The co-expression of MMP-9 and Tenascin-C is significantly associated with the progression and prognosis of pancreatic cancer
Xu et al. Diagnostic Pathology
The co-expression of MMP-9 and Tenascin-C is significantly associated with the progression and prognosis of pancreatic cancer
Yingqiang Xu 0 2
Zhonghu Li 0 2
Peng Jiang 2
Guo Wu 2
Kai Chen 2
Xi Zhang 2
Xiaowu Li 1 2
0 Equal contributors
1 No. 30 Gaotanyan Street, Shapingba District, Chongqing 400038 , China
2 Department of Hepatobiliary Surgery Institute, Southwest Hospital, Third Military Medical University , Chongqing , China
Background: Matrix metalloproteinase-9 (MMP-9) and Tenascin-C (TN-C) have been shown to be involved in the metastasis of many tumors. The purpose of this study was to determine the relationship between the co-expression of these two molecules and the clinical prognosis of pancreatic cancer. Methods: We investigated the expression of TN-C and MMP-9 in 103 pancreatic cancer tissues by immunohistochemistry and used statistical analyses to investigate the correlations of individual expression or coexpression of these two molecules with clinicopathological parameters and survival of pancreatic cancer. Results: The expression of MMP-9 and TN-C were increased in pancreatic cancer. The co-expression of MMP-9 and TN-C was also detected. The expression of MMP-9 and TN-C were correlated with vascular invasion, lymph node invasion, liver metastases and TNM stage. The co-expression of MMP-9 and TN-C was significantly related to the pancreatic cancer metastases. The individual overexpression of MMP-9 or TN-C significantly decreased the overall survival rates. The co-expression of MMP-9 and TN-C had the lowest overall survival rates. The co-expression of MMP-9 and TN-C was an independent predictor of survival for pancreatic cancer patients. Conclusions: Co-expression of MMP-9 and TN-C was associated with poorer prognosis and was found to be an independent predictor of survival.
MMP-9; TN-C; ECM; Prognosis; Pancreatic cancer
Pancreatic cancer is one of the most aggressive and
metastatic malignant tumors [
]. Previous studies have
shown that various extracellular matrix (ECM) proteins
are involved in the cancer progression and prognosis.
However, the role of the ECM proteins on the prognosis
of patients with pancreatic cancer remains unclear.
The ECM is well known for playing a key role in
inflammation, lesions, tissue repair, tumor invasion and metastasis
]. Numerous proteins that constitute the
ECM show a close association with tumor tissue, such as
Tenascin-C (TN-C) and Matrix metalloproteinases
]. TN-C is a complex multifunctional protein,
which can influence cellular behavior directly via cell
surface receptors or indirectly by binding to other matrix
]. This induces angiogenesis and promotes cell
migration . The fibrillar TN-C (fTN-C) is primarily
expressed in tumor extracellular matrix, and fTN-C matrix
formation requires the participation of MMPs and may play
a role in promoting cancer metastasis [
overexpression can impact the progression and prognosis of
various malignant tumors, including glioma , gastric cancer
], breast carcinoma [
], and Merkel cell carcinoma
]. However, the role of TN-C on the prognosis of
patients with pancreatic cancer remains unclear. MMPs are
a group of zinc-binding endopeptidases and can degrade
multiple components of the ECM and regulate ECM
]. MMPs are associated with cancer growth
and are considered as the prime candidates during tumor
invasion, metastasis and angiogenesis [
of the ECM is an essential step in tumor invasion and
metastasis, and the MMP-9 was able to degrade the ECM
and promote tumor cell metastasis. In previous studies,
MMPs have been shown to be involved in TN-C formation,
but the relationship of MMP-9 and TN-C in pancreatic
cancer is still not clear.
Although TN-C and MMP-9 have been thought to
be related to invasion and metastasis of pancreatic
], it is unclear whether there is a
coexpression of MMP-9 and TN-C in pancreatic cancer.
In this study, we investigated the levels of TN-C and
MMP-9 in pancreatic cancer tissues by
immunohistochemistry and analyzed the correlations of the individual
expression of MMP-9 and TN-C with clinicopathological
parameters and survival of pancreatic cancer patients
using statistical analysis methods. Then, we investigated
the relationship between the co-expression of these two
molecules and the clinical prognosis of pancreatic cancer.
A total of 103 Chinese patients (67 males and 36 females)
with a median age of 56 years (range 36-86 years)
underwent surgery at the Department of Hepatobiliary Surgery
Institute, Southwest Hospital, Third Military Medical
University, China, for pancreatic cancer from January
2007 to June 2010. All patients underwent curative
resection by pancreaticoduodenectomy and pylorus-preserving
pancreaticoduodenectomy with lymph node dissection.
Patients who had received neoadjuvant or adjuvant radio
or chemotherapy were excluded in this study! All of the
tumors were invasive ductal adenocarcinomas
histologically, including 9 (8.7 %) well-differentiated, 67 (65 %)
moderately differentiated and 27 (26.2 %) poorly
differentiated cases, 15 (14.5 %) tumors were found in head of
pancreas and 88 (85.5 %) in body. Vascular invasion and
lymph node metastasis were observed in 36 (34.9 %)
tumors and 41 (39.8 %), respectively. The number of
patients with pT1, pT2, pT3, and pT4 tumors was 17
(16.5 %), 31 (30.1 %), 51 (49.5 %), and 4 (3.9 %),
respectively. All patients were assessed by
ultrasonography, radiography and computed tomography every 3
months after discharge. New lesions detected by imaging
were considered indicative of relapse. The median
followup period was 13 months (range 3-49 months), the
median survival was 11 months. During this period, 13
patients experienced recurrence of liver disease, 1 patient
experienced recurrence of lung disease, 91 patients were
dead. In addition, 6 normal pancreatic samples were
collected from the donors for liver transplantation.
This study was approved by the Ethics Committee of
the Southwest Hospital, and all patients provided written
A total of 103 pancreatic cancer specimens were fixed in
formalin, embedded in paraffin and cut into 3-
micrometer serial sections. The deparaffinized sections were
trypsinized (0.05 % trypsin with 0.05 % Triton X-100 in TBS)
for 20 min. Slides were heated at 120 °C in an autoclave in
10 mM sodium citrate (pH 6.0) for 130 s and cooled to
room temperature. After blocked with 10 % goat serum in
Superblock, each section was incubated separately with
anti-TN-C (Sigma; HPA004823) at 20 μg/ml and
antiMMP-9 (Proteintech; 10375-2-AP) at 8 μg/ml at 4 °C for
18-24 h. The sections were incubated with anti-mouse/
rabbit immunoglobulins (Dako EnVisionTM System;
K5007) for 60 min at 37 °C. After washing four to five
times (15 min each) with Triton-TBS, the slides were
processed in the Ventana-automated stainer according to
the manufacturer's instructions. The
immunoperoxidase3, 3-diaminobenzidine-stained slides were subsequently
counterstained with hematoxylin and mounted with a
The immunostained sections were evaluated by two
independent investigators without knowledge of the
patients’ identity and clinical status. The investigators that
scored the sections are trained pathologist. Ten random
fields were selected, and expression was evaluated in
1,000 tumor or stromal cells (100 cells per field) with an
image analyzer (MetaMorph Imaging System version
6.0), then the slide were scored as 0, 1, 2, 3 and 4 if the
percentage of positive cells were less than 5 %, 6-25 %,
26-50 %, 51-75 % and 76-100 %, respectively. The
staining intensity of MMP-9 and TN-C was scored as 0, 1, 2
and 3 according to the intensity of positive staining
color. In the end, the final score was evaluated by the
multiplications of the scores of positive cells and
intensity of positive staining: 1+ (multiplication 1-4), 2+
(multiplication 5-8) and 3+ (multiplication 9-12) (Fig. 1a;
Table 1). The staining of fTN-C was further determined
based on the fibril shape of TN-C. The staining intensity
of fTN-C was positive and negative respectively (Fig. 1b).
For statistical analysis and to reduce intraobserver
variability, the immunohistochemical scores were further
grouped into the following two categories: negative
(grade 0 or 1+) or positive (grade 2+ or 3+).
All statistical analyses were performed using the IBM
SPSS Statistics 19.0 software. Group differences were
statistically analyzed using the χ2 test. Correlation of
TN-C with MMP-9 was calculated by Pearson χ2 test.
The Kaplan-Meier method was used to analyze survival
and the log-rank test was used to estimate differences in
survival. Prognostic factors were examined using
univariate and multivariate analyses, the multivariate analysis by
a Cox proportional hazards regression model with a
Forward LR method. All reported P values were two-sided,
and P < 0.05 was considered statistically significant. All
statistical analyses were completed under the guidance of
experienced experts in the Statistics Department.
The co-expression of MMP-9 and TN-C in pancreatic cancer
MMP-9 and TN-C have been associated with tumor
invasion and metastasis. To determine the relationship
between pancreatic cancer and the expression of MMP-9
“0-3”was refer to the staining score, “-”: negative, “+”: positive
and TN-C, we analyzed the expression levels of MMP-9
and TN-C in 103 pancreatic cancers and 6 normal
pancreatic samples by immunohistochemistry. We found
that the expression of MMP-9 (stain ratio: 88 %) and
TN-C (stain ratio: 74 %) was increased in most of
pancreatic cancers compared with normal pancreatic
samples (stain ratio: 0). MMP-9 and TN-C is primarily
overexpressed in the stroma but exists in part in the
cytoplasm of tumor cells. We also detected the MMP-9
and TN-C can be expressed in the same tumor region
through serial section, and identified as co-expression
(Fig. 1). Moreover, the pancreatic cancer samples that
experienced recurrence of liver metastases had a
stronger expression of MMP-9 and TN-C. Of the 103
pancreatic cancers, the expression levels of MMP-9 were high
in 61 samples and low in 42 samples, and the expression
of TN-C was high in 56 samples and low in 47 samples
(Table 1). In addition, as shown in Table 2, the
expression levels of MMP-9 were significantly correlated with
TN-C in 103 pancreatic cancer specimens (P < 0.001).
Interestingly, a tube-shaped fTN-C was found in 28
samples, primarily focused on the vascular invasion,
lymph node and liver metastases samples (Fig. 1, Table 1).
Furthermore, the co-expression of MMP-9 and TN-C was
positive in 43 samples, and 12 samples had hepatic
metastasis (Table 1). These results suggest that the
expression of MMP-9 and TN-C is increased in pancreatic cancer
and that the co-expression of these two molecules may be
associated with the distant metastasis of pancreatic cancer.
Co-expression of MMP-9 and TN-C was correlated with
lymph node metastasis, vascular invasion and hepatic
To investigate whether MMP-9 and TN-C are involved
in the metastasis of pancreatic cancer, we further
analyzed the relationship between the expression levels of
MMP-9 and TN-C and the clinical characteristics of the
pancreatic cancer patients. As shown in Table 3, the
overexpression of individual MMP-9 or TN-C was
correlated with lymph node metastasis (P = 0.019, χ2 = 5.49
and P = 0.021, χ2 = 5.23), vascular invasion (P = 0.049, χ2 =
3.87 and P = 0.024, χ2 = 5.07), hepatic metastasis (P =
0.009, χ2 = 6.47 and P = 0.003, χ2 = 8.63) and TNM stage
(P = 0.019, χ2 = 13.56 and P = 0.012, χ2 = 14.73). The
coexpression of MMP-9 and TN-C was correlated with
lymph node metastasis (P < 0.001, χ2 = 13.15), vascular
invasion (P = 0.003, χ2 = 8.53), hepatic metastasis (P < 0.001,
χ2 = 15.64) and TNM stage (P < 0.001, χ2 = 25.06). The
formation of fTN-C was involved in lymph node metastasis
(P = 0.008, χ2 = 7.02), vascular invasion (P = 0.015, χ2 =
5.86), hepatic metastasis (P = 0.003, χ2 = 8.87) and TNM
stage (P = 0.005, χ2 = 16.74). These results suggested that
the co-expression of MMP-9 and TN-C are correlated
with lymph node metastasis, vascular invasion and hepatic
metastasis in pancreatic cancer.
Co-expression of MMP-9 and TN-C indicates a poorer
prognosis in pancreatic cancer patients
To determine the effects of MMP-9 and TN-C on the
survival rates of pancreatic cancer patients, we used Log-rank
test analyses. Kaplan-Meier survival curves showed that the
individual overexpression of MMP-9, TN-C or fTN-C
significantly decreased overall survival (P = 0.005, P = 0.019,
P = 0.033, respectively) (Fig. 2a-c). The median survival of
individual overexpression of MMP-9, TN-C or fTN-C were
11 months, 11 months, 10 months separately, the median
survival of co-expression of MMP-9 and TN-C was 9
months. Furthermore, compared to individual
overexpression of MMP-9, TN-C or fTN-C, the co-expression of
MMP-9 and TN-C had the lowest overall survival and
1-year survial rate (P < 0.001) (Fig. 2d, Table 4).
Co-expression of MMP-9 and TN-C is an independent
predictor of survival in pancreatic cancer
To determine the independent risk factors of survival,
we used univariate and multivariate analyses. We found
that the individual expression or co-expression of
MMP9 and TN-C were risk factors affecting survival of
patients by the univariate analysis. In contrast, multivariate
analysis showed that only the co-expression of MMP-9
and TN-C was an independent predictor for survival of
patients (P = 0.042) (Table 5).
The extracellular matrix (ECM) plays an important role
in tumor growth, invasion and metastasis [
previous study demonstrated that fTN-C matrix formation
required participation of MMPs and plays a role in
promoting cancer metastasis [
]. In this study, we only
focus on the relation of MMP-9, total TN-C and fTN-C
in pancreatic cancer invasion, we found that the
expression of MMP-9 and TN-C was increased in all
pancreatic cancer tissues compared with normal pancreatic
tissue. In fact, TN-C was primarily overexpressed in the
stroma but also existed in the cytoplasm of tumor cells.
This finding was consistent with previous research
]. Present studies have shown that MMPs
not only induce TN-C expression but also promote the
formation of fTN-C matrices. In 103 pancreatic cancer
specimens, 43 samples exhibited the co-expression and
co-location of MMP-9 and TN-C. In addition, the
MMP-9 expression was significantly correlated with
TNC expression, this co-expression and co-location of
MMP-9 with TN-C in pancreatic cancer may imply that
MMP-9 helps TN-C incorporate into the stroma to form
tube-shaped structures named “tubular matrix channels”
or “vasculogenic mimicry”, which guide tumor cells to
migrate to vascular invasion . It should be noted that
cancer-fibroblast cell interaction leads to a “fibrillar”
organization of extracellularely deposited Tn-C, However,
this is not a real fibrillogenesis but is due to a molecular
interaction of tenascin-C with other molecules including
fibrillary, therefore we interpret the “fibril like” stromal
depositions as “tubular matrix channels” or “vasculogenic
]. Stromal deposition of TN-C seems to
be associated with an “activated” tumor stroma / presence
of cancer-associated fibroblasts (CAFs) as indicated by
alpha smooth muscle actin (ASMA) positivity [
Interestingly, in this study, the tube-shaped structures of
fTN-C that were found in 28 pancreatic cancer samples
were primarily focused on pancreatic cancer with vascular
invasion, lymph node and liver metastases. Therefore, the
co-expression of MMP-9 and TN-C may promote tumor
metastasis and thus affect the progression of pancreatic
Pancreatic cancer is characterized by aggressive
behavior, poor prognosis, and low survival rate [
whether the expression of TN-C and MMP-9 is involved
in the clinical prognosis of pancreatic cancer patients is
unclear. Present studies have shown that TN-C is a
predictor of survival in colorectal , breast [
cervical cancers , but not in pancreatic cancer [
contrast, the MMP-9 in the prognosis of pancreatic cancer
patients is still controversial [
]. Especially, Lekstan
A found that the formation of lymph node metastases was
characterized by the higher concentrations of MMP9 in
pancreatic cancer [
], Gardian K also found a high
expression of MMP9 at more advanced tumors in a
meaningful research about microenvironment elements [
nevertheless, both of them had an absence of survival
analysis. In this study, we found that overexpression of
MMP9, TN-C and fTN-C was correlated with lymph node
metastasis, vascular invasion, hepatic metastasis and TNM
stage, but not differentiation, this is little inconsistent with
previous study by Juuti et al. [
], which shows that TN-C
is correlated with differentiation. This inconsistent may be
original from that the fTN-C was not distinguished in
their study. In addition, the co-expression of MMP-9 and
TN-C was significantly associated with lymph node
metastasis, vascular invasion and hepatic metastasis.
Univariate analysis showed that MMP-9, TN-C, and fTN-C
expression levels, co-expression of MMP-9 and TN-C
were risk factors and affected patient survival. Multivariate
analysis showed that the co-expression of MMP-9 and
TN-C was an independent predictor for the survival of
1-year survial rate (%)
The bold values indicate P-values less than 0.05. pL lymphatic invasion, pV
pathological vessel status, pT T-factor, pStage TNM stage (UICC)
pancreatic cancer patients. Kaplan-Meier survival curves
showed that the individual overexpression of MMP-9,
TN-C or fTN-C significantly decreased the overall
survival and that co-expression of MMP-9 and TN-C had the
lowest overall survival. Therefore, the co-expression of
these two molecules may indicate a poorer prognosis in
pancreatic cancer patients.
Our study provided the real close clinical evidences
between MMP-9 and TN-C in pancreatic carcinoma, we
further showed fTN-C had tube-like structures in 28
pancreatic cancer patients, interestingly, most of them
experienced vascular invasion, which suggests its possible
functions in angiogenesis or metastasis. The underlying
mechanisms and the cellular effects of the co-expressions
and fTN-C were worth studying, which would be our next
work. However, this study had several limitations. First,
this data was from a single-center. Second, this study was
retrospective and only patients who underwent curative
resection were included. Third, the co-expression study
via immunohistochemistry (semi-quantitative) may not
precise enough. These questions would be solved in our
In conclusion, we demonstrated that MMP-9 and
TNC were overexpressed in pancreatic cancer and were
associated with tumor progression and prognosis. The
coexpression of these two molecules was associated with
poorer prognosis and was found to be an independent
predictor of survival. Combining with previous research,
it may provide a novel biomarker and potential
therapeutic targets in human pancreatic cancer.
In this study, via using immunohistochemistry and
clinical survival analysis we investigated the expression
patterns of TN-C and MMP-9 and their correlations of
individual expression or co-expression with
clinicopathological parameters and survival of pancreatic cancer.in
103 pancreatic cancer tissues, we found that the elevated
expressions of TN-C and MMP-9 were related to the
pancreatic cancer metastases and the low survival rates,
The co-expression of MMP-9 and TN-C had the lowest
overall survival rates, The co-expression of MMP-9 and
TN-C was an independent predictor of survival for
pancreatic cancer patients.
The authors declare no conflict interest.
XWL designed the study, YQX and ZHL carried out the experiments and
drafted the manuscript, ZHL and PJ participated in the statistical analysis,
GW, XZ, KC and YQX collected the clinical data, all authors read and
approved the final manuscript.
This work was supported by the project of National Natural Science Fund
(81272363, 81430063), the National 863 Project of China (No. 2012AA02A201)
and the Chongqing Natural Science Fund Project (2012JB1032). We thank Dr.
Yazhou Wu and Dr. Lin Liu (Statistics Department, Military Preventive
Medicine, Third Military Medical University) for their statistical help.
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