ORIGINAL ARTICLE

Altered Chemokine Receptor Expressionin Papillary Thyroid Cancer

Hernan E. Gonzalez,1,2 Andrea Leiva,1,3 Hugo Tobar,1,3 Karen Bohmwald,1,3 Grace Tapia,2

Javiera Torres,4 Lorena M. Mosso,5 Susan M. Bueno,6 Pablo Gonzalez,6

Alexis M. Kalergis,1,6 and Claudia A. Riedel1,3

Background: Papillary thyroid cancer (PTC), the most prevalent type of differentiated thyroid carcinoma, dis-plays a strikingly high frequency of lymph node metastasis (LNM). Recent data suggest that chemokines canplay an important role in promoting tumor progression and metastatic migration of tumor cells. Here we haveevaluated whether PTC tissues express a different pattern of chemokine receptors and if the expression of thesereceptors correlates with LNM.Methods: We assessed by immunohistochemistry and flow cytometry the expression of the chemokine receptorsCCR3, CCR7, and CXCR4 in tumor and nonmalignant thyroid tissues from patients suffering from PTC. Ex-pression of these receptors in PTC was correlated with the clinical pathological condition of PTC.Results: Our data show a significant enhancement of CCR3 (2.5 times higher, p¼ 0.038) and CXCR4 (1.7 timeshigher, p¼ 0.02) expression in PTC tissues as determined by immunohistochemical staining, and of CCR3(3.5 times higher, p< 0.002) in the plasma membrane as determined by flow cytometric analyses, compared tocontrols. In addition, while CCR3 (100%) and CXCR4 (90%) were present in both tumor and control thyroidtissues, expression of CCR7 was scarcely detected in PTC cells (5–10%) and not found in control cells. CXCR4expression correlated with the classical variant of PTC ( p< 0.035) and extranodal extension ( p< 0.010) in pa-tients with LNM.Conclusions: Our data support the notion that CCR3, CCR7, and CXCR4 are increasingly expressed in tumorcells from PTC and that CXCR4 expression in PTC could be a potential marker for enhanced tumor aggres-siveness.

Introduction

Thyroid cancer is the most common endocrine malig-nancy, causing over 37,000 new cases and 1590 deaths

each year in the United States (1). Papillary thyroid cancer(PTC) is the most frequent form of thyroid cancer, making upas much as 80% of all thyroid cancers (2). In the last decade,numerous studies have reported a rising incidence of PTC (3–5). A remarkable feature of this malignancy is the high fre-quency of lymph node metastasis (LNM) (6,7). The frequencyof LNM in PTC is close to 50%, reaching up to 80% whensubclinical PTC is considered (6,7). The presence of LNM is

associated with a greater risk of recurrent disease (8,9), and incases of advanced disease it can increase the probability ofdeath up to 38% to 69% (10,11).

The high frequency of LNM in PTC contrasts with thelow frequency of hematogenous dissemination, suggestingpreferential metastatic spread via the lymphatic pathway.Nevertheless, till date, the factors regulating the metastaticprocess in PTC remain poorly understood. Recently, it hasbeen shown that chemoattractant molecules secreted fromtarget organs and the specific pattern of chemoattractant re-ceptors expressed on tumors cells can define the destination oftumor metastases (12). Therefore, the selective trafficking of

1Millennium Nucleus on Immunology and Immunotherapy. b AU12Department b AU2of Oncological Surgery, The Pontifical Catholic University of Chile, Marcoleta, Santiago, Chile.3Laboratory of Cell Biology and Pharmacology, Department of Biological Sciences, Faculty of Health Sciences, Andres Bello University,

Republica, Santiago, Chile.4Department of Pathology, The Pontifical Catholic University of Chile, Santiago, Chile.5Department of Endocrinology and Rheumatology, The Pontifical Catholic University of Chile, Santiago, Chile.6Department of Molecular Genetics and Microbiology, Faculty of Biological Sciences, The Pontifical Catholic University of Chile, Portugal,

Santiago, Chile.

THYROIDVolume 19, Number 9, 2009ª Mary Ann Liebert, Inc.DOI: 10.1089=thy.2008.0432

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PTC metastases to lymph nodes could be a result of a par-ticular expression of chemoattractant receptors by PTC cells.Hence, expression of those chemoattractant receptors couldpromote the migration of tumor cells in response to a gradientof chemoattractant molecules derived from lymph nodes. Theidentification of these molecules and their receptors could beconsidered as markers for those PTC that are more likely toundergo LNM. Candidates for chemoattractant receptorsare chemokine receptors. Chemokines contribute to the de-velopment and progression of cancer (13,14) and tumor me-tastasis (12,15,16). Chemokines are a family of about 50chemotactic proteins (8–10 kDa) classified into four highlyconserved groups—CXC, CC, C, and CX3C—based on theposition of their first two cysteines adjacent to the amino-terminus (17–19). These molecules stimulate cell movementduring inflammation, as well as the homeostatic transport ofhematopoietic stem cells, lymphocytes, and dendritic cells(20–22). The activity of chemokines is mediated by theirbinding to a large family of seven-transmembrane G protein–coupled receptors (18 in humans) (23), which promote re-ceptor internalization (24,25) and the signaling leading tothe transcription of genes required for cell motility, invasion,interaction with the extracellular matrix, and cell survival(17,24–26).

Altered expression of chemokine receptors has been re-ported in several tumors, including breast cancer (12), ma-lignant melanoma (27,28), lung cancer (29), and squamous cellcarcinoma of the head and neck (30). For thyroid cancer, ex-pression of chemokine receptors CXCR4 and CCR7 has beenevaluated (31–33). CXCR4 has been particularly studied dueto its previous involvement in tumor growth, survival, andspread to other tissues (32–33). CCR7 is also expressed inthyroid carcinoma cell lines (TPC-1) and thyroid cancer tis-sues (31,33), which is thought to contribute to tissue invasionand cellular proliferation (31). CCR3 is another receptor as-sociated with development, progression, and aggressivenessof several types of cancer (34,35).

The objective of this study was to evaluate the expressionand cellular localization of CCR3, CCR7, and CXCR4 in thy-roids of patients suffering from PTC and to determine itscorrelation with LNM and clinical pathological features. Weobserved that thyroid cells from PTC patients show an alteredexpression of chemokine receptors. CCR3, CCR7, and CXCR4were overexpressed in PTC tissues and always found mainlyin the intracellular region of PTC cells. The expression of thesethree receptors was independent of whether the patient de-veloped LNM or not. However, the expression of CXCR4correlated with the classical variant of PTC and extranodalextension.

Materials and Methods

Patients and tissue samples

Thirty patients with suspicious thyroid nodules had aconfirmed diagnosis of PTC by fine needle aspiration inthe Hospital Clınico de la Pontificia Universidad Catolica deChile. All those patients, included in this study, signed awritten informed consent approved by the ethical committeeof the hospital before they underwent surgery. Preoperativeultrasound staging of the neck determined if the patientshad macroscopic LNM. Total thyroidectomy was performedin all patients and lymph node dissection (central compart-

ment or comprehensive) only in those with ultrasonographicevidence of macroscopic adenopathy. Presence of metastasiswas defined as patients with macroscopic pathologicallyconfirmed metastasis. Tumor samples and matched non-malignant tissue (control) were obtained immediately aftertotal thyroidectomy. The control tissue was obtained fromthe tumor-free contralateral lobe of the same patient fromwhom the tumor sample was obtained. After surgery,pathology report was obtained for each patient. Tumorcharacteristics and staging for each patient are shown in

b T1Table 1.

Immunohistochemistry

For immunohistochemistry analyses, fresh thyroid tissuesobtained from patients were fixed in 4% paraformaldehyde in140 mM sodium chloride (NaCl), 3 mM potassium chloride(KCl), 0.01 M sodium phosphate, and 1.8 mM potassiumphosphate at pH 7.4 (phosphate buffered saline [PBS]) over-night at 48C. The tissues were dehydrated using an ethanoland xylol battery. Briefly, tissues were kept in 70% ethanolthree times for 20 minutes, 95% ethanol three times for 20minutes, 100% ethanol three times for 30 minutes, 34% xy-lol=66% ethanol for 30 minutes, 50% xylol=50% ethanol for 30minutes, 66% xylol=34% ethanol for 30 minutes, and finally in100% xylol three times for 30 minutes. The dehydrated tissueswere embedded first in 50% xylol=50% paraffin for 1 hour at568C, then in 100% paraffin overnight at 568C, and finally in100% paraffin for 1 hour at 568C. The tissues were sliced into5 mm sections using a microtome (Leica, RM2235; Wetzlar,Hesse, Germany). These thyroid sections were deparaffinizedusing a battery of alcohols (100% xylol three times for 10minutes, 100% ethanol three times for 10 minutes, 95% etha-nol three times for 2 minutes, and 70% ethanol three times for2 minutes). The sections were then mounted on silanizedmicroscope slides and rehydrated in 140 mM NaCl, 3 mMKCl, and 20 mM tris at pH 7.4 (tris-buffered saline). The en-dogenous peroxidase activity in the thyroid sections wasblocked using 6% hydrogen peroxide in tris-buffered salinefor 15 minutes at room temperature. Nonspecific binding wasblocked using a blocking solution (5 mM EDTA, 1% fish gel-atin, 1% bovine serum albumin [BSA] IgG free, 2% horse se-rum) in a wet chamber for 1 hour at room temperature. Thethyroid tissue sections were incubated overnight at 48C withprimary specific antibodies prepared in blocking solution di-luted 10 times. The primary antibodies used were monoclonalanti-human CCR3 antibody (MAB155, 1:1000 dilution; R&DSystems, Minneapolis, MN), monoclonal anti-human CCR7(1:250 dilution; R&D Systems), and monoclonal anti-humanCXCR4 (MAB173, 1:250 dilution; R&D Systems). The sectionswere rinsed six times for 5 minutes with blocking solutiondiluted 10 times and incubated with a secondary biotinylatedantibody provided in the LSABþ System-HRP kit (cat. no.K0679) (DakoCytomation, Glostrup, Hovedstaden, Denmark)for 1 hour at 48C. The sections were washed six times for5 minutes with blocking solution diluted 10 times.

Immunohistochemistry was revealed by substrate-chromogen solution provided by the DakoCytomation kit. Toidentify cells and nuclei, tissues were stained with hematox-ylin (Merck, Darmstadt, Hesse, Germany). The immunohis-tochemistry for each antibody was quantified using theImage-Pro Plus program version 6.0 (Media Cybernetic Inc.,

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Bethesda, MD). Briefly, at least three representative picturesof each thyroid tissue section were taken and analyzed withImage-Pro Plus program. The value obtained from eachquantification analysis was normalized by the number ofnuclei in this picture and was referred as mean intensityvalue (MIV). To normalize the expression of chemokine re-ceptors in the tumor relative to the matched nonmalignanttissue, the ratio of MIV of tumor tissue to MIV of the matchnonmalignant tissue for each patient was calculated. Todetermine the differences in chemokine receptor expression,an average of the ratio was calculated, referred to as relativeexpression.

Flow cytometry analysis of CCR3, CCR7, and CXCR4

For flow cytometry analysis, tumor and control thyroidtissue were processed immediately after surgery. Briefly,thyroid tissues were kept in RPMI medium without supple-ments (cat. no. 31800-89) (Gibco�, Carlsbad, CA) and disso-

ciated manually with a scalpel at room temperature. Duringthis procedure, the adipose and connective tissues were re-moved from the sample tissue and the latter passed through asyringe (tip G5). The cell suspension obtained was incubatedwith primary antibodies. For CCR3 detection, a monoclonalanti-human CCR3 antibody (MAB155, 500mg=mL; R&DSystems) diluted 1:200 and unlabelled streptavidin (S-888,10 mg=mL; Invitrogen, Carlsbad, CA) diluted 1:500 wereused b AU3. Cells were centrifuged at 250 g (Micromax RF IECCentra CL3R termo IEC; b AU4Waltham, MA) for 6 minutes andrinsed two times with PBS and 3% BSA (PBS=BSA).Then the cells were incubated with 1.5 mg=mL of an anti-ratbiotinylated secondary antibody (BA-4000; Vector, Burlin-game, CA) diluted 1:200 for 30 minutes at 48C. After incuba-tion cells were rinsed with PBS=BSA and incubated with0.2 mg=mL streptavidin-PerCP (cat. no. 554064; BD Phar-mingen, San Diego, CA) diluted 1:300 in PBS=BSA. ForCCR7 detection, a monoclonal anti-human CCR7 antibody(MAB197, 500mg=mL; R&D Systems) diluted 1:100 was used,

Table 1. Clinical and Histopathological Patient Characteristics

Patientno. Sex Age Multifocal

Invasion ofthyroid capsule

Extrathyroidalextension

Surgicalmargin

Vascularinvasion

Extranodalextension T N M Stage

1 F 64 � � � � � � T1 N0 M0 I2 M 51 � � � � � � T1 N0 M0 I3 F 77 � � � � � � T1 N0 M0 I4 F 28 � � � � � � T1 N0 M0 I5 F 37 � þ þ � � � T1 N0 M0 I6 F 61 � � � � � � T1 N0 M0 I7 F 48 � þ � � � � T1 N1a M0 III8 M 54 þ � � � � � T1 N1b M0 IVA9 M 28 þ þ � � þ � T1 N1b M0 IVA

10 F 35 � þ � � þ � T1 N1a M0 III11 M 41 þ þ þ þ þ þ T1 N1b M0 IVA12 F 35 � � � � � � T1 N0 M0 I13 F 49 � þ � � þ � T2 N1a M0 III14 F 57 þ þ � � þ � T2 N1b M0 IVA15 F 48 þ þ � � � þ T1 N1a M0 III16 F 47 � þ þ � � � T1 N0 M0 I17 F 53 � þ þ þ � � T1 N0 M0 I18 F 39 þ þ � � þ þ T2 N1b M0 IVA19 F 50 � þ � � � � T1 N0 M0 I20 M 25 þ � � � � � T1 N1b M0 III21 M 35 þ þ � � þ þ T1 N1b M0 IVA22 F 53 þ þ þ � þ � T2 N1a M0 III23 F 48 � þ � � þ � T2 N1b M0 IVA24 F 44 � � � � � � T1 N1a M0 III25 F 56 � � � � � � T1 N0 M0 I26 F 53 þ � � � � � T1 N0 M0 I27 F 36 � þ � � � � T1 N1a M0 III28 F 50 � þ � � � � T2 N1a M0 III29 M 40 � � � � � � T1 N0 M0 I30 F 45 þ þ þ � � þ T2 N1b M0 IVA

All patients had histologically confirmed papillary thyroid cancer (PTC). F, female; and M, male. The cancer stage classification wasaccording to AJCC TNM staging system, 6th edition. T, primary tumor; N, regional lymph nodes; M, distant metastasis. T1 is a tumor 2 cm orless in greatest dimension limited to the thyroid. T2 is a tumor more than 2 cm but not more than 4 cm in greatest dimension limited to thethyroid. N0, absence of regional lymph node metastasis; N1a, presence of metastasis to level VI (pretracheal b AU10, paratracheal, and prelaryngeallymph nodes); N1b, presence of metastasis to unilateral, bilateral, or contralateral cervical or superior mediastinal lymph nodes. M0, absenceof distant metastasis. The histopathology characteristics were classified with þ or � according to the presence or absence of the condition,respectively. Multifocal refers to the presence of more than one foci of tumor in the thyroid gland. Invasion of thyroid capsule is the invasionfor the tumor cells. Extrathyroidal extension refers to the presence of tumor cells beyond the thyroid capsule with invasion of subcutaneoussoft tissue or other local structures. Surgical margin denotes the presence of tumor in the surgical resection margin of the thyroid gland.Vascular invasion is the presence of tumor cells in the blood vessels of the thyroid gland. Extranodal extension implies the presence ofproliferating malignant cells outside the capsule of an involved lymph node.

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and an anti-mouse IgG made in goat labeled with R-phycoerythrin (cat. no. 31861; Pierce, Rockford, IL) diluted1:200 was used as a secondary antibody. For CXCR4 detec-tion, a monoclonal anti-human CXCR4 antibody (MAB172,500 mg=mL; R&D Systems) diluted 1:100 was used, and ananti-mouse IgG made in goat labeled with R-phycoerythrin(cat. no. 31861; Pierce) diluted 1:200 was used as a secondaryantibody. The incubations of cells with antibodies were per-formed in PBS=BSA for 45 minutes at 48C. The expressionof CCR3, CCR7, and CXCR4 at the cell surface was analyzedon a FACSCalibur (Becton Dickinson Instrument Systems,Sparks, MD). Flow cytometry analysis was performed usingthe program CellQuest Pro (BD Biosciences, San Jose, CA) andWinmdi 2.8 (AU5 c San Diego, CA). The mean fluorescence intensity(MFI) of CCR3, CCR7, and CXCR4 in the tumor cells wasdivided by MFI of these receptors in matched nonmalignanttissue for each patient.

Statistical analyses

All statistical analyses were performed using GraphPadPrism version 4.0 (GraphPad Software, San Diego, CA) andSPSS Statistic 17.0 for Windows. The statistical analyses for

both immunohistochemistry and flow cytometry were doneby Wilcoxon test. Differences between patients with andwithout LNM was analyzed by the Mann-Whitney test.Correlation of positive staining of CCR3, CCR7, and CXCR4with clinicopathologic features ( b T2Table 2) was done by t-test orMann-Whitney test depending on whether the samples hadnormal distribution or not. The Kruskal-Wallis test was usedto statistically analyze the stage of disease.

Results

CCR3 and CXCR4 are overexpressed in PTC

Expression of CCR3, CCR7, and CXCR4 was analyzedin PTC and control tissue by immunohistochemicalstaining using specific antibodies for these receptors( b F1Fig. 1). CCR3 staining was observed in all tissues, bothcontrol and PTC samples (Fig. 1a–d). Near to 100% oftumor cells in PTC samples stained for CCR3 (Fig. 1c).The intensity of CCR3 staining in all tissues was quantified(see Materials and Methods) and referred to as MIV. Thequantitative analysis of CCR3 indicated that its expressionwas approximately 2.5 times higher in PTC than in controltissues (for control, MIV� SEM¼ 445.08� 124.47; for PTC,

Table 2. Correlation of Clinicopathological Features with Immunohistochemical Staining

of Chemokine Receptors for Papillary Thyroid Cancer

CCR3 CCR7 CXCR4

Factors # p # p # p

Age <45 years 7 0.0919 2 0.8186 6 0.2526>45 years 12 5 10

Sex Male 4 0.9257 1 0.2824 3 0.5732Female 15 6 13

Histology Classic variant 16 0.6474 7 — 13 0.0345*Other variant 3 0 3

Tumor size <1 cm 5 0.5088 1 0.3541 3 0.5871>1 cm 14 6 13

Focality Unifocal 11 0.5439 5 0.8020 9 0.2087Multifocal 8 2 7

LNM Yes 10 0.2660 4 0.9154 10 0.3716No 9 3 6

Hyperplasia Yes 4 0.5097 3 0.6505 4 0.7720No 15 4 12

Capsule invasion Yes 11 0.3491 4 0.9614 10 0.4040No 8 3 6

Extrathyroid extension Yes 5 0.8144 1 0.2651 5 0.1901No 14 6 11

Vascular invasion Yes 6 0.0797 2 0.8177 5 0.1872No 13 5 11

Extranodal extension Yes 3 0.9519 1 0.6329 3 0.0101*No 16 6 13

Chronic thyroiditis Yes 8 0.3423 1 0.3972 7 0.3278No 11 6 9

Stage I 9 0.3336** 3 0.9823** 6 0.6773**

II 4 2 4IV 6 2 6

p denotes p-value obtained after t-test or Mann-Whitney test. # denotes the number of patients who have the clinical pathological condition.LNM, lymph node metastasis. * denotes significant differences ( p< 0.05). Negative expression was considered when there was no staining forthe receptor in the PTC tissue. Positive expression was considered when there was staining in the PTC tissue. **denotes p-value obtained afterKruskal-Wallis test for statistic analysis of all three PTC stages and chemokine receptor immunohistochemical staining.

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MIV� SEM¼ 1070.636� 259.316; p¼ 0.038, n¼ 19). Re-presentative slides for CCR7 staining are shown in Figure 1f–i.Staining was detected in one normal thyroid tissue and inseven PTC samples. Even though CCR7 expression was sig-nificantly different between PTC and normal tissue (control,MIV�SEM¼ 0.378� 0.378; PTC, 7.106� 3.007; p¼ 0.017,n¼ 16) (Fig. 1f–j), it was scarcely detected in thyroid cells fromPTC (5–10% of tumor cells stained for CCR7). Representativeslides for CXCR4 staining are shown in Figure 1k–n. Stainingwas observed in all samples, both control and PTC. CXCR4-derived signal was present in approximately 90% of tumorcells in PTC tissues. CXCR4 staining was significantly higherin PTC than in control tissues (control, MIV� SEM¼ 313.68�79.11; PTC, 525.17� 84.205; p¼ 0.02, n¼ 16) (Fig. 1k–o). Therelative expression of chemokine receptors was comparedamong patients with and without LNM (F2 c Fig. 2 and Table 2).None of CCR3, CCR7, and CXCR4 expression correlated withLNM (Fig. 2). Potential associations between the relative ex-pression of CCR3, CCR7, and CXCR4 seen by immunohisto-chemical staining and the patients’ clinicopathologic featureswere evaluated (Table 2). While CCR3 and CCR7 expressionswere independent of all these clinical features, a positivecorrelation was found between CXCR4 expression and theextranodal extension in patients with LNM and the classicalvariant of PTC type.

The qualitative analysis of the immunohistochemicalstaining for each chemokine receptor showed that all recep-tors were present mainly in the cytoplasm, in both control andPTC tissues (Fig. 1). We measured by flow cytometry the ex-pression of CCR3, CCR7, and CXCR4 at the plasma mem-

brane of PTC and control cells. Cells from PTC and normalthyroid tissues were prepared as described in Materials andMethods. CCR3 expression at the plasma membrane showeda 3.5-fold increase in PTC cells compared to control cells ( b F3Fig.3A). However, no significant correlation could be demon-strated between CCR3 expression at the cell surface and thepatients’ clinicopathologic characteristics (data not shown).On the other hand, CCR7 expression at the plasma membranewas similar for PTC and control cells (Fig. 3D). Similarly,surface expression of CXCR4 was equivalent between PTCand control cells (Fig. 3G). Representative histograms for thethree receptors in normal cells and PTC are shown (Fig. 3B, C,E, F, H, and I).

Discussion

Chemokines and their receptors contribute to migrationand proliferation of malignant cells during metastatic pro-cesses (14). Here we have analyzed the expression of threechemokine receptors (CCR3, CCR7, and CXCR4) in thyroidsamples obtained from PTC patients. Our results showed asignificant increase in the expression of these receptors inPTC samples compared to normal thyroid tissue from thesame patient. To our knowledge, this is the first report of asignificant increase in CCR3 expression in PTC compared tomatched nonmalignant tissue samples (control), both byimmunohistochemistry (Fig. 1e) and flow cytometry analyses(Fig. 3A). Consistent with previous reports (31,32), PTCtissues significantly expressed CXCR4 and CCR7, support-ing the notion that these two receptors can be potential

FIG. 1. CCR3, CCR7, and CXCR4 show a

4C c

significantly increased expression in papillary thyroid cancer (PTC). Re-presentative pictures of immunohistochemistry slides show chemokine receptor expression at low (�10) (a, c, f, h, k, and m)and high (�100) magnification (b, d, g, i, l, and n) for matched nonmalignant control tissues and PTC tissues. CCR3expression in control (a, b) and PTC tissues (c, d), CCR7 expression in control (f, g) and PTC tissues (h, i), CXCR4 expressionin control (k, l) and PTC tissues (m, n) are shown. The number of patients analyzed was n¼ 19 for CCR3 and 16 each forCCR7 and CXCR4. Scale bar¼ 26mm. Quantitative analyses of staining intensity for CCR3 (e), CCR7 (j), and CXCR4 (o) wereperformed as described in Materials and Methods. Values plotted represent the relative expression of each receptor, con-sisting of the average of the intensity staining of the tumor normalized by the intensity staining of each respective controlsample. *p< 0.05 by Wilcoxon test.

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markers for PTC. We found that near 100% and 90% of tumorcells overexpressed CCR3 (Fig. 1c) and CXCR4 (Fig. 1m),respectively, while CCR7 was scarcely expressed (less than5% of tumor cells in PTC stained for CCR7) (Fig. 1h). WhileCCR3 (Fig. 1a) and CXCR4 (Fig. 1k) were found in 25% ofnormal thyroid cells, CCR7 was absent in these cells (Fig. 1f ).We observed in some PTC tissues and in matched nonma-lignant tissue the presence of infiltrated cells that also stainedfor these three receptors.

Chemokine receptors potentially promote tumor progres-sion and LNM. A recent study reported that the expression ofCXCR4 in PTC correlated with larger tumor size (33). In thesame article the authors reported that CCR7 correlated withextrathyroidal extension, angiolymphatic invasion, and LNM(33). In our study, no statistically significant correlations couldbe demonstrated between the expression of CCR3, CCR7, orCXCR4 by PTC and LNM (Fig. 2). However, we observed atendency for an increased CXCR4 expression by PTC samplesderived from patients with LNMs. It is possible that thistendency would become statistically significant if the numberof patients analyzed is increased.

Further, we found that CXCR4 expression correlated withextranodal extension and classic variant PTC (Table 2), sug-gesting that CXCR4 expression could be related to moreaggressive tumors and negative prognosis (36,37). The ex-pression of CCR3 and CCR7 showed no significant correlationwith any of the pathological parameters analyzed (Table 2).However, the expression of chemokine receptors might bepossibly linked to other clinical features not assessed in thisstudy, such as the risk of recurrent disease. To approach this

issue, appropriate prospective follow-up of patients is re-quired, which is currently taking place in our hospital. Inother types of cancer CCR3 expression is shown to correlatewith LNM (34,38), and CCR7 (33,39) and CXCR4 (32) withthyroid carcinoma. It is also possible that the increased ex-pression of CCR3, CCR7, or CXCR4 in PTC is a consequenceof tumor-associated inflammatory environment, as has beenshown for other types of tumors (34). Regardless of the cause,altered expression of CCR3, CCR7, or CXCR4 could likelyassociate with the acquisition of a malignant potential, as seenfor other tumors such as T-cell lymphoma (40), kidney cancer(34), and glioblastoma (35).

Flow cytometry analyses showed an increased expressionof CCR3 on the surface of PTC-derived cells (Fig. 3A). Incontrast, no significant changes were observed for CCR7 andCXCR4 expression on PTC compared to control cells (Fig. 3Dand G). On the other hand, immunohistochemistry analysesshowed that CCR3, CCR7, and CXCR4 were strongly ex-pressed intracellularly both in PTC and in control samples.These observations are consistent with the notion that ac-tive chemokine receptors are normally located intracellularly(24,25). Receptor internalization seems to be necessary totrigger the signal transduction pathways induced by thechemokine binding to the receptor (25,41,42). A similar in-tracellular pattern for CCR3 location has been previouslydescribed for dendritic cells (43), lymphocytes (44), and forCXCR4 and CCR7 in thyroid cancer (31,33). Further func-tional studies for CCR3, CCR7, and CXCR4 in PTC arerequired to evaluate whether their intracellular location isrelated to their activity.

FIG. 2. Metastatic PTC shows equivalent CCR3, CCR7, and CXCR4 expression than nonmetastatic PTC. Quantita-tive analysis for the relative expression of CCR3 (A), CCR7 (B), and CXCR4 (C) in PTC tissues derived from patients withor without lymph node metastasis (LNM). Data were obtained from tissue slides, analyzed by immunohistochemistry,and quantified as described in the Materials and Methods section. Data shown are means� SEM of several independentexperiments for CCR3 (n¼ 19), CCR7 (n¼ 16), and CXCR4 (n¼ 16). No statistical differences were found by Mann-Whitneytest.

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In summary, this report shows for the first time a significantincrease of CCR3 expression in PTC, as compared to controlthyroid tissues. Our data indicate that at least three chemo-kine receptors, CCR3, CCR7, and CXCR4, are likely involvedin the tumorogenic process of PTC and support the notion thata particular pattern of chemokine receptor expression couldbe related to PTC.

Acknowledgments

We thank Dr. Oslando Gutierrez for helping us with thestatistical analyses. This work was supported by the Millen-nium Nucleus on Immunology and Immunotherapy (P04=030-F) and the Biomedical Research Consortium, Chile.

Disclosure Statement

The authors declare that no competing financial interestsexist.AU6 c

References

1. American Cancer Society 2008 Cancer Facts & Figures.Atlanta, GA: American Cancer Society. b AU7

2. Nikiforov YE 2008 Thyroid carcinoma: molecular pathwaysand therapeutic targets. Mod Pathol 21 Suppl 2:S37–S43.

3. Enewold L, Zhu K, Ron E, Marrogi AJ, Stojadinovic A,Peoples GE, Devesa SS 2009 Rising thyroid cancer incidencein the United States by demographic and tumor character-istics, 1980–2005. Cancer Epidemiol Biomarkers Prev 18:784–791.

4. Leenhardt L, Grosclaude P, Cherie-Challine L 2004 Increasedincidence of thyroid carcinoma in France: a true epidemic orthyroid nodule management effects? Report from the FrenchThyroid Cancer Committee. Thyroid 14:1056–1060.

5. Arai T, Yamashita T, Urano T, Masunaga A, Itoyama S, ItohK, Shiku H, Sugawara I 1995 Preferential reduction ofnm23-H1 gene product in metastatic tissues from papillaryand follicular carcinomas of the thyroid. Mod Pathol 8:252–256.

FIG. 3. Flow cytometry b AU11analysis for CCR3, CCR7, and CXCR4 in PTC. Cell surface expression for CCR3, CCR7, and CXCR4in control and PTC samples was determined by flow cytometry. The mean fluorescence intensity (MFI) for the three receptorsin tumor cells was normalized to the MFI shown by each receptor on the respective control sample, for each patient. Theaverage of these normalized values was calculated, plotted, and referred to as relative MFI. (A) CCR3, *p< 0.002, n¼ 10; (D)CCR7, p> 0.5, n¼ 5; (G) CXCR4, p> 0.5, n¼ 5. Statistical analysis was done using Wilcoxon test. Representative forward andside-scatter analyses with their respective histograms are shown for control (B, E, and H) and tumor cells (C, F, and I) forCCR3, CCR7, and CXCR4, respectively. Gray areas in the histograms correspond to immunodetection of each chemokinereceptor and the empty areas to the respective staining controls.

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6. Noguchi S, Noguchi A, Murakami N 1970 Papillary carci-noma of the thyroid. II. Value of prophylactic lymph nodeexcision. Cancer 26:1061–1064.

7. Noguchi S, Noguchi A, Murakami N 1970 Papillary carci-noma of the thyroid. I. Developing pattern of metastasis.Cancer 26:1053–1060.

8. Mazzaferri EL, Kloos RT 2001 Clinical review 128: currentapproaches to primary therapy for papillary and follicularthyroid cancer. J Clin Endocrinol Metab 86:1447–1463.

9. Rossi RL, Cady B, Silverman ML, Wool MS, Horner TA 1986Current results of conservative surgery for differentiatedthyroid carcinoma. World J Surg 10:612–622.

10. Samaan NA, Maheshwari YK, Nader S, Hill CS Jr., SchultzPN, Haynie TP, Hickey RC, Clark RL, Goepfert H, IbanezML, Litton CE 1983 Impact of therapy for differentiatedcarcinoma of the thyroid: an analysis of 706 cases. J ClinEndocrinol Metab 56:1131–1138.

11. Tubiana M, Schlumberger M, Rougier P, Laplanche A,Benhamou E, Gardet P, Caillou B, Travagli JP, Parmentier C1985 Long-term results and prognostic factors in patientswith differentiated thyroid carcinoma. Cancer 55:794–804.

12. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME,McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL,Mohar A, Verastegui E, Zlotnik A 2001 Involvement ofchemokine receptors in breast cancer metastasis. Nature410:50–56.

13. Baggiolini M 2001 Chemokines in pathology and medicine.J Intern Med 250:91–104.

14. Frederick MJ, Clayman GL 2001 Chemokines in cancer.Expert Rev Mol Med 3:1–18.

15. Sun YX, Wang J, Shelburne CE, Lopatin DE, ChinnaiyanAM, Rubin MA, Pienta KJ, Taichman RS 2003 Expression ofCXCR4 and CXCL12 (SDF-1) in human prostate cancers(PCa) in vivo. J Cell Biochem 89:462–473.

16. Kato M, Kitayama J, Kazama S, Nagawa H 2003 Expressionpattern of CXC chemokine receptor-4 is correlated withlymph node metastasis in human invasive ductal carcinoma.Breast Cancer Res 5:R144–R150.

17. Raman D, Baugher PJ, Thu YM, Richmond A 2007 Role ofchemokines in tumor growth. Cancer Lett 256:137–165.

18. Matsukawa A, Lukacs NW, Hogaboam CM, Chensue SW,Kunkel SL 2001 IIIAU8 c . Chemokines and other mediators, 8AU8 c .Chemokines and their receptors in cell-mediated immuneresponses in the lung. Microsc Res Tech 53:298–306.

19. Balkwill F 2004 Cancer and the chemokine network. Nat RevCancer 4:540–550.

20. Beider K, Abraham M, Peled A 2008 Chemokines and che-mokine receptors in stem cell circulation. Front Biosci 13:

6820–6833.21. Rossi D, Zlotnik A 2000 The biology of chemokines and their

receptors. Annu Rev Immunol 18:217–242.22. Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R,

Matsushima K, Miller LH, Oppenheim JJ, Power CA 2000International union of pharmacology. XXII. Nomenclaturefor chemokine receptors. Pharmacol Rev 52:145–176.

23. Baggiolini M 1998 Chemokines and leukocyte traffic. Nature392:565–568.

24. Allen SJ, Crown SE, Handel TM 2007 Chemokine: receptorstructure, interactions, and antagonism. Annu Rev Immunol25:787–820.

25. Neel NF, Schutyser E, Sai J, Fan GH, Richmond A 2005Chemokine receptor internalization and intracellular traf-ficking. Cytokine Growth Factor Rev 16:637–658.

26. Locati M, Deuschle U, Massardi ML, Martinez FO, Sironi M,Sozzani S, Bartfai T, Mantovani A 2002 Analysis of the geneexpression profile activated by the CC chemokine ligand5=RANTES and by lipopolysaccharide in human monocytes.J Immunol 168:3557–3562.

27. Murakami T, Maki W, Cardones AR, Fang H, Tun KyiA, Nestle FO, Hwang ST 2002 Expression of CXC chemokinereceptor-4 enhances the pulmonary metastatic potential ofmurine B16 melanoma cells. Cancer Res 62:7328–7334.

28. Cardones AR, Murakami T, Hwang ST 2003 CXCR4enhances adhesion of B16 tumor cells to endothelial cellsin vitro and in vivo via beta(1) integrin. Cancer Res 63:6751–6757.

29. Takanami I 2003 Overexpression of CCR7 mRNA in non-small cell lung cancer: correlation with lymph node metas-tasis. Int J Cancer 105:186–189.

30. Wang J, Xi L, Hunt JL, Gooding W, Whiteside TL, Chen Z,Godfrey TE, Ferris RL 2004 Expression pattern of chemokinereceptor 6 (CCR6) and CCR7 in squamous cell carcinoma ofthe head and neck identifies a novel metastatic phenotype.Cancer Res 64:1861–1866.

31. Sancho M, Vieira J, Casalou C, Mesquita M, Pereira T,Cavaco BM, Dias S, Leite V 2006 Expression and functionof the chemokine receptor CCR7 in thyroid carcinomas.J Endocrinol 191:229–238.

32. Yasuoka H, Kodama R, Hirokawa M, Takamura Y, MiyauchiA, Sanke T, Nakamura Y 2008 CXCR4 expression in papillarythyroid carcinoma: induction by nitric oxide and correlationwith lymph node metastasis. BMC Cancer 8:274 b AU9.

33. Wagner PL, Moo TA, Arora N, Liu YF, Zarnegar R, Scog-namiglio T, Fahey TJ III 2008 The chemokine receptorsCXCR4 and CCR7 are associated with tumor size andpathologic indicators of tumor aggressiveness in papillarythyroid carcinoma. Ann Surg Oncol 15:2833–2841.

34. Johrer K, Zelle-Rieser C, Perathoner A, Moser P, Hager M,Ramoner R, Gander H, Holtl L, Bartsch G, Greil R, ThurnherM 2005 Up-regulation of functional chemokine receptorCCR3 in human renal cell carcinoma. Clin Cancer Res 11:

2459–2465.35. Kouno J, Nagai H, Nagahata T, Onda M, Yamaguchi H,

Adachi K, Takahashi H, Teramoto A, Emi M 2004 Up-regulation of CC chemokine, CCL3L1, and receptors, CCR3,CCR5 in human glioblastoma that promotes cell growth.J Neurooncol 70:301–307.

36. Yamashita H, Noguchi S, Murakami N, Kawamoto H,Watanabe S 1997 Extracapsular invasion of lymph nodemetastasis is an indicator of distant metastasis and poorprognosis in patients with thyroid papillary carcinoma.Cancer 80:2268–2272.

37. Yamashita H, Noguchi S, Murakami N, Toda M, Uchino S,Watanabe S, Kawamoto H 1999 Extracapsular invasion oflymph node metastasis. A good indicator of disease recur-rence and poor prognosis in patients with thyroid micro-carcinoma. Cancer 86:842–849.

38. Sugasawa H, Ichikura T, Tsujimoto H, Kinoshita M, MoritaD, Ono S, Chochi K, Tsuda H, Seki S, Mochizuki H 2008Prognostic significance of expression of CCL5=RANTES re-ceptors in patients with gastric cancer. J Surg Oncol 97:

445–450.39. Sancho M, Vieira JM, Casalou C, Mesquita M, Pereira T,

Cavaco BM, Dias S, Leite V 2006 Expression and function ofthe chemokine receptor CCR7 in thyroid carcinomas. J En-docrinol 191:229–238.

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THY-2008-0432-Gonzalez_1P.3D 08/03/09 10:02am Page 8

40. Kleinhans M, Tun-Kyi A, Gilliet M, Kadin ME, Dummer R,Burg G, Nestle FO 2003 Functional expression of the eotaxinreceptor CCR3 in CD30þ cutaneous T-cell lymphoma. Blood101:1487–1493.

41. New DC, Wong YH 2003 CC chemokine receptor-coupledsignalling pathways. Sheng Wu Hua Xue Yu Sheng Wu WuLi Xue Bao (Shanghai) 35:779–788.

42. Zimmermann N, Conkright JJ, Rothenberg ME 1999 CCchemokine receptor-3 undergoes prolonged ligand-inducedinternalization. J Biol Chem 274:12611–12618.

43. Beaulieu S, Robbiani DF, Du X, Rodrigues E, Ignatius R, WeiY, Ponath P, Young JW, Pope M, Steinman RM, Mojsov S2002 Expression of a functional eotaxin (CC chemokineligand 11) receptor CCR3 by human dendritic cells. J Im-munol 169:2925–2936.

44. Forster R, Kremmer E, Schubel A, Breitfeld D, Klein-schmidt A, Nerl C, Bernhardt G, Lipp M 1998 Intra-

cellular and surface expression of the HIV-1 coreceptorCXCR4=fusin on various leukocyte subsets: rapid internali-zation and recycling upon activation. J Immunol 160:1522–1531.

Address correspondence to:Claudia A. Riedel, Ph.D.

Laboratorio de Biologıa Celular y FarmacologıaDepartamento de Ciencias Biologicas

Facultad de la SaludUniversidad Andres Bello

Republica 217Santiago

Chile

E-mail: riedel@unab.cl

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AUTHOR QUERY FOR THY-2008-0432-GONZALEZ_1P

AU1: Please note that affiliations on the title page of the article should be in English. Hence names of depart-ments, institutions, etc. have been translated. Please verify if these and the locations added are correct.Also the affiliation notes have been renumbered so that authors with the same affiliation have the samenote number.

AU2: Please mention location for Millennium Nucleus on Immunology and Immunotherapy.AU3: Change ok for clarity?AU4: Please provide name of manufacturer.AU5: Please mention name of manufacturer. If mentioned earlier in the article, please delete the location here

and retain only manufacturer name.AU6: Disclosure statement accurate? If not, please amend as needed.AU7: Reference entry ok as edited? Please verify.AU8: Please clarify what III and 8 refer to.AU9: Please provide page number range for the article.AU10: Please verify spelling changed.AU11: In Fig 3, labels are not clear. Please provide the same.

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