Abstract
OBJECTIVE To examine the expression of vascular endothelial growth factor C (VEGF-C) in human esophageal squamous cell carcinoma (ESCC), and to clarify its role in lymphatic metastasis in ESCC patients.
METHODS Esophageal carcinoma EC9706 cells and samples from 49 patients with primary ESCC were investigated by using S-P immunohistochemistry (IHC), the semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) methods for VEGF-C expression.
RESULTS VEGF-C positive expression was found in EC9706 cells through IHC, ISH and RT-PCR. Positive IHC for VEGF-C was observed in 36 of 49 cases of ESCC. There was a significant difference between the expression of VEGF-C in a lymph-node-positive group compared to a node-negative group (χ2=4.7, P<0.05). Positive ISH for VEGF-C mRNA was observed in 23 of 49 cases of ESCC. There was a significant difference between the expression of VEGF-C in the lymph-node-positive group and node-negative group (χ2=31.3, P<0.01). The expression of VEGF-C was significantly higher in the lymph-node-positive group compared to the node-negative group. Of 49 ESCC tissues, RT-PCR for VEGF-C mRNA was observed positively in 29 cases. There was a significant difference between the expression of VEGF-C in the lymph-node-positive group and node-negative group (χ2=23.3, P<0.01). The expression of VEGF-C was significantly higher in the lymph-node-positive group compared to the node-negative group. Expressions of VEGF-C were not significantly associated with age, gender, and pathological grade. There was a relationship between VEGF-C mRNA expressions by RT-PCR and ISH (χ2=18.5, P<0.01) in ESCC cases, but with no significant difference between the two methods.
CONCLUSION VEGF-C expression may induce lymphangiogenesis in human ESCC. There was a close correlation between VEGF-C expression and lymph node metastasis. VEGF-C can serve as a useful prognostic factor for ESCC patients.
keywords
- esophageal squamous cell carcinoma (ESCC)
- esophageal cancer EC9706 cells
- vascular endothelial growth factor C (VEGF-C)
- lymphatic metastasis
- immunohistochemistry (IHC)
- RT-PCR
- in situ hybridization (ISH)
Esophageal carcinoma is a common cause of death throughout the world including China, especially in the Taihang mountain range. The lymphatic system is the primary pathway of metastasis for ESCC, and the extent of lymph node involvement is a key prognostic factor for the outcome of patients. However, the mechanism of lymphatic metastasis remains unclear.
Lymphangiogenesis, the development of new lymph vessels, is a relatively new area of clinical investigation. Recent studies show that vascular endothelial growth factor C (VEGF-C) has been identified as a new member of the VEGF family, and is believed to be the only lymphangiogenesis factor in the VEGF family. It activates both vascular endothelial growth factor receptor 2 (VEGFR-2) and VEGFR-3[1]. However, few studies have been conduced regarding VEGF-C expression in ESCC tissues. The purpose of the study was to detect the expression of VEGF-C and its association with lymph node metastasis in ESCC with IHC, ISH and RT-PCR techniques. Furthermore, the relationship of VEGF-C to the clinicopathological features was investigated.
METERIALS AND METHODS
Materials
Reagents
TRIzol Reagent was purchased from Invitrogen Corporation, USA. A TaKaRa One Step RNA PCR Kit (AMV) was purchased from the TaKaRa Biotechnology (Dalian) Co. Ltd. The PCR primers for VEGF-C, β-actin and probe for VEGF-C were obtained from the Beijing AuGCT Biotechnology Co. Ltd. VEGF-C rabbit anti-human polyclonal antibodies and a S-P immunohistochemical staining kit (SP9001) were purchased from the Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd.
Clinical data
The resected specimens from 49 cases of ESCC were obtained from the Anyang Tumor Hospital, Henan, China between September to November of 2004. The carcinoma tissues and corresponding normal tissue were immediately placed in liquid nitrogen at -80°C until used for RT-PCR. Of the 49 cases of ESCC, 25 were male, 24 were female, with a mean age of 58.3 years, ranging from 44 to 76 years. The patients received no prior radiotherapy or chemotherapy. All the specimens were clearly classified by experienced pathologists. Routine pathological diagnosis showed that grade I, II and III were 14, 23 and 12 cases respectively. Among them, 20 cases presented with lymph node metastasis, and 29 cases had no lymph node metastasis.
Cell culture
The human esophageal carcinoma EC9706 cell line was kindly provided by the Chinese Academy of Medical Sciences. The cells were grown in a monolayer culture containing humidified 5% CO2 in air at 37°C. They were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 100 U/ml and penicillin and 100 mg/L streptomycin (pH 7.2-7.4).
Methods
RNA isolation and reverse transcriptasel PCR amplification
Total RNA was extracted from ESCC tissue samples stored at -80°C and form the EC9706 cells by using the TRIzol Reagent according to the procedures described in the kit. The quality of the isolated RNA was determined by gel electrophoresis, and the concentration and purity of RNA were determined by the A260/280 ratio. Amplification of VEGF-C and β-actin as an internal control in each reaction was carried out by PCR with the following primer pairs described previously[2]. The primers of VEGF-C that yield 229 bp and the sequences were 5’-AAG GAG GCT GGC AAC ATA AC-3’ (forward) and 5’-CCA CAT CTG TAG ACG GAC AC -3’(reverse). The primer for β-actin that yielded a 302 bp product as follows: 5’-TCC TCC CTG GAG AAG AGC TA-3’ (forward), 5’-TCA GGA GGA GCA ATG ATC TTG-3’ (reverse).
In situ hybridization
Sections (6 μm thick) of the tissue and the prepared cell slides for hybridization were stained according to Chen et al[3]. The oligonucleotide probe for the VEGF-C sequence was 5’-GTC ATG GAA TCC ATC TGT TGA GT-3’ as described previously[4]. The biotin-labeled VEGF-C cDNA anti-sense probes were all modified by sulphur.
Immunohistochemistry
ESCC tissues were sectioned at 4 μm. After deparaffinization with xylene and dehydration with graded ethanol, the tissue sections and cell slides were incubated in PBS containing 30 ml/L H2O2 to remove endogenous peroxidases, and then in PBS containing 0.1 mol/L citrate to saturate the nonspecific binding sites. After incubation with VEGF-C rabbit antihuman polyclonal antibodies at 1:150 dilution, the sections were treated with instant S-P immunohistochemical reagents and then incubated in a buffer solution containing 3,3-diaminobenzidine tetrahydrochloride (DAB) and H2O2 for visualization, followed by dehydration and mounting procedures. Microscopic examination of the sections was then performed. Colorectal cancer sections were defined as immunohistochemically positive according to the reagent guide. Omission of the primary antibody was used as a negative control.
Statistical analysis
All statistical calculations were carried out using the statistical software package SPSS (Statistical Package for the Social Sciences) for Windows 10.0 (SPSS Inc., Chicago, IL, USA). Each figure was presented as the mean ± SD. The Chi-square test or Student’s t-test was used to analyze data. Values with a P < 0.05 were considered to be statistically significant.
RESULTS
Expression of VEGF-C in the EC9706 cell line
EC9706 cells expressed and secreted VEGF-C as assessed by immunohistochemical staining, ISH, and RT-PCR. The brown positive staining granules for VEGF-C were identified in the cytoplasm of EC9706 cells by IHC(Fig.1). VEGF-C mRNA (blue-purple granules) was found to be located in the cytoplasm of the EC9706 cells by ISH (Fig.2). VEGF-C mRNA positive bands were found in EC9706 cells by RT-PCR (Fig.3).
Expression of VEGF-C in EC9706 cells by IHC. The brown positive staining granules for VEGF-C were identified in the cytoplasm of the carcinoma cells(SP, ×200).
Expression of VEGF-C mRNA in EC9706 cells by ISH. There were blue-purple granules in the cytoplasm(×400).
Expression of VEGF-C mRNA in EC9706 cells by RTPCR. M: 100 bp DNA marker.
Relationship between the expression of VEGF-C and pathological features of ESCC
Positive staining was defined as the presence of VEGF-C immunoreactivity in at least 30% of the tumor cells[5]. Positive IHC for VEGF-C was observed in 36 of 49 cases of ESCC (Fig. 4). The positive rate of VEGF-C with the cases of metastatic lymph nodes and nonmetastatic lymph node, was 90.0% (18/20) and 62.1% (18/29) respectively. There was a signifcant difference between the expression of VEGF-C in the lymph-node-positive group and the node-negative group (χ2=4.7, P<0.05). The expression of VEGF-C in ESCC cases was significantly higher in the lymph node-involved group than in the node-negative group by IHC.
Expression of VEGF-C in ESCC tissues by IHC. The brown positive staining granules for VEGF-C were identified in the cytoplasm of carcinoma cells(SP, ×400).
In 29 of the 49 cases of ESCC, VEGF-C mRNA was detected in tumor tissues by RT-PCR (Fig.5). There was a significant difference between the expression of VEGF-C in the lymph node-involved group compared to the lymph node non-involved group (χ2=23.3, P<0.01). The expression of VEGF-C mRNA was significantly higher in the lymph nodeinvolved group compared to the lymph node noninvolved group utilizing RT-PCR.
Expression of VEGF-C mRNA in ESCC tissues by RT-PCR. N: negative; M: 100 bp DNA marker; T: tumor; P: peritumor.
Of 49 ESCC cases, ISH for VEGF-C mRNA was observed positively in 23 cases (Fig. 6). Most cases with metastatic lymph nodes showed positive staining for VEGF-C (19 of 20; 95.0%), compared with the nonmetastatic lymph node group (4 of 29; 13.8%). There was significantly higher staining in the carcinomas with the lymph node-metastatic group compared to the group without lymph node metastasis (χ2=31.3, P<0.01). The expression of VEGF-C mRNA was significantly higher in the lymph node-involved group than in the group without lymph node involvement by the ISH method. The expressions of VEGF-C were not significantly associated with age, gender, or pathological grade. Comparison of VEGF-C positive expression in ESCC cases among clinicopathologic features is shown in Table 1.
Expression of VEGF-C mRNA in ESCC tissues by ISH. There were blue-purple granules in the cytoplasm(×200).
Correlation between the clinicopathologic parameters and the VEGF-C expression by 3 methods in ESCC.
Correlation between the expression of VEGF-C by RT-PCR and ISH in ESCC
In this study, VEGF-C mRNA in ESCC was detected by RT-PCR and ISH methods. There was a relationship between expressions by RT-PCR and ISH (χ2=18.5, P<0.01), with no significant differences between them (χ2=2.5, P>0.05). (Table 2)
Relationship between the expression of VEGF-C by RT-PCR and ISH.
DISCUSSION
The VEGF family is comprised of the only growth factors that are specific for vascular endothelial cells. These factors consist of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and PlGF. Among them, VEGF-C is a potent stimulator of lymphangiogenesis (the growth of lymphatic vessels) both in vitro and in vivo. Recently, it has been reported that VEGF-C is expressed in several solid tumors including gastric cancer[4], lung cancer[5], breast cancer[6], thyroid carcinoma, prostate cancer, colorectal carcinoma and cervical cancer[7,8]. The over-expression of VEGF-C was related to lymphatic spread.
VEGF-C has been implicated in the regulation of tumor lymphangiogenesis and enhancement of lymphatic invasion. It was initially identified as a ligand of the VEGF receptor-3 (VEGFR-3), which at the time was considered an “orphan” receptor that showed sequence similarity to VEGFR-2 and VEGFR-3. Because expression of VEGFR-3 is largely restricted to the lymphatic endothelium, the major function of VEGF-C appears to be the regulation of lymphatic vessel growth. It is thought that VEGF-C plays a role in the maintenance of the lymphatic endothelium, and over-expression of VEGF-C has been found to induce lymphatic endothelial proliferation in the skin of transgenic mice[9]. These results indicate that VEGF-C is a lymphangiogenic factor. Overexpression of VEGF-C transgenes in experimental tumors revealed a direct correlation between lymphangiogenesis and lymph-node metastasis[10]. The majority of clinical studies show a strong positive relationship between the expression of VEGF-C and lymph node metastasis. VEGF-C promotes growth of tumor cells, which correlates with the growth of lymphatic vessels around tumors and the intralymphatic spread of cancer. The expression of VEGF-C in tumor cells is closely associated with lymph node metastasis[11].
In the current study, EC9706 cells all displayed VEGF-C positive expression through IHC, ISH and RT-PCR. The results correlated with the highly metastatic EC9706 cell line’s invasive character. In addition, the findings of the present study demonstrated a positive correlation of VEGF-C expression with lymph node metastasis in ESCC cases. A strong correlation was found between VEGF-C protein and mRNA expression and metastasis in ESCC by IHC, ISH, and RT-PCR. The results were consistent with those of previous reports[7,8]. All the tissue expressions of VEGF-C were not significantly associated with age, gender, and pathological grade. These results are different from those Onogawa et al.[12] in gastric carcinoma and Hanrahan et al.[7] in colorectal cancer. It maybe that VEGF-C is expressed differently in different kinds of tissues. The cases with VEGF-C expression without lymph node involvement are possibly a pre-clinical status. In contrast, cases with lymph node involvement without VEGF-C expression are likely to belong to selective expression of VEGF-C.
Many experimental studies have indicated that the lymphangiogeneic factors (VEGF-C and VEGF-D) can stimulate lymphangiogenesis in tumors by binding to their receptors (VEGFR-3) on the lymphatic endothelial cells, and induce proliferation and growth of new lymphatic capillaries, then enhance the incidence of lymph node metastasis in animal models. Some studies indicate that VEGF-C levels in primary tumors correlate with lymph node metastasis. Moreover, patients with some cancers expressing high levels of VEGF-C were reported to have poorer prognoses than those with tumors expressing low levels of VEGF-C. In a minority of studies, expressions of VEGF-C do not correlate with lymphatic involvement or cancer progression[13]. Despite these uncertainties, there is abundant evidence from experimental and clinicopathologic studies to justify targeting the VEGF-C/VEGFR-3 lymphangiogenic signaling pathway in the clinic. This will be a priority for the future[14].
Tumor tissues consist of multiple cell types including tumor cells and host, stromal, endothelial and infiltrating cells. RT-PCR of mRNA isolated from bulk tissues represents the average amount of mRNA for all the cells in the sample, and cannot be used to determine if a specific mRNA is derived from normal or tumor cells. RNA from a few contaminating cells may be amplified during RT-PCR and obscure tumor specific alterations. RT-PCR analysis cannot reveal any site-dependent differential expression of VEGF-C. In contrast, ISH and IHC can identify the cellular source as well as reveal intratumor heterogeneity in expression. Comparison of the two methods for VEGF-C mRNA, showed there was a correlation between the expression by RT-PCR and by ISH, without a statistically significant difference between them. So this finding suggests that the ISH method is a better way to detect VEGF-C mRNA expression in ESCC tissues.
In conclusion, this study has demonstrated that the vascular endothelial growth factor-C may play key role in tumor cell lymphatic metastases. There is an association with expression of VEGF-C and lymph node metastasis in ESCC cases. VEGF-C may become a target for the treatment of ESCC by some methods[15]. Furthermore the examination of VEGF-C may be useful in predicting lymph node metastasis for ESCC patients.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to Dr. Sanshen Zhang and his group members of the Anyang Tumor Hospital for assistance in tissue collections.
Footnotes
This work was supported by a grant from the National Natural Science Foundation of China (No.30470779) and the Henan Innovation Project for University Prominent Research Talents (No.2006KYCX016)
- Received January 17, 2007.
- Accepted February 25, 2007.
- Copyright © 2007 by Tianjin Medical University Cancer Institute & Hospital and Springer
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