Characterization of Macrophage Subpopulations in Colon Cancer Using Tissue Microarrays

Characterization of macrophage subpopulations in coloncancer using tissue microarrays

D Sickert, D E Aust,1 S Langer,1 I Haupt,1 G B Baretton1 & P DieterInstitute of Physiological Chemistry and 1Institute of Pathology, Medical Faculty Carl Gustav Carus, University of

Technology Dresden, Dresden, Germany

Date of submission 5 March 2004Accepted for publication 7 June 2004

Sickert D, Aust D E, Langer S, Haupt I, Baretton G B & Dieter P

(2005) Histopathology 46, 515521

Characterization of macrophage subpopulations in colon cancer using tissue microarrays

Aims: To determine the pattern of macrophage infil-tration in colon cancers and its correlation withclinicopathological characteristics.Methods and results: Colon cancers from 100 patientswere arrayed into a tissue microarray (TMA). Fourcores per tumour were taken: three from the invasionfront (IF) and one from the tumour surface (TS).Macrophages were quantified by immunohistochemis-try with antibodies to the PG-M1, KP-1, MRP8, MRP14and MRP8 14 antigens. The number of macrophageswas significantly higher in the TS cores than in the IFcores and both tumour sites showed a higher number

of macrophages than the normal mucosa. The numberof macrophages decreased in higher stage tumours.The different tumour-associated macrophage (TAM)subpopulations were positively correlated with eachother.Conclusions: The increased number of macrophages incancers compared with normal colon mucosa indicatesthat macrophages are attracted to the tumour site.However, decreasing macrophages in higher stagecolon cancers suggest that this attraction decreaseswith tumour progression.

Keywords: colon cancer, KP-1, MRP14, MRP8, MRP8 14, PG-M1, tissue microarray, tumour-associatedmacrophages

Abbreviations: IF, invasion front; TAM, tumour-associated macrophage; TMA, tissue microarray; TNF, tumournecrosis factor; TS, tumour surface; VEGF, vascular endothelial growth factor

Introduction

There is considerable controversy as to whethertumour-associated macrophage (TAM) subpopulationspromote or inhibit tumour progression.1 It is wellknown that macrophages orchestrate immune res-ponses through the secretion of cytokines, chemokinesand proteolytic enzymes.2 A model for the functionalheterogeneity observed in macrophage populations atinflammatory sites in vivo suggests that cytokineproduction at the site of inflammation might be duelargely to newly recruited monocytes, while phago-cytosis and tissue remodelling are the work of late-stage macrophages.3 Moreover, macrophages can

present processed foreign antigens to primed T lympho-cytes, allowing the enhancement or inhibition of aspecific immune response.4

Although previous studies have shown that theabove-mentioned functions of macrophages probablyplay a role in anti-tumour response in colon cancer,57

there is no good evidence for tumour regression due toTAMs. On the contrary, recent studies have pointed outthat tumours are able to escape the effects of cytotoxicmacrophages through the secretion of anti-inflamma-tory cytokines.8 Siegert et al.9 detected a significantreduction of oxygen radical production in macrophagesafter incubation with cell culture supernatants derivedfrom colon cancer cell lines.

Additionally, TAMs in colon cancer have angiogenicpotential due to TAM-derived cytokines such as tumournecrosis factor (TNF)-a and vascular endothelialgrowth factor (VEGF).1013 Analyses of colon cancer

D.S. and D.E.A. contributed equally to this work.

Address for correspondence: Dr Daniela E Aust, Institute of

Pathology, TU Dresden, Fetscherstr. 74, D-01307 Dresden, Germany.

e-mail: [email protected]

2005 Blackwell Publishing Limited.

Histopathology 2005, 46, 515521. DOI: 10.1111/j.1365-2559.2005.2129.x

xenografts indicate that macrophages assist in tumourinvasion through the expression of lytic enzymes (e.g.matrix metalloproteinase 2) digesting the extracellularmatrix.14 Furthermore, Niemi et al.15 hypothesize thatapolipoprotein E expression by macrophages surround-ing the colon tumour area may modulate epithelialintegrity and thus contribute to tumour growth.Previous findings of Hauptmann et al.16 suggest thatdifferent macrophage phenotypes are associated withdifferent regions of colorectal carcinoma and differenteffects on tumour cells.

Macrophages are a heterogeneous population of cellsderived from blood-borne monocytes that migrate intotissues, where they undergo differentiation dependenton the microenvironment.

We used a panel of five antibodies to determine theinfiltration of human colon carcinomas and normalcolon mucosa by monocytes macrophages. Panmacrophage markers, PG-M1 and KP-1, characterizeTAMs, with PG-M1 showing more restricted reactivityto cells of the monocyte macrophage lineage thanKP-1. Monocytes macrophages can also be detectedby differentiation-associated antigens belonging to theCa2+-binding S100 protein family, namely MRP8,MRP14 and MRP8 14. MRP8- and MRP14-proteinexpression is restricted to distinct stages of monocyticmaturation17 and appears very early at the site ofinflammation.18 Mature tissue macrophages do notexpress these proteins. S100-calgranulins (MRP8,MRP14) and calprotectin (MRP8 14) are abundantlyexpressed in myeloid cells (monocytes macrophages,neutrophils) and have been associated with variousinflammatory diseases19 as well as carcinomas.8,2022

These cells can be divided into an active inflammatorytype (MRP14+, MRP8 14+) expressing proinflamma-tory cytokines such as TNF-a as well as oxygen radicalsand a chronic inflammatory type (MRP8+).8

We designed a tissue microarray (TMA) comprising100 well-characterized colon cancers. Previous studieshave demonstrated that TAMs provide insight intomolecular mechanisms important in carcinogene-sis.23,24 There was scepticism that, due to tumourheterogeneity, tissue cores used for the microarraymight not be representative of the biological propertiesof the entire tumour. Therefore, several groups havecompared 0.6-mm cores from tumours with the corres-ponding conventional large sections. These analyseshave demonstrated excellent agreement (84100%)between conventional tissue sections and single cores.Such results indicate that tumour heterogeneity does notinfluence the predictive power of the TMA results.23,2527

To date, no data exist on the use of TMAs for the analysisof infiltrating immune cells in cancer.

The aim of our study was to determine the morpho-logical pattern of macrophage infiltration in coloncancers and to correlate it with clinicopathologicalcharacteristics.

Materials and methods

clinical materials

One hundred colon cancers were selected from thepathology archives, reviewed, and two representativeparaffin blocks chosen for the TMA. On each block anarea from the tumour surface (TS) and one from theinvasion front (IF) were marked. In addition, normalcolonic mucosa was marked on resection margins(n 39). All the cancers were staged according theInternational Union Against Cancer (UICC) and gradedaccording to World Health Organization criteria.28,29

The clinicopathological characteristics associated withthese samples are listed in Table 1.

tissue microarrays

TMAs were constructed by randomly selecting one core(diameter 0.6 mm, length 0.7 mm) from the TS andthree cores from the IF region. Tissue cylinders werepunched out of two different donor blocks and placedinto a recipient paraffin block with defined arraycoordinates (1.0 mm distance between the cores) using

Table 1. Clinical characteristics

Male n 59Age* 68 12 years

UICC stageI n 15II n 38III n 39IV n 8

Grade1 n 12 n 4423 n 233 n 32

Mucinous cancers n 20

*Mean age in years SD.

UICC, International Union Against Cancer.

516 D Sickert et al.

2005 Blackwell Publishing Ltd, Histopathology, 46, 515521.

a tissue microarrayer (Beecher Instruments, SilverSpring, MD, USA).

immunohistochemistry

For immunohistochemistry, 4-lm sections of themicroarray block were cut and transferred to glassslides using a paraffin-sectioning aid system (Instrume-dics Inc., Hackensack, NJ, USA). After deparaffinizationand hydration, the slides were treated with 1% H2O2for 15 min at room temperature to abolish endogenousperoxidase activity. Standard indirect immunoperoxi-dase procedures were used for immunohistochemistry(ABC KIT-Elite; Vector Laboratories, Burlingame, CA,USA). A panel of five primary monoclonal antibodiesfor macrophage subtypes, i.e. PG-M1 (CD68), KP-1(CD68) (Dako Corp., Carpinteria, CA, USA) and MRP8,MRP14 and MRP8 14 (27E10) (Dianova, Hamburg,Germany) were applied overnight at 4C. Antigenretrieval was carried out for PG-M1 and MRP14(microwave pretreatment, 15 min, 600 W, dilution1 : 100 and 1 : 400) and MRP8 14 [pronase pre-treatment, 15 min, 5% (v v) in TBSbuffer pH 7,2,37C, dilution 1 : 30). KP-1 and MRP8 antibodies wereused without antigen retrieval (dilution 1 : 400 and1 : 1200). The reaction was visualized with diam-

inobenzidine, and slides were counterstained withhaematoxylin (Figure 1). The primary antibody wasomitted for the negative control. Internal positivecontrols (spleen, granulation tissue, tonsil) were inclu-ded in every TMA block.

The percentage of tumour cells in each tissue corewas recorded semiquantitatively in five categories(0, no tumour cells; a, > 025%; b, > 2550%;c, > 5075%; and d, > 75100% tumour cells percore). Stained macrophages and neutrophils werescored quantitatively (details are given in Table 2).Lymphocytes were recorded semiquantitatively in fourcategories: no lymphocytes, small number of lympho-cytes, moderate number of lymphocytes and largenumber of lymphocytes.

statistics

The score results for tumour cells and different macro-phage subgroups were converted into interval means(Table 2) using Microsoft Excel 2000 (9.0). All subse-quent statistical anayses were done with these values.Statistical significance of differences was determined bypaired t-test, t-test for unpaired samples or U-test. AP-value of 0.05 was considered significant. Statis-tical analysis was performed using the Spearman rank

Figure 1. Photomicrographs of five successive

invasion front cores of colon cancer immuno-

histochemically stained for: a, 27E10

(MRP8 14); b, KP-1; c, MRP14; d, MRP8;e, PG-M1.

Macrophages in colon cancer 517


correlation for comparison between pairs of several cellgroups. All statistical tests were done with SPSS 11.0.1(LEAD Technologies, Haddonfield, NJ, USA).

Results

Although there was a significant variation of tumourcell content within the cores, the number of macro-phages was independent of the percentage of tumourcells within the cores (Figure 2). In TS cores macro-phages increased slightly with increasing tumour cellcontent until the tumour cell content reached appro-ximately 50%; in cores with a higher tumour cellcontent, the number of macrophages decreased(KP-1, PG-M1, MRP8, not significant; PG-M1 (c versusd, P 0.013). No significant correlation betweennumber of macrophages and tumour cell content wasfound in IF cores with different tumour cell contentswith the exception of MRP8 14 (b versus c, P 0.001). Significant differences between the threeIF cores were found in the number of infiltratingmacrophages, ranging from 19% (PG-M1) to 52%(MRP14) deviation in the number of macrophagesfrom core to core, indicating that there is significantheterogeneity in immune cell infiltration. However,within one case the deviation between the IF coresremained constant.

Comparison between tumour tissues and normalmucosa revealed a significantly higher density of allmacrophage subsets in carcinoma tissue than innormal mucosa. The number of macrophages in TSand IF cores from the same patient correlated posi-tively with each other (KP-1, P 0.0001; PG-M1,P 0.006; MRP8, P 0.001; MRP14, P 0.001;

MRP8 14, P 0.0001). However (except for KP-1+macrophages) monocyte macrophage densities werehigher in TS than in IF cores: PG-M1+ (P 0.005),MRP8+ (P 0.031), MRP14+ (P 0.091) andMRP8 14+ (P 0.135) TAMs (Figure 3). All macro-phage subpopulations investigated in our study werestatistically significantly correlated with each other.

Table 2. Score indices of interval-means of positively stainedmacrophages

PG-M1, KP-1 MRP8, MRP14, 27E10

ScorePositivemacrophages

Interval-means Score

Positivemacrophages

Interval-means

0 0 0 0 0 0

1 13 2 1 13 2

2 420 12 2 415 9,5

3 2140 30,5 3 1630 23

4 4160 50,5 4 3145 38

5 61100 80,5 5 4660 53

6 101140 120,5 6 6199 80

70

60

50

40

30

20

10

00 a b c d

TSKP-1PG-M1MRP8MRP14MRP8/14

IF

KP-1PG-M1MRP8MRP14MRP8/14

Percentage of tumour cells

0 a b c dPercentage of tumour cells

Num

ber o

f mac

roph

ages

70

60

50

40

30

20

10

0

Num

ber o

f mac

roph

ages

Figure 2. Comparison of number of macrophages between cores with

different tumour cell content. TS, Tumour surface; IF, invasion front.

60

50

40

30

20

10

KP-1

PG-M1

MRP8 MRP14 MRP8/14

0

0.60

1

Num

bher

of m

acro

phag

es

0.00

3

0.00

50.

000

0.03

10.

001

0.09

10.

000

0.13

50.

001

*

Figure 3. Comparison between tumour surface (TS), invasion front

(IF) and normal mucosa (N) with regard to macrophage infiltration.

*Significances of mean value comparison. , TS; hatched, IF; h, N.



We found a higher number of lymphocytes withinnormal mucosa than in TS (P 0.0001) and IF cores(P 0.0001). The number of lymphocytes in TS andIF cores correlated positively with each other. Thenumber of lymphocytes decreased with increasedtumour cell percentage (not significant).

Positive correlations were demonstrated betweenthe number of lymphocytes and the number ofmacrophage subsets: KP-1 (TS, P < 0.05), PG-M1(TS, P < 0.01, IF, P < 0.01), MRP8 (TS, P < 0.01),MRP14 (TS, P < 0.01, IF, P < 0.01) and MRP8 14(trend to significance).

The densities of KP-1+ and PG-M1+ macrophages inTS and IF cores correlated positively with advancedtumour stage (not significant). Contrary to this result,MRP8+, MRP14+ and MRP8 14+ monocytes macro-phages correlated inversely with advanced T-stage(in IF, MRP14 P < 0.05, MRP8 14 P < 0.01)

The comparison between the different UICC stagesrevealed that KP-1+ and PG-M1+ macrophagesincreased with larger tumour size, but decreased inmetastatic tumours. For KP1+ TAMs in TS cores, therewas a significant increase in UICC stage 2 tumourscompared with UICC stage 1 tumours (P 0.043), anda significant decrease in UICC stage 4 tumours whencompared with UICC stage 3 tumours (P 0.013). InIF cores, the number of KP1+ macrophages decreasedsignificantly in stage 4 tumours when compared withUICC stage 3 tumours (P 0.015). In contrast, theMRP8-, MRP14- and MRP8 14-expressing mono-cytes macrophages did not show any differencesbetween different tumour UICC stages (Figure 4).

Although there was no correlation between thenumber of infiltrating macrophages and tumour grade,the comparison between mucinous and non-mucinouscancers showed a higher infiltration for all subsets ofTAMs in non-mucinous adenocarcinomas with theexception of KP-1. However, this difference betweenboth cancer types reached statistical significance onlyfor MRP14+ TAMs (P 0.004). In addition, non-mucinous tumours showed a higher infiltration withlymphocytes than mucinous cancers (TS, P 0.038;IF, P 0.001).

The density of all macrophage subtypes was a littlehigher within tumours without necrosis than intumours showing necrosis.

Discussion

In this paper, we describe the morphological patterns ofmacrophage infiltration in colon cancer using TMAs.TMAs have been used successfully for immunohisto-chemical studies focused on carcinogenesis,23,24,30

showing that the tissue cores are highly representativeof the whole tumour.23,2527 These studies, however,were not dependent on the tumour cell content of theindividual cores. When starting this study, we did notknow whether the macrophage infiltration in tumourswould be dependent on the tumour cell content of thecores, thus limiting the representativity of the TMA.However, our findings have shown that the infiltrationwith macrophages is independent of tumour cellpercentages within the individual cores. Since macro-phage infiltration is known to be heterogeneous withinan individual tumour, multiple cores were taken fromeach tumour to allow for the expected heterogeneity.By arraying several cores per case and using a largenumber of cases, we ensured that our TMA results arerepresentative of the tumours studied.

We found increased numbers of the different TAMsubgroups in tumour tissue compared with normalcolonic mucosa, indicating that TAMs are attracted tothe tumour site. These results may be explained by thefinding of Suzuki et al.,31 who reported a positivecorrelation between macrophage numbers along the

TS60

50

40

30

20

10

0KP-1 PGM-1 MRP-8 MRP-14 MRP-8/14

11234 8 8 8

1111 131337 3428 37 35

37 353838 28

8

76

1234 8 8 8

1415 151538 3834 38 38

39 373939 37

14

88

234

IF60

50

40

30

20

10

0KP-1 PGM-1 MRP-8 MRP-14 MRP-8/14

1234

UICC

Num

ber o

f mac

roph

ages

Num

ber o

f mac

roph

ages

KP-1

PG-M

1M

RP8

MR

P14

MR

P8/1

4

UICC

KP-1

PG-M

1M

RP8

MR

P14

MR

P8/1

4

Figure 4. Relationship of tumour-associated macrophage

subgroups with International Union Against Cancer (UICC) stage.

The inserted table gives the number of cases per UICC stage.

TS, Tumour surface; IF, invasion front.



invasion front of colon cancers and the expression ofcell adhesion molecules on tumour microvessels.Hemmerlein et al.32 found similar effects in advancedstage renal cancer.

In addition to the expression of cell adhesionmolecules in endothelial cells of tumour microvessels,MRP8+ and MRP14+ monocytes macrophages play aregulatory role in the transendothelial migration ofhuman leucocytes and a modulatory role in inflam-matory responses.33 In our study, the number ofMRP8+, MRP14+ and MRP8 14+ macrophages wasclosely correlated with the number of CD68+ macro-phages. These data may be explained by MRP8+,MRP14+ and MRP8 14+ monocytes macrophagesmigrating continuously into the tumour area andmaturing there due to tumour-induced signals.MRP8+, MRP14+ and MRP8 14+ monocytes macro-phages have been shown to inhibit tumour cellproliferation22 due to the release of cytotoxic agentssuch as oxygen radicals.34,35 A previous study bySiegert et al.9 showed a significant reduction in oxygenradical production in macrophages after incubationwith cell culture supernatants derived from coloncancer cell lines. This is in line with our findingsthat the number of monocytes macrophages with theacute and chronic inflammatory phenotype (MRP8+,MRP14+, MRP8 14+ macrophages) decreased withincreasing tumour size, while the overall number ofCD68+ macrophages increased with increasingtumour size. These tumour-primed macrophagesmay have failed to mature normally into tissuemacrophages. This may result in altered functions,e.g. decreased production of reactive oxygen speciesand tumour cytotoxicity.36,37

We have observed that lymphocyte density wasmuch higher within normal mucosa than in tumourtissue. With increased tumour cell percentage andwithin mucinous tumours the density of lymphocytesdecreased. Saio et al.38 showed that highly proapop-totic TAMs induce lymphocyte apoptosis in murinetumours. Additionally, MRP8 14 protein has cytotoxicactivity against lymphocytes and induces lymphocyteapoptosis.39 These functional data are corroborated byour morphological finding of decreasing lymphocyteinfiltration in combination with increasing mono-cyte macrophage densities.

In conclusion, this is the first study showingthat cytotoxic macrophage subpopulations (MRP8+,MRP14+, MRP8 14+ macrophages) and lymphocytesdecrease with increasing tumour size, suggesting adecreasing anti-tumour response in higher stage coloncancers, which is in line with the results of previouslypublished functional studies. Further morphological

and functional studies are needed to elucidate thecomplex relation between immune cells and tumours.

References

1. Konur A, Kreutz M, Knuchel R, Krause SW, Andreesen R. Three-

dimensional co-culture of human monocytes and macrophages

with tumor cells: analysis of macrophage differentiation and

activation. Int. J. Cancer 1996; 66; 645652.

2. Schweyer S, Hemmerlein B, Radzun HJ, Fayyazi A. Continuous

recruitment, co-expression of tumour necrosis factor-alpha and

matrix metalloproteinases, and apoptosis of macrophages in gout

tophi. Virchows Arch. 2000; 437; 534539.

3. Riches DW. Signalling heterogeneity as a contributing factor in

macrophage functional diversity. Semin. Cell Biol. 1995; 6; 377

384.

4. Baxevanis CN, Voutsas IF, Tsitsilonis OE, Gritzapis AD, Sotiriadou

R, Papamichail M. Tumor-specific CD4+ T lymphocytes from

cancer patients are required for optimal induction of cytotoxic T

cells against the autologous tumor. J. Immunol. 2000; 164;

39023912.

5. Ohtani H, Naito Y, Saito K, Nagura H. Expression of costimu-

latory molecules B7-1 and B7-2 by macrophages along invasive

margin of colon cancer: a possible antitumor immunity? Lab.

Invest. 1997; 77; 231241.

6. Nakayama Y, Nagashima N, Minagawa N et al. Relationships

between tumor-associated macrophages and clinicopathological

factors in patients with colorectal cancer. Anticancer Res. 2002;

22; 42914296.

7. Herbeuval JP, Lambert C, Sabido O et al. Macrophages from

cancer patients: analysis of TRAIL, TRAIL receptors, and colon

tumor cell apoptosis. J. Natl Cancer Inst. 2003; 95; 611621.

8. Hemmerlein B, Markus A, Wehner M, Kugler A, Zschunke F,

Radzum HJ. Expression of acute and late-stage inflammatory

antigens, c-fms, CSF-1, and human monocytic serine esterase 1,

in tumor-associated macrophages of renal cell carcinomas.

Cancer Immunol. Immunother. 2000; 49; 485492.

9. Siegert A, Denkert C, Leclere A, Hauptmann S. Suppression of the

reactive oxygen intermediate production of human macrophages

by colorectal adenocarcinoma cell lines. Immunology 1999; 98;

551556.

10. Zhu GH, Lenzi M, Schwartz EL. The Sp1 transcription factor

contributes to the tumor necrosis factor-induced expression of

the angiogenic factor thymidine phosphorylase in human colon

carcinoma cells. Oncogene 2002; 21; 84778485.

11. Etoh T, Shibuta K, Barnard GF, Kitano S, Mori M. Angiogenin

expression in human colorectal cancer: the role of focal

macrophage infiltration. Clin. Cancer Res. 2000; 6; 35453551.

12. Barbera-Guillem E, Nyhus JK, Wolford CC, Friece CR, Sampsel

JW. Vascular endothelial growth factor secretion by tumor-

infiltrating macrophages essentially supports tumor angiogene-

sis, and IgG immune complexes potentiate the process. Cancer

Res. 2002; 62; 70427049.

13. Bamba H, Ota S, Kato A, Kawamoto C, Matsuzaki F. Effect of

prostaglandin E1 on vascular endothelial growth factor produc-

tion by human macrophages and colon cancer cells. J. Exp. Clin.

Cancer Res. 2000; 19; 219223.

14. Aharinejad S, Abraham D, Paulus P et al. Colony-stimulating

factor-1 antisense treatment suppresses growth of human tumor

xenografts in mice. Cancer Res. 2002; 62; 53175324.

15. Niemi M, Hakkinen T, Karttunen TJ et al. Apolipoprotein E and

colon cancer. Expression in normal and malignant human



intestine and effect on cultured human colonic adenocarcinoma

cells. Eur. J. Intern. Med. 2002; 13; 3743.

16. Hauptmann S, Zwadlo-Klarwasser G, Hartung P, Klosterhalfen B,

Kirkpatrick CJ, Mittermayer C. Association of different macro-

phage phenotypes with infiltrating and non-infiltrating areas of

tumorhost interface in colorectal carcinoma. Pathol. Res. Pract.

1994; 190; 159167.

17. Roth J, Goebeler M, van den Bos C, Sorg C. Expression of calcium-

binding proteins MRP8 and MRP14 is associated with distinct

monocytic differentiation pathways in HL-60 cells. Biochem.

Biophys. Res. Commun. 1993; 191; 565570.

18. Kerkhoff C, Klempt M, Sorg C. Novel insights into structure and

function of MRP8 (S100A8) and MRP14 (S100A9). Biochim.

Biophys. Acta 1998; 1448; 200211.

19. Nacken W, Roth J, Sorg C, Kerkhoff C. S100A9 S100A8:Myeloid representatives of the S100 protein family as prominent

players in innate immunity. Microsc. Res. Tech. 2003; 60; 569

580.

20. Endress H, Freudenberg N, Fitzke E, Grahmann PR, Hasse J,

Dieter P. Infiltration of lung carcinomas with macrophages

of the 27E10-positive phenotype. Lung Cancer 1997; 18; 3546.

21. Komatsu K, Kobune-Fujiwara Y, Andoh A et al. Increased

expression of S100A6 at the invading fronts of the primary

lesion and liver metastasis in patients with colorectal adeno-

carcinoma. Br. J. Cancer 2000; 83; 769774.

22. Hauptmann S, Zwadlo-Klarwasser G, Jansen M, Klosterhalfen B,

Kirkpatrick CJ. Macrophages and multicellular tumor spheroids

in co-culture: a three-dimensional model to study tumorhost

interactions. Evidence for macrophage-mediated tumor cell proli-

feration and migration. Am. J. Pathol. 1993; 143; 14061415.

23. Kononen J, Bubendorf L, Kallioniemi A et al. Tissue microarrays

for high-throughput molecular profiling of tumor specimens.

Nat. Med. 1998; 4; 844847.

24. Schraml P, Kononen J, Bubendorf L et al. Tissue microarrays for

gene amplification surveys in many different tumor types.

Clin. Cancer Res. 1999; 5; 19661975.

25. Bubendorf L. High-throughput microarray technologies: from

genomics to clinics. Eur. Urol. 2001; 40; 231238.

26. Torhorst J, Bucher C, Kononen J et al. Tissue microarrays for

rapid linking of molecular changes to clinical endpoints. Am. J.

Pathol. 2001; 159; 22492256.

27. Rimm DL, Camp RL, Charette LA, Costa J, Olsen DA, Reiss M.

Tissue microarray: a new technology for amplification of tissue

resources. Cancer J. 2001; 7; 2431.

28. Wittekind C, Meyer HJ, Bootz F. TNM Klassifikation maligner

Tumoren. Berlin: Springer Verlag, 2002.

29. Hamilton SR, Aaltonen LA. Tumours of the digestive system. Lyon:

IARC Press, 2000.

30. Tzankov A, Zimpfer A, Pehrs AC et al. Expression of B-cell

markers in classical Hodgkin lymphoma: a tissue microarray

analysis of 330 cases. Mod. Pathol. 2003; 16; 11411147.

31. Suzuki Y, Ohtani H, Mizoi T et al. Cell adhesion molecule

expression by vascular endothelial cells as an immune inflam-matory reaction in human colon carcinoma. Jpn J. Cancer Res.

1995; 86; 585593.

32. Hemmerlein B, Scherbening J, Kugler A, Radzun HJ. Expression

of VCAM-1, ICAM-1, E- and P-selectin and tumour-associated

macrophages in renal cell carcinoma. Histopathology 2000; 37;

7883.

33. Kerkhoff C, Eue I, Sorg C. The regulatory role of MRP8 (S100A8)

and MRP14 (S100A9) in the transendothelial migration of

human leukocytes. Pathobiology 1999; 67; 230232.

34. Mahnke K, Bhardwaj R, Sorg C. Heterodimers of the calcium-

binding proteins MRP8 and MRP14 are expressed on the surface

of human monocytes upon adherence to fibronectin and colla-

gen. Relation to TNF-alpha, IL-6, and superoxide production.

J. Leukoc. Biol. 1995; 57; 6371.

35. Bhardwaj RS, Zotz C, Zwadlo-Klarwasser G et al. The calcium-

binding proteins MRP8 and MRP14 form a membrane-associated

heterodimer in a subset of monocytes macrophages present inacute but absent in chronic inflammatory lesions. Eur. J.

Immunol. 1992; 22; 18911897.

36. Siziopikou KP, Harris JE, Casey L, Nawas Y, Braun DP. Impaired

tumoricidal function of alveolar macrophages from patients

with non-small cell lung cancer. Cancer 1991; 68; 10351044.

37. Alleva DG, Burger CJ, Elgert KD. Tumor growth increases

Ia-macrophage synthesis of tumor necrosis factor-alpha and

prostaglandin E2: changes in macrophage suppressor activity.

J. Leukoc. Biol. 1993; 53; 550558.

38. Saio M, Radoja S, Marino M, Frey AB. Tumor-infiltrating

macrophages induce apoptosis in activated CD8(+) T cells by a

mechanism requiring cell contact and mediated by both the cell-

associated form of TNF and nitric oxide. J. Immunol. 2001; 167;

55835593.

39. Kerkhoff C, Hofmann HA, Vormoor J et al. Binding of two nuclear

complexes to a novel regulatory element within the human

S100A9 promoter drives the S100A9 gene expression. J. Biol.

Chem. 2002; 277; 4187941887.



Documents

Characterization of Macrophage Subpopulations in Colon Cancer Using Tissue Microarrays