ONCOLOGY LETTERS 4: 755-758, 2012
Abstract. Mast cells (MCs) and regulatory T cells (Tregs) are
the important components of the inflammatory infiltrating
leukocytes in most malignant tumors. Our study was designed
to investigate the infiltrating correlation between MCs and
Tregs and clarify their prognostic significance in gastric
cancer (GC). A total of 60 fresh GC tissues were collected
and tumor-infiltrating leukocytes were isolated by gradient
centrifugation. Tryptase and Foxp3 were used as markers for
MCs and Tregs, respectively. The expression of tryptase and
Foxp3 was determined in tumor-infiltrating leukocytes using
flow cytometry. The expression of tryptase and Foxp3 were
positively correlated. The increased infiltration of MCs corre-
lated significantly with advanced stage of GC. The infiltration
of MCs into the tumor may increase the number of Tregs.
Tryptase is a promising marker to stratify GC patients into
different risk groups.
Gastric cancer (GC), a common malignancy of the digestive
tract worldwide, remains largely incurable (1,2). It is one of
the leading causes of tumor-associated mortality in a number
of developing Asian countries despite declining morbidity
and mortality among patients with GC in the majority of
developed countries (3,4). Tumor-induced immunosuppres-
sion, which hinders the cytotoxic responses of T lymphocytes
and natural killer cells, is a principal problem to be solved in
cancer immunotherapy (5,6). There are various immunosup-
pressive strategies employed by tumors in which regulatory
T cells (Tregs) play a pivotal role (7).
Tregs are a distinct lymphocyte lineage with immunosup-
pressive potential in maintaining immunological tolerance.
The transcription factor Foxp3 is a unique marker for Tregs
and is indispensable for their development (8,9)
Mast cells (MCs) are a group of long-lived heterogeneous
cells originating from the bone marrow. They were recognized
by their modulating activities in inflammation and angiogen-
esis until recent investigations revealed their roles in shaping
adaptive immune responses (10). MCs have been found to accu-
mulate in the tumor microenvironment through the SCF/c-kit
signaling pathway, leading to the aggravation of inflammation
and immunosuppression in the tumor microenvironment (11).
The complex reciprocal correlation between MCs and Tregs
determines the functions of both cell types. This interaction of
MCs with Tregs dictates the intensity of tumor-associated inflam-
mation and thus either promotes or inhibits tumor growth (10).
In numerous experimental models and human specimens, it
has been observed that the increase in the number of MCs occurs
in a number of tumors (12). However, there is little evidence of a
clear correlation between the number of MCs and Foxp3 expres-
sion in human GC. The purpose of this study was to assess the
infiltrating correlation between MCs and Tregs in GC.
Patients and methods
Patients and specimens. Tumor samples were obtained from
60 patients with pathologically confirmed gastric adenocarci-
noma from the Department of Gastrointestinal Surgery, Union
Hospital (Wuhan, China). None of the patients accepted anti-
cancer therapy prior to surgical resection. Clinical stages were
classified according to the 7th UICC TNM staging system.
Fresh GC tissues were used for the isolation of tumor-infil-
trating leukocytes, as previously described (13). The clinical
characteristics of all patients are summarized in Table I. The
research protocol was approved by the Institutional Review
Board of Tongji Medical College of Huazhong University of
Science and Technology (Wuhan, China). Written and oral
consents were obtained from the patients prior to the collec-
tion of samples.
Flow cytometry analysis. Tumor-infiltrating leukocytes were
stained extracellularly with specific antibodies against human
Increased numbers of gastric-infiltrating mast cells
and regulatory T cells are associated with tumor
stage in gastric adenocarcinoma patients
YIBIN ZHAO, KE WU, KAILIN CAI, RONGLIN ZHAI, KAIXIONG TAO, GUOBIN WANG and JILIANG WANG
Department of Gastrointestinal Surgery and Minimal Invasive Surgery, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan 430022, P.R. China
Received March 23, 2012; Accepted July 5, 2012
Correspondence to: Dr Jiliang Wang, Department of Gastro-
intestinal Surgery and Minimal Invasive, Union Hospital, Tongji
Medical College, Huazhong University of Science and Technology,
No. 1277 Jiefang Road, Hubei, Wuhan 430022, P.R. China
Key words: gastric cancer, mast cells, regulatory T cells,
TNM stage, coexpression
ZHAO et al: INCREASED NUMBERS OF MAST CELLS AND REGULATORY T CELLS IN GASTRIC CANCER
CD45, CD3 and CD4 (BD Biosciences, Franklin Lakes,
NJ, USA), fixed and permeabilized with Perm/Fix solution
(eBiosciences, San Diego, CA, USA) and finally stained
intracellularly with anti-Tryptase (Millipore, Billerica, MA,
USA) and anti-Foxp3 (eBiosciences). Tryptase and Foxp3 were
used as the markers for MCs and Tregs, respectively. Samples
were acquired on an LSR II (BD Biosciences) and data were
analyzed with DIVA software (BD Biosciences).
Statistical analysis. Results are expressed as mean ± SEM.
Spearman's rank correlation coefficient test and ANOVA were
carried out as indicated. P<0.05 was considered to indicate a
statistically significant result. All analyses were performed
using SPSS v12.0 software (Chicago, IL, USA).
Tryptase and Foxp3 expression were positively correlated in
human GC. Representative dot plots for Foxp3 and tryptase
expression are shown in Fig. 1. Tryptase and Foxp3 expres-
sion were positively correlated (R=0.898, P<0.01; Fig. 2).
Correlations were determined by Spearman's rank correlation
Tryptase and Foxp3 coexpression was associated with TNM
stage in human GC. The development and progression of
cancer are known to be regulated by various oncogenes and
tumor suppressor genes. We analyzed 60 primary GC patients
for the expression of tryptase by ANOVA. As shown in Fig. 3, a
high level of tryptase expression was associated with advanced
tumor stage, which is a marker of poor prognosis.
Table I. Clinical characteristics and the stages of the patients.
Characteristics Number (%)
Gender (male/female) 39/21 (65/35)
Age, years (range) 30-75
Figure 1. Numbers of Tregs and MCs are increased in tumor tissues. Single-
cell suspensions of normal (right) and tumor (left) tissues were prepared
from fresh human GC stained with relevant antibodies. Tregs and MCs were
analyzed by flow cytometry. The specimens were from 60 patients with GC
(see Table I and Materials and methods). The results of one patient are shown.
(A) Tregs (Foxp3) in T cells (gated CD4 cells) from the tumor were analyzed
by flow cytometry. Numbers on the plots are the proportions of Foxp3+ cells
in the CD4+ cells. (B) MCs in CD45+CD3-CD14-SSChigh cells. Numbers
on the plots are the proportions of tryptase+ cells in the CD45+CD3-CD14-
SSChigh cells. MC, mast cell; Treg, regulatory T cell; GC, gastric cancer.
Figure 2. Correlation between MCs and Tregs. MCs were positively corre-
lated with Tregs (R=0.898, P<0.01). Patients with more MCs had more Tregs.
The percentage of tryptase expression = the percentage of tryptase in tumor
tissue - the percentage of tryptase in normal tissue. The percentage of Foxp3
expression = the percentage of Foxp3 in tumor tissue - the percentage of
Foxp3 in normal tissue. MC, mast cell; Treg, regulatory T cell.
Figure 3. Correlation between number of MCs and stage of GC in the total
set of subjects. The percentages of tryptase expression are separated into four
groups by the TNM stage. The short bar indicates the mean value in each
group. MC, mast cell; GC, gastric cancer.
ONCOLOGY LETTERS 4: 755-758, 2012
Our study revealed that an increased number of MCs in GC
patients is correlated with a higher frequency of Foxp3 expres-
sion. Numerous studies have documented an increased level
of Foxp3 during GC progression (14,15). However, the role of
MCs in GC remains unclear.
Ribatti et al (16) demonstrated that MC density correlates
with progression of patients with gastric carcinoma. This
means that the density of MCs is positively correlated with the
development of the disease from stage I to stage IV. However,
there have been reports of a protective role for MCs in human
cancer. For instance, in a multivariate analysis of colorectal
cancer patients, high counts of eosinophils and MCs predicted
longer survival (17). MC tryptases activate the nuclear
peroxisome proliferator-activated receptor-γ (PPAR-γ); the
expression of PPAR-γ is associated with improved clinical
outcome in colon cancer (18). Our findings imply a complex
correlation between the increased number of infiltrating MCs
and advanced stages of GC patients.
These results suggest that higher numbers of MCs are
associated with poor outcomes, which is similar to the results
of previous studies reported in GC and other tumors (19-23).
The present study not only provides support for the correla-
tion between MCs and the stages of GC, but also focused on
mechanisms other than angiogenesis, which has already been
demonstrated (16). We studied the correlation between Tregs
and MCs in order to provide an explanation of the influence
of MCs on the stage of GC. Several studies have researched
the immune suppression mechanism of MCs in tumors (11).
However, to date, no study has directly demonstrated the
correlation between the increased frequency of MCs and
higher levels of Foxp3 in human GC. Our data demonstrate
that MCs may affect the progression of GC, partially via
interaction with Tregs. Studies in liver cancer reported similar
results (32). Numerous researchers consider IL-9 to be critical
factor in this interaction (24-26).
In several types of cancer, an increased level of Foxp3+
Tregs has been detected in tumor tissues and peripheral
blood, consistent with their presumed function in immuno-
suppression (27,28). Much concern has also been attached to
the roles of Foxp3 in human GC. There is a link between
the concentration of Tregs and patient survival in GC (29,30).
Recently, a study partly explained the mechanisms of the
weakened immune reactions in GC based on the overexpres-
sion of Foxp3 (31). Yuan et al (32) demonstrated a mechanism
by which tumor-infiltrating Tregs with increased Foxp3
expression mediate immune suppression via COX-2/PGE2
production in the GC microenvironment. Furthermore, Tregs
with higher levels of Foxp3 were able to suppress the prolif-
eration of autologous CD4+CD25-T cells. The suppression
of the effector T-cell response was reversed by COX inhibi-
tors and PGE2 receptor-specific antagonists. In 2011, Yuan
et al (33) performed further research on Tregs in GC and
found that GC cells induce the development of Tregs via the
production of TGF-β, by which the existence of cross-talk
between the tumor and immune cells may regulate antitumor
immune responses. Our research first confirmed that the
expression of Foxp3 in tumor-infiltrating T lymphocytes was
higher in the GC tissues compared with normal tissues. We
also identified links between MCs and Foxp3, providing a
new strategy of targeting Tregs and Foxp3. As mentioned, GC
cells induce human CD4+Foxp3+ Tregs through the produc-
tion of TGF-β (34), and as MCs secrete TGF-β, we speculate
that one of the ways in which MCs affect Tregs is their
secretion of TGF-β, thus explaining the correlation between
MCs and Foxp3 at the molecular level, and providing a new
support and research direction for the immunosuppressive
effects of MCs.
In conclusion, our results reveal that the frequency of MCs
and the level of Foxp3 are increased in tumors compared with
normal tissues. The significant correlation between MCs and
Foxp3 may be considered to support the hypothesis that MCs
play a role in immunosuppression in GC and may be, at least
partially, responsible for their prognosis. These results are
significant and may provide promising clinical treatments for
cancer, in at least in three aspects. First, these findings show
the significance of MCs in GC and provide a probable mecha-
nism by which MCs affect GC development, thus providing
references for the application of MC-regulating drugs. Second,
there are few studies concerning the correlation between MCs
and Foxp3 and the present study linked them and provided
new insights into the mechanism of immune suppression.
Third, we confirmed the close correlation between MCs and
Tregs, making a foundation for the further study of the detailed
mechanisms. Therefore, further studies should be performed
to explore the mechanism of the correlation between MCs,
Foxp3 and GC.
This study was supported by the Natural Science Foundation
of Hubei Province and the Biological Targeted Therapy
Laboratory Foundation of Hubei Province (02.03.2011-6).
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