ANTICANCER RESEARCH 27: 1693-1700 (2007)

Characterization of Macrophage Subpopulations and Microvessel

Density 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

Abstract. Background: The role of tumor associated

macrophages (TAMs) in tumor angiogenesis and
inflammation and the interactions between TAMs and tumor
cells as well as lymphocytes appear to be critical factors in the
development and progression of cancer. Patients and Methods:
Carcinomas of the gastrointestinal tract have been analysed by
tissue microarrays. TAMs and vessels were characterized by
immunohistochemistry using the antibodies PG-M1, KP1,
MRP8, MRP14, MRP8/14 and CD31, CD34, respectively.
Results: The number of all macrophages was significantly
higher and lymphocyte densities were lower in tumor tissues
than in tumor-free tissues. The MRP-antibodies identified a
minority population of macrophages and a low numbers of
these macrophages tended to occur in more advanced cancers.
There was a positive correlation between the number of
macrophages and the number of microvessels in all tumors,
but no correlation between macrophages and vessel counts in
tumor-free tissues. Conclusion: The results indicated a
suppressed immune response towards the tumors. The observed
common characteristics regarding macrophage attraction,
lymphocyte suppression and microvessel density suggested that
these mechanisms are regulated similarly in all carcinomas of
the GI-tract.
In previous studies tumor-associated macrophages (TAMs)
have been characterized with the antibodies KP1 or PG-M1
directed against the CD68-epitope (1-4). In addition to the
CD68 markers, TAMs can be identified by differentiation

*Both authors contributed equally to this work.

Correspondence to: Dr. Daniela E. Aust, Institute of Pathology, TU
Dresden, Fetscherstr.74, D-01307 Dresden, Germany. Tel: +49
351 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.

0250-7005/2007 $2.00+.40

antigens expressed during the early or late phase of

inflammation. MRP14-positive macrophages appear in the
acute inflammatory phase, MRP8-postive macrophages in
the late stage of inflammation. The monocytes /
macrophages expressing the heterodimer MRP8/14 play a
role in the active phase of chronic inflammation (1, 2). The
formation of the MRP8/14 heterodimer is correlated with
cellular activation such as activation of NADPH oxidase
leading to the release of superoxide anions (3).
Furthermore, these macrophages are thought to inhibit
tumor cell proliferation (4) and may play a role in antitumor
cytotoxicity in human lung carcinomas (2).
The functions of the TAM subtypes are highly variable
and depend on their maturation and differentiation. It has
been hypothesized that reprogramming of TAMs occurs in
the tumor microenvironment as a result of tumor-driven
activation (5). Tumor infiltrating macrophages are
stimulated by tumor-derived and T-cell-derived cytokines to
acquire a polarized M2 phenotype secreting IL (interleukin)4, IL-10 and IL13 (6). These polarized TAMs could promote
tumor growth and progression, stroma formation, adaptive
immunity, angiogenesis and metastasis (6, 7). In contrast,
TAMs are also directly involved in the antitumor response
by exerting antibody-dependent cytotoxicity and by
producing cytotoxic substances (1). However, these
tumoricidal effects of TAMs can be suppressed by tumorderived regulatory molecules that can modulate and subvert
macrophage activities to minimize anti-tumor effects and to
favor tumor growth and progression (8, 9). The lack of
macrophages may inhibit angiogenesis and, as a
consequence, induce tumor cell death (10, 11).
It can be postulated, that immunosuppressive
mechanisms and angiogenesis are central to tumor
formation and progression and thus the microenvironment
within different tumor types may have very similar
characteristics. The general consensus from published
immunohistological studies is that the number of
macrophages is substantial, but varies greatly between the
different tumors (12). Moreover, the pathological
significance of TAMs in human cancer tissues has remained

1693

ANTICANCER RESEARCH 27: 1693-1700 (2007)

Table I. Clinical characteristics.
Organs

Oral
cavity

Pharynx

Number of
patients (m/f)*

22/5

15/3

51/5

Age**

5913

5510

Adenocarcinomas
Squamous cell
carcinomas
pT-stage+
1
2
3
4
pTx
pN-stage+
0
1
2
3
pNx
G / grading+
1
1-2
2
2-3
3/4

Esophagus Stomach

Small
intestine

Colon

Rectum Appendix

Anus

Liver

Gall
bladder

Pancreas

47/28

10/11

59/41

36/21

7/12

4/11

20/9

12/36

27/21

639

6911

6212

6812

679

7310

5817

6110

6810

6410

57

12

29

48

48

29

75id

100m

27

18

27

15

13
8
0
6
0

4
9
3
2
0

21
13
21
1
0

17
37
14
6
1

0
0
4
2
0

3
20
65
12
0

8
19
30
0
0

0
3
3
6
0

5
7
1
0
2

7
10
8
1
3

10
21
14
2
1

1
11
33
3
0

10
7
6
0
4

4
4
7
2
1

33
22
0
0
1

32
25
9
5
4

3
3
0
0
0

53
25
21
0
1

31
11
9
0
6

4
3
1
0
4

4
1
1
0
9

9
3
0
0
17

9
8
2
0
29

15
27
5
0
1

3
1
15
5
3

0
0
10
4
4

4
0
28
2
22

5
5
21
11
32

1
0
2
0
3

1
0
44
23
32

1
1
36
13
6

1
0
8
0
3

2
1
9
0
3

7
0
19
1
2

2
2
26
6
12

1
2
21
11
13

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

controversial. Up to date, there are no published data

regarding macrophage subtype recruitment into different
tumors and organs. This study was therefore undertaken to
quantify monocyte / macrophage subtype infiltration and
microvessel density in tissue microarrays (TMA) of
gastrointestinal carcinomas using specific markers against
human monocytes / macrophages (KP1, PG-M1, MRP8,
MRP14, MRP8/14) and endothelial cells (CD31, CD34).
The quantitation of macrophage subtypes and microvessels
might elucidate the impact of inflammatory cells on tumor
growth and angiogenesis.

invasion front (IF) was marked. In addition, tumor-free tissue was

marked on resection margins. All the carcinomas were staged
according to UICC and graded according to WHO criteria (13).
The clinico-pathological characteristics associated with these
samples are listed in Table I.

Patients and Methods

Immunohistochemistry. For immunohistochemistry, 4 m sections

of the microarray block were cut and transferred to glass slides
using a paraffin-sectioning aid system (Instrumedics Inc.,
Hackensack, NJ, USA). After deparaffinization and hydration,
the slides were treated with 1% H 2O 2 for 15 minutes at room
temperature to abolish endogenous peroxidase activity. Standard
indirect immunoperoxidase procedures were used for
immunohistochemistry (ABC KIT-Elite, Vector Laboratories,
Burlingame, CA, USA). A panel of five primary mouse

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 two
representative blocks from each were chosen for the TMA. On
each block an area from the tumor surface (TS) and one from the

1694

Tissue microarrays. TMA were constructed by acquiring one core

(diameter 0.6 mm, length ca. 0.7 mm) from the TS and three cores
from the IF region. Tissue cylinders were punched out of two
different donor blocks and placed into a recipient paraffin block
with defined array coordinates (1.0 mm distance between the
cores) using a tissue microarrayer (Beecher Instruments, Silver
Spring, Maryland, USA).

Sickert et al: Macrophages in Gastrointestinal Tract 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 monoclonal
antibodies clone JC10A (anti-CD31) and clone QBEnd10 (antiCD34) (Dako Corp., Carpinteria, CA, USA) for 90 minutes at
room 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 (pronase
pretreatment, 15 minutes, 5% pronase in TBS-buffer (TRISbuffered saline solution with TWEEN 20; Dako Corp.,
Carpinteria, CA, USA) pH 7,6, 37C, dilution 1:30). KP1- and
MRP8-antibodies were used without antigen retrieval (dilution
1:400 and 1:1200). The reaction was visualized with
diaminobenzidine, and slides were counterstained with
hematoxylin. The primary antibody was omitted for the negative
control. Internal positive controls (spleen, granulation tissue and
tonsil) were included in every TMA block.
The percentage of tumor cells in each tissue core was recorded
semiquantitatively 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. Lymphocytes
were easily identified on a morphological basis and recorded
semiquantitatively in four categories: no lymphocytes, sparse
lymphocytic infiltrate, moderate lymphocytic infiltrate and dense
lymphocytic infiltrate. Microvessel density was assessed by counting
endothelial cells, endothelial cell clusters and vessels. The
occurence of necrosis within the tumor cores was also recorded
semiquantitatively in four categories: no necrosis, necrosis
comprises <25% of tumor area, necrosis comprises 25 to 50% of
tumor area and necrosis comprises >50% of tumor area.
Statistics. The results were standardized for common investigations
of malignancies of different organs. A sample with standardized
values has the mean value 0 and the standard deviation 1 and was
calculated with the formula:
X-
XSN=

(XSN: standardized sample value, X: number of macrophages of

one patient, : mean value of all macrophages of each subtype
from one organ and the same tumor area, : standard deviation).
The statistical significance of the differences was determined by
paired t-test, t-test for unpaired samples (macrophages) or U-test
and Wilcoxon-test respectively (lymphocytes). A p-value of 0.05 or
less was considered significant. Statistical analysis was performed
using the Spearman rank correlation for comparison between pairs
of several cell groups. All statistical tests were done with SPSS
11.0.1 (SPSS-GmbH, Munich, Germany).

Results
Quantitation of macrophage infiltration. In all organs of the
GI-tract, tumor areas showed higher numbers of
macrophages than the tumor-free tissue, with the exception
of KP1+ TAMs in the pharynx and KP1+ and MRP8+
TAMs in the liver.

The mean values of all adenocarcinoma specimens

revealed significantly higher densities of KP1+ and PG-M1+
TAMs in the TS, IF and tumor-free tissue compared to
mean values of all squamous cell carcinoma specimens
(Figure 1). In particular, the adenocarcinomas of the
esophagus had higher densities of CD68+ macrophages
than squamous cell carcinomas (in TS PG-M1: p=0.02).
The macrophage densities, in particular MRP8+ and
MRP14+ TAMs, were higher within the TS- than in the IFcore of adenocarcinomas.
In squamous cell carcinomas, macrophages expressing
KP1 and PG-M1 were detected at lower densities within TS
than within IF.

Correlation of the markers. All macrophage subtypes in the

tumor tissues investigated in our study were statistically
significantly correlated with each other (MRP8 and
MRP14 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 in
58% 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 ratio
was higher within the TS than in the IF (pharynx p=0.050,
esophagus p=0.005, stomach p=0.036). In the squamous
cell carcinomas a significantly higher MRP8 : PG-M1 ratio
was observed than in the adenocarcinomas (TS p=0.018 and
IF p=0.004). Also, there was a higher MRP8 : PG-M1 ratio
in squamous cell carcinomas than in adenocarcinomas of
the esophagus whereas the corresponding tumor-free tissue
of 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 the
tumor tissues of the small intestine, rectum, anus, liver, gall
bladder and pancreas revealed a higher MRP8: PG-M1
ratio in the tumor-free tissues than in the carcinomas (liver
TS versus tumor-free tissue: p=0.003, IF versus tumor-free
tissue: p=0.007) (Figure 2).
Macrophage density and tumor size (pT-stage). MRP+
macrophage subtype counts were slightly lower in advanced
pT-stage carcinomas according to UICC in the oral cavity,
esophagus, colon, rectum, anus, liver and pancreas (colon
in IF MRP14: p<0.05, MRP8/14: p<0.01 and pancreas
MRP14: p<0.05). All organs considered, higher pT-stage
was associated with higher numbers of KP1+ TAMs
(p<0.05) and lower numbers of MRP14+ and MRP8/14+
TAMs (p<0.05). PG-M1+ macrophages did not show any
association with pT- tage.

1695

ANTICANCER RESEARCH 27: 1693-1700 (2007)

Figure 1. Comparison between squamous cell carcinomas and adenocarcinomas with regard to macrophage subtype infiltration; tumor surface (TS),
invasion front (IF) and tumor-free tissue (N). *significant difference from mean value of tumor-free tissue. **significant difference between mean value
of TS and IF, respectively; V: significant difference between the mean values of adenocarcinomas and squamous cell carcinomas.

Macrophage density and lymph-node status. In adenocarcinomas, lower densities of MRP8+, MRP14+ and
MRP8/14+ TAMs were associated with the presence of
lymph node metastases (MRP8/14 in the TS p<0.05,
MRP14 in the IF: p<0.01). However, in squamous cell
carcinomas, higher densities of MRP8+ and MRP8/14+
macrophages were associated with the presence of lymph
node metastases (MRP8 in the TS p<0.01). In both
carcinoma types, the density of KP1+ and PG-M1+
macrophages did not show any difference between cases
with and without lymph node metastases.
Macrophage density and tumor necrosis. The density of all
macrophage subtypes in adenocarcinomas was increased
within tumors showing necrosis compared to tumors without
necrosis (stomach MRP14 p=0.030, colon MRP8/14
p=0.050, all organs together KP1 p=0.047, MRP8/14
p=0.011).
Macrophages density and lymphocytes. We found higher
numbers of lymphocytes within tumor-free tissues than in

1696

tumor tissue in all organs of the GI-tract, with the exception

of the esophagus and pancreas (Figure 3). The number of
lymphocytes decreased with increased tumor cell percentage
in the individual cores. The density of lymphocytes
correlated negatively with the occurrence of lymph node
metastases and higher pT-stage in the adenocarcinomas (in
the IF of all adenocarcinomas together p<0.01, colon
p<0.01 and rectum p<0.05). No correlation was revealed
for squamous cell carcinomas.
Microvessel density. The number of vessels in the
adenocarcinomas was significantly higher than in the
squamous cell carcinomas (p<0.05). This observation was
also made in the esophagus, where the adenocarcinomas
showed significantly higher vessel counts than the squamous
cell carcinomas (p<0.05). However, this difference was not
observed in corresponding tumor-free tissue.
Analysis of the correlation between macrophages and
microvessels in the carcinomas showed that higher number
of macrophages were associated with a higher number of
vessels stained with CD31- and CD34-antibodies (Table II).

Sickert et al: Macrophages in Gastrointestinal Tract Carcinomas

Figure 2. MRP8 : PG-M1 ratio.

Figure 3. Mean value of lymphocyte scores.

1697

ANTICANCER RESEARCH 27: 1693-1700 (2007)

Table II. Correlation of macrophage subtypes and microvessels stained with CD31 in the invasion front.
Squamous carcinomas

Adenocarcinomas

Oral Pharynx Anus EsoEso- Stomach Small Colon Rectum Appendix Liver Gall Pancreas All squamous All adenocavity
phagus phagus
intestine
bladder
carcinomas carcinomas
together
together
KP1
(+)
PG-M1 (+)
MRP8
+
MRP14 +
MRP8-14 ++

()
0
0
0
+

+
+
+
(+)
+

(+)
0
(+)
(+)
(+)

(+)
0
(+)
0
(+)

0
(+)
()
()
0

0
()
0
(+)
(+)

(+)
0
(+)
(+)
++

(+)
++
++
++
+

0
0
0
0
0

0
0
(+)
0
(+)

++
0
(+)
(+)
++

+
0
0
0
(+)

(+)
(+)
++
+
++

++
++
+
+
++

spearman correlation: 0: significance p>0.5; (+),(): positive and negative trend; 0.05<p<0.5, respectively; +,++: significant or highly significant
correlation p<0.05 and p<0.01, respectively.

However, no correlation was found between macrophages

and vessel counts in tumor-free tissue.
Whereas the number of microvessels in the TS seemed to
be independent from the pT-stage, the number of
microvessels in IF tended to be lower in higher pT-stages
(the differences were significant for the oral cavity,
esophagus, colon, pancreas p<0.05).
The vessel density in all carcinomas was significantly
decreased in tumors showing necrosis compared to tumors
without necrosis (p=0.05).
The number of microvessels correlated positively with the
lymphocyte density in carcinomas of all organs (p<0.01).

Discussion
There have been numerous reports regarding the
recruitment of macrophages into tumors, all these studies
have been conducted with diverse methodology and have
led to controversial results (1, 2, 14-18). To the best of our
knowledge, this is the first study investigating carcinomas of
the GI-tract using immunohistochemistry on tissue
microarrays allowing the comparison of macrophage and
microvessel density in different tumors to be made on the
same microscopical slide.
All the investigated tumors share common characteristics
with regard to infiltrating immune cells and microvessel
density. Infiltrating macrophages comprise a major
component of the immune cell infiltrate in carcinomas of
the GI-tract. TAMs overall are significantly more frequent
in tumor than in tumor-free tissue, corroborating the finding
of functional studies that monocytes / macrophages are
recruited into the tumor areas by signals from the tumor
microenvironment such as hypoxia (19-21). Hemmerlein et
al. hypothesized, that local hypoxia was more likely
responsible for tumor necrosis than macrophage-driven
cytotoxicity, because in their functional studies they did not
find direct evidence of a macrophage-derived cytotoxic

1698

effect (1). Our data corroborate these findings in that only

small numbers of the inflammatory monocytes /
macrophages, especially of MRP8/14+ cells, which are able
to produce cytotoxic factors, were found in all tumors. Also,
the inverse correlation of MRP+ macrophages with the
advanced pT-stage rather indicates a suppressed cytotoxic
macrophage response against tumor cells. Furthermore, the
ratio of MRP8 to PG-M1 was higher in tumor-free tissue
than in the corresponding carcinomas, indicating that the
percentage of inflammatory MRP8+ macrophages
compared to the percentage of PG-M1+ TAMs was lower
in the carcinomas than in the tumor-free tissues and that
probably the tumoricidal effects of these TAMs were
consequently reduced.
Additionally, a significantly positive correlation between
all macrophage subtypes in all tumors of the GI-tract was
found. These positive correlations suggest that macrophages
expressing MRP-proteins immigrate continuously into the
tumor areas, then undergo a tumor-induced change in
phenotype during the monocyte to macrophage maturation.
Probably, the tumor-primed macrophages fail to undergo a
normal maturation compared to tissue macrophages. This
may also be associated with changes in macrophage function
such as a decreased production of reactive oxygen species,
nitric oxide and tumor cytotoxicity (6, 22, 23).
Current research suggests that the cellular basis for
tumor-induced immune dysfunction includes the generation
of immunoregulatory macrophages that inhibit Th1 cell
responses (24, 25). Much attention has recently been
devoted to the role of NADPH-dependent reactive oxygen
metabolites or hydrogen peroxide, produced by MRP8/14+
monocytes/macrophages as potential inhibitors of tumor
infiltrating lymphocytes (5, 26). Although functional data
cannot be gathered from a morphological study, our data
may suggest that macrophages are not responsible for the
decreased number of lymphocytes within carcinomas
compared to tumor-free tissues, because a decreased

Sickert et al: Macrophages in Gastrointestinal Tract Carcinomas

number of lymphocytes in our study was associated with a

decreased number of TAMs in any given tumor. It may be
more likely that the decreased densities of lymphocytes in
carcinomas is induced by the tumor itself rather than by the
TAMs. The decrease of lymphocytes seemed to be common
to all the tumors of the GI-tract, suggesting that
lymphocyte-suppression may be a general strategy to subvert
immune responses against tumors (7). A high lymphocyte
density was also found to be associated with high vessel
counts in all organs, corroborating the data of a previous
study, raising the possibility that the lymphatic endothelial
cells may actively recruit lymphocytes (27).
While there was no correlation between macrophages and
vessel counts in tumor-free tissue, macrophages and vessel
density were significantly positively correlated with each other
in all GI-tumors indicating a close relationship between
macrophages and angiogenesis. Recently, there is increasing
evidence that low oxygen tumor areas contribute to the
accumulation of macrophages. TAMs accumulate in poorly
vascularized, hypoxic and necrotic areas and subsequently
produce angiogenic factors (7, 28, 29). Hypoxic stimulation
of tumor areas results in rapid increase of the hypoxia
inducible factor (HIF)-2 protein levels in macrophages, which
mediate the hypoxic regulation of a number of specific genes,
such as VEGF, via their interaction with hypoxia response
elements (30, 31). The potential importance of angiogenic
gene expression in macrophages is supported by the
association of high levels of CD68+ macrophage infiltration
with high vascular density and poor survival in breast (30) and
lung carcinoma (32). Our data indicate a close association
between TAMs and angiogenesis, because there was a
significantly positive correlation between macrophages and
vessels in all the carcinomas of the GI-tract.
Despite the similar characteristics of all the investigated
carcinomas of the GI-tract demonstrating the fundamental
properties of immune cells in tumors, differences between
adenocarcinomas and squamous cell carcinomas were
found. The comparison between these two tumor types
reflected higher amounts of CD68+ macrophages and a
lower MRP8:PG-M1 ratio in the adenocarcinomas
compared to the squamous cell carcinomas. The differences
in TAM-infiltration between tumors of the GI-tract may be
caused by their histological differentiation, because the set
of cytokines produced by tumor cells varies with their
origin and genetic alterations. Thus, histological
differentiation might be an important determinant of the
relative distribution of TAMs, which is reflected in the
varying numbers of macrophages in different tumor types
(6, 33-36). The level of macrophage infiltration seemed to
be specific for the individual organs. Functionally related
organs, such as the stomach, small intestine, colon and
rectum showed a similar number of macrophages as well as
a constant MRP8: PG-M1 ratio.

Our results suggest that the small numbers of the

inflammatory MRP+ macrophages and the decrease
numbers of the lymphocytes in the carcinomas indicate a
suppressed immune response towards all carcinomas of the
GI-tract. Additionally, the different tumors of the GI-tract
show common characteristics with regard to recruitment
and differentiation of macrophages as well as to the
association between TAMs and vessels, indicating that
macrophage attraction, and vessel formation (angiogenesis)
are regulated in a similar way in all these tumors.
Functional studies are needed to examine the relationship
of the different macrophage-subtypes to lymphocytesuppression and angiogenesis.

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Received March 21, 2006

Revised December 19, 2006
Accepted January 2, 2007