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Correlationship of elevated FOXP3 expression with increased
lymph node metastasis of gastric cancer
WANG Li-hong, SU Lin, WANG Jing-tong
Department of Gastroenterology, Peking University People's Hospital Beijing, 100044,
China
correspondence author:
WANG Jing-tong: Department of Gastroenterology, Peking University People's
Hospital Beijing, 100044, China
telephone number :13910632219
fax number:88324473
E-mail:[email protected]
1
Original article
Correlationship of elevated FOXP3 expression with increased
lymph node metastasis of gastric cancer
WANG Li-hong, SU Lin, WANG Jing-tong
Department of Gastroenterology, Peking University People's Hospital Beijing, 100044,
China (WANG Li-hong, SU Lin, WANG Jing-tong)
Key words: FOXP3, gastric cancer, metastasis, prognosis
Background FOXP3 was thought to express in the T-cell lineage exclusively until
recently when FOXP3 was shown to be expressed by cancer cells. It was indicated
that FOXP3 may play a wider role in biology by endowing tumor cells with immune
suppressive activity. However, researches between FOXP3 and lymph node metastasis
of gastric cancer were relatively infrequent, so the present work was aimed to
investigate the relationship between FOXP3 expression and lymph node metastasis in
human gastric cancer.
Methods A total of 122 gastric cancer patients were enrolled in this study, and gastric
tumor specimens and lymph nodes were acquired. Thirty patients who had chronic
superficial gastritis diagnosed by gastroscopy contemporaneously in the Peking
University People's Hospital were chosen randomly as the control
group.Immunohistochemistry was performed to evaluate FOXP3 expression. A
survival analysis on the 122 patients was then performed. Then, NCI-N87 cell lines
was used to confirm FOXP3 expression in gastric carcinoma cells. Finally, evaluation
of FOXP3 expression in gastric tumor and peritumor tissues in 12 patients were
conducted using immunohistochemistry and Western blot. A χ2 test or Fisher exact
test (bilateral) was conducted to compare the percentage of positive percentage
staining between groups. Kaplan-Meier analysis was performed for survival analysis.
Results FOXP3 was expressed by gastric cancer cells and peritumor epithelial cells.
FOXP3 expression was increased in primary tumors （58.2%）than that in control
group（26.7%）. In the lymph-node metastasis group, the incidence of lymph node
metastasis which was less than 60% had a significant upregulation of FOXP3 in
primary tumors and lymph nodes. However, the frequency of FOXP3 expression had
no relationship with survival.
Conclusion FOXP3 probably has a relationship with lymph node metastasis of gastric
cancer.
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FOXP3与胃癌临床病理特点的关系分析
背景：FOXP3曾被认为仅在T细胞系中表达。目前的研究表明，FOXP3可在癌细胞
中表达,在肿瘤细胞的免疫抑制过程中可能起重要作用。目前关于FOXP3与胃癌淋
巴结转移关系的研究较少。本文的目的在于探讨FOXP3的表达与胃癌淋巴结转移
之间的关系。
方法：收集北京大学人民医院经手术切除的122例胃癌患者的原发灶和淋巴结标
本，以及30例同时期经胃镜活检并经病理证实为慢性浅表性胃炎患者的胃黏膜标
本做对照，行FOXP3免疫组织化学染色。对胃癌患者进行随访，并进行生存分析。
然后，用NCI-N87细胞系证明FOXP3在胃癌细胞中的表达。最后选取12例胃癌患者
的原发灶及癌旁组织，用免疫组织化学染色及Western blot检测FOXP3的表达。
组间比较采用χ2检验或Fisher精确概率法（双侧），生存分析采用Kaplan-Meier
法。
结果：FOXP3在胃癌细胞和癌旁上皮细胞中表达。FOXP3在胃癌原发灶（58.2%）
中的表达较无瘤胃黏膜（26.7%）中的表达上调。有淋巴结转移的患者，不论其
原发灶还是淋巴结，淋巴结转移率小于60%的FOXP3的表达都呈现上调趋势。然而
单因素分析显示胃癌原发灶及林巴结中FOXP3的表达不是影响生存期的主要因
素。
结论：FOXP3可能与胃癌淋巴结转移相关
关键词：FOXP3；胃癌；转移；预后
3
T Regulatory Cells (Tregs) play an important role in the suppression of tumor
immunity and induction of dominant transplantation tolerance [1]. Although the
mechanisms of immunosuppression by Tregs are still unclear, it has been reported that
Tregs can inhibit the function of effector T cells indirectly, via secretion of
immune-suppressive cytokines [2,3]. Tregs accumulated in tumors has been
demonstrated in several human cancers and in some cases they were correlated with
reduced survival [4-8]. In several malignancies, the quantity of Tregs in
tumor-infiltrating lymphocytes (TILs) is significantly higher than that in normal
tissues [6,8-11]. Recently, in murine models, many studies have shown that depletion or
inhibition of Tregs can enhance anti-tumor immunity[12,13].
FOXP3, a member of the forkhead family of transcription factors, is widely accepted
as the best marker for Tregs identification in human [14,15]. It is critically involved in
the development and function of CD25+ regulatory T cells [16,17].
FOXP3 was thought to express in the T-cell lineage exclusively until recently, when
FOXP3 was shown to be expressed by cancer cells: in melanoma, glioma, et al [18-21].
It was indicated that FOXP3 may play a wider role in biology by endowing tumor
cells with immune suppressive activity. However, researches between FOXP3 and
gastric cancer were relatively infrequent.
Despite the worldwide decline in incidence and mortality of gastric carcinoma along
the second half of the 20th century, it is still the 4th most common cancer and the 2nd
leading cause of cancer-related deaths in the world. Prognosis of gastric carcinoma
patients depends on several variables, of which lymph node metastasis is one of the
most important reasons . The objective of this study was to ascertain whether FOXP3
expression in gastric cancer cells, then to investigate the correlationship between
FOXP3 expression and gastric cancer lymph node metastasis and prognosis.
METHODS
Patients
A total of 122 gastric cancer patients confirmed by pathology at the Peking University
People's Hospital were enrolled from Jan 2000 to Dec 2004. The mean age was
62.1±14.3 years old. Among them, eighty-nine patients were men and 33 patients were
women. None had previously received radiotherapy, chemotherapy or other medical
interventions before surgery. All patients were followed up until December 2009. Of
the 122 patients, 44 died, 15 survived and 63 patients were lost to follow-up. The
control group comprised of contemporaneous 30 patients who had chronic superficial
gastritis diagnosed by gastroscopy in the Peking University People's Hospital
randomly(simple random sampling). They were 59.3±10.4 years old (mean±SD).
Among them, 16 patients were men and 14 patients were women. We obtained all 122
patients’ primary tumors and lymph nodes(87 had regional lymph node metastasis) and
30 patients’ non-neoplastic gastric mucosa. The tumor and peritumor tissues of 12
patients (2 cm away from the tumor) were also acquired, for paraffin-embedding and
for storage at -80°C. This study was approved by the ethics committee of the Peking
University, and written informed consent was obtained from all patients at study entry.
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Gastric cancer culture
NCI-N87 gastric carcinoma cells were preserved by Institute of Clinical Molecular
Biology, Peking University People's Hospital. Cells were cultured in culture dish. and
maintained in Dulbecco's Modified Eagle Medium/Ham's F12 (1:1, v/v) culture
medium supplemented with 10% foetal Bovine serum, biotin (8 μ g/ml),
pantothenate (4 μ g/ml), and penicillin/streptomycin (10 μ l/ml) at 37ºC in a
humidified atmosphere with 5% CO2.
Immunohistochemistry
Paraffin-embedded, formalin-fixed gastric cancer tumor tissues, peritumor tissues,
lymph nodes, and healthy control gastric mucosa tissues were cut into 4-µm sections
and placed on polylysinecoated slides. FOXP3 staining was conducted using the
avidin-biotin-peroxidase complex method. Briefly, each paraffin section was
deparaffinized, followed by antigen retrieval with Epitope Retrieval Solution (10
mmol citrate buffer (pH 6.0)) in a pre-heated water bath at 98°C for 10min, and
endogenous peroxidase was blocked using 3% hydrogen peroxide. Subsequently,
sections were incubated overnight with the diluted (1:100 in PBS,
Biolegend)biotinylated mouse antihuman FOXP3 antibody in a humidified chamber
at 4°C. Thereafter, the sections were incubated with streptavidin conjugated
horseradish peroxidase (Zymed) for 30 min, followed by development with
3,30-diaminobenzidine (Zymed) for 5 min, and counter-staining with hematoxylin.
Negative control was performed by using appropriate serum controls for the primary
antibody, colon carcinoma tissue was used as positive control. Tissue sections were
counterstained with haematoxylin and permanently mounted.
NCI-N87 Cell climbing film were made, FOXP3 staining was conducted using the
avidin-biotin-peroxidase complex method (FOXP3 antibody diluted by PBS, 1:200,
Biolegend).
Quantitative evaluation of FOXP3
Semi-quantitative method was used. Under ordinary optical microscope, five different
perspectives were randomly selected at a magnification of 400. The percentage of
positive cells were calculated, 0%, 10%, 10-15%, 51-80% and >80% positive cells
were marked as 0, 1, 2, 3, and 4, respectively. Staining intensity was observed . No
coloring, slightly yellow, brown yellow and tan stains were marked as 0,1, 2 and 3,
respectively. Finally, the product of staining intensity and positive cell percentage
points were calculated. ≤ 3 was defined as negative, and ≥ 4 as positive. Reviews of
pathological films and staining points were conducted by two pathologists blinded to
the clinical details.
Western blot
Whole-cell lysates of tumor and peritumor tissues were prepared by sonication in
RIPA lysate containing protease inhibitors. Samples were heated to 100 ℃, and 60 µl
were subjected to SDS-PAGE using 12% Bis-Tris gels then transferred to
Immobilon-P membranes. The membranes were blocked with 5% non fat dry milk in
1TBS-T, then probed with 1 µl/ml anti-FOXP3 (Abcam) overnight at 4°C, followed
by a 1/2000 dilution of secondary antibody, goat anti-rabbit-Ig–HRP conjugate (Santa
Cruz) for 1 h at room temperature. Whole-cell lysates of human PBMC was positive
5
control. The bands were analyzed with Quantityone software. Subsequently,
membranes were washed in TBS-T and re-probed with rabbit antibody to β-actin
(Santa Cruz), as described above.
Statistical analysis
A χ2 test or Fisher exact test (bilateral) was conducted to compare the percentage of
positive percentage staining between groups. The Spearman correlation test was
conducted to evaluate relationships. Kaplan-Meier analysis was performed for
survival analysis. Test level α = 0.05, a P value less than 0.05 was considered
statistically significant. All statistical analyses were performed with SPSS 13.0.
RESULTS
FOXP3 was expressed by gastric cancer cells and peritumor epithelial cells
Samples were performed by immunohistochemistry, including 30 non-neoplastic
gastric mucosas, 122 primary tumors and lymph nodes, another 12 patients’ primary
tumors and peritumor tissues. In addition to the expected staining of Tregs in gastric
cancer tissue, we found that some tumor cells and most peritumor epithelial cells
express FOXP3. To exclude false positive results, firstly, we repeated the experiment
twice and obtained identical results including negative and positive controls. Then, we
use NCI-N87 cell lines to confirm FOXP3 expression in gastric carcinoma cells. We
found that FOXP3 was expressed in the nucleus of Tregs and peritumor cells,while in
gastric carcinoma cells it was mainly expressed in nucleus and partly in cytoplasm.
Upregulation of FOXP3 in gastric primary tumor cells and relationship between
FOXP3 and clinico-pathologic features of gastric cancer patients
We counted FOXP3+ cells in five independent fields at 400×magnification. The
frequency of FOXP3+ cells in gastric cancer was increased compared to control
group(P = 0.001), consistent with many previous studies (Table 1). We analyzed the
relationship between FOXP3 expression in primary tumor and the clinicopathologic
characteristics, including age, gender, tumor size, localization, macroscopic
classification, degree of differentiation, TNM classification and lymph-node
metastasis, et al. We found that FOXP3 expression was related to the incidence of
lymph node metastasis (P = 0.007) exclusively (Table 2).
Differences of FOXP3 expression in lymph nodes between metastasis group and
non-metastasis group are not significant
In the metastasis group, FOXP3 expression in lymph nodes (33.3%) were higher than
that in non-metastasis group(20.0%), although the differences are not significant
statistically(P=0.144).
Frequency of FOXP3 expression in metastatic lymph nodes exhibited significant
difference with incidence of lymph node metastasis
FOXP3 expression in metastatic lymph nodes had no relationship with the clinical and
pathological features, such as tumor localization, TNM classification, and
macroscopic classification (Table 2). However, it was related to incidence of lymph
node metastasis significantly.
FOXP3 protein expression between tumor tissues and peritumor tissues was not
significant
Western blot was used to detect FOXP3 protein expression in gastric tumor tissues
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and peritumor tissues. There was no significant difference statistically(P = 0.349,
Figure 2).
FOXP3 expression in primary tumors and lymph nodes had no relationship with
survival
Fifty-nine patients’ follow-up data were aquired. Survival analysis was performed on
59 patients and the result confirmed that the frequency of FOXP3 in the primary
tumor (P = 0.610) and lymph nodes (P = 0.588) had no relationship with survival
(Figure 3) .
DISCUSSION
It is previously believed that FOXP3 expression is strictly limited to Tregs. Recently,
some studies have shown that some solid tumor cells also expressed FOXP3. Some
express in the nucleus and some in cytoplasm. In the present study, we found that
FOXP3 was not only expressed in Tregs nucleus, but also expressed in gastric
carcinoma cytoplasm, nucleus and peritumor cytoplasm.
In this study, upregulation of FOXP3 expression in gastric primary tumor was
significantly higher than that in the control group, as previous studies have shown.
Upregulation of FOXP3 played an important role in the tumorigenesis of gastric
cancer. It confirmed the hypothesis that FOXP3 do goods to help tumor escape from
immuno-surveillance.
Kono et al. showed that increased proportion of FOXP3 positive cells was
significantly restored to levels comparable to normal healthy donors [22], following
curative resection for gastric cancer. These strongly suggested that the tumor-related
factors could induce and expand FOXP3 positive cells accumulation in cancer-bearing
hosts, and the mechanism of which needs to be clarified. The tumor
microenvironment is comprised of many agents, such as H.pylori [23], PGE2 and
COX-2 [24] which may contribute to the accumulation of FOXP3 positive cells. More
attention should be paid to the microenvironment of tumor tissues, which can help
prevent accumulation of FOXP3 positive cells. They also indicated that
CD4+CD25highFOXP3+ cells were increased with tumor stage. Some other studies
had also shown that elevated quantity of intratumoral FOXP3 positive cells were
related to the unfavorable outcomes in gastric cancer patients [25,26]. However, in our
study we found that upregulation of FOXP3 in gastric primary tumors had no
relationship with clinicalpathological features except incidence of metastasis of lymph
node. We also found that, in the metastatic lymph nodes, FOXP3 expression had no
relationship with clinicalpathological features except incidence of metastasis of lymph
node either.Our results demonstrated that FOXP3 might have a relationship with the
progress of lymph node metastasis. The exact mechanism are not clear.
Shen et al . showed that higher intratumoral infiltrated Foxp3+ Treg numbers are
assosiated with adverse prognosis in resectable gastric cancer[27].Our results show that
FOXP3 positive cells neither in gastric primary tumor nor in lymph node had no
significant impact on survival, suggesting that it may not be an important independent
factor which impacts prognosis. Previously, Haas et al. reported that stromal
regulatory T-cells were associated with favorable prognosis in gastric cancer of the
cardia[28]. As such, FOXP3 may plays different roles in different gastric cancers.
7
In conclusion, FOXP3 may play an important role in the process of malignant gastric
tumorigenesis. The mechanism of FOXP3 accumulation in tumor cells needs to be
further elucidated.
Figure 1
(A) FOXP3 expressed in Tregs infiltrating in the tumor stroma (Black arrow).
(B) FOXP3 expressed in the cytoplasma of poorly differentiated (B1) and moderately
differentiated (B2) tumor cells (Black arrow).
(C) FOXP3 expressed in the cytoplasm of peritumor epithelial cells (Black arrow).
(D) Normal control(tissue).
Figure2. FOXP3 protein expression in tumor tissues (T) and peritumor tissues (P).
Figure3. Survival analysis of FOXP3 in the primary tumors and lymph nodes
Table 1. FOXP3 expression in primary gastric tumors compared to the control group.
Foxp3
Control group
Primary tumour
n
positive
P
30
122
8（26.7%） 0.002
71
（58.2%）
8
Table 2. Relationship between FOXP3 of primary gastric tumors and the clinicopathological
characteristics. Relationship between FOXP3 expression of lymph nodes and the clinicopathological
characteristics in the lymph node metastasis group.
Clinico-pathological data
Age
≤60
＞60
Gender
female
male
Tumor size(cm)
＜4
≥4
Tumor localisation
cardia
body
pylorus
Macroscopic classification
Early gastric cancer
Advanced gastric cancer
Primary tumor
Lymph node
group.
n
positive
metastasis
n
positive
P
48
74
30(62.5%)
41(55.4%)
0.438
35
52
11(31.4%)
18(34.6%)
0.757
36
86
23(63.9%)
48(55.8%)
0.410
25
62
10(40.0%)
19(30.6%)
0.402
39
83
26(66.7%)
45(54.2%)
0.194
17
70
9(52.9%)
20(28.6%)
0.054
21
29
72
10(47.6%)
19(65.5%)
42(58.3%)
0.448
17
20
50
6(35.3%)
3(15.0%)
20(40.0%)
0.132
9
113
5(55.6%)
66(58.4%)
0.867
3
84
1(33.3%)
28(33.3%)
0.743
5(50.0%)
26(63.4%)
40(56.3%)
0.658
7
26
54
2(28.6%)
10(38.5%)
17(31.2%)
0.794
26(61.9%)
45(56.3%)
0.547
9
78
3(33.3%)
26(33.3%)
0.655
24(68.6%)
47(54.0%)
0.141
30(68.2%)
17(39.5%)
0.007
44
43
19(43.2%)
10(23.3%)
0.049
Degree of differentiation
well- differentiated
10
Moderately- differentiated
41
Poor- differentiated
71
TNM classification
42
Ⅰ+Ⅱ
80
Ⅲ+Ⅳ
Lymph-node metastasis
absent
35
present
87
Incidence of lymph node metastasis
＜60%
44
≥60%
43
P
the number of FOXP3 positive lymph node
*Incidence of lymph node metastasis= the number of total lymph node obtained by surgery
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