Expressions of CCR7 and CXCR4 Are Associated with Differentiation in Gastrointestinal Cancer
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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
Differentiation
level
Well 5 1
0.696
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:
CCR7 forward, 5’-CTTCTTCAGTGGCATGCTCCT-
A-3’; reverse, 5’-GCTGAGACAGCCTGGACGAT-3’;
CXCR4 forward, 5’-CAGTGGCCGACCTCCTCTT-3’;
reverse, 5’-CAGTTTGCCACGGCATCA-3’; GAPDH for-
ward, 5’-CACATGGCCTCCAAGGAGTAAG-3’; re-
verse, 5’-TGAGGGTCTCTCTCTTCCTCTTGT-3’.
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