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Contents lists available at ScienceDirect

Acta Histochemica

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xpression pattern of RAGE and IGF-1 in the human fetal ovary andvarian serous carcinoma

na Poljicanin a, Natalija Filipovic a, Tanja Vukusic Pusic b, Violeta Soljic c, Ana Caric a,irna Saraga-Babic a, Katarina Vukojevic a,∗

Laboratory for Early Human Development, Department of Anatomy, Histology and Embryology, School of Medicine, University of Split, Soltanska 2, 21000plit, CroatiaDepartment of Gynecology, University Hospital in Split, Spinciceva 1, 21000 Split, CroatiaDepartment of Pathology, Cytology and Forensic Medicine, University Hospital in Mostar, Kralja Tvrtka bb, 88 000 Mostar, Bosnia and Herzegovina

r t i c l e i n f o

rticle history:vailable online xxx

eywords:AGE

GF-1uman fetusvaryerous ovarian carcinoma

a b s t r a c t

The expression pattern of RAGE and IGF-1 proteins in different ovarian cell lineages was histologicallyanalyzed in six fetal, nine adult human ovaries, and nine serous ovarian carcinomas (OSC) using immuno-histochemical methods. Mild expression of IGF-1 in ovarian surface epithelium (Ose) and oocytes in the15-week human ovaries increased to moderate or strong in the stromal cells, oocytes and follicular cellsin week 22. Occasional mild RAGE expression was observed in Ose during week 15, while strong expres-sion characterized primordial follicles in week 22. In the reproductive human ovary, IGF-1 was mildlyto moderately expressed in all ovarian cell lineages except in theca cells of the tertiary follicle whereIGF-1 was negative. RAGE was strongly positive in the granulosa cells and some theca cells of the ter-tiary follicle, while negative to mildly positive in all cells of the secondary follicle. In the postmenopausalhuman ovary IGF-1 and RAGE were mildly expressed in Ose and stroma. In OSC, cells were stronglypositive to IGF-1 and RAGE, except for some negative stromal cells. Different levels of IGF-1 and RAGE co-expression characterized fetal ovarian cells during development. In reproductive ovaries, IGF-1 and RAGE

were co-localized in the granulosa and theca interna cells of tertiary follicles, while in postmenopausalovaries and OSC, IGF-1 and RAGE were co-localized in Ose and OSC cells respectively. Our results indi-cate that intracellular levels of IGF-1 and RAGE protein might regulate the final destiny of the ovariancell populations prior and during folliculogenesis, possibly controlling the metastatic potential of OSCas well.

ntroduction

Ovarian cancers represent a great challenge for scientistsecause their etiology and pathogenesis is still poorly understoodespite the existence of numerous studies. The most common his-ological type of epithelial ovarian cancer is high grade ovarianerous carcinoma (OSC) which is also a common lethal gyneco-

Please cite this article in press as: Poljicanin A, et al. Expression pattercarcinoma. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acth

ogical malignancy among women (Jemal et al., 2010). In order tonderstand better the histogenesis of the epithelial ovarian cancers,

t is important to emphasize that during embryonic development

Abbreviations: IGF-1, insulin-like growth factor 1; Ose, ovarian surface epithe-ium; RAGE, the receptor for advanced glycation endproducts; OSC, serous ovarianarcinomas.∗ Corresponding author at: Head of Laboratory for Early Human Development,epartment of Anatomy, Histology and Embryology, School of Medicine, Universityf Split, Soltanska 2, 21000 Split, Croatia.

E-mail address: katarina.vukojevic@mefst.hr (K. Vukojevic).

ttp://dx.doi.org/10.1016/j.acthis.2015.01.004065-1281/© 2015 Elsevier GmbH. All rights reserved.

© 2015 Elsevier GmbH. All rights reserved.

the ovarian surface epithelium (Ose) develops from pluripotentcells of the celomic epithelium, and has the same origin as Mül-lerian ducts that develop into Fallopian tubes, endometrium andendocervix in adults. During ovarian carcinogenesis, Ose has theability to acquire Müllerian cell phenotype and develop intoserous (Fallopian tube), endometrioid (endometrium) and muci-nous (endocervix) cancer (Shih Ie and Kurman, 2004). The highgrade OSC is thought to arise de novo from a single layer of squa-mous or cuboidal cells that cover the surface of the ovary, but alsofrom epithelial inclusion cysts that develop during the period ofpostovulatory healing (Cramer et al., 1983; Cramer et al., 1983). Inline with this is the “incessant ovulation hypothesis” that attemptsto explain the increased probability of ovarian cancer appearanceby repeated minor damage of Ose due to continuous ovulations

n of RAGE and IGF-1 in the human fetal ovary and ovarian serousis.2015.01.004

(Cooke et al., 2003). On the other hand, a study of Barker et al. (2008)proposes a theory of developmental origin of cancer (Buhimschiet al., 2009; Gebhardt et al., 2006). Unlike other tumors, wheretumor progression leads to de-differentiation of epithelial cells,

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pithelial ovarian cancer cells usually display a higher level of dif-erention than the surface epithelial cells from which the tumorriginated (Shih Ie and Kumaran, 2004). This higher scale of differ-ntiation level of epithelial tumor cells could also be explained byhe aberrant activation of embryonic pathways in ovarian canceristogenesis or even by reactivation of embryonic stem cells (Carict al., 2014). Compelling evidence also demonstrated that fuelingnflammation in the tumor microenvironment creates a tumor-romoting milieu, which in turn favors proliferation and survivalf cancer cells (Rojas et al., 2010). It was estimated that almost 25%f all cancers are somehow associated with chronic infection and

nflammation (Rojas et al., 2010) as some reliable evidence existsoncerning infection and inflammation effects on tumor growth invarian cancer pathogenesis (Liliac et al., 2012).

Recent evidence indicates that two new markers, which arenvolved in normal cell proliferation and differentiation, mightlso have an important role in cancer pathogenesis. One of thems insulin-like growth factor 1 (IGF-1) the major mediator of theffects of growth hormone. It also plays an important role in cellroliferation, differentiation and cell survival (Hilmi et al., 2008;akar et al., 2005), particularly in prenatal growth through itsnti-apoptotic effects in various pathophysiological conditions (Leoith and Roberts, 2003). Studies on human tissue cultures indi-ate that IGF-1, as a major determinant of growth, operates in thevary during the prenatal and postnatal periods (Baker et al., 1993;aughaday, 1989; Yakar et al., 2005). Studies on the IGF-1 knockut mice showed that they are infertile (Baker et al., 1993). It iselieved that disturbances in any of the IGF signaling pathwayay be responsible for the development and progression of tumors

Yakar et al., 2005). Interest in IGF-1 and its effects on carcinogen-sis has increased recently because high serum concentrations ofGF-1 are associated with an increased risk of breast, prostate, col-rectal, and lung cancers (Furstenberger and Senn, 2002). So far,

mmunohistochemical expression of IGF-1 in human fetal ovariesnd OSC has not been investigated (Le Roith and Roberts, 2003;akar et al., 2005). Previous investigations on human fetal ovariesnd tumor tissues are either scarce or explore only certain ovarianell lines.

The receptor for advanced glycation and products RAGE is aultifunctional receptor of the immunoglobulin superfamily, that

inds to various ligands and is mainly expressed on the surface ofmmune cells, neurons, activated endothelial and vascular smooth

uscle cells, bone forming cells, and a variety of cancer cellsLogsdon et al., 2007). RAGE mediates responses to cell damagend stress conditions, activates programs responsible for acute andhronic inflammation, and is involved in a number of pathologi-al diseases (Riehl et al., 2009; Schmidt and Stern, 2001) includingrogression of cancer (Lu et al., 2010 Zhang et al., 2013). RAGE

s highly expressed during development, especially in the brain,ut its expression level decreases in adult tissues (Rojas et al.,010). By now, a possible role of RAGE in tissue of reproductiveuman ovary was demonstrated only in a study of Diamanti Kan-arakis (Diamanti-Kandarakis et al., 2007). RAGE expression haseen extensively investigated in many cancer types (Logsdon et al.,007; Rojas et al., 2010), disclosing that its expression is dictated byhe accumulation of damage-associated molecules. So far, immuno-istochemical expression of RAGE in human fetal and adult ovariesas not been investigated, thus the possible role of RAGE in ovariesemains unknown.

Our knowledge on the molecular function of IGF-1 and RAGEuring neoplastic transformation and malignant progression is lim-

ted. However, recent experimental data including in vitro, in vivo

Please cite this article in press as: Poljicanin A, et al. Expression pattercarcinoma. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acth

nd clinical studies, support a direct link between RAGE and IGF- activation and processes of cell proliferation, survival, cancerell migration, and invasion (Abe and Yamagishi, 2008; Le Roithnd Roberts, 2003; Liliac et al., 2012; Logsdon et al., 2007). Recent

PRESSemica xxx (2015) xxx–xxx

studies have shaped the conceptual framework in which cancer isviewed as a disorder that is closely related to early human devel-opment (Caric et al., 2014; Naora, 2005). Our study investigatesthe expression pattern of IGF-1 and RAGE during the most inten-sive period of proliferation and folliculogenesis in the human ovaryduring fetal development (developmental weeks 15 and 22), inthe reproductive and postmenopausal ovaries as well as in ovar-ian serous carcinoma. Investigations on the possible changes inIGF-1 and RAGE protein expression pattern during normal ovar-ian maturation and in carcinogenesis may clarify their role in thedevelopment of ovarian cancer, thus introducing them as targets fortherapeutic intervention and risk assessment. As tumor develop-ment can be regarded as a deviant form of organogenesis, aberrantactivation of developmental pathways in ovarian cancer histogene-sis can highlight the intimate relationship between developmentalplasticity and malignant transformation.

Materials and methods

Human material and tissue processing

The study was approved by the Ethical and Drug Committee ofthe School of Medicine University of Split (#2181-198-03-04/10-11-0074) in accordance with the Helsinki Declaration (Williams,2008). Ovaries of six normal human fetuses (developmental weeks15 and 22), were obtained after spontaneous abortions from thearchives of Department of Anatomy, Histology and Embryology.All tissues were morphologically normal, without signs of macer-ations and every tenth section was stained with Hematoxylin andEosin to establish tissue preservation. The crown-rump length wasused to determine the corresponding age of fetuses (O’Rahilly andGardner, 1971) and correlated with the menstrual data. Five repro-ductive and four postmenopausal ovaries from healthy women(aged 45–53 and 56–63, respectively, who underwent oophorec-tomy and total abdominal hysterectomy for benign conditions),and nine high grade serous ovarian cancers (material after firstsurgery prior to chemotherapy) were archival materials from theDepartment of Pathology, Cytology and Forensic Medicine, Uni-versity Hospital in Split, Split, Croatia. Histological grading of thetumors was performed according to the classification of the WorldHealth Organization (WHO) (Ellerman et al., 2007). Tissue sampleswere paraffin embedded and processed as we described previously(Bartling et al., 2006; Bucciarelli et al., 2002; Poljicanin et al., 2013).

Immunohistochemical staining

Immunohistochemistry was performed as described in previ-ous studies (Sparvero et al., 2009; Zhang et al., 2003) with somemodifications. After deparaffinization and rehydration, endoge-nous peroxidase activity was prevented by incubation with 3%H2O2 for 15 min. Non-specific binding sites were blocked using1% normal goat serum (Santa Cruz Biotechnology, Santa Cruz, CA,USA) and diluted in phosphate-buffered saline. Sections were sub-mitted to a water bath antigen retrieval step, immersed in DakoTarget Retrieval Solution (S2367 Dako Cytomatiom, Carpinteria,CA, USA) in a microwave oven at 97 ◦C for 15 min. After cooling toroom temperature slides were washed with PBS and then incubatedwith primary antibody for 1 h: goat anti-human IGF-1 antibody(AF-291-NA; diluted at 1:100, R&D Systems, Minneapolis, MN,USA) and rabbit anti-RAGE antibody (abcam3611; diluted at 1:500,Abcam, Cambridge, UK). Primary antibody binding was followed by

n of RAGE and IGF-1 in the human fetal ovary and ovarian serousis.2015.01.004

incubation with biotinylated swine-anti-mouse, rabbit, goat anti-body and with streptavidin–biotin peroxidase conjugate (K0690;LSAB+ System-HRP Dako Cytomation), both for 30 min. Antibodycomplexes were developed after the addition of a buffered DAB

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Table 1Immunoreactivity to IGF-1 and RAGE antibodies in the human ovary during the 15th and 22nd week of development.

Antibody Dev. week Ovarian surface epithelia Stromal cells Primordial follicle

Oocyte Follicular cells

IGF-1 15 + − + −22 − ++ ++/+++* −/++

RAGE 15 −/+* − − −−/ + * ++/+++* −/+++*

S moderate to mild, moderate or strong (*).

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Table 2Immunoreactivity to IGF-1 and RAGE antibodies in the reproductive human ovary.

Ovarian cell population Reproductive

IGF-1 RAGE

Ovarian surface epithelia/OSC cells + −/+Stromal cells +/++ +Primary follicle

Oocyte + +Granulosa cells ++ +++

Secondary follicleOocyte + +Granulosa cells −/+ +++Theca cells − −/+

Tertiary follicleOocyte / /Granulosa cells ++ +++Theca cells ++ −/+++*

TI

S

22 −/+*

trong reactivity (+++), moderate reactivity (++), mild reactivity (+), no reactivity (−), reactivity from negative, mild or

ubstrate (K3468; Liquid DAB+ Substrate chromogen system Dakoytomation), for 10 min. Sections were than counterstained withematoxylin and mounted for light microscope viewing (OlympusX40, Tokyo, Japan). Cells reacting with the primary antibody hadrown stained cytoplasm or nucleus. Positive control for IGF-1 anti-ody was liver tissue and for RAGE antibody was smooth muscle

n blood vessels. As negative control, the primary antibodies werexcluded from the staining procedures.

riple immunofluorescence staining

Sections were deparaffinized and rehydrated as we described formmunohistochemistry, but after antigen retrival the primary anti-odies were incubated together (1 h) to examine the relationshipf IGF-1 and RAGE in the same sections. Slides were then rinsed

n PBS and incubated for 1 h with an appropriate combination ofecondary antibodies: Rhodamine Red TM-conjugated donkey anti-abbit (711-295-152; diluted at 1:300, Jackson ImmunoResearchaboratories, West Grove, PA, USA) and FITC-conjugated donkeynti-sheep (713-095-147; diluted at 1:200, Jackson ImmunoRe-earch PA, USA). All primary and secondary antibodies were dilutedn DAKO antibody diluent with background reducing componentsS3022; Dako, Carpinteria CA, USA). Nuclei were counterstainedith DAPI and coverslipped (Immuno-Mount, Shandon, Pittsburgh,

A, USA). Sections were analyzed with a fluorescence microscopeOlympus BX51, Tokyo, Japan). Digital images were taken with cam-ra (SPOT Insight, Diagnostic Instruments, USA) using the OlympusellA software and assembled by Adobe Photoshop (Adobe Sys-ems, San Jose, CA, USA).

emi-quantification

The intensity of staining of ovarian structures was semi-uantitatively organised into four groups according to the staining

ntensity: the absence of any reactivity (−), a mild reactivity+), moderate reactivity (++), strong reactivity (+++) (Tables 1–3).onsidering inter-observer variations, three investigators blindlynalyzed the staining intensity.

esults

ealthy fetal and adult ovary

Please cite this article in press as: Poljicanin A, et al. Expression pattercarcinoma. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acth

In week 15 of development, the ovary is covered by a layer ofuboidal epithelium and underlying thin dense connective layer,he tunica albuginea. The ovarian stroma is highly cellular, with

able 3mmunoreactivity to IGF-1 and RAGE antibodies in the postmenopausal human ovary and

Ovarian cell population Postmenopausal

IGF-1

Ovarian surface epithelia/OSC cells +++ *

Stromal cells +++ *

trongreactivity (+++), moderate reactivity (++), mild reactivity(+), noreactivity (−), reactivity from negative, mild or moderate

Strong reactivity (+++), moderate reactivity (++), mild reactivity (+), noreactivity (−), reactivity from negative, mild or

moderate to mild, moderate or strong (*), structure absent in the tissue section (/).

nests of primary oogonia partially enveloped by follicular epithe-lium. The epithelium of cortical sex cords extends from the surfaceepithelium into the underneath mesenchyme (Fig. 1a). Within thesex cords, some oogonia are arrested in the prophase of meio-sis and are called oocytes. Most oocytes appear in the form offollicles, except a small number of “lost oocytes” trapped in thesurface epithelium (Fig. 2a). In week 22 of development the ovar-ian surface epithelium is a single layer of flat to cuboidal epithelialcells–mesothelium (Fig. 1c), and oocytes within stroma are fullyenveloped by follicular cells (Fig. 1d). In the healthy reproductiveovary, the ovarian surface epithelium is also a monolayer of flatto cuboidal epithelium (Fig. 1e), attached to the basement mem-brane which lies on a thin layer of connective tissue. Beneath thetunica albuginea, the ovarian follicles in various stages of matura-tion (Fig. 2c) are embedded in the connective tissue stroma. Theoocyte within the primary follicle is surrounded by one or twolayers of granulosa cells, while granulosa cells in the secondaryand tertiary follicle are surrounded by the theca interna and thecaexterna cells (Fig. 2c).

Ovarian serous carcinomas

At histological examination, OSC displayed solid or papil-

n of RAGE and IGF-1 in the human fetal ovary and ovarian serousis.2015.01.004

lary growth with marked nuclear atypia and variations in thenuclear size, showing also high mitotic activity. The ovarianstroma is diffusely invaded by OSC cells and disseminated with

ovarian serous carcinomas (OSC).

OSC

RAGE IGF-1 RAGE

+ +++ ++++ −/ + + +* −/ + + +*

to mild, moderate or strong (*), structure absent in the tissue section (/).

Please cite this article in press as: Poljicanin A, et al. Expression pattern of RAGE and IGF-1 in the human fetal ovary and ovarian serouscarcinoma. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.01.004

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Fig. 1. (a,b) Section through the ovary of a 15-week human fetus. Cells of the ovarian stroma (Os) and follicular cells (f) are not IGF-1 positive, while ovarian surface epithelium(Ose) cells (arrows) and oocytes (arrowheads) are mildly positive to IGF-1 (mildly brown stained cytoplasm), cortical cords (asterisks). (c,d) Section through the ovary of a22-week human fetus: ovarian surface epithelium (Ose) cells are IGF-1 negative while ovarian stroma (Os) cells (arrow) and oocytes (arrowhead) are moderately to stronglyIGF-1 positive. Some follicular cells (f) are IGF-1 positive and some are negative. IGF-1 positive cells have brown-stained cytoplasm or nuclei. (e,g) Section through the healthyovary in reproductive period (ro): ovarian surface epithelium (Ose) and ovarian stroma (Os) cells are mildly to moderately IGF-1 positive (arrows). In the primary follicle (f)granulosa cells (GC) are moderately positive to IFG-1, while granulosa cells (GC) in the secondary follicle (g) are negative to mildly positive to IGF-1. Theca interna cells (TIC)of the secondary follicle are IGF-1 negative. (h) Section through the healthy postmenopausal ovary (po): ovarian surface epithelium (Ose) and ovarian stroma (Os) cells aremildly to moderately IGF-1 positive (arrows). (i) Section through an invasive serous ovarian carcinoma (OSC): while some OSC cells are negative other are strongly positiveto IGF-1 (arrows). Immunostaining to IGF-1, scale bar on a = 40 �m; on b–i = 20 �m.

Fig. 2. (a) Section through the ovary of a 15-week human fetus: ovarian surface epithelium (Ose) cells are occasionally mildly RAGE positive (arrow) while cells of the ovarianstroma (Os), follicular cells (f) and oocytes (arrowheads) are not RAGE positive. (b) Section through the ovary of a 22-week human fetus: while some ovarian stroma (Os)cells are mildly RAGE positive, oocytes (arrowheads) and some follicular cells (f) are moderately to strongly RAGE positive. RAGE positive cells have brown-stained cytoplasmor nuclei. (c) Section through the healthy ovary in reproductive period (ro): In the tertiary follicle granulosa cells (GC) are strongly positive to RAGE (arrows), while thecainterna cells (TIC) are negative to strongly positive to RAGE (arrows). (d) Section through the healthy postmenopausal ovary (po): ovarian surface epithelium (Ose) andovarian stroma (Os) cells are mildly RAGE positive (arrows). (e,f) Section through an invasive serous ovarian carcinoma (OSC): the majority of OSC cells have strong nuclearpositivity to RAGE (arrows), while OSC cells in mitosis (black arrowheads) have strong cytoplasmic positivity; apoptotic bodies (white arrowhead). Immunostaining to RAGE;scale bar = 20 �m.

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ononuclear gigantic cells exhibiting prominent nucleoli, andmmune and inflammatory cells infiltrations (Figs. 1i and 2e–f).

mmunohistochemical staining with IGF-1

During week 15 of development, immunohistochemical stain-ng with IGF-1 and DAB disclosed mild expression of IGF-1 in thevarian surface epithelium and oocytes enabling prevalence of anti-poptotic properties which are important for their growth andurvival. No staining of stromal and follicular cells was detectedndicating the importance of other cell processes rather than pro-iferation and growth in those cell lines (Table 1, Fig. 1a,b). Contraryo this finding, in developmental week 22 IGF-1 was moderatelyxpressed in the stromal cells and moderately to strongly in theocytes, while the ovarian surface epithelium was negative to IGF-. Expression of follicular cells varied from negative to moderate inifferent parts of ovarian tissue (Table 1, Fig. 1c,d). IGF-1 expres-ion increased only in the stromal cells and the oocytes, indicatinghat in developmental week 22 the ovarian surface epitheliumlowed down its proliferation rate, while this process was inten-ified in other ovarian cell lines, especially in the oocytes. In theeproductive ovary, IGF-1 was mildly expressed in the ovarian sur-ace epithelium and oocytes, while it was mildly to moderatelyxpressed in the ovarian stroma and granulosa cells of primarynd tertiary follicles. In the secondary follicle, oocytes were some-imes absent due to the process of atresia. In the theca cells andn some granulosa cells we noted some IGF-1 negative cells, whilether granulosa cells were mildly positive (Table 1; Fig. 1e–g). In theostmenopausal ovary, IGF-1 was mildly to moderately expressed

n both the ovarian surface epithelium and ovarian stroma (Table 1;ig. 1h). In the ovarian serous carcinoma, IGF-1 was also stronglyxpressed in both ovarian surface epithelium and ovarian stroma,ut approximately one third of the cells in ovarian stroma were

GF-1 negative (Table 1; Fig. 1i).

mmunohistochemical staining with RAGE

In week 15 of development, immunohistochemical stainingith RAGE and DAB, showed negative to mild expression of RAGE

n the ovarian surface epithelium, while no staining of stromal, fol-icular cells and oocytes was observed (Table 1, Fig. 2a). Later in

eek 22 of development, RAGE displayed negative to mild expres-ion in the ovarian surface epithelium and stroma, while oocytesnd follicular cells showed strong expression of RAGE. Occasionally,ollicular cells were RAGE negative (Table 1; Fig. 2b).

In the reproductive ovary, RAGE was negative or was mildlyxpressed in the ovarian surface epithelium and theca cells of theecondary follicle. Mild expression of RAGE was observed in thetromal cells and oocytes of primary and secondary follicle, whiletrong expression was detected in the granulosa cells during allaturation stages of follicles. Although theca cells of the tertiary

ollicles also displayed strong expression, some cells were RAGEegative (Table 1; Fig. 2c). In the postmenopausal ovary, RAGE wasildly expressed in both ovarian surface epithelium and ovarian

troma (Table 1; Fig. 2d). In the ovarian serous carcinoma, OSC cellsere strongly RAGE positive in both the ovarian surface epithelium

nd ovarian stroma, but occasionally some OSC cells in the ovariantroma were RAGE negative (Table 1; Fig. 2e,f). In the inflammatorynfiltrations in OSC, neutrophils had moderate RAGE expression inheir cytoplasm (Fig. 2e).

riple immunofluorescence staining with IGF-1, RAGE and

Please cite this article in press as: Poljicanin A, et al. Expression pattercarcinoma. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acth

API

Immunofluorescence staining with IGF-1, RAGE and DAPIuclear stain were used to determine their possible co-expression

PRESSemica xxx (2015) xxx–xxx 5

pattern. While some ovarian surface epithelium cells in the 15-week human ovary were co-localizing IGF-1 and RAGE positivity,cells of the ovarian stroma, follicular cells, and oocytes were onlyIGF-1 positive (Fig. 3a). In the 22-week human ovary, strong cyto-plasmic co-expression of IGF-1 and RAGE was seen in some nests ofoocytes, while some oocytes were only IGF-1 positive (Fig. 3b). Inthe granulosa cells of the follicles in the reproductive ovary, IGF-1and RAGE were strongly co-localized in the cytoplasm, while thecainterna cells strongly co-localized IGF-1 in cytoplasm and RAGEin nuclei of the same cells (Fig. 3c). Cells of the ovarian surfaceepithelium and ovarian stoma were not co-localizing IGF-1 andRAGE. After merging of immunostaining of the two markers in thepostmenopausal ovary, IGF-1 and RAGE were co-localizing in theovarian surface epithelium cells, while the ovarian stroma cells didnot co-localize IGF-1 and RAGE (Fig. 3d). In the invasive serous ovar-ian tumor, OSC cells had strong nuclear co-localization of IGF-1 andRAGE (Fig. 3e).

Discussion

IGF-1 and RAGE seems to play a critical role in the growth andsurvival of many cell types. Both factors promote the cell prolifera-tion through Akt/Wnt signaling pathways that play a critical role inthe cell proliferation (Gong, 2013; Vanamala et al., 2010). In partic-ular, IGF-1 promotes cells from G1 to S phase of cell cycle thusenhancing cell proliferation (Liang and Slingerland, 2003). Anti-apoptotic effect of IGF-1 is achieved through activation of p53 geneaccording to humans and rodent experimental models of inducedcolon cancer (Sanders et al., 2004). Similarly, RAGE limits apopto-sis through a p53 dependent mitochondrial pathway (Kang et al.,2010). Blocade of IGF-1 receptor concurrently attenuates the down-stream Akt/Wnt signaling pathways (Vanamala et al., 2010) thusinhibiting apoptosis.

In our study on the expression of IGF-1 protein in the cells ofthe human fetal ovary, we showed that the distinct expressionof IGF-1 protein depended on the fetal age, or type of examinedovarian cell line. The described temporal and spatial expression ofIGF-1 protein in oocytes and follicular cells of the human fetus is inaccordance with intense proliferative activity that is characteristicof the studied period of fetal ovarian development. Additionally,the intense apoptotic activity was shown in our previous studyfor the same developmental period (Poljicanin et al., 2013). In thereproductive ovary, the expression pattern of IGF-1 protein varieddepending on the follicles’ developmental stage and their des-tiny to undergo either atresia or further maturation and ovulation.These results are in accordance with the findings of Buyalos (1995)who also found stage dependent IGF-1 expression in oocytes ofthe human ovary. An important role of IGF-1 gene in the develop-ment of ovarian follicle was confirmed by a study on geneticallymodified mice, in which follicular development was stopped in theearly antral stage (Hu et al., 2004). Additionaly, numerous stud-ies on experimental animals showed that granulosa cells are themain source of IGF-1, which plays an important role in the selec-tion of follicles that will undergo the process of ovulation (Murphyet al., 1987; Samaras et al., 1993). The epithelium in the repro-ductive and postmenopausal ovary seems to be under the directinfluence of stromal cells that secrete IGF-1 responsible for stimu-lated proliferation. Interactions between stromal cells and ovarianepithelium seem to be important not only for normal functioning ofthe reproductive ovary, but also for the formation of serous ovariancarcinomas (Cramer et al., 1983; Slot et al., 2006), as also shown

n of RAGE and IGF-1 in the human fetal ovary and ovarian serousis.2015.01.004

in studies on breast, bladder, cervix, colon, prostate and ovariancarcinoma (Pritchett et al., 1989). On the other hand, we noticedan increased pattern of IGF-1 expression in the ovarian surfaceepithelium in reproductive and postmenopausal ovary. IGF-1 was

Please cite this article in press as: Poljicanin A, et al. Expression pattern of RAGE and IGF-1 in the human fetal ovary and ovarian serouscarcinoma. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acthis.2015.01.004

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Fig. 3. (a) Section through the ovary of a 15-week human fetus: while some ovarian surface epithelium (Ose) cells are co-localizing IGF-1 and RAGE positivity (arrow), cellsof the ovarian stroma (Os), follicular cells (f) and oocytes (arrowheads) are only IGF-1 positive. (b) Section through the ovary of a 22-week human fetus: while some oocytesin ovarian stroma (Os) are only IGF-1 positive (arrow), some nests of oocytes have strong cytoplasmic co-localization of IGF-1 and RAGE (arrowheads). (c) Section throughthe healthy ovary in reproductive period (ro): In the tertiary follicle granulosa cells (GC) at the luminal part IGF-1 and RAGE are strongly co-localized in the cytoplasm(arrowheads), while theca interna cells (TIC) strongly co-localize IGF-1 in the cytoplasm and RAGE in nuclei of the same cells (arrows). (d) Section through the healthypostmenopausal ovary (po): ovarian surface epithelium (Ose) cells co-localize IGF-1 and RAGE (arrowheads), while ovarian stroma (Os) cells do not co-localize IGF-1 andRAGE (arrows). (e) Section through an invasive serous ovarian carcinoma (OSC): OSC cells have strong nuclear co-localization of IGF-1 and RAGE (arrowheads). Tripleimmunofluorescence staining to IGF-1 (green), RAGE (red) and DAPI (blue), Scale bar = 20 �m. (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

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specially intensified and strong in OSC, thus indicating the loss oformal control of its expression during aging and persistence ofroliferative activity in postmenopausal ovary that might be asso-iated with malignant progression of OSC. This is also in line withome earlier studies (Anderson et al., 2009; Chan et al., 2000) andupports the contention that an imbalance in the IGF system mayesult in ovarian pathology. The possible role of the IGF pathwayn the development of ovarian tumors was also observed in in vitrotudies that disclosed IGF-1 effects on increased growth of tumorells and their invasiveness (Chakrabarty et al., 2006; Khandwalat al., 2000), on acceleration of tumor progression from precan-erous lesions to invasive (Furstenberger and Senn, 2002) and anncrease of cancer metastatic potential (Smith et al., 2000). It washown that knock-down of IGF-1 receptor using IGF-1R siRNA sup-ressed cell proliferation (Vanamala et al., 2010). Furthermore, thexpression of the IGF-1 receptor and the presence of genetic varia-ions of IGF1, IGFBP1 and IGFBP3 genes were shown to be associatedith an increased risk of ovarian cancer (Berns et al., 1992; Terry

t al., 2009), allowing the cancerous cells to oppose the cytotoxichemotherapeutic drugs or radiotherapy (Smith et al., 2000). Addi-ionally, in the present study, a small number of apoptotic bodiesas also observed in the tumor tissue, which is in line with our

revious study that showed a small index of apoptotic activity inhe OSC samples (expression of caspase-3 and Apototic Inducingactor – AIF) (Caric et al., 2014). That might suggest the role ofGF-1 in preventing cancer cells to undergo programmed cell deathnd that predominance of stimulated proliferation over apoptosiseads to malignant tumor progression. Other studies also reportedhat cancer cells frequently take an advantage of the IGF signalingChan et al., 2000; Diebold et al., 1996; Malaguarnera and Belfiore,014) which is a key player in the induction and maintenance ofell stemness, thus contributing to the growth and expansion ofancer stem-like cells (Reya et al., 2001). In addition, as sugesstedn the study of Gong (2013) IGF-1 and RAGE were overexpressedn the microenvironment of pancreatic cancer cells, leading toncontrolled cancer cell proliferation, unorganized angiogenesisnd evasion of apoptosis. Thus the role they played in the cellu-ar growth and survival may depend on different mechanisms, notnly on the effect on the apoptotic machinery, but also on the cellycle. Namely, the production of growth factors promoting the cel-ular growth is a key determinant in the acquisition of a malignanthenotype (Hilmi et al., 2008). In regard to this, IGF-1 as a growth

actor may be one of the factors involved in the malignant pheno-ype of ovarian tumors, and its aberrant regulation may contributeo the malignant transformation of ovarian epithelium.

Our study on the expression of RAGE in human fetal ovariesisclosed that in week 15 of development RAGE was only occa-ionally expressed in the ovarian surface epithelium, while ineek 22 of development expression of RAGE spread to all ovarian

tructures. Some studies suggested an association between highoncentrations of intrafollicular soluble RAGE and poor embryoevelopment after ovarian stimulation for intracytoplasmic sperm

njection (Bonetti et al., 2013). Additionally, RAGE was expressedt the sites of fetal tissue injury (Buhimschi et al., 2009) and waslevated in women with preeclampsia (Cooke et al., 2003). So far,mmunohistochemical expression of RAGE during developmentas been described only in the central nervous system of cul-ured embryonic rat neurons (Hori et al., 1995). In the reproductivevary, RAGE was strongly expressed in the granulosa cells duringll maturation stages of follicles and in the theca cells of the ter-iary follicles. Since RAGE expression is normally down-regulatedn adult life, it may have a role in the decline of ovarian reproductive

Please cite this article in press as: Poljicanin A, et al. Expression pattercarcinoma. Acta Histochemica (2015), http://dx.doi.org/10.1016/j.acth

apabilities during ageing (Stensen et al., 2014). Additionally, sometudies suggested a possibility that RAGE–VEGF interactions maye related to reproductive dysfunction in aging ovaries, and thatoluble RAGE might be a biological indicator of reproductive state

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(Fujii and Nakayama, 2010). Since RAGE concentration is elevatedin polycystic ovaries, it could also contribute to ovarian dysfunction(Diamanti-Kandarakis et. al., 2007).

In the postmenopausal ovary, RAGE was mildly expressed inboth ovarian surface epithelium and ovarian stroma, while it wasstrong in most of OSC and stromal cells. Similarly, overexpression ofRAGE ligands characterized most types of solid tumors (Ellermanet al., 2007; Gebhardt et al., 2006), while RAGE mutation or lossof function in pancreatic ductal adenocarcinoma promoted cellgrowth and inhibited apoptosis (Gong, 2013). Contrary, blockade ofthe RAGE expression pathway in mouse tumor models and in vitrostudies decreased cell proliferation and metastasis thus providingthe concept of RAGE as a potent inducer of carcinoma cells pro-liferation and also implying the role of RAGE in the cell cycle andcarcinoma development (Taguchi et al., 2000). Additionally, besidesthe role of RAGE in cell proliferation, it also initiates a variety ofcell signaling pathways that regulate important cellular functions,including survival, migration, motility and invasiveness (Logsdonet al., 2007). However, few studies reported that RAGE is involvedin tumour-suppressive functions in different cell types, (Stav et al.,2007), while in the study of Bartling et al. (2006) re-expressionof RAGE in lung tumour cell lines was shown to reduce their pro-liferatative activity and displayed diminished tumour growth inathymic mice.

Furthermore, RAGE is involved in immune response modula-tion, since RAGE-knockout animals are less predisposed to acuteinflammation and carcinogen-induced tumor development (Kanget al., 2010). Namely, activation of RAGE leads to activation ofNF-kappaB and tumor cell proliferation which additionally propa-gate and maintain inflammation (Turovskaya et al., 2008). Targetedknockdown of RAGE in the tumor cell, leads to higher apoptosisrate, decreased autophagy and tumor cell survival. Contrary, over-expression of RAGE in the tumor cell increases autophagy, anddecreases apoptosis and tumor cell viability (Kang et al., 2010).RAGE limits apoptosis through a p53-dependent mitochondrialpathway and directly link inflammatory mediators in the tumormicroenvironment and resistance to apoptosis (Kang et al., 2010).In addition, RAGE ligands obtained from caracinoma cells can influ-ence a variety of cell types within the tumor microenvironment,including leukocytes, fibroblasts, and vascular cells, leading toinflammation, increased fibrosis, and angiogenesis (Logsdon et al.,2007) In the present study, we found an increased number of lym-phocytes within the nests of malignantly transformed cells in OSCwhich characterizes also other carcinomas such as breast cancerand melanoma (Anderson et al., 2009). Several studies have shownthat the presence of lymphocyte infiltration in ovarian cancerconfers a survival advantage (Leffers et al., 2010). Namely, they pro-mote mesenchymal cell growth by secretion of TGF-� (Andersonet al., 2009) thus causing growth of higher grade OSC, as OSC cellsunderwent epithelial to mesenchymal transition. Furthermore, itwas shown that RAGE mediated leukocyte activation may be associ-ated with higher RAGE expression in cancer (Bucciarelli et al., 2002;Sparvero et al., 2009). Among immune cells, neutrophils may playan important protumorigenic role by favoring neoangiogenesis andsuppressing anti-tumor immune effects (Rojas et al., 2010). Inter-estingly, in our study, some neutrophils had moderate cytoplasmicRAGE expression, while some lymphocytes displayed nuclear RAGEpositivity. In line with this finding are studies showing that lympho-cyte infiltrations in the tumor microenvironment are important, asthey enhance proliferation and survival of cancer cells. It has beenshown that functional RAGE is present on the plasma membraneof human neutrophils and that engagement of RAGE impairs neu-

n of RAGE and IGF-1 in the human fetal ovary and ovarian serousis.2015.01.004

trophil functions (Collison et al., 2002). Our findings may indicatethe role of RAGE as an important mediator of OSC cell injury inthe setting of RAGE positive lymphocytes and neutrophils that caninterfere with antitumor immunity, promoting angiogenesis and

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upporting tumor growth. This is in accordance with Rage knock-ut mice studies, providing genetic evidence for a role of Rage

n linking inflammation and cancer (Riehl et al., 2009). Likewise,ncreasing experimental data suggest that the RAGE expressionathway may be a valuable contributor to inflammation relatedumorigenesis through distinct signaling mechanisms (Rojas et al.,010). This may also involve the activation of processes that mightromote tumor anti-apoptotic activity, since we found strong co-xpression of IGF-1 as an anti-apoptotic factor and RAGE in our OSCells. However, the association between inflammation and tumorrowth and associated lymphocytes infiltrations still remains elu-ive.

onclusion

In summary, our, data have shown characteristic IGF-1 andAGE expression patterns in human ovaries during fetal develop-ent and reproductive and postmenopausal periods which were

tage and cell type dependent. Strong co-expression of IGF-1 andAGE in same OSC cells indicate the role of these factors as keylayers that control the growth and cell survival by their effect onhe cell cycle and anti-apoptotic activity. These effects togetherith inflammatory responses may in part contribute to carci-

oma invasion, chemoresistance and metastasis. Since IGF-1 andAGE are important components of ovarian epithelial and stro-al cells which promote molecular interactions in OSC, they may

erve as a target for development of new diagnostic and prognos-ic markers. Additionally, it is important to identify the dominant

echanisms by which these factors influence cell cycle and sur-ival so that chemotherapeutic strategies against IGF-1 and RAGEromoted cancers can be designed. This approach would strike twoital compartments during ovarian carcinogenesis: the malignantlyransformed OSC cells and inflammatory response, but furthern vivo studies are necessary to explore these possibilities.

onflict of interest

None of the authors have competing interests.

cknowledgements

This work was supported by the Ministry of Science, Educationnd Sports of the Republic of Croatia (grant No. 021-2160528-507).

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