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DifferentialCXCReceptorExpressioninColorectalCarcinomas

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Differential CXC Receptor Expression in ColorectalCarcinomas

C. Rubie*, O. Kollmar*, V. O. Frick*, M. Wagner�, B. Brittner*, S. Graber� & M. K. Schilling*

Introduction

Colorectal cancer (CRC) is currently ranging among thethree most frequent malignancies in Western industrialnations. Although the 5-year survival rate for patientswith early stage and local CRC approaches nearly 90%,survival is dramatically decreased by local recurrence andthe development of distant metastases primarily affectingthe liver as the predominant cause of CRC-related mor-tality [1–3].

Certain organs are much more likely to be metastaticdestinations than others and lately various cancer-relatedstudies indicate that specific chemokine receptors may beinvolved in the molecular mechanisms that controlmetastasis in the early stages of cancer development [4].Chemokine receptors are linked to seven-transmembraneheterotrimeric G proteins and divided into CXCR andCCR receptors that mediate various pro- and anti-inflam-matory responses by interacting with the two major CXC

and CC subfamilies of chemokines respectively [5, 6].Based on the presence or absence of the amino acidsequence Glu-Leu-Arg (ELR) preceding the first con-served cysteine amino acid residue in the primary struc-ture of CXC chemokines, this chemokine family isfurther subdivided into ELR+ and ELR) chemokines.The ELR motif is important for ligand ⁄ receptor interac-tions and the regulation of CXC chemokine-inducedangiogenesis [7].

The ELR+ CXC chemokines such as CXCL8 (interleu-kin 8), CXCL1-3 (growth-related oncogenes a, b and c),CXCL6 (granulocyte chemotactic protein-2), CXCL7(neutrophil-activating protein-2) and CXCL5 (epithelialneutrophil-activating protein 78) are potent promoters ofangiogenesis [8, 9]. To date, CXCR1 and 2 are the onlymammalian receptors for ELR+ chemokines. WhileCXCL6, CXCL5 and CXCL8 bind to both receptors, allother ELR+ chemokines activate and interact highlyselective by CXCR2 [10].

*Department of General-, Visceral-, Vascular-

and Pediatric Surgery, University of the

Saarland; �Institute of Pathology, University of

the Saarland; and �Institute of Medical

Biometrics, Epidemiology, and Medical

Informatics (IMBEI), University of the Saarland,

Homburg ⁄ Saar, Germany

Received 7 January 2008; Accepted in revisedform 19 July 2008

Correspondence to: C. Rubie, PhD, Labor fur

Allgemein-, Viszeral-, Gefaß- und

Kinderchirurgie, Universitatsklinikum des

Saarlandes, Chirurgische Klinik, Gebaude 57,

66421 Homburg ⁄ Saar, Germany.

E-mail: [email protected]

Abstract

In this study, we aimed to assess the expression profile of chemokine receptorsCXCR1–4 in inflammatory and malignant colorectal diseases and correspond-ing hepatic metastases of synchronous and metachronous origin to elucidatetheir role in colorectal cancer (CRC) progression and metastasis. Chemokinereceptor expression was assessed by quantitative real-time PCR, immunohisto-chemistry (IHC) and Western blot analysis in resection specimens frompatients with ulcerative colitis (UC, n = 25), colorectal adenomas (CRA,n = 8), different stages of CRC (n = 48) as well as colorectal liver metastases(CRLM) along with their corresponding primary colorectal tumours (n = 16).While none of the chemokine receptors were significantly upregulated ordownregulated in UC or CRA tissues, CXC receptors 1, 2 and 4 demonstrateda significant increase in expression in all tumour stages of CRC specimenswith CXCR4 correlating with tumour grading (P < 0.05). On the other hand,CXCR3 showed no significant upregulation in either tumour stage, but signif-icant overexpression in CRLM. While CXCR4 demonstrated significant upreg-ulation in both tumour entities, IHC analysis revealed that the predominatecell type expressing CXCR4 in CRC is represented by tumour cells, whereasin CRLM the majority of positive CXCR4 signals is due to hepatocytes alongthe tumour invasion front. In conclusion, our findings show a very differentialexpression pattern of the four receptors in colorectal carcinomas and their cor-responding liver metastases with prominent expression profiles that indicate apotential role in the pathogenesis of CRC.

C L I N I C A L I M M U N O L O G Y doi: 10.1111/j.1365-3083.2008.02163.x..................................................................................................................................................................

� 2008 The Authors

Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 68, 635–644 635

The major receptor that has been identified for CXCchemokines which lacks the ELR motif (ELR)) isCXCR3 [11]. ELR) CXC chemokines like CXCL4 (plate-let factor-4) and interferon-inducible CXC chemokinesare potent angiostatic factors that can inhibit neovascular-ization mediated by ELR+ chemokines and other angio-genic factors like basic fibroblast growth factor andvascular endothelial growth factor [7]. Angiostatic CXCchemokines mediate inhibition of angiogenesis exclu-sively through binding and activation of CXCR3, whichwas originally identified on murine endothelial cells [12].

Although the homeostatic chemokine CXCL12 is anon-ELR+ CXC chemokine that signals via CXCR4, theCXCL12–CXCR4 system has been implicated in promot-ing angiogenesis and metastasis [13, 14]. In infectious dis-ease, CXCR4 is also used by the human immunodefiencyvirus to gain entry to cells [15]. With respect to colorectalcarcinoma, CXCR4 has recently been suggested as a riskfactor for the development of colon carcinoma microme-tastases [16] and recent results of our group indicated acorrelation between CXCR4 expression and the develop-ment of colorectal liver metastases (CRLM) [17, 18].

Despite increasing knowledge about the involvement ofCXC receptors in the invasion and dissemination of variouscancer types, the precise mechanisms determining theprocess of organ-selective metastasis of CRC cells remainunclear. Therefore, we aimed to conduct a comparativeanalysis of the RNA and protein expression profile ofchemokine receptors CXCR1–4 in inflammatory andmalignant colorectal diseases along with their correspond-ing hepatic metastases to elucidate a potential associationwith CRC progression and the development of CRLM.

Materials and methods

Patients. Surgical specimens and corresponding normaltissue from the same samples were collected from patientswho underwent surgical resection at our departmentbetween 2003 and 2006. Our patient collectives comprisedpatients with ulcerative colitis (UC, n = 25) and colorectaladenomas (CRA, n = 8) (Table 1), colorectal carcinomas ofdifferent cancer stages (n = 48) with different metastaticpotentials according to 8 T1-, 15 T2-, 15 T3- and 10T4-stage CRC patients, respectively, and synchronous ormetachronous CRLM (n = 16) (Table 2). Adjacent,disease-free tissue samples served as control groups respec-tively. Informed consent for tissue procurement wasobtained from all patients. The study was approved by theethics commission of the Arztekammer of the Saarland.The clinical variables presented in Tables 1 and 2 wereobtained from the clinical and pathological records and arein accordance with the UICC ⁄ TNM classification [19].

Tissue preparation. Tissue samples were collectedimmediately after resection, snap frozen in liquid nitrogenand then stored at )80 �C until they were processed

Table 1 Clinical characteristics of patients with ulcerative colitis and

colorectal adenomas.

Factor

UC

(n = 25)

CA

(n = 8)

Localization of colitis or adenoma respectively

Colon 25 6

Rectum 0 2

Gender

Male 18 5

Female 7 3

Age, yeara 52.4 (23–79) 65.3 (41–75)

Adipositas

Positive 1 5

Negative 24 3

UC, ulcerative colitis; CA, colorectal adenoma.aMedian with range in parentheses.

Table 2 Clinical characteristics of patients with colorectal carcinomas

and colorectal liver metastases.

Factor

CRC

(n = 48)

CRLM

(n = 16)a

Localization of primary tumour

Colon 22 6

Rectum 26 8

Gender

Male 29 7

Female 19 7

Age, yearb 63.7 (47–78) 60.3 (41–76)

Hepatitis (A,B or C)

Positive 6 2

Negative 42 12

Liver cirrhosis

Positive 1 0

Negative 47 14

Adipositas

Positive 12 4

Negative 36 10

Largest tumour diameter (cm)b 4.6 (1.2–9.1) 4.2 (1.5–5.5)

TNM stage of primary tumour

I 8 1

II 15 2

III 15 10

IV 10 1

Grading

I 0 0

II 21 4

III 26 9

Lymphatic permeation

Positive 27 10

Negative 21 4

Vascular invasion

Positive 6 5

Negative 42 9

Chemotherapy before operation 0 2

Radiotherapy before operation 0 2

TNM, tumour-node-metastasis; CRC, colorectal carcinoma; CRLM,

colorectal liver metastases.a16 CRLM originating from 14 CC patients.bMedian with range in parentheses.

636 CXC Receptor Expression in Colorectal Malignancies C. Rubie et al...................................................................................................................................................................


Journal compilation � 2008 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 68, 635–644

under nucleic acid sterile conditions for RNA and proteinextraction. Tumour samples were taken from vital areas ofhistopathologically confirmed (M.W.) adenocarcinomasand liver metastases respectively. As corresponding nor-mal tissue we used adjacent unaffected mucosa, 2–3 cmdistal to the resection margin from the same resected ade-nocarcinoma or liver specimen respectively. All tissuesobtained were reviewed by an experienced pathologist(M.W.) and examined for the presence of tumour cells. Asminimum criteria for usefulness for our studies, we onlychose tumour tissues in which tumour cells occupied amajor component (>80%) of the tumour sample.

Single-strand cDNA synthesis. Total RNA was isolatedusing RNeasy columns from Qiagen (Hilden, Germany)according to the manufacturer’s instructions. RNA integ-rity was confirmed spectrophotometrically and by electro-phoresis on 1% agarose gels. For cDNA synthesis 5 lgof each patient total RNA sample were reverse-tran-scribed in a final reaction volume of 50 ll containing 1·TaqMan RT buffer, 2.5 lM ⁄ l random hexamers,500 lM ⁄ l each dNTP, 5.5 mM ⁄ l MgCl2, 0.4 U ⁄ll RNaseinhibitor and 1.25 U ⁄ll Multiscribe RT. All RT-PCRreagents were purchased from Applied Biosystems (FosterCity, CA, USA). The reaction conditions were 10 min at25 �C, 30 min at 48 �C and 5 min at 95 �C.

Real-time PCR. All quantitative real-time PCR(Q-RT-PCR) assays containing the primer and probe mixwere purchased from Applied Biosystems and utilizedaccording to the manufacturer’s instructions. PCR reac-tions were carried out using 10 ll 2· Taqman PCR Uni-versal Master Mix No AmpErase� UNG and 1 ll geneassay (Applied Biosystems), 8 ll RNase-free water and1 ll cDNA template (50 mg ⁄ l). The theoretical basis ofthe Q-RT assays is described in detail elsewhere [20]. Allreactions were run in duplicates along with no templatecontrols and an additional reaction in which reversetranscriptase was omitted to assure the absence of geno-mic DNA contamination in each RNA sample. For thesignal detection, ABI Prism 7900 sequence detector(Applied Biosystems, Foster City, CA, USA) was pro-grammed to an initial step of 10 min at 95 �C, followedby 40 thermal cycles of 15 s at 95 �C and 10 min at60 �C and the log-linear phase of amplification was mon-itored to obtain CT values for each RNA sample.

Gene expression of all target genes was analysed inrelation to the levels of the slope matched housekeepinggenes cyclophilin C (cyc C) and phosphomannomutase I[21]. As reporting of data obtained from raw CT valuesinappropriately represent the variations, we converted theindividual CT values to the linear form as follows:

Fold difference = 2)(mean CT

pathological tissue–mean CT

cali-

brator) = 2)delta CT.

Hence, the normal tissue became the 1 · sample andall other quantities were expressed as an n-fold differencerelative to this tissue.

Isolation of total protein. Protein lysates from frozentissues were extracted with the radioimmunoprecipitationcell lysis and extraction buffer from Pierce (Rockford, IL,USA). Total protein content was assessed by using BCAprotein assay reagent kit (Pierce).

Immunohistochemistry. Operative specimens were rou-tinely fixed in formalin and subsequently embedded inparaffin. Before staining, 4-lm thick paraffin-embeddedtissue sections were mounted on Superfrost Plus slides,deparaffinized and rehydrated in graded ethanol to deion-ized water. The sections were microwaved with an anti-gen retrieval solution (Target Retrieval, Dakocytomation,Carpinteria, CA, USA) and after blocking of endogenousperoxidase activity with 3% hydrogen peroxide, the sec-tions were sequential treated with avidin and biotin(Avidin ⁄ Biotin blocking kit; Vector Laboratories Inc.,Burlingame, CA, USA). In addition, the specimens werefurther blocked for 30 min at room temperature withnormal goat or rabbit serum. Overnight incubation at4 �C with primary rabbit anti-human CXCR1 antibody(1:70, sc-988; Santa Cruz Biotechnology, Santa Cruz,CA, USA), rabbit anti-human CXCR2 antibody (1:60,sc-682; Santa Cruz Biotechnology), rabbit anti-humanCXCR3 antibody (1:150, HP003189; Atlas AntibodiesAB, Stockholm, Sweden) and goat polyclonal anti-humanCXCR4 antibody (1:300, AB1671; Abcam, Cambridge,UK), respectively, was followed by incubation ofsecondary biotinylated goat anti-rabbit IgG antibody orbiotinylated rabbit anti-goat IgG antibody and theavidin-biotin-peroxidase reaction (Vectastain ABC ELITEKit; Vector Laboratories Inc.). After colour reactionwith aminoethylcarbazol solution (Merck, Darmstadt,Germany), tissues were counterstained with haematoxy-lin. Negative controls were performed in all cases omit-ting primary antibody.

Western blots. Chemokine receptors were detectedwith anti-CXCR1 (1:500, sc-988 rabbit anti-human;1:10,000, BioRad cat. 170-6515 goat anti-rabbit HRP;BioRad, Munich, Germany), anti-CXCR2 (1:500, sc-682rabbit anti-human; 1:10,000, BioRad cat. 170-6515 goatanti-rabbit HRP; BioRad), anti-CXCR3 (1:500, sc-9900goat anti-human; 1:10,000, sc-2350 bovine anti-goatHRP; Santa Cruz Biotechnology) and anti-CXCR4(1:500, Serotec, AHP442 rabbit anti-human; Serotec,Oxford, UK; 1:5000, BioRad cat. 170-6515 goat anti-rabbit HRP; BioRad). Bands were visualized by ECLWestern blotting analysis systems (Amersham Bioscienc-es, Piscataway, NJ, USA) and quantified densitometrical-ly. Human cell lysates HL-60 (SC-2209; Santa CruzBiotechnology), Imgenex cell lysates A375 and HeLa(Imgenex, San Diego, CA, USA) served as positivecontrols.

Laser capture microdissection. Laser microbeam micro-dissection (LMM) was employed for obtaining puretumour cell and pure normal cell samples for subsequent

C. Rubie et al. CXC Receptor Expression in Colorectal Malignancies 637..................................................................................................................................................................



genetic analysis. LMM was performed on three samplesfor each tissue type for CXCR1–4. Histochemical stain-ing was used on cryo sections before microdissection.Specimen preparation, microdissection and catapultingwere performed following a laser pressure catapultingprotocol according to the manufacturer’s instructions(P.A.L.M. Microlaser Technologies, Bernried, Germany).RNA was extracted using the P.A.L.M. RNA extractionkit and for reverse transcription the invitrogen reversetranscription kit (Invitrogen Life Technologies,Karlsruhe, Germany) was applied. Subsequently, quanti-tative PCR analysis was performed.

Calculations and statistical methods. Chemokine recep-tor expression profiles in the different groups are shownas mean and standard error of the mean (SEM). Statisticalcalculations were done with the MedCalc software pack-age (MedCalc software, Mariakerke, Belgium)[22]. Whereappropriate, either the Student’s t-test or the Wilcoxon’srank sum test was applied to test for group differences ofcontinuous variables. A P-value of 0.05 or less was con-sidered significant.

Results

CXC receptor expression in patients with UC and CRA

Quantitative real-time PCR analysis revealed a trendtoward a higher upregulation for chemokine receptors 1–4in UC tissues. However, this difference was not significant.Accordingly, we observed no significant upregulation ordownregulation of mRNA expression in CRA tissuespecimens analysed (Fig. 1A).

As our results represent pooled mean expression levels,we also analysed the differences between gene expressionsfrom individual matched normal ⁄ pathological samples.The results obtained from this type of analysis corre-sponded well with the results presented in Fig. 1A.

Our RNA expression results were confirmed on theprotein level for all CXC receptors under investigationas assessed by Western blot analysis and subsequentdensitometric measurements (Fig. 1B). For CXC recep-tors 1–4 the protein level revealed no significant differ-ence in band intensity between the normal andpathological tissues of UC and CRA thus correspondingwell with our TaqMan data. Likewise, we observed nosignificant difference in band intensity between the UCand CRA tissues.

CXC receptor expression in colorectal carcinomas related to

tumour stage

In all T-stages of the CRC specimens analysed mRNAexpression of CXC receptors 1, 2 and 4 was significantlyupregulated (P < 0.05) (Fig. 2A). In contrast, CXCR3showed no significant upregulation in any tumour stage.

Relative CXC receptor expression in UC and CRAtissues, RNA level

0

2

4

6

8

10

12

A

B

CXCR1 CXCR2 CXCR3 CXCR4

n-fo

ld g

ene

expr

essi

on r

elat

ed to

corr

espo

ndin

g no

rmal

tiss

ue

CRA tissue UC tissue

kDa

50

40

Control CRA UC

HL60 N P N P

CXCR4

Figure 1 CXC receptor expression in ulcerative colitis (UC) and CRA

specimens as determined by (A) quantitative real-time PCR (Q-RT-

PCR) and (B) Western blot analysis. (A) Q-RT-PCR data are expressed

as mean ± standard error of the mean (SEM), *P < 0.05, n = 25 and 8

respectively. Fold increase above one indicates receptor overexpression

in affected tissues related to unaffected neighbour tissues. (B) Total cell

lysates of pathological (P) and corresponding normal tissues (N) of one

representative patient of UC and CRA respectively were immunoblot-

ted with antibodies specifically recognizing chemokine receptor

CXCR4. Cell line HL60 served as a positive control for the detection

of CXCR4.

Relative CXC receptor expression in different T-stagesof CRC tissues, RNA level

0

2

4

6

8

10

12

14

A

B


n-fo

ld g

ene

expr

essi

on r

elat

ed to

corr

espo

ndin

g no

rmal

tiss

ue CRC T1 CRC T2 CRC T3 CRC T4*

*

**

*

*

** *

**

*

Control T stage 2 T stage 4

HL60 N P N PkDa

50

40CXCR4

Figure 2 CXC receptor expression in colorectal carcinoma (CRC) speci-

mens as determined by (A) quantitative real-time PCR (Q-RT-PCR)

and (B) Western blot analysis. (A) Q-RT-PCR data are expressed as

mean ± SEM, *P < 0.05, n = 8, 15, 15 and 10 respectively. Fold

increase above one indicates receptor overexpression in affected tissues

related to unaffected neighbour tissues. (B) Total cell lysates of tumour

(P) and corresponding normal tissues (N) of one representative patient

of CRC T-stages 2 and 4 were immunoblotted with antibodies specifi-

cally recognizing chemokine receptor CXCR4. Cell line HL60 served as

a positive control for the detection of CXCR4.




Microdissection of tumour and normal cells in threespecimens of each tumour stage, followed by subsequentRT-PCR gene expression analysis confirmed that theexpression profile corresponded with the results presentedin Fig. 2A.

Protein expression of CXC receptors 1-4 was con-firmed by Western blot analysis for all CRC T-stages asrepresentatively demonstrated for CXCR4 in T-stages 2and 4 (Fig. 2B).

CXC receptor expression in colorectal liver metastases

Next, we analysed CXC receptor mRNA and proteinexpression in CRLM of synchronous and metachronousorigin and the corresponding CRC tissues where themetastases originated from.

While CXCR1 and 2 mRNA expression showed noupregulation in the CRLM, both receptors presented asignificant up to eightfold higher expression in the corre-sponding primary tumours of the colon and rectum ofour patient cohort (P < 0.05) (Fig. 3A). The mRNAexpression pattern of CXCR1 and 2 showed a decreasewith increasing T-stages. It may be speculated thatCXCR1 and 2 mRNA expression is associated with earlyonset of CRC. Thus, both receptors may have some

meaning for the development of CRC, but may not be asimportant for the maintenance of the cancerous stage.

In contrast, we found no significant CXCR3 upregula-tion in the corresponding colorectal primary tumours,but a significant CXCR3 upregulation in the CRLM(P < 0.05) (Fig. 3A). Analysis of CXCR4 expressionrevealed significant upregulation in the CRLM and in thecorresponding CRC respectively (P < 0.05), as presentedin Fig. 3A.

Protein expression profiles of all CXC receptor pro-teins as determined by Western blot analysis widely con-firmed the RNA results in the CRLM and in thecorresponding primary colorectal tumours. For CXCR3and CXCR4 our protein data corresponded very wellwith our RNA data showing significantly increasedCXCR3 and CXCR4 expression levels in the CRLM ofsynchronal and metachronal origin. Moreover, in severalpatients we detected a significant CXCR4 protein upreg-ulation in the CRLM compared with the correspondingprimary colorectal tumours (P < 0.05), as shown for onerepresentative patient in Fig. 3B.

Analysis of CXC receptor expression by IHC

Detection of CXC receptor expression was assessed byimmunohistochemical staining in 48 CRC and 16 CRLMspecimens and corresponding normal tissues for theexpression of CXCR1–4. Antibody specificity was con-firmed in preliminary assays, where the application ofblocking peptides removed any specific signal. Immuno-staining of the CRC tissues revealed positive staining in78% (37 ⁄ 48) for CXCR1, 88% (42 ⁄ 48) for CXCR2,56% (26 ⁄ 48) for CXCR3 and 62% (30 ⁄ 48) for CXCR4respectively. Immunostaining of the CRLM tissuesrevealed positive staining in 28% (4 ⁄ 16) for CXCR1,38% (6 ⁄ 16) for CXCR2, 94% (15 ⁄ 16) for CXCR3 and88% (14 ⁄ 16) for CXCR4 respectively. The totalnumber of leucocytes per five high-power fields (using·40 – HPF objective magnification) was determined.Cells were considered positive, when they demonstratedstrong and exclusive labelling. Staining for CXCR1–4 inleucocytes was heterogeneous for the four receptors. Thus,the number of leucocytes positive for CXCR1–4 variedbetween 2 and 75 cells per five HPF.

Immunostaining with CXCR1-specific antibodiesrevealed weak insular signals in mesenchymal cells withintumour neighbour tissues of CRC and intense insular sig-nals in mesenchymal cells within CRC specimens(Fig. 4A, B). In tumour neighbour tissues of CRLM wedetected no substantial CXCR1 reactivity. Only insularpositive signals were found in some hepatocytes(Fig. 4C). Within CRLM specimens, we observed lowstaining intensities restricted to singular leucocytes only(Fig. 4D). CXCR2 staining revealed weak to mediumstaining intensities in mesenchymal areas within

Relative CXC receptor expression in CRLM, RNA level

0

2

4

6

8

10

12

A

B


n-fo

ld g

ene

expr

essi

on

CRLM corresponding CRC tissue

*

*

**

Up regulation

*

HL60 N P N PkDa

50

40

Control Syn CRLM CRC

CXCR4

Figure 3 CXC receptor expression in colorectal liver metastases

(CRLM) and corresponding colorectal primary tumours as determined

by (A) quantitative real-time PCR (Q-RT-PCR) and (B) Western blot

analysis. (A) Q-RT-PCR data are expressed as mean ± SEM, *P < 0.05,

n = 9. Fold increase above one indicates receptor overexpression in

CRLM related to hepatic neighbour tissues and in colorectal primary

tumours related to colorectal neighbour tissues. The ratio normal liver

tissue ⁄ normal colorectal tissue corresponds to 7.2 for CXCR1 and

CXCR2, 1.1 for CXCR3 and 2.2 for CXCR4. (B) Total cell lysates of

tumour (P) and corresponding normal tissues (N) of one representative

patient of each tissue type were immunoblotted with antibodies specifi-

cally recognizing chemokine receptor CXCR4. Cell line HL60 served as

a positive control for the detection of CXCR4.




unaffected CRC neighbour tissues and medium to intensestaining intensities mainly concentrated in mesenchymalcell areas within CRC specimens (Fig. 5A, B). Withinunaffected CRLM neighbour tissues weak staining ofinsular hepatocytes and infiltrating leucocytes wasobserved (Fig. 5C) and within CRLM specimens wedetected weak staining mainly concentrated in leucocytes(Fig. 5D).

For CXCR3 no substantial immunostaining wasdetected within unaffected CRC neighbour tissues orCRC tissue specimens with the exception of some insularpositive signals in mesenchymal cells (Fig. 6A, B).Within corresponding normal tissues of CRLM weobserved weak staining intensities generally localized in

hepatocytes in the vicinity of relatively large vessels asshown in Fig. 6C. In contrast, we detected intensivestaining of mainly mesenchymal cells but also in tumourcells within CRLM specimens (Fig. 6D).

While CXCR4 displayed rather weak staining intensi-ties in singular leucocytes within tumour neighbour tis-sues of CRC (Fig. 7A), we observed a ratherheterogeneous expression pattern for CXCR4 with med-ium signal intensities in varying zonal distribution local-ized in the cytoplasm of tumour cells within the CRCspecimens (Fig. 7B).

Within unaffected CRLM neighbour tissues, CXCR4showed weak to medium staining in the cytoplasm ofnormal liver cells (Fig. 7C). However, in CRLM

A B

C D

Figure 4 Detection of CXCR1 protein

expression in representative CRC and CRLM

specimens as assessed by immunohistochemi-

cal staining with CXCR1-specific antibodies.

A and B refer to CRC and C and D refer to

CRLM tissues. (A) Weak immunostaining in

mesenchymal cells within tumour neighbour

tissues of CRC, (B) intense insular signals in

mesenchymal cells within CRC specimens,

(C) no substantial reactivity in unaffected

corresponding CRLM tissues with the excep-

tion of a few positive signals in hepatocytes

and (D) low staining intensities in infiltrat-

ing leucocytes (original magnification ·200

and ·400).

A B

C D

Figure 5 Detection of CXCR2 protein expres-

sion in representative CRC and CRLM speci-

mens as assessed by immunohistochemical

staining with CXCR2-specific antibodies. A

and B refer to CRC and C and D refer to

CRLM tissues. (A) Weak to medium staining

intensities in mesenchymal areas within unaf-

fected CRC neighbour tissues, (B) medium to

intense staining mainly concentrated in mesen-

chymal cell areas within CRC specimens, (C)

weak staining of insular hepatocytes and infil-

trating leucocytes within unaffected CRLM

neighbour tissues and (D) weak staining mainly

concentrated in leucocytes within CRLM speci-

mens (original magnification ·200 and ·400).




specimens we detected strong CXCR4 staining intensitiesmainly accumulated in a streak of hepatocytes along thetumour invasion front as demonstrated in Fig. 7D.

Clinicopathological correlations

We compared various clinicopathological factors such astumour grading, lymphatic or vascular invasion, pre-existing conditions such as hepatitis, adipositas and cir-rhosis as well as neoadjuvant noxes, age and gender tothe expression of chemokine receptors CXCR1–4. ForCXCR4 we detected a correlation with age. CRC patientsolder than 70 years expressed significantly more CXCR4

than CRC patients younger than 70 years (P < 0.05).Furthermore, we observed a significant correlationbetween CXCR4 expression and tumour grading(P < 0.05).

Discussion

Recently, our group demonstrated in a mouse model ofhepatic and extrahepatic metastasis a correlation betweenCXCR2 expression and angiogenesis and tumour growth[23, 24]. To date, various studies have demonstrated theimportance of angiogenic ELR+ chemokines in mediatingtumour-derived angiogenesis [8, 9, 25, 26]. The primary

A B

C D






CRLM tissues. (A) No substantial immuno-

staining within unaffected CRC neighbour

tissues with the exception of a few positive

signals in mesenchymal cells, (B) weak insu-

lar signals mainly concentrated in mesenchy-

mal cells of CRC specimens, (C) weak

paravascular staining of hepatocytes within

corresponding normal tissues of CRLM and

(D) intensive staining of mainly mesenchymal

cells and insular tumour cells within CRLM

specimens (original magnification ·200 and

·400).

A B

C D






CRLM tissues. (A) Weak staining intensities

in singular leucocytes within tumour neigh-

bour tissues of CRC, (B) medium staining

with varying zonal distribution in tumour

cells within CRC specimens, (C) weak to

medium staining in the cytoplasm of normal

liver cells within unaffected CRLM neigh-

bour tissues and (D) strong staining intensi-

ties in hepatocytes within the tumour

invasion front of CRLM specimens (original

magnification ·200 and ·400).




functional chemokine receptor responsible for mediatingthe proangiogenic effects of ELR+ CXC chemokines dur-ing tumourigenesis is CXCR2 [27]. For instance, Keaneet al. demonstrated in a murine model of lung cancerthat depletion of CXCR2 as well as treatment withanti-CXCR2 inhibited tumour growth and angiogenesis[28]. Other studies demonstrated that application ofanti-CXCR2 antibodies significantly inhibited neovascu-larization in cornea micropocket assays and in humanpancreatic cancer cell lines [29]. Accordingly, humanCRC tissue specimens and CRLM investigated in thisstudy showed elevated CXCR1 and 2 expression levels.However, CXCR1 and 2 overexpression in CRLM speci-mens was only observed on the protein level as deter-mined by Western blot analysis. In IHC assays wedetected only weak staining, which was mainly concen-trated in leucocytes. As the expression of chemokinereceptors CXCR1 and CXCR2 correlated with theamount of inflammatory infiltrate, this could explain thelow-expression ratios in the metastases. The inconsistencyin expression between the RNA and protein level mightbe due to post-transcriptional and post-translational con-trol mechanisms. Thus, our results are in line with a sur-vey, which examined CXCR2 expression in human coloncarcinoma cell lines with different metastatic potential[30], demonstrating increased CXCR2 protein but notmRNA in metastatic compared with non-metastaticcolon carcinoma cells. While we demonstrated thatCXCR1 and 2 expressions were highly upregulated onthe mRNA level in all T-stages of CRC specimens, IHCstudies revealed that this upregulation mainly resultedfrom mesenchymal cells. In contrast, CXCR3 showed nosignificant upregulation in either tumour stage of CRCon the mRNA level. These data corresponded very wellwith our IHC assays, which detected no substantialimmunostaining within CRC tissue specimens. CXCR3is an essential receptor for the recruitment of naturalkiller cells, plasmacytoid monocytes and myeloid den-dritic cell precursors to inflamed lymph nodes (LN)under physiological as well as under pathological condi-tions [31, 32]. Although CXCR3 interacts with angio-static chemokines like CXCL4 and interferon-inducibleCXC chemokines CXCL9, CXCL10 and CXCL11 tomediate angiostatic activity [33], recent reports suggest acritical role for CXCR3 in LN metastasis of human andmouse melanoma cells [34, 35]. With respect to coloncarcinoma, a very recent study [36] demonstrated thatCXCR3 expression significantly correlated with lympha-tic invasion and suggested a critical role for CXCR3 inthe metastasis of colon cancer cells to LN. In line withthese results we observed significant CXCR3 overexpres-sion in the CRLM of our patient cohort on the RNA andprotein level as well as in our IHC assays, which detectedintensive staining in mesenchymal and tumour cells. Thisoverexpression may indicate a putative role for CXCR3

in the process of colorectal metastasis to the liver, but itmay also reflect the high extent of deregulation and alter-ation which is generally related to the ferocious malig-nity of metastatic cells.

For CXCR4 we observed highly upregulated mRNAand protein levels in all T-stages of CRC tissues. Accord-ingly, our IHC results confirmed that this upregulationresulted from tumour cells. These findings are well inline with various recent surveys indicating that theCXCL12–CXCR4 ligand–receptor pair does not onlypromote angiogenesis but also metastasis of tumour cellsin an angiogenesis-independent manner. For instance, inrenal cell carcinoma cell lines and patient specimens aswell as in breast cancer and Non-small cell lung carci-noma specimens CXCR4 was found expressed on tumourcells without mediating tumour-associated angiogenesis[13, 14, 37]. Accordingly, there was no decline in pri-mary tumour-associated angiogenesis in breast or NSCLCtumours of mice that were treated with neutralizing anti-CXCR4 antibodies [13, 14]. Yet, the animals showed asignificant increase of metastases in an organ-specificmanner, which suggests a role for CXCR4 in the meta-static process by defining the metastatic destination.Hence, many other studies have documented an involve-ment of CXCR4 in the metastasis of different cancertypes such as pancreatic, thyroid and bladder cancer [38–40], neuroblastoma [41] or non-Hodgkin lymphoma [42].

Despite a controversial role of the CXCL12 ⁄ CXCR4system in tumour biology, the vast majority of recentpublications emphasized its promoting role in angiogenicactivities and metastasis. With respect to CRC, CXCR4expression was recently associated with recurrence andsurvival in CRC patients [43] and the outgrowth of coloncarcinoma micrometastases [16]. In the context of thesestudies, our group has recently found some indicationsfor a correlation between CXCR4 expression and thedevelopment of CRLM [17, 18]. Also in this study,mRNA and protein assays demonstrated CXCR4 overex-pression in CRLM tissues. However, IHC stainingrevealed that this overexpression mainly resulted fromhepatocytes along the tumour invasion front which repre-sented the majority of positive CXCR4 signals. It maybe speculated that the high density of receptor expressionalong the tumour invasion front may be a stimulativesignal for the tumour to further expand as the tumoursurrounding tissue may also contribute to the metastasisprocess. These results are consistent with previousfindings of our group [44], which demonstratedCXCR4 staining in hepatocellular carcinoma tissuesmainly restricted to non-cancerous hepatocytes.

In conclusion, our study represents a comprehensiveanalysis of the expression profile of the chemokinereceptors CXCR1–4. We observed no significant upreg-ulation or downregulation of expression in the non-malignant colorectal diseases, UC and CRA, conditions




often preceding the development of colorectal malignan-cies. CXCR1 and 2 expression was significantly upregu-lated only in the primary tumour and mainly expressedby mesenchymal cells. No upregulation of CXCR1 and2 was observed in CRLM corresponding to low stainingintensities restricted to singular leucocytes only. CXCR3expression was significantly upregulated only in CRLMcorresponding to intensive staining of mesenchymal andtumour cells within CRLM specimens. No substantialCXCR3 immunostaining was detected within CRC tis-sue specimens corresponding to the RNA and proteinlevel, which showed no CXCR3 upregulation in pri-mary tumours. However, CXCR4 was significantly up-regulated in the primary tumour and in the CRLMmainly expressed by tumour cells in the primarytumour, while in CRLM, we detected strong CXCR4staining intensities mainly accumulated in a streak ofhepatocytes along the tumour invasion front. Conse-quently, we observed a very differential expression pat-tern of the four receptors in colorectal carcinomas andtheir corresponding liver metastases with prominentexpression profiles that indicate a potential role in thepathogenesis of CRC. Thus, our data encourage func-tional tests to further investigate the CXC receptor sig-nalling pathways as therapeutic targets in CRC and thedevelopment of CRLM.

Acknowledgment

We thank C. Weber and B. Kruse for excellent technicalassistance.

References

1 Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics,

2000. CA Cancer J Clin 2000;50:7–33.

2 McLeod HL, McKay JA, Collie-Duguid ES, Cassidy J. Therapeutic

opportunities from tumor biology in metastatic colon cancer. Eur JCancer 2000;36:1706–12.

3 Booth RA. Minimally invasive biomarkers for detection and staging

of colorectal cancer. Cancer Lett 2007;249:87–96.

4 Zlotnik A. Involvement of chemokine receptors in organ-specific

metastasis. Contrib Microbiol 2006;13:191–9.

5 Rossi D, Zlotnik A. The biology of chemokines and their receptors.

Annu Rev Immunol 2000;18:217–42.

6 Zlotnik A, Yoshie O. Chemokines: a new classification system and

their role in immunity. Immunity 2000;12:121–7.

7 Strieter RM, Polverini PJ, Kunkel SL et al. The functional role of

the ELR motif in CXC chemokine-mediated angiogenesis. J BiolChem 1995;270:27348–57.

8 Smith DR, Polverini PJ, Kunkel SL et al. Inhibition of interleukin

8 attenuates angiogenesis in bronchogenic carcinoma. J Exp Med

1994;179:1409–15.

9 Arenberg DA, Keane MP, DiGiovine B et al. Epithelial-neutrophil

activating peptide (ENA-78) is an important angiogenic factor in

non-small cell lung cancer. J Clin Invest 1998;102:465–72.

10 Addison CL, Daniel TO, Burdick MD et al. The CXC cemokine

receptor, CXCR2, is the putative receptor for ELR+ CXC chemoki-

ne-induced angiogeneic activity. J Immunol 2000;165:5269–77.

11 Loetscher M, Gerber B, Loetscher P et al. Chemokine receptor spe-

cific for IP10 and Mig: structure, function and expression in acti-

vated T-lymphocytes. J Exp Med 1996;184:963–9.

12 Yang J, Richmond A. The angiostatic activity of interferon-induc-

ible protein-10 ⁄ CXCL10 in human melanoma depends on binding

to CXCR3 but not to glycosaminoglycan. Mol Ther 2004;9:

846–55.

13 Muller A, Homey B, Soto H et al. Involvement of chemokine recep-

tors in breast cancer metastasis. Nature 2001;410:50–6.

14 Phillips RJ, Burdick MD, Lutz M, Belperio JA, Keane MP,

Strietew RM. The stromal derived factor –1 ⁄ CXCL12-CXC

chemokine receptor 4 biological axis in non-small cell lung metas-

tases. Am J Respir Crit Care Med 2003;167:1667–86.

15 Singh S, Malik BK, Sharma DK. Targeting HIV-1 through molecu-

lar modeling and docking studies of CXCR4: leads for therapeutic

development. Chem Biol Drug Des 2007;69:191–203.

16 Zeelenberg IS, Ruuls-Van Stalle L, Roos E. The chemokine receptor

CXCR4 is required for outgrowth of colon carcinoma micrometasta-

ses. Cancer Res 2003;63:3833–9.

17 Rubie C, Oliveira V, Kempf K et al. Involvement of chemokine

receptor CCR6 in colorectal cancer metastasis. Tumour Biol2006;27:166–74.

18 Rubie C, Oliveira Frick V, Wagner M et al. Chemokine expression

in hepatocellular carcinoma versus colorectal liver metastases. World

J Gastroenterol 2006;41:6627–33.

19 UICC. TNM Classification of Malignant Tumors, 5th edn. New York:

Wiley-Liss, 1997.

20 Bustin SA. Absolute quantification of mRNA using real time

reverse transcription polymerase chain reaction assays. J Mol Endocri-nol 2000;25:169–93.

21 Rubie C, Kempf K, Hans J et al. Housekeeping gene variability in

normal and cancerous colorectal, pancreatic, esophageal, gastric and

hepatic tissues. Mol Cell Probes 2005;19:101–9.

22 Schoonjans F, Zalata A, Depuydt CE, Comhaire FH. MedCalc: a

new computer program for medical statistics. Comp Meth Pro Biomed

1995;48:257–62.

23 Kollmar O, Scheuer C, Menger MD, Schilling MK. Macrophage

inflammatory protein-2 promotes angiogenesis, cell migration and

tumor growth in hepatic metastasis. Ann Surg Oncol 2006;13:263–

75.

24 Kollmar O, Junker B, Rupertus K, Menger MD, Schilling MK.

Studies on MIP-2 and CXCR2 expression in a mouse model of

extrahepatic colorectal metastasis. Eur J Surg Oncol 2007;33:803–11.

25 Strieter RM, Burdick MD, Mestas J, Gomperts B, Keane MP,

Belperio JA. Cancer CXC chemokine networks and tumour angio-

genesis. Eur J Cancer 2006;42:768–78.

26 Moore BB, Arenberg DA, Addison CL, Keane MP, Polverini PJ,

Strieter RM. CXC chemokines mechanism of action in regulating

tumor angiogenesis. Angiogenesis 1998;2:123–34.

27 Keane MP, Burdick MD, Xue YY, Lutz M, Belperio JA, Strieter

RM. The chemokine receptor CXCR2 mediates the tumorigenic

effects of ELR+ CXC chemokines. Chest 2004;125:133S.

28 Keane MP, Belperio JA, Xue YY, Burdick MD, Strieter RM.

Depletion of CXCR2 inhibits tumor growth and angiogenesis in a

murine model of lung cancer. J Immunol 2004;172:2853–60.

29 Wente MN, Keane MP, Burdick MD et al. Blockade of the chemo-

kine receptor CXCR2 inhibits pancreatic cancer cell-induced angio-

genesis. Cancer Lett 2006;241:221–7.

30 Li A, Varnea ML, Singh RK. Constitutive expression of growth reg-

ulated oncogene (gro) in human colon carcinoma cells with different

metastatic potential and its role in regulating their metastatc phe-

notype. Clin Exp Metastasis 2004;21:571–9.

31 Cella M, Jarrossay D, Facchetti F et al. Plasmacytoid monocytes

migrate to inflamed lymph nodes and produce large amounts of

type I interferon. Nat Med 1999;5:919–23.




32 Martin-Fontecha A, Thomsen LL, Brett S et al. Induced recruitment

of NK cells to lymph nodes provides IFN-gamma for T(H)1 prim-

ing. Nat Immunol 2004;5:1260–5.

33 Romagnani P, Annunziato F, Lasagni L et al. Cell cycle-dependent

expression of CXC chemokine receptor 3 by endothelial cells medi-

ates angiostatic activity. J Clin Invest 2001;107:53–63.

34 Robledo MM, Barolom RA, Longo N et al. Expression of functional

chemokine receptors CXCR3 and CXCR4 on human melanoma

cells. J Biol Chem 2001;276:45098–105.

35 Kawada K, Sonoshita M, Sakashita H et al. Pivotal role of CXCR3

in melanoma cell metastasis to lymph nodes. Cancer Res

2004;64:4010–7.

36 Kawada K, Hosogi H, Sonoschita H et al. Chemokine receptor

CXCR3 promotes colon cancer metastasis to lymph nodes. Oncogene2007;26:4679–88.

37 Schrader AJ, Lechner O, Templin M et al. CXCR4 ⁄ CXCL12

expression and signalling in kidney cancer. Br J Cancer

2002;86:1250–6.

38 Koshiba T, Hosotani R, Miyamoto Y et al. Expression of stromal

cell-derived factor 1 and CXCR4 ligand receptor system in pancre-

atic cancer: a possible role for tumor progression. Clin Cancer Res2000;6:3530–5.

39 Hwang JH, Chung HK, Kim DW et al. CXC receptor 4 expression

and function in human anaplastic thyroid cancer cells. J Clin Endo-

crinol Metab 2003;88:408–16.

40 Eisenhardt A, Frey U, Tack M et al. Expression analysis and poten-

tial functional role of the cxcr4 chemokine receptor in bladder can-

cer. Eur Urol 2005;47:111–7.

41 Geminder H, Sagi-Assif O, Goldberg L et al. A possible role for

CXCR4 and its ligand, the CXC chemokine stromal cell-derived

factor–1 in the development of bone marrow metatases in neuro-

blastoma. J Immunol 2001;167:4747–57.

42 Bertolini F, Dell’Agnola C, Mancuso P et al. CXCR4 neutralization,

a novel therapeutic approach for Non-Hodgkin’s lymphoma. Cancer

Res 2002;62:3106–12.

43 Kim J, Takeuchi H, Lam ST et al. Chemokine receptor CXCR4

expression in colorectal cancer patients increases the risk for recur-

rence and for poor survival. J Clin Oncol 2005;23:2744–53.

44 Rubie C, Frick VO, Wagner M et al. Enhanced expression and clin-

ical significance of CC-chemokine MIP-3alpha in hepatocellular car-

cinoma. Scand J Immunol 2006;63:468–77.


