Abstract. Background: The role of tumor associatedmacrophages (TAMs) in tumor angiogenesis andinflammation and the interactions between TAMs and tumorcells as well as lymphocytes appear to be critical factors in thedevelopment and progression of cancer. Patients and Methods:Carcinomas of the gastrointestinal tract have been analysed bytissue microarrays. TAMs and vessels were characterized byimmunohistochemistry using the antibodies PG-M1, KP1,MRP8, MRP14, MRP8/14 and CD31, CD34, respectively.Results: The number of all macrophages was significantlyhigher and lymphocyte densities were lower in tumor tissuesthan in tumor-free tissues. The MRP-antibodies identified aminority population of macrophages and a low numbers ofthese macrophages tended to occur in more advanced cancers.There was a positive correlation between the number ofmacrophages and the number of microvessels in all tumors,but no correlation between macrophages and vessel counts intumor-free tissues. Conclusion: The results indicated asuppressed immune response towards the tumors. The observedcommon characteristics regarding macrophage attraction,lymphocyte suppression and microvessel density suggested thatthese mechanisms are regulated similarly in all carcinomas ofthe GI-tract.

In previous studies tumor-associated macrophages (TAMs)have been characterized with the antibodies KP1 or PG-M1directed against the CD68-epitope (1-4). In addition to theCD68 markers, TAMs can be identified by differentiation

antigens expressed during the early or late phase ofinflammation. MRP14-positive macrophages appear in theacute inflammatory phase, MRP8-postive macrophages inthe late stage of inflammation. The monocytes /macrophages expressing the heterodimer MRP8/14 play arole in the active phase of chronic inflammation (1, 2). Theformation of the MRP8/14 heterodimer is correlated withcellular activation such as activation of NADPH oxidaseleading to the release of superoxide anions (3).Furthermore, these macrophages are thought to inhibittumor cell proliferation (4) and may play a role in antitumorcytotoxicity in human lung carcinomas (2).

The functions of the TAM subtypes are highly variableand depend on their maturation and differentiation. It hasbeen hypothesized that reprogramming of TAMs occurs inthe tumor microenvironment as a result of tumor-drivenactivation (5). Tumor infiltrating macrophages arestimulated by tumor-derived and T-cell-derived cytokines toacquire a polarized M2 phenotype secreting IL (interleukin)-4, IL-10 and IL13 (6). These polarized TAMs could promotetumor growth and progression, stroma formation, adaptiveimmunity, angiogenesis and metastasis (6, 7). In contrast,TAMs are also directly involved in the antitumor responseby exerting antibody-dependent cytotoxicity and byproducing cytotoxic substances (1). However, thesetumoricidal effects of TAMs can be suppressed by tumor-derived regulatory molecules that can modulate and subvertmacrophage activities to minimize anti-tumor effects and tofavor tumor growth and progression (8, 9). The lack ofmacrophages may inhibit angiogenesis and, as aconsequence, induce tumor cell death (10, 11).

It can be postulated, that immunosuppressivemechanisms and angiogenesis are central to tumorformation and progression and thus the microenvironmentwithin different tumor types may have very similarcharacteristics. The general consensus from publishedimmunohistological studies is that the number ofmacrophages is substantial, but varies greatly between thedifferent tumors (12). Moreover, the pathologicalsignificance of TAMs in human cancer tissues has remained

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*Both authors contributed equally to this work.

Correspondence to: Dr. Daniela E. Aust, Institute of Pathology, TUDresden, Fetscherstr.74, D-01307 Dresden, Germany. Tel: +49351 4583004, Fax: +49 351 4584328, e-mail: Daniela.Aust@uniklinikum-dresden.de

Key Words: Tissue microarray, tumor-associated macrophages,gastrointestinal cancer, inflammation, angiogenesis, MRP8,MRP14, MRP8/14.

ANTICANCER RESEARCH 27: 1693-1700 (2007)

Characterization of Macrophage Subpopulations and MicrovesselDensity in Carcinomas of the Gastrointestinal Tract

DENISE SICKERT1*, DANIELA E. AUST2*, SILKE LANGER2, GUSTAVO B. BARETTON2 and PETER DIETER1

1Institute for Physiological Chemistry and 2Institute for Pathology,Medical Faculty Carl Gustav Carus, University of Technology Dresden, D-01307 Dresden, Germany

0250-7005/2007 $2.00+.40

controversial. Up to date, there are no published dataregarding macrophage subtype recruitment into differenttumors and organs. This study was therefore undertaken toquantify monocyte / macrophage subtype infiltration andmicrovessel density in tissue microarrays (TMA) ofgastrointestinal carcinomas using specific markers againsthuman monocytes / macrophages (KP1, PG-M1, MRP8,MRP14, MRP8/14) and endothelial cells (CD31, CD34).The quantitation of macrophage subtypes and microvesselsmight elucidate the impact of inflammatory cells on tumorgrowth and angiogenesis.

Patients and Methods

Clinical materials. Cancers of the oral cavity (n=27), pharynx(n=18), esophagus (n=56), stomach (n=75), small intestine(n=25), colon (n=100), rectum (n=57), appendix (n=19), anus(n=15), liver (n=29), gall bladder (n=60) and pancreas (n=48)were selected from the pathology archives, reviewed and tworepresentative blocks from each were chosen for the TMA. Oneach block an area from the tumor surface (TS) and one from the

invasion front (IF) was marked. In addition, tumor-free tissue wasmarked on resection margins. All the carcinomas were stagedaccording to UICC and graded according to WHO criteria (13).The clinico-pathological characteristics associated with thesesamples are listed in Table I.

Tissue microarrays. TMA were constructed by acquiring one core(diameter 0.6 mm, length ca. 0.7 mm) from the TS and three coresfrom the IF region. Tissue cylinders were punched out of twodifferent donor blocks and placed into a recipient paraffin blockwith defined array coordinates (1.0 mm distance between thecores) using a tissue microarrayer (Beecher Instruments, SilverSpring, Maryland, USA).

Immunohistochemistry. For immunohistochemistry, 4 m sectionsof the microarray block were cut and transferred to glass slidesusing a paraffin-sectioning aid system (Instrumedics Inc.,Hackensack, NJ, USA). After deparaffinization and hydration,the slides were treated with 1% H2O2 for 15 minutes at roomtemperature to abolish endogenous peroxidase activity. Standardindirect immunoperoxidase procedures were used forimmunohistochemistry (ABC KIT-Elite, Vector Laboratories,Burlingame, CA, USA). A panel of five primary mouse

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Table I. Clinical characteristics.

Organs Oral Pharynx Esophagus Stomach Small Colon Rectum Appendix Anus Liver Gall Pancreascavity intestine bladder

Number of 22/5 15/3 51/5 47/28 10/11 59/41 36/21 7/12 4/11 20/9 12/36 27/21patients (m/f)*

Age** 5913 5510 639 6911 6212 6812 679 7310 5817 6110 6810 6410

Adenocarcinomas - - 29 75id 6 100m 57 12 - 29 48 48

Squamous cell 27 18 27 - - - - - 15 - - -carcinomas

pT-stage+

1 13 4 21 17 0 3 8 0 5 7 10 12 8 9 13 37 0 20 19 3 7 10 21 113 0 3 21 14 4 65 30 3 1 8 14 334 6 2 1 6 2 12 0 6 0 1 2 3pTx 0 0 0 1 0 0 0 0 2 3 1 0

pN-stage+

0 10 4 33 32 3 53 31 4 4 9 9 151 7 4 22 25 3 25 11 3 1 3 8 272 6 7 0 9 0 21 9 1 1 0 2 53 0 2 0 5 0 0 0 0 0 0 0 0pNx 4 1 1 4 0 1 6 4 9 17 29 1

G / grading+

1 3 0 4 5 1 1 1 1 2 7 2 11-2 1 0 0 5 0 0 1 0 1 0 2 22 15 10 28 21 2 44 36 8 9 19 26 212-3 5 4 2 11 0 23 13 0 0 1 6 113/4 3 4 22 32 3 32 6 3 3 2 12 13

*f, female, m, male; **mean age in year standard deviation; +according to TNM 1997; idstomach: 52 carcinomas of the intestinal type and 23carcinomas of the diffuse type; mcolon: 20 mucinous and 80 non-mucinous carcinomas.

monoclonal antibodies for macrophage subtypes, i.e., PG-M1,KP1 (anti-CD68, Dako Corp., Carpinteria, CA, USA) and 8-5C2(anti-MRP8), S36.48 (anti-MRP14), 27E10 (anti-MRP8/14)(Dianova, Hamburg, Germany) were applied over night at 4C.Endothelial cells were highlighted with the mouse monoclonalantibodies clone JC10A (anti-CD31) and clone QBEnd10 (anti-CD34) (Dako Corp., Carpinteria, CA, USA) for 90 minutes atroom temperature. Antigen retrieval was conducted for PG-M1,MRP14 and CD34 (microwave pretreatment, 15 minutes, 600 W,dilution 1:100, 1:400 and 1:100) and MRP8/14, CD31 (pronasepretreatment, 15 minutes, 5% pronase in TBS-buffer (TRIS-buffered saline solution with TWEEN 20; Dako Corp.,Carpinteria, CA, USA) pH 7,6, 37C, dilution 1:30). KP1- andMRP8-antibodies were used without antigen retrieval (dilution1:400 and 1:1200). The reaction was visualized withdiaminobenzidine, and slides were counterstained withhematoxylin. The primary antibody was omitted for the negativecontrol. Internal positive controls (spleen, granulation tissue andtonsil) were included in every TMA block.

The percentage of tumor cells in each tissue core was recordedsemiquantitatively in five categories (0: no tumor cells, a: >0-25%,b: >25-50%, c: >50-75% and d: >75-100% tumor cells per core).Stained macrophages were scored quantitatively. Lymphocyteswere easily identified on a morphological basis and recordedsemiquantitatively in four categories: no lymphocytes, sparselymphocytic infiltrate, moderate lymphocytic infiltrate and denselymphocytic infiltrate. Microvessel density was assessed by countingendothelial cells, endothelial cell clusters and vessels. Theoccurence of necrosis within the tumor cores was also recordedsemiquantitatively in four categories: no necrosis, necrosiscomprises 50% of tumor area.

Statistics. The results were standardized for common investigationsof malignancies of different organs. A sample with standardizedvalues has the mean value 0 and the standard deviation 1 and wascalculated with the formula:

XSN=

(XSN: standardized sample value, X: number of macrophages ofone patient, : mean value of all macrophages of each subtypefrom one organ and the same tumor area, : standard deviation).The statistical significance of the differences was determined bypaired t-test, t-test for unpaired samples (macrophages) or U-testand Wilcoxon-test respectively (lymphocytes). A p-value of 0.05 orless was considered significant. Statistical analysis was performedusing the Spearman rank correlation for comparison between pairsof several cell groups. All statistical tests were done with SPSS11.0.1 (SPSS-GmbH, Munich, Germany).

Results

Quantitation of macrophage infiltration. In all organs of theGI-tract, tumor areas showed higher numbers ofmacrophages than the tumor-free tissue, with the exceptionof KP1+ TAMs in the pharynx and KP1+ and MRP8+

TAMs in the liver.

The mean values of all adenocarcinoma specimensrevealed significantly higher densities of KP1+ and PG-M1+

TAMs in the TS, IF and tumor-free tissue compared tomean values of all squamous cell carcinoma specimens(Figure 1). In particular, the adenocarcinomas of theesophagus had higher densities of CD68+ macrophagesthan squamous cell carcinomas (in TS PG-M1: p=0.02).

The macrophage densities, in particular MRP8+ andMRP14+ TAMs, were higher within the TS- than in the IF-core of adenocarcinomas.

In squamous cell carcinomas, macrophages expressingKP1 and PG-M1 were detected at lower densities within TSthan within IF.

Correlation of the markers. All macrophage subtypes in thetumor tissues investigated in our study were statisticallysignificantly correlated with each other (MRP8 andMRP14 were positively correlated with each other in 75%,MRP8 and PG-M1 in 71%, MRP8/14 and KP1 in 63%,MRP8/14 and MRP8 in 63%, and PG-M1 and MRP14 in58% of the cores).

MRP8: PG-M1 ratio. With the exception of the oral cavity,in all organs of the upper GI-tract the MRP8 : PG-M1 ratiowas higher within the TS than in the IF (pharynx p=0.050,esophagus p=0.005, stomach p=0.036). In the squamouscell carcinomas a significantly higher MRP8 : PG-M1 ratiowas observed than in the adenocarcinomas (TS p=0.018 andIF p=0.004). Also, there was a higher MRP8 : PG-M1 ratioin squamous cell carcinomas than in adenocarcinomas ofthe esophagus whereas the corresponding tumor-free tissueof both tumor types of the esophagus showed similar ratios(Figure 2).

The MRP8: PG-M1 ratio in stomach, small intestine,colon, rectum adenocarcinomas showed constant values.

The comparison between the tumor-free tissues and thetumor tissues of the small intestine, rectum, anus, liver, gallbladder and pancreas revealed a higher MRP8: PG-M1ratio in the tumor-free tissues than in the carcinomas (liverTS versus tumor-free tissue: p=0.003, IF versus tumor-freetissue: p=0.007) (Figure 2).

Macrophage density and tumor size (pT-stage). MRP+

macrophage subtype counts were slightly lower in advancedpT-stage carcinomas according to UICC in the oral cavity,esophagus, colon, rectum, anus, liver and pancreas (colonin IF MRP14: p

Macrophage density and lymph-node status. In adeno-carcinomas, lower densities of MRP8+, MRP14+ andMRP8/14+ TAMs were associated with the presence oflymph node metastases (MRP8/14 in the TS p

Sickert et al: Macrophages in Gastrointestinal Tract Carcinomas

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Figure 2. MRP8 : PG-M1 ratio.

Figure 3. Mean value of lymphocyte scores.

However, no correlation was found between macrophagesand vessel counts in tumor-free tissue.

Whereas the number of microvessels in the TS seemed tobe independent from the pT-stage, the number ofmicrovessels in IF tended to be lower in higher pT-stages(the differences were significant for the oral cavity,esophagus, colon, pancreas p

number of lymphocytes in our study was associated with adecreased number of TAMs in any given tumor. It may bemore likely that the decreased densities of lymphocytes incarcinomas is induced by the tumor itself rather than by theTAMs. The decrease of lymphocytes seemed to be commonto all the tumors of the GI-tract, suggesting thatlymphocyte-suppression may be a general strategy to subvertimmune responses against tumors (7). A high lymphocytedensity was also found to be associated with high vesselcounts in all organs, corroborating the data of a previousstudy, raising the possibility that the lymphatic endothelialcells may actively recruit lymphocytes (27).

While there was no correlation between macrophages andvessel counts in tumor-free tissue, macrophages and vesseldensity were significantly positively correlated with each otherin all GI-tumors indicating a close relationship betweenmacrophages and angiogenesis. Recently, there is increasingevidence that low oxygen tumor areas contribute to theaccumulation of macrophages. TAMs accumulate in poorlyvascularized, hypoxic and necrotic areas and subsequentlyproduce angiogenic factors (7, 28, 29). Hypoxic stimulationof tumor areas results in rapid increase of the hypoxiainducible factor (HIF)-2 protein levels in macrophages, whichmediate the hypoxic regulation of a number of specific genes,such as VEGF, via their interaction with hypoxia responseelements (30, 31). The potential importance of angiogenicgene expression in macrophages is supported by theassociation of high levels of CD68+ macrophage infiltrationwith high vascular density and poor survival in breast (30) andlung carcinoma (32). Our data indicate a close associationbetween TAMs and angiogenesis, because there was asignificantly positive correlation between macrophages andvessels in all the carcinomas of the GI-tract.

Despite the similar characteristics of all the investigatedcarcinomas of the GI-tract demonstrating the fundamentalproperties of immune cells in tumors, differences betweenadenocarcinomas and squamous cell carcinomas werefound. The comparison between these two tumor typesreflected higher amounts of CD68+ macrophages and alower MRP8:PG-M1 ratio in the adenocarcinomascompared to the squamous cell carcinomas. The differencesin TAM-infiltration between tumors of the GI-tract may becaused by their histological differentiation, because the setof cytokines produced by tumor cells varies with theirorigin and genetic alterations. Thus, histologicaldifferentiation might be an important determinant of therelative distribution of TAMs, which is reflected in thevarying numbers of macrophages in different tumor types(6, 33-36). The level of macrophage infiltration seemed tobe specific for the individual organs. Functionally relatedorgans, such as the stomach, small intestine, colon andrectum showed a similar number of macrophages as well asa constant MRP8: PG-M1 ratio.

Our results suggest that the small numbers of theinflammatory MRP+ macrophages and the decreasenumbers of the lymphocytes in the carcinomas indicate asuppressed immune response towards all carcinomas of theGI-tract. Additionally, the different tumors of the GI-tractshow common characteristics with regard to recruitmentand differentiation of macrophages as well as to theassociation between TAMs and vessels, indicating thatmacrophage attraction, and vessel formation (angiogenesis)are regulated in a similar way in all these tumors.Functional studies are needed to examine the relationshipof the different macrophage-subtypes to lymphocyte-suppression and angiogenesis.

References

1 Hemmerlein B, Markus A, Wehner M, Kugler A, Zschunke Fand Radzun HJ: Expression of acute and late-stageinflammatory antigens, c-fms, CSF-1, and human monocyticserine esterase 1, in tumor-associated macrophages of renal cellcarcinomas. Cancer Immunol Immunother 49: 485-492, 2000.

2 Endress H, Freudenberg N, Fitzke E, Grahmann PR, Hasse Jand Dieter P: Infiltration of lung carcinomas with macrophagesof the 27E10-positive phenotype. Lung Cancer 18: 35-46, 1997.

3 Goebeler M, Roth J, Henseleit U, Sunderkotter C and Sorg C:Expression and complex assembly of calcium-binding proteinsMRP8 and MRP14 during differentiation of murinemyelomonocytic cells. J Leukoc Biol 53: 11-18, 1993.

4 Hauptmann S, Zwadlo-Klarwasser G, Jansen M, Klosterhalfen Band Kirkpatrick CJ: Macrophages and multicellular tumorspheroids in co-culture: a three-dimensional model to study tumor-host interactions. Evidence for macrophage-mediated tumor cellproliferation and migration. Am J Pathol 143: 1406-1415, 1993.

5 Whiteside TL: 22. Immune responses to malignancies. J AllergyClin Immunol 111: 677-686, 2003.

6 Mantovani A, Sozzani S, Locati M, Allavena P and Sica A:Macrophage polarization: tumor-associated macrophages as aparadigm for polarized M2 mononuclear phagocytes. TrendsImmunol 23: 549-555, 2002.

7 Balkwill F and Mantovani A: Inflammation and cancer: back toVirchow? Lancet 357: 539-545, 2001.

8 Shimura S, Yang G, Ebara S, Wheeler TM, Frolov A andThompson TC: Reduced infiltration of tumor-associatedmacrophages in human prostate cancer: association with cancerprogression. Cancer Res 60: 5857-5861, 2000.

9 O'Sullivan C, Lewis CE, Harris AL and McGee JO: Secretionof epidermal growth factor by macrophages associated withbreast carcinoma. Lancet 342: 148-149, 1993.

10 Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R andSorg C: Macrophages and angiogenesis. J Leukoc Biol 55: 410-422, 1994.

11 Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J andHarris AL: Association of macrophage infiltration withangiogenesis and prognosis in invasive breast carcinoma.Cancer Res 56: 4625-4629, 1996.

12 al-Sarireh B and Eremin O: Tumour-associated macrophages(TAMS): disordered function, immune suppression andprogressive tumour growth. J R Coll Surg Edinb 45: 1-16, 2000.

Sickert et al: Macrophages in Gastrointestinal Tract Carcinomas

1699

13 Wittekind C, Meyer HJ and Bootz F: TNM Klassifikationmaligner Tumoren: Springer Verlag, 2002.

14 Owen MR and Sherratt JA: Modelling the macrophage invasionof tumours: effects on growth and composition. IMA J MathAppl Med Biol 15: 165-185, 1998.

15 Nakayama Y, Nagashima N, Minagawa N, Inoue Y, Katsuki T,Onitsuka K, Sako T, Hirata K, Nagata N and Itoh H:Relationships between tumor-associated macrophages andclinicopathological factors in patients with colorectal cancer.Anticancer Res 22: 4291-4296, 2002.

16 Takanami I, Takeuchi K and Kodaira S: Tumor-associatedmacrophage infiltration in pulmonary adenocarcinoma:association with angiogenesis and poor prognosis. Oncology 57:138-142, 1999.

17 O'Sullivan C and Lewis CE: Tumour-associated leucocytes:friends or foes in breast carcinoma. J Pathol 172: 229-235, 1994.

18 Ohno S, Inagawa H, Soma G and Nagasue N: Role of tumor-associated macrophage in malignant tumors: should thelocation of the infiltrated macrophages be taken into accountduring evaluation? Anticancer Res 22: 4269-4275, 2002.

19 Suzuki Y, Ohtani H, Mizoi T, Takeha S, Shiiba K, Matsuno Sand Nagura H: Cell adhesion molecule expression by vascularendothelial cells as an immune/inflammatory reaction in humancolon carcinoma. Jpn J Cancer Res 86: 585-593, 1995.

20 Hemmerlein B, Scherbening J, Kugler A and Radzun HJ:Expression of VCAM-1, ICAM-1, E- and P-selectin andtumour-associated macrophages in renal cell carcinoma.Histopathology 37: 78-83, 2000.

21 Kerkhoff C, Eue I and Sorg C: The regulatory role of MRP8(S100A8) and MRP14 (S100A9) in the transendothelialmigration of human leukocytes. Pathobiology 67: 230-232, 1999.

22 Konur A, Kreutz M, Knuchel R, Krause SW and Andreesen R:Three-dimensional co-culture of human monocytes andmacrophages with tumor cells: analysis of macrophagedifferentiation and activation. Int J Cancer 66: 645-652, 1996.

23 Mantovani A, Schioppa T, Biswas SK, Marchesi F, Allavena Pand Sica A: Tumor-associated macrophages and dendritic cellsas prototypic type II polarized myeloid populations. Tumori 89:459-468, 2003.

24 Elgert KD, Alleva DG and Mullins DW: Tumor-inducedimmune dysfunction: the macrophage connection. J LeukocBiol 64: 275-290, 1998.

25 Saio M, Radoja S, Marino M and Frey AB: Tumor-infiltratingmacrophages induce apoptosis in activated CD8(+) T cells by amechanism requiring cell contact and mediated by both the cell-associated form of TNF and nitric oxide. J Immunol 167: 5583-5593, 2001.

26 Kerkhoff C, Hofmann HA, Vormoor J, Melkonyan H, Roth J,Sorg C and Klempt M: Binding of two nuclear complexes to anovel regulatory element within the human S100A9 promoterdrives the S100A9 gene expression. J Biol Chem 277: 41879-41887, 2002.

27 Kerjaschki D, Regele HM, Moosberger I, Nagy-Bojarski K,Watschinger B, Soleiman A, Birner P, Krieger S, Hovorka A,Silberhumer G, Laakkonen P, Petrova T, Langer B and RaabI: Lymphatic neoangiogenesis in human kidney transplants isassociated with immunologically active lymphocytic infiltrates.J Am Soc Nephrol 15: 603-612, 2004.

28 Schoppmann SF, Birner P, Stockl J, Kalt R, Ullrich R, CaucigC, Kriehuber E, Nagy K, Alitalo K and Kerjaschki D: Tumor-associated macrophages express lymphatic endothelial growthfactors and are related to peritumoral lymphangiogenesis. Am JPathol 161: 947-956, 2002.

29 Liss C, Fekete MJ, Hasina R, Lam CD and Lingen MW:Paracrine angiogenic loop between head-and-neck squamous-cell carcinomas and macrophages. Int J Cancer 93: 781-785,2001.

30 Leek RD, Talks KL, Pezzella F, Turley H, Campo L, BrownNS, Bicknell R, Taylor M, Gatter KC and Harris AL: Relationof hypoxia-inducible factor-2 alpha (HIF-2 alpha) expression intumor-infiltrative macrophages to tumor angiogenesis and theoxidative thymidine phosphorylase pathway in human breastcancer. Cancer Res 62: 1326-1329, 2002.

31 Talks KL, Turley H, Gatter KC, Maxwell PH, Pugh CW,Ratcliffe PJ and Harris AL: The expression and distribution ofthe hypoxia-inducible factors HIF-1alpha and HIF-2alpha innormal human tissues, cancers, and tumor-associatedmacrophages. Am J Pathol 157: 411-421, 2000.

32 Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H, TalksK, Pezzella F, Gatter KC and Harris AL: Relation of hypoxiainducible factor 1 alpha and 2 alpha in operable non-small celllung cancer to angiogenic/molecular profile of tumours andsurvival. Br J Cancer 85: 881-890, 2001.

33 O'Byrne KJ and Dalgleish AG: Chronic immune activation andinflammation as the cause of malignancy. Br J Cancer 85: 473-483, 2001.

34 Sica A, Saccani A and Mantovani A: Tumor-associatedmacrophages: a molecular perspective. Int Immunopharmacol2: 1045-1054, 2002.

35 Mantovani A, Bottazzi B, Sozzani S, Peri G, Allavena P, DongQG, Vecchi A and Colotta F: Cytokine regulation of monocyterecruitment. Arch Immunol Ther Exp (Warsz) 43: 149-152,1995.

36 Wilson J and Balkwill F: The role of cytokines in the epithelialcancer microenvironment. Semin Cancer Biol 12: 113-120, 2002.

Received March 21, 2006Revised December 19, 2006

Accepted January 2, 2007

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