Journal of Cancer Therapy, 2013, 4, 49-53 Published Online January 2013 (
Expressions of CCR7 and CXCR4 Are Associated with
Differentiation in Gastrointestinal Cancer
Chunhui Lu, Shiwen Chen, Feng Xu, Yiwen Chen, Qing Zhang, Yong Li*
Department of Oncology, Gongli Hospital, Shanghai, China.
Email:,,,,, *
Received November 28th, 2012; revised December 30th, 2012; accepted January 9th, 2013
Purpose: The chemokine receptors CCR7 and CXCR4 have been shown to play an important role in cancer invasion
and metastasis. This study was aimed to investigate CCR7 and CXCR4 expressions and evaluate the association be-
tween their expressions and the clinicopathological features in gastrointestinal cancer. Method: 27 paired tissue sam-
ples from patients who had curative surgery for gastrointestinal cancer were obtained. Quantitative real-time PCR, im-
munochemistry assay and western blot analysis were carried out to investigate the expressions of CCR7, CXCR4 ex-
pressions in gastrointestinal cancer. Results: The cancer tissues expressed significant higher level of CCR7 (P = 0.000)
and CXCR4 (P = 0.000) protein than the adjacent normal mucosa. Expressions of CCR7 (P = 0.002) and CXCR4 (P =
0.003) protein in cancer tissues exhibited significant correlation with differentiation in gastrointestinal cancer. Conclu-
sion: Expressions of CCR7 and CXCR4 protein were associated with differentiation in gastrointestinal cancer. CCR7
and CXCR4 may be predictive factors for poor prognosis in patients with gastrointestinal cancer.
Keywords: Chemokine Receptor; CCR7; CXCR4; Gastrointestinal Cancer
1. Introduction
Chemokines belong to small-molecule chemoattractive
cytokine family and are categorized into four groups
(CXC, CC, CX3C, and C) [1-3]. Usually, chemokines are
molecules that are structurally and functionally similar to
growth factors. They bind to G-protein-coupled receptors
on leukocytes and stem cells, and work through guanine-
nucleotide-binding (G) proteins to initiate intracellular
signaling cascades that promote migration towards the
chemokine source [1-3].
Chemokine receptors are seven-transmemberane re-
ceptors coupled to G-proteins, all with their N-terminus
outside the cell surface, three extracellular and three in-
tracellular loops as well as a C-terminus in the cytoplasm.
One of the intracellular loops of the chemokine receptors
couples with heterotrimeric G-proteins, and that mediate
ligand binding to the receptor which initiates signal
transduction cascade [4].
To date, at least 20 chemokine receptors (CCR1-11,
CXCR1-7, XCR1, and CX3CR1) have been identified.
Chemokines and their receptors have been known to play
important roles in inflammation, infection, tissue injury,
allergy, cardiovascular diseases, and malignant tumors
Perhaps one of the most important roles that chemokines
and the chemokine receptors have is in regulating metas-
tasis. Chemokine receptors may potentially facilitate tu-
mor dissemination at each step of metastasis, including
adherence of tumor cells to endothelium, extravasation
from blood vessels, metastatic colonization, angiogenesis,
proliferation, and protection from the host response via
activation of key survival pathways [6,7].
Recent study also indicated that different cancers ex-
press varying combinations of CC and CXC chemokine
receptors. Chemokine receptors may direct lymphatic
and hematogenous spread and may additionally influence
the sites of metastatic growth of different tumors [8]. The
mechanisms involved in lymph node metastasis haven’t
been fully understood, whereas, more and more evidence
has showed a correlation between chemoreceptors CCR7,
CXCR4 and various types of malignancy. High expres-
sion of CCR7 and CXCR4 are often associated with
faster progression and poorer prognosis of the malignant
In gastrointestinal cancer, Previous studies have showed
these two receptors might increase the metastatic pheno-
type and the risk of poor survival [9,10]. However, in-
stead of pure laboratory study on gastro-colorectal cancer
cell lines, few reports had focused on the CCR7, CXCR4
expressions in clinical work. Therefore, in this study, we
*Corresponding author.
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Expressions of CCR7 and CXCR4 Are Associated with Differentiation in Gastrointestinal Cancer
investigated CCR7 and CXCR4 expressions in gastro-
colorectal tumor specimens to evaluate the association
between their expressions and the clinicopathological
features of gastrointestinal cancer.
2. Patients and Methods
2.1. Patients Enrollment and Tissue Samples
This study was approved by the Research Ethics Com-
mittee of Gongli Hospital, Shanghai Pudong New Area.
China. Written informed consent was obtained from all
of the patients enrolled in this study. All specimens were
handled and made anonymous according to the ethical
and legal standards.
In this study, paired tumour specimens were obtained
from 27 patients who underwent radical surgery for gas-
tro-colorectal cancer in our hospital from July to De-
cember 2010. The baseline characteristics of enrolled
patients were as showed in Table 1. Tumor specimens
were obtained at the time of surgery and reserved in
pathological laboratory in a 80˚C refrigerator. None of
the patients had received radiotherapy or chemotherapy
prior to surgery.
2.2. Immunohistochemistry Assay
Sections of 4-um thickness were obtained from repre-
sentative central and para-tumor areas of each tumor
specimen and were mounted on to glass slides for im-
munostaining. Briefly, after being sealed in goat serum
for 20 minutes the sections were incubated with mouse
CCR7 (abcam®, ab32527) and CXCR4 (abcam®, ab2074)
antibody at 4˚C overnight, then with horseradish peroxi-
dase-labled antimouse immunoglobulin (Sigma®,
A6154) for 20 minutes, followed by incubation with
0.05% 3,39-diaminobenzidine tetrahydrochloride solu-
tion at 37˚C for 1 hour. Finally, the slides were counter-
stained with Mayer’s hematoxylin and mounted in an
aqueous mounting medium. At each step, the slides were
washed carefully in phosphate-buffered saline (pH 7.4). The
Immunohistochemistry results were divided into 5 grades
including negative and positive (+~4+). The results were
evaluated by 2 pathologists independently. There was the
Table 1. Baseline characteristics of enrolled patients.
Clinical grade
II (B-C1) III (C2) P
Age 59.47 ± 9.52 55.63 ± 14.120.414
Male 9 4
Gender Female 10 4 0.983
Poor 7 4
Middle 7 3
Well 5 1
additional third pathologist for judgments in case of the
former 2 pathologists holding diverse opinion. The fol-
lowing were judgment criteria for immunohistochemistry
assay: 1) Cells with buffy cytoplasma were recognized as
positive staining; 2) One central and four corner of 10 ×
10 visual field of every section would be observed to
counting the positive cells for grading; 3) The positive
cell ratio of 0% - 20%, 20% - 40%, 40% - 60%, 60% -
80% and 80% - 100% was regarded as Grade Negative
and Grade +~4+ repectively.
2.3. Western Blot Analysis
Western blotting was also used to detect CCR7 and
CXCR4 protein. The whole specimens were separated
into central and para-tumor areas and sonicated re-
spectively. The cells were collected by centrifugation,
washed in phosphate-buffered saline (PBS), and lysed by
the addition of SDS sample buffer [62.5 mM Tris-HCl
(pH 6.8), 6% (w/v) SDS, 30% glycerol, 125 mM DTT,
and 0.03% (w/v) bromophenol blue]. Equal amounts of
protein from each sample were electrophoresed on 10%
SDS-polyacrylamide gels and transferred to nitrocellu-
lose membranes. The membranes were blocked for 1
hour with Tris-buffered saline (TBS) containing 5% (w/v)
milk and 0.1% Tween, and then incubated with the pri-
mary antibody CCR7 (abcam®, ab32527) and CXCR4
(abcam®, ab2074) overnight at 4˚C. The blots were
washed with TBS containing Tween, incubated with hor-
seradish peroxidase-labled antimouse immunoglobulin
(Sigma®, A6154) for 1 hour at 37˚C, then add ECL
solution to record the image.
2.4. Realtime-PCR
200 mg separated central or para-tumor sample from
each sonicated specimen was weighed to extracting total
RNA (Sangon total RNA extracting kit-SK1352, China)
for realtime-PCR on PRISM®7900HT. Every sample was
tested 3 times and the average value was calculated as
the results. The primers and fluorescent probes were de-
signed and synthesized by Sangon Biotech (Shanghai)
CO. Ltd. PCR was performed under the following con-
ditions: an initial cycle of denaturation at 94˚C for 2
minutes, followed by 21 - 23 cycles of denaturation at
92˚C for 45 seconds; annealing at 60˚C for 60 seconds;
extension at 72˚C for 60 seconds; and a final extension at
72˚C for 5 minutes. The sequences for qRT-PCR primers
were as follows:
A-3’; reverse, 5’-GCTGAGACAGCCTGGACGAT-3’;
Copyright © 2013 SciRes. JCT
Expressions of CCR7 and CXCR4 Are Associated with Differentiation in Gastrointestinal Cancer 51
2.5. Statistical Analysis
The quantitative data were measured as average and
standard deviation. The association between the variables
was tested using the chi-square test or T-test (quantitative
data). Probability values of <0.05 were considered stati-
stically significant. The statistic analysis was completed
with PASW Statistics 18 Software.
3. Results
3.1. CCR7 and CXCR4 Protein Expression in
Gastro-Colorectal Cancer
Of all 27 paired specimens, CCR7 and CXCR4 protein
were both detected in central and para tumor tissue by
either immunohistochemistry assay (Figure 1) or western
blot analysis (Figure 2). The central-tumor tissue ex-
pressed significant higher level of CCR7 (χ2 = 47.455, P =
Figure 1. Immunohistochemistry stain of CCR7 and
CXCR4 in gastro-colorectal cancer (×200). Positive expres-
sion of CCR7 in gastro-colorectal cancer with immunos-
taing (A,B); Positive expression of CXCR4 in gastro- colo-
rectal can c e r with immunostaing (C,D).
Figure 2. Expression of CCR7 and CXCR4 proteins in
gastro-colorectal cancer tissues and adjacent normal mu-
cosa by Western blot analysis. No significance was found
between expression of CCR7 and CXCR4 proteins in
gastro-colorectal cancer tissues and adjacent normal mu-
cosa. “N” refers to adjacent normal mucosa; “T” refers to
gastro-colorectal cancer tissues.
0.000) and CXCR4 (χ2 = 47.600, P = 0.000) protein than
para-tumor tissue. However, CCR7 and CXCR4 protein
expression between Stages II and III patients showed no
variance (Table 2).
While comparing patients with various tumor differ-
entiation levels, the CCR7 and CXCR4 protein ex-
pression of central tumor samples exhibited significant
variance as demonstrated in Table 3.
3.2. CCR7 and CXCR4 mRNA Expression in
Gastro-Colorectal Cancer
The realtime-PCR showed no mRNA expression differ-
ence between central and para tumor tissue for CCR7
(2.215 ± 0.462 vs. 1.962 ± 0.660, P = 0.109) and CXCR4
(1.543 ± 0.836 vs. 1.483 ± 1.197, P = 0.832). Based on
clinical stage and tumor differentiation level, the statistic
analysis showed none of them was a factor associated
with CCR7 and CXCR4 mRNA expression (Table 4).
4. Discussion
This study analyzed the chemokine receptor CCR7,
CXCR4 expressions in small series of human gastro-
colorectal cancer specimens. Our results showed both
CCR7 and CXCR4 protein expressions were signifi-
cantly higher in cancer tissues than in adjacent normal
tissues. Furthermore, CCR7 and CXCR4 protein ex-
pressions were significantly lower in better-differentiated
tumor. This finding demonstrated that CCR7 and CX-
CR4 are involved in gastro-colorectal cancer progression
just as in many other malignancies, meanwhile the fact
that their expressions are associated with tumor diffe-
rentiation also indicated CCR7 and CXCR4 may be
predictive factors for faster progression and poorer prog-
nosis of the diseases.
Our study also revealed that the protein and mRNA
expressions of CCR7, CXCR4 showed no difference
among patients with different stage. This finding does
not mean the denial of commonly accepted opinion of the
relevance of CCR7, CXCR4 expression with clinical
stage in various types of cancer. For example, Schi-
manski reported that CXCR4 in colorectal cancer was
significantly associated with advanced UICC tumor
stages [11]. The aim of our study was restrictively con-
fined to candidates who were suitable for radical opera-
tion. That means all enrolled patients were stage II or III
for gastric cancer and stage Duke’s B-C1 or C2 for colo-
rectal cancer and with no distant metastasis. In other
words, the tumor progression was at similar level. Theo-
retically, if surveying a wider stage of the cancer at the
time of diagnosis, the results might be different.
The CCR7 and CXCR4 have been considered as pos-
sible targets of anti-cancer drugs. Luo has reported that
the blockage of CXCR4-SDF1 combination can inhibit
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Expressions of CCR7 and CXCR4 Are Associated with Differentiation in Gastrointestinal Cancer
Copyright © 2013 SciRes. JCT
Table 2. Grading of CCR7 and CXCR4 protein expression between different clinical stage.
Central-tumor Para-tumor
Clinical Stage II (B-C1) III (C2) χ2, P II (B-C1) III (C2) χ2, P
+ 0 0 17 (62.96%) 8 (29.63%)
2+ 6 (22.22%) 3 (11.11%) 2 (7.41%) 0
3+ 7 (25.93%) 4 (14.81%) 0 0
4+ 6 (22.22%) 1 (3.70%)
0 0
+ 0 0 17 (62.96%) 8 (29.63%)
2+ 6 (22.22%) 2 (7.41%) 2 (7.41%) 0
3+ 8 (29.63%) 5 (18.52%) 0 0
4+ 5 (18.52%) 1 (3.70%)
0 0
Table 3. Grading of CCR7 and CXCR4 protein expression among various tumor cell differentiation.
Central-tumor Para-tumor
Differentiation Poor Middle Well χ2, P Poor Middle Well χ2, P
+ 0 0 0 10 9 6
2+ 0 5 4 1 1 0
3+ 4 5 2 0 0 0
4+ 7 0 0
0 0 0
+ 0 0 0 10 9 6
2+ 0 4 4 1 1 0
3+ 5 6 2 0 0 0
4+ 6 0 0
0 0 0
Table 4. CCR7, CXCR4 mRNA expression based on clinical stage and differentiation level.
Clinical stage
II (B-C1) III (C2)
χ2, P
central 2.171 ± 0.484 2.319 ± 0.417 0.228, 0.633
para 1.939 ± 0.748 2.017 ± 0.417 0.477, 0.490
central 1.537 ± 0.920 1.558 ± 0.648 0.282, 0.595
para 1.669 ± 1.377 1.042 ± 0.363 3.454, 0.063
Differentiation level
Poor middle well
χ2, P
central 2.158 ± 0.593 2.155 ± 0.273 2.419 ± 0.458 1.137, 0.566
para 1.806 ± 0.442 1.990 ± 0.821 2.200 ± 0.733 1.673, 0.433
central 1.769 ± 1.127 1.324 ± 0.551 1.494 ± 0.579 0.623, 0.732
para 1.585 ± 1.711 1.207 ± 0.544 1.757 ± 0.891 2.019, 0.364
tumor cell growth and metastasis [12]. Our study found
the deviation in expression of CCR7 and CXCR4 at pro-
tein level did not exhibit at mRNA level as well among
various differentiation cancers. This phenomenon indi-
cated that the difference in translation of mRNA might
be a key step for the variant expression of CCR7 and
Expressions of CCR7 and CXCR4 Are Associated with Differentiation in Gastrointestinal Cancer 53
CXCR4. For anti-cancer therapy, inhibiting the transla-
tion of CCR7, CXCR4 mRNA may be another effective
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