary ovarian cancer 359

cells stimulated with IL-17 and IFN-G can be induced to secrete CXCL9 and CXCL10 360

(Kryczek et al. 2009), while CXCL11 (and CXCL9) expression has been detected in serous 361

ovarian carcinoma cell lines and tissue by immunohistochemistry and reverse transcriptase 362

polymerase chain reaction (Furuya et al. 2007). These CXCR3-binding chemokines can 363

inhibit endothelial cell migration and proliferation, attract cells involved in innate and 364

adaptive immunity, and increase MHC class I processing by tumour cells, thereby enhancing 365

recognition by the immune system (Groom & Luster 2011, Wilke et al. 2011) (Figure 3). 366

CXC-mediated regulation of the EOC microenvironment appears to be primarily due 367

to CXCL9 and CXCL10; the anti-tumour effect of CXCL11 in EOC is reportedly less 368

efficient in mounting an anti-tumour response (Lasagni et al. 2003, Furuya et al. 2007). 369

Similarly to expression of CXCL9 and CXCL10 ligands, increased expression of CXCR3 has 370

been reported on the surface of epithelial ovarian cancer cells (Furuya et al. 2007) and clear-371

cell ovarian cancer cells (Furuya et al. 2011). CXCR3 is has found to be expressed by 372

endothelial cells while undergoing G2M and late S phases of the cell cycle (Romagnani et al. 373

2001). Although the expression of CXCR3 in multiple cell-types and specific cell cycle 374

phases have been identified (Romagnani et al. 2001), their biological relevance has yet to be 375

conclusively demonstrated (Lasagni et al. 2003, Giuliani et al. 2006, Gacci et al. 2009, 376

Campanella et al. 2010). 377

4.4 Angiogenic chemokine ligands can promote tumour development 378

Pathological angiogenesis involves multiple cell types and growth factors, and is 379

observed in wound healing, arthritis, diabetic retinopathy. It is closely related to chronic 380

inflammation and is an important event in the progression and spread of cancer (Keeley et al. 381

2008). ELR+ chemokines including CXCL8 (IL-8), CXCL5 (ENA-78), and CXCL1(GRO-α) 382

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are known to induce endothelial migration and proliferation through binding to CXCR2, 383

leading to metastatic spread (Mukaida & Baba 2012). They can also act indirectly via the 384

expression of CXCR2 on leukocytes, leading to the secretion of pro-angiogenic factors such 385

as vascular endothelial growth factor (VEGF) (Kiefer & Siekmann 2011). All three CXC 386

chemokines have the ability to attract polymorphonuclear leukocytes including neutrophils 387

(Mukaida & Baba 2012). 388

The significance of inflammation in the potential initiation and progression of EOC 389

makes CXCL5 a potentially important (and largely unreported) factor in this disease. CXCL5 390

is expressed in both lung and pancreatic cancer, and is upregulated in endometriosis and 391

ovarian carcinoma where it can attract neutrophils (Wislez et al. 2004, Furuya et al. 2007, 392

Frick et al. 2008, Li et al. 2011). Once present, neutrophils secrete a broad spectrum of 393

proteases including cathepsin G that can cleave CXCL5; N-terminal truncation of CXCL5 394

significantly enhances the chemotactic potency of CXCL5 both in vitro and in vivo, (Wuyts 395

et al. 1999, Mortier et al. 2010). Neutrophils isolated from the peripheral blood of ovarian 396

cancer patients also produce increased reactive oxygen species, and display increased 397

adhesion to ovarian cancer cells through enhanced CD11b/CD18 expression on their cell 398

surfaces (Klink et al. 2008). The adhesion of neutrophils to endothelial cells initiates 399

remodelling of the endothelium, making it easier for tumour cells to metastasize (Wu et al. 400

2001, De Larco et al. 2004, Klink et al. 2008). Figure 4 attempts to summarise the influence 401

of neutrophils and modified CXCL5 in the metastasis of ovarian tumour cells. 402

Both CXCL8 (Schutyser et al. 2002) and CXCL1 (Bolitho et al. 2010) are 403

upregulated in ascites fluid, whilst CXCL8 protein and anti-CXCL8 antibodies are also 404

present in serum from advanced EOC patients (Lokshin et al. 2006). Stimulation by CXCL8 405

of various ovarian tumour cell lines leads to their proliferation in vitro (Wang et al. 2005), 406

and is correlated with increased metastatic potential in SKOV3 cells (Yin et al. 2011). 407

CXCL8 also has a direct role in angiogenesis, stimulating capillary tube formation via 408

CXCR1 and CXCR2 (Li et al. 2003). In the context of advanced EOC, development of 409

ascites and possible metastasis is promoted by angiogenesis through the key regulator VEGF 410

(Masoumi Moghaddam et al. 2011). In an early study to determine the genes expressed in 411

angiogenesis, five different human ovarian carcinomas were implanted into the peritoneal 412

cavities of nude mice (Yoneda et al. 1998). The formation of ascites was associated with the 413

expression of VEGF and CXCL8, and this was inversely associated with survival. 414

Similarly to CXCL5, CXCL1 has chemotactic activity for neutrophils and its N-415

terminal truncation leads to enhanced chemotaxis in vitro (Wuyts et al. 1999). Over-416

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expression of CXCL1 in vitro also results in increased cell proliferation (Bolitho et al. 2010). 417

CXCL1 has been shown to promote malignant transformation of epithelial cells through a 418

complex RAS pathway-mediated transformation, which induces normal stroma to “re-419

program” and become tumourogenic (Yang et al. 2006). Silencing of CXCL1 using siRNA 420

eliminated RAS induced transformation (Yang et al. 2006). In the same study, CXCL1 421

expression was elevated in both serum and tumour tissue from ovarian cancer patients (Yang 422

et al. 2006). CXCL1 (in combination with CCL18) has also been suggested as a serum 423

biomarker of ovarian cancer (Wang et al. 2011). Multivariate ELSIA gave a sensitivity of 424

92% and specificity of 97% for detecting ovarian cancers, which was significantly higher 425

than values obtained using CA 125 alone (Wang et al. 2011). 426

In normal ovarian function, both CXCL8 and CXCL1 are produced by the ovulating 427

follicle to attract CXCR1+ and CXCR2

+ leukocytes to assist with ovulation (Karstrom-428

Encrantz et al. 1998). Using mouse models of peritoneal ovarian cancer, it has been shown 429

that matrix metalloprotease-1 (MMP1) activation of the G protein–coupled receptor, 430

protease-activated receptor-1(PAR1), is also an important stimulator of angiogenesis and 431

metastasis in EOC (Agarwal et al. 2008). The paracrine control of chemokine production by 432

CXCR1 and CXCR2 receptors on ovarian carcinoma endothelial cells is stimulated by the 433

MMP1-PAR1 signalling system (Agarwal et al. 2010). After MMP1-PAR1 stimulation, it 434

was shown that ovarian carcinoma cells both in vitro and in mice secreted CXCL8 and 435

CXCL1, with introduction of X1/2pal-i3 pepducin completely inhibiting angiogenesis 436

(Agarwal et al. 2010). Inhibition of this pathway blocks tumour growth and metastasis in 437

breast cancer models (Yang et al. 2009); the MMP1-PAR1-CXCR1/2 pathway may therefore 438

be a new target for preventing EOC-related angiogenesis. 439

5. Direct involvement of the ELR- chemokine CXCL12 in metastatic spread 440

In early studies to determine the influence of chemokine gradients on the metastatic 441

spread of ovarian tumours, it was shown that the receptor CXCR4 induced calcium flux and 442

could direct migration of tumour cells (Scotton et al. 2001, Scotton et al. 2002). The 443

expression of CXCR4 mRNA was detected in 6 ovarian cancer cell lines, ascites (19 of 20 444

samples), and primary ovarian tumours (8 of 10 samples). The study showed that the cell 445

lines displayed chemotaxis toward CXCL12, which was also present at high levels in ovarian 446

cancer ascites fluid from 63 patients (Scotton et al. 2001). However, not all tumour cells in 447

the primary ovarian tumours expressed CXCR4 mRNA suggesting that other factors present 448

in the tumour microenvironment contributed to receptor expression. After in vitro stimulation 449

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of the ovarian cancer cell line IGROV with CXCL12, cells could invade through Matrigel 450

toward a CXCL12 gradient (Scotton et al. 2002). This also promoted the activation of 451

Akt/protein kinase B, biphasic phosphorylation of p44/42 mitogen-activated protein kinase, 452

and induction of the cytokine TNF-α (Scotton et al. 2002). 453

The metastatic spread of EOC tumour cells to the peritoneal mesothelium also 454

involves the CXCL12/CXCR4 axis (Kajiyama et al. 2008, Miyanishi et al. 2010). 455

Furthermore, a significant correlation has been shown between CXCR4 expression in both 456

primary and metastatic ovarian tumours, as well as lymph node metastases (Jiang et al. 457

2006). In a mouse model of ovarian cancer siRNA-mediated silencing of CXCL12 reduced 458

tumour growth in vivo, and a CXCR4 antagonist (AMD3100) could increase T-cell-mediated 459

anti-tumour responses (Righi et al. 2011). Human primary ovarian tumour cells also express 460

CXCL12 and VEGF following exposure to the hypoxic tumour microenvironment (Kryczek 461

et al. 2005). In this in vivo model, CXCL12 and VEGF promoted angiogenesis and the 462

migration of human vascular endothelial cell expressing CXCR4 (Kryczek et al. 2005). 463

Importantly, CXCL12 and CXCR4 are not detected in normal ovarian tissues (Jiang et al. 464

2006, Kajiyama et al. 2008), even in women with a family history of ovarian cancer (Scotton 465

et al. 2002). 466

Recently, sequential genetic changes were found in the TP53 allele as well as the 467

CXCR4 gene locus (chromosome 2q21.3) of human transformed ovarian surface epithelial 468

(TOSE) cell lines (Archibald et al. 2012). These were investigated to determine early events 469

in the development of serous EOC. Following the screening of 50 serous EOC patient 470

biopsies, the mutated TP53 expression gene signature (with decreased TP53 target gene 471

expression) was associated with activation of Epidermal Growth Factor Receptor (EGFR) 472

pathways, as well as high expression of CXCR4 genes (with corresponding mRNA and 473

protein). This suggests that these events may occur early in the development of EOC 474

(Archibald et al. 2012). Amongst other recently discovered genetic changes to potentially 475

upregulate CXCR4 expression in EOC is the abrogation of Breast Cancer Metastasis 476

Suppressor 1 (BRMS1) (Sheng et al. 2012). Suppression of BRMS1 by short-hairpin RNA 477

transfection of OVCAR3 cells lines significantly enhanced CXCR4 expression at the mRNA 478

and protein levels, with enhanced expression mediated by the nuclear factor-κB (NF- κB) 479

signalling pathway (Sheng et al. 2012). 480

Figure 5 offers a simplistic summary of the immunosuppressive tumour 481

microenvironment in EOC. The lack of angiogenic ELR- chemokines results in unchecked 482

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16

angiogenesis due to the presence of Treg cells, whilst CXC chemokines and their receptors, in 483

particular the CXCL12/CXCR4 axis, can promote tumour metastasis. 484

485

6. CXC chemokines as future therapeutic targets for EOC 486

Due to the importance of CXC chemokine signalling, there are active development 487

strategies to exploit these pathways to reduce the morbidity and mortality associated with 488

EOC. One strategy involves the stimulation of Th17 cells through the use of vaccines or 489

immunotherapy to stimulate IFN-G and IL-17, resulting in elevated CXCL9 and CXCL10 490

levels (Munn 2009, Cannon et al. 2011, Goyne et al. 2011). However, animal or clinical trials 491

are yet to be reported. The use of CXC chemokine inhibitors and antagonists for a variety of 492

different cancers, including EOC (Zlotnik et al. 2011, Balkwill 2012), is emerging as a 493

promising strategy for new treatments. 494

Of the CXC chemokine ligands and receptors involved in EOC, inhibition of CXCR4 495

has received a great deal of attention with several molecules found that are able to antagonize 496

activity in response to CXCL12 (Barbieri et al. 2010). In two separate studies involving 497

mouse models of EOC, AMD3100 (a clinically approved CXCR4 inhibitor) significantly 498

reduced ovarian cancer cell growth (Ray et al. 2011, Righi et al. 2011). In mice, 499

administration of AMD3100 significantly reduced intraperitoneal dissemination and 500

angiogenesis (measured by vessel density with the tumour), increased T-cell mediated anti-501

tumour immune responses and apoptosis, and reduced FoxP3+

Treg cell numbers within the 502

tumour (Righi et al. 2011). Subsequently it was shown that AMD3100 blocked 503

CXCL12/CXCR4 binding, resulting in prolonged survival and reduced tumour growth in 504

mice with disseminated ovarian cancer (Ray et al. 2011). The use of this important 505

chemokine inhibitor in human trials is yet to be reported. 506

7. Conclusion 507

A significant body of evidence indicates the involvement of chemokines in epithelial 508

ovarian cancer and suggests that initiation, progression and metastasis are strongly influenced 509

by the CXC chemokines. In particular, their roles in inflammation, angiogenesis and immune 510

cell recruitment have all been shown to exert profound influence in the tumour 511

microenvironment; moreover, there is a complex interplay of these factors that influences 512

clinical outcomes. Research to exploit these chemokine signalling pathways, and translate the 513

findings to a clinical environment, remains at a very early stage. Relevant studies in murine 514

models using the inhibitor AMD3100 targeting the CXCR4/CXCL12 axis have been 515

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reported, but there are no human trials yet reported. Similarly, the enhancement of CXCL9 516

and CXCL10 expression through stimulation of Th17 cells, or CXCL5 inhibitor development 517

to prevent neutrophil adhesion and potential metastasis, are also at an early stage. 518

The groundwork for understanding the combined action and mechanisms of CXC 519

chemokine activity in EOC is underway. Exploitation of CXC chemokine signalling 520

pathways to reduce EOC morbidity, and mortality remains as a future avenue to potentially 521

improve outcomes for patients with this disease. 522

523

Acknowledgments & Funding: 524 This work was supported by the Ovarian Cancer Research Foundation and the Victorian 525

Government’s Operational Infrastructure Support Program. 526

527

Declaration of interest: 528 The authors have nothing to disclose.529

530

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899 900

901

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25

Figure 1. Chemokine ligands and receptor: Intramolecular disulfide bonds form between cysteine C1-C3 and 902

C2-C4 pairs, leading to the formation of the characteristic triple-stranded beta-sheet structures (with the 903

exception of the ‘C’ chemokines subgroup, which has only one C-C pair). CXC chemokines are further grouped 904

according to the presence of the “ELR” Glu-Leu-Arg motif. Chemokines bind to their cognate G-protein-905

coupled receptors (GPCRs), comprising seven-transmembrane-spanning domains and belonging to the class A 906

rhodopsin-like family. 907

908 Figure 2. An overview of CXC chemokine involvement in normal wound repair (expanded details found 909

in (Romagnani et al. 2004). (A). Generation of blood clot and neutrophil recruitment: (A1) Growth factors, 910

cytokines and chemokines are released by clotted platelets, whilst CXCL4, 5, and 7 initiate neutrophil 911

recruitment to the wound site; (A2) Epithelial cells at the wound site express CXCL8, whilst endothelial cells 912

express CXCL1; (A3) Neutrophils expressing CXCR2 migrate into the wound site along the CXCL1 / CXCL8 913

chemotactic gradient. (B). Recruitment of lymphocytes & Monocytes: (B1) Epithelial cells continue to 914

express CXCL8, promoting the growth of new blood cells. Epithelial cells also express CXCL 9, 10, and 11 to 915

limit and regulate endothelial cell proliferation and new blood vessel growth; (B2) CXCL9 and 10 recruit 916

lymphocytes during wound healing; (B3) Ongoing CXCL1 expression by endothelial cells promotes continued 917

neutrophil recruitment, while CXCL9, 10, and 11 regulate blood vessel growth and the migration of 918

proliferating endothelial cells; (B4) CXCL4 helps promote and maintain blood clotting. 919

920 Figure 3. Promotion of a favourable outcome in EOC involving CXC chemokines. (1) Th17 cells (without 921

Treg suppression in this example) secrete IL17 and anti-tumour promoting IFN-G, after induction by tumour 922

associated macrophages secreting IL-1B (Kryczek et al. 2009); (2) IL-17 secretion and IFN-G induce tumour 923

cells to secrete CXCL9 and CXCL10; (3) Angiostatic chemokines establish a gradient whereby adaptive and 924

innate immune cells migrate to the tumour site. A high CD8+ T-cell to low CD4+ T-cell and Treg ratio can lead 925

to positive outcomes in EOC (Sato et al. 2005). 926

927

Figure 4 Modification of CXCL5 increases chemotactic potency and neutrophil recruitment. (1) As in 928

normal wound healing, neutrophils containing CXCR2 continue to migrate to the wound site toward CXCL1 929

and 8; (2) Neutrophils secrete cathepsin G and cleave CXCL5, increasing chemotactic potency. Enhanced 930

CD11b/CD18 expression on the neutrophil surfaces increases adhesion to endothelial tumour cells initiates 931

remodelling of the endothelium, making it easier for tumour cells to metastasize. 932

933

Figure 5. Promotion of an unfavourable outcome in EOC involving CXC chemokines. (1) Tumour 934

associated macrophages secrete CCL22 to attract Treg cells. This results in suppression of Th17 cells, thereby 935

reducing IL-17 and IFN-G and impairing production of CXCL9 and 10 (Kryczek et al. 2009) CXCL1 and 8 936

continue to be produced, enhancing angiogenesis (Romagnani et al. 2004); (2) CXCL12 production by tumour 937

cells and enhanced CXCR4 expression by epithelial cancer cells facilitates metastasis; (3) The lack of 938

angiostatic chemokines and presence of Treg cells prevents immune cell migration to the immunosuppressive 939

tumour site (Sato et al. 2005). 940

941

942

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* Epi – epithelial cell expression , Endo – endothelial cell expression

Table 1. General overview of CXC chemokines involved in the regulation of angiogenesis and lymphocyte recruitment.

CXC L ELR Previous

nomenclature CXC R

Receptor

expression* Function General Origin Common Disease Ref.

CXCL1

CXCL2

CXCL3

+

GROa/MGSA-a,

GROb/MGSA-b,

GROg/MGSA-g

CXCR2 Epi/Endo Chemotaxis of neutrophils. Endothelial cell migration and proliferation

Neutrophils Melanoma, gastric and

colorectal cancers, ovarian (Cuenca et al. 1992, Wuyts et

al. 1999, Eck et al. 2003)

CXCL4

+ PF4 CXCR3B Endo Anti-angiogenic, inhibits tumour vascularisation

Thymus, Natural

Killer cell,

monocytes

Melanoma, ovarian cancer

(Gottlieb et al. 1988, Lasagni

et al. 2003, Struyf et al. 2007, Callesen et al. 2010, Ha et al.

2011)

CXCL5

+ ENA-78 CXCR2 Epi/endo Angiogenesis, promotes tumour growth and

metastasis Neutrophils

Lung, pancreatic, ovarian

cancers

(Wislez et al. 2004, Furuya et

al. 2007, Frick et al. 2008,

Furuya et al. 2011, Li et al. 2011)

CXCL6 +

Granulocyte chemotactic protein

2

CXCR1,

CXCR2 Epit/endo Angiogenesis Neutrophils Gastrointestinal tumour (Gijsbers et al. 2005)

CXCL7 + NAP-2, PBP CXCR2, Epi/endo

Mediates angiogenesis via MAPK and mTOR

pathway

Neutrophils, platelets,

monocytes Pancreatic cancers (Matsubara et al. 2011)

CXCL8 + IL-8

CXCR1, CXCR2

Epi/endo Neutrophil chemoattractant and activating factor. Angiogenic agent

Neutrophils Gastric carcinoma, ovarian

cancer (Eck et al. 2003, Yigit et al.

2011)

CXCL9 -

Monokine induced

by IFN-G (MIG) CXCR3 Endo

Attract tumour infiltrating lymphocytes, inhibit endothelial cell migration and

proliferation

Thymus, natural killer cell,

widespread

Gastric, colorectal cancers (Musha et al. 2005, Ohtani et

al. 2009)

CXCL10

- IFN-G-inducible protein 10 (IP-10)

CXCR3 Endo Promote anti-tumour effects by attracting T-lymphocytes

Thymus, natural

killer cell,

widespread

Gastric, colorectal,

endometrial, ovarian

cancers

(Musha et al. 2005, Son et al.

2007b, Ohtani et al. 2009,

Smirnova et al. 2012)

CXCL11

-

IFN-inducible T-

cell a chemoattractant (I-

TAC)

CXCR3, CXCR7

Endo/Epi Inhibit endothelial cell migration and proliferation

Thymus, natural killer cell

Clear cell ovarian cancers (Furuya et al. 2011)

CXCL12

- SDF-1a/b CXCR4,

CXCR7 Endo/Epi

Neovascularisation during fetal organogenesis. Tumour cell migration and

proliferation

Widespread Breast, colorectal

carcinoma, endometrial,

ovarian cancers

(Jiang et al. 2006, Akishima-

Fukasawa et al. 2009,

Gelmini et al. 2009, Hassan et

al. 2009)

CXCL13 + BLC/BCA-1 CXCR5 - Migration of T-cells B cells Human lung cancer (de Chaisemartin et al. 2011)

CXCL14 + BRAK/bolekine Unknown Unknown

Migration of T-cells, chemoattraction of NK

cells in epithelium of the endometrium Monocytes Endometrial cancer (van der Horst et al. 2012)

CXCL16

+

SRPSOX;

CXCLG16; SR-

PSOX

CXCR6 Epi

Soluble CXCL16 enhances proliferation and

migration. Trans-membrane CXCL16 inhibits

proliferation

Activated T cells

Glioma, colorectal

carcinoma, renal cancer, prostate cancer, breast

cancer

(Ludwig et al. 2005, Hojo et

al. 2007, Darash-Yahana et

al. 2009, Gutwein et al. 2009,

Deng et al. 2010)

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1 1 the emerging role of cxc chemokines in epithelial ovarian

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