and Gastric Cancer
Angiogenesis (blood vessel growth), lymphangiogenesis (lymph system growth) are all intrinsically connected with lymphedema and share many of the same genes. We have several pages on both processes.
May 23, 2008
Cancer Research Vol. 5, 1823-1829, July 1999
© 1999 American Association for Cancer Research
Second Department of Surgery, School of Medicine [Y. Y., H. F., S. F., I. N., E. B., K. T., K. M.], and Experimental Therapeutics Cancer Research Institute [Y. E., T. S.], Kanazawa University, Kanazawa 920; Virology Division, National Cancer Center Research Institute, Tokyo 104 [K. S.]; and Cancer Institute Hospital, Tokyo [S. O.], Japan
Neogenesis of lymphatic vessel and lymphatic invasion is frequently found in the stroma of cancers, but the mechanisms of this phenomenon remain unclear. Vascular endothelial growth factor C (VEGF-C) is known to be the only growth factor for the lymphatic vascular system, and its receptor has been identified as Flt4. To clarify the mechanism of lymphatic invasion in cancer, we studied the expression of VEGF-C and flt4 genes in gastric cancer tissues. VEGF-C mRNA was mainly expressed in primary tumors (15 of 32; 47%), but the frequency of VEGF-C mRNA expression was low in normal mucosa (4 of 32; 13%). In primary tumors, there was a significant relationship between VEGF-C and flt4 mRNA expression. In contrast, Flt4 was mainly expressed on the lymphatic endothelial cells but not in cancer cells. A strong correlation was found between VEGF-C expression and lymph node status, lymphatic invasion, venous invasion, and tumor infiltrating patterns. Cancer cells in the lymphatic vessels frequently showed intracytoplasmic VEGF-C immunoreactivity. Furthermore, there was a close correlation between VEGF-C tissue status and the grade of lymph node metastasis.
Patients with high expression of VEGF-C protein had a significantly poorer prognosis than did those in low VEGF-C expression group. By the Cox regression model, depth of wall invasion, lymph node metastasis, and VEGF-C tissue status emerged as independent prognostic parameters, and the VEGF-C tissue status was ranked third as an independent risk factor for death. These results strongly suggest that cancer cells producing VEGF-C may induce the proliferation and dilation of lymphatic vessels, resulting in the development of invasion of cancer cells into the lymphatic vessel and lymph node metastasis.
Gastric cancer is still a leading cause of death worldwide (1) . Despite surgeons’ efforts to remove lymph node metastasis by aggressive resection, many patients have died of lymph node metastasis (2, 3, 4) . Actually, >80% of patients with advanced gastric cancer have lymph node metastasis, and the remote lymph nodes, such as the para-aortic nodes, are involved in 20% of gastric cancers. Accordingly, control of lymph node metastasis is the most important strategy for the treatment of gastric cancer.
However, it is very difficult to predict or to diagnose the precise lymph node metastasis preoperatively or intraoperatively (5) . The accuracy of diagnosis of lymph node metastasis by computed tomography and ultrasonography is reported to be low, with a rate of 10–20% (6) . New methods to increase the accuracy rates of detection of lymph node metastasis using molecular biological techniques have been reported. In gastric cancer, we reported previously that the expressions of c-erbB-2 (7) , epidermal growth factor receptor (8) , c-met (9) , autocrine motility factor receptor (10) , and urokinase-type plasminogen activator and its receptor (10) are closely associated with lymph node metastasis. These proteins are now believed to have a great role in the intravasation of cancer cells into lymphatic capillary through destruction of stromal elements and lymphatic vessel or high motile activity. However, the expressions of these genes are not associated with the proliferation of lymphatic vessels. Lymph node metastasis is strongly related with the lymphatic invasion in the primary tumor, and the proliferation and dilation of lymphatic vessels are frequently found in the stroma of gastric cancer with nodal involvement.
Here, we report the close relationship between VEGF-C expression and lymph node metastasis in gastric cancer. To the best of our knowledge, there is no published article on the relationship between VEGF-C tissue status and lymph node metastasis in cancer. This study suggests that the detection of VEGF-C protein in the primary gastric cancer may represent a potential risk of lymph node metastasis and poor prognosis.
MATERIALS AND METHODS
The nine human cancer cell lines used in this study were from the following sources: prostatic adenocarcinoma PC-3 and gastric carcinomas MKN-28, MKN-45, KATO III, MKN-7, and AZ521 were from the Japanese Cancer Research Resources Bank; gastric carcinomas KKLS and NKPS were kindly provided by Dr. M. Mai (Cancer Research Institute, Kanazawa University, Kanazawa, Japan); and gastric cancer cell line TMK-1 was a kind gift from E. Tahara (Department of Pathology, Hiroshima University, Hiroshima, Japan). All cell lines except for AZ521 were maintained in RPMI 1640 supplemented with 10% heated-inactivated fetal bovine serum (Life Technologies, Inc.) at 37°C and 5% CO2; AZ521 was incubated with MEM with 10% fetal bovine serum.
and Tumor Samples.
One hundred seventeen patients with primary gastric cancer who were diagnosed and treated in the Department of Surgery II, Kanazawa University Hospital, between 1990 and 1997 were entered into the study. All of the patients underwent gastrectomy with lymph node dissection. Among them, 25 patients had early gastric cancer (T1 tumor), in which the tumor invasion is confined to the mucosa or submucosa.
All of the resected primary tumors and regional lymph nodes were histologically examined by H&E staining according to the general rules of the Japanese Classification of Gastric Carcinoma (12) .
Immediately after resection of primary tumor, samples of 5 mm in diameter were taken from 32 primary tumors and each adjacent normal mucosa, and these samples were stored at -80°C. In addition, small pieces of tissue 5–8 mm in diameter were sampled from the primary tumor and fixed in acetone at -20°C overnight, dehydrated in acetone at 4°C for 15 min and acetone at room temperature for 15 min, and then cleared in methyl benzoate for 30 min and in xylene in a vacuum evaporating emitter (AMeX method; Ref. 13 ).
Anti-VEGF-C (C-20) and anti-Flt4 antibodies (15 , 21) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-VEGF-C antibody (C-20) is an affinity-purified goat polyclonal antibody that is raised against a peptide corresponding to amino acids 136–155 mapping at the COOH terminus of the VEGF-C precursor of human origin. Anti-Flt4 antibody is an affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to amino acids 1279–1298 mapping at the COOH terminus of the precursor form of Flt4 of human origin.
Immunohistochemistry was performed as follows. Paraffin sections (3–4 µm thick) were deparaffinized and then placed in a solution of absolute methanol and 2% hydrogen peroxide for 30 min. They were subsequently washed in distilled water, rinsed for PBS, and treated with 10-fold diluted blocking serum (rabbit) for 20 min for blocking of nonspecific reaction. The slides were then incubated overnight at 4°C in a humidified chamber with anti-VEGF-C antibody diluted 1:100 in PBS. After the overnight treatment, the slides were incubated with biotinylated goat antirabbit IgG for 20 min (Vectastain ABC kits; Vector Laboratories, Burlingame, CA) and then with premixed ABC (Vector Laboratories) reagent for 20 min. Immunostaining was performed by incubating the slides in diaminobenzidine (DAKO) solution containing 0.06 mM diaminobenzidine and 2 mM hydrogen peroxide in 0.05% PBS (pH 7.6) for 5 min. After chromogen development, the slides were washed, counterstained with methyl green, dehydrated with alcohol and xylene, and mounted in a routine fashion. Negative control were performed in all cases by omitting the first antibody. Only cases in which at least 20% of tumor cells were immunoreactive were scored as positive.
RT-PCR analysis was carried out according to the modifications by Conboy et al. (14) . Briefly, total RNAs were extracted from primary gastric cancer and the normal mucosa, using Isogen (Nippon Gene, Tokyo, Japan; Ref. 16 ). The prepared RNA (1 µg) was mixed with the oligo(dT) (50 pmol), incubated for 15 min at 68°C, and then quickly chilled in an ice bath for 5 min. The RNA samples were reverse-transcribed at 42°C for 60 min into first-strand cDNA in reverse transcription solution [50 mM Tris-HCl (pH 8.3), 40 mM KCl, 8 mM MgCl2, 0.5 mM each dNTP, 225 µg/ml BSA, 5 mM DTT, 20 units of RNasin (Promega, Madison, WI), and 4 units of avian myeloblastosis virus reverse transcriptase (Life Sciences, St. Petersburg, FL)] with a total volume of 20 µl. The cDNA samples were incubated at 95°C for 5 min to inactivate the reverse transcriptase and then chilled. The samples were amplified by the addition of 80 µl of PCR mixture [50 mM Tris-HCl (pH 8.3), 40 mM KCl, 8 mM MgCl2, 0.5 mM each dNTP, 50 pmol of each sense and antisense primer, and 2.5 units of Taq polymerase (Takara, Tokyo)]. The amplification was performed for 1.5 min at 94°C, 2 min at 48°C, and 2 min at 72°C for 3 cycles, followed by 25 cycles of 40 s at 94°C, 1.5 min at 48°C, and 1.3 min at 72°C. The products were electrophoresed on 2% agarose gels and then transferred to a nylon membrane filter. The transferred products were hybridized overnight to a 32P-end-labeled probe specific for the target cDNA fragment (Southern blotting). The autoradiogram was exposed for 4–5 h with two intensifying screens at -80°C.
Specific primers for the VEGF-C gene, targeting an 867-bp fragment, were: VEC-S3, 5'-AGTTTTGCCAATCACACTTCCTG-3'; and VEC-A3, 5'-GTCATTGGCAGAAAACCAGT-CTT-3'. The probe oligonucleotide was VEC-A2 (5'-CTTTCTGTACATTCACAGGCACA-3'). Primers for the flt4 gene were: Flt-4-1, 5'-AGCCATTCATCAACAAGCCT-3'; and Flt-4-2, 5'-GGCAACAGCTGGATGTCATA-3' (PCR product, 298 bp). The probe was Flt-4-probe (5'-TTCCTTTCCAACCCCTTCCTGGTGCACATC-3'; Ref. 17 ). As internal standards, the ß-actin gene and the transferrin receptor gene were used. Primer pairs specific to the ß-actin gene (18) , targeting a 865-bp fragment, were used: ß-act-5, 5'-TTGAAGGTAGTTTCGTGGAT-3'; and ß-act-11, 5'-GAAAATCTGGCACCACACCTT-3'. ß-act-7 (5'-ACTGACTACCTCATGAAGAT-3') was used as the probe. Primers for transferrin receptor (TFR), targeting a 415-bp fragment, were: TFR2-S, 5'-ACAGACTCTACATGTAGGAT-3'; and TFR4-A, 5'-AAACCTTGAAGTTGCTGGTA-3'. The probe was TFR4-P (5'-TATCCCTCTAGCCATTCAGT-3'; Ref. 19 ).
Twelve µl of protein sample (total protein 20 µg) were mixed with 6 µl of sample buffer [50 mM Tris-HCl (pH 6.5), 10% glycerol, 2% SDS, and 0.1% bromphenol blue] prior to separation by 10% SDS-polyacrylamide gel. After completion of electrophoresis, samples were transferred to polyvinylidene difluoride membrane filters (Immobilon; Millipore, Bedford, MA).
Immunoreactivity were performed using anti-VEGF antibody (C-20) and anti-VEGFR-3 (Flt4) antibody as primary antibodies and a peroxidase-conjugated secondary antibody. The transferred samples were conjugated with anti-VEGF-C antibody (C-20; 1:1000), anti-Flt4 antibody (1:1000), and anti-ß-actin monoclonal antibody (Sigma Chemical Co., St. Louis, MO) for 2 h at room temperature. As a negative control, primary VEGF-C antibody, which was preabsorbed with synthetic VEGF-C peptide (Santa Cruz Biotechnology) overnight at a ratio of 1:1, was reacted with the membrane filter. VEGF-C peptide was used as a positive control. After washing with TBS-Tween 20 with 0.05% skim milk to block a non-specific reaction, membranes were incubated with peroxidase-conjugated Affipure F(ab')2 fragment rabbit antigoat IgG F(ab') fragment (Jackson ImmunoResearch Laboratory, Inc.; 1:5000), and antirabbit IgG horseradish peroxidase linked F(ab') fragment (Amersham Life Science; 1:5000) for VEGF-C, Flt4, and ß-actin for 40 min at room temperature, respectively. The antigen and antibody complexes were detected using ECL Western blotting detection reagent (Amersham Life Science).
Presentation and Statistical Analysis.
All statistical calculations were carried out using SPSS statistical software. Data are presented as means ± SD. The 2 test was used to analyze data. The outcomes of the different groups of patients were compared by the generalized Wilcoxon test. A multivariate model using Cox stepwise regression analysis was used to evaluate the statistical strength of independent association between selected covariates and patient survival. Values with a P of 0.05 were considered to be statistically significant.
and flt4 mRNA Expression in Gastric Cancer Cell
Among eight gastric cancer cell lines, NKPS, TMK-1, AZ521, and MKN-45 expressed VEGF-C mRNA, but VEGF-C expression was not detected in KATO III, MKN-7, KKLS, and MKN-28 (Fig. 1) . However, flt4 mRNA was expressed from KKLS only. PC-3 overexpressed not only VEGF-C but also flt4 mRNA (Fig. 1)
and Flt4 Protein Expression in Gastric Cancer and Normal
Mucosa (Western Blot Analysis).
In gastric cancer cell lines, C-20 recognized main band of Mr 58,000 precursor of VEGF-C and proteolytic forms of Mr 31,000, 29,000, and 21,000 molecules (Fig. 3) . In the clinical specimens, a Mr 31,000 molecule was predominant (Fig. 4) , and the intensity of VEGF-C expression in cancer tissue was usually stronger than that of the normal counterpart. By preabsorption of VEGF-C antibody with synthetic VEGF-C peptide, Mr 55,000 and 31,000 bands, corresponding to VEGF-C peptides and VEGF-C, respectively, disappeared.
DISCUSSIONVEGF family is the only growth factor that is specific for vascular endothelial cells, and it consists of VEGF-A, VEGF-B, and VEGF-C. Among them, VEGF-C is ranked first as a lymphoangiogenic factor (20) , which induces proliferation of lymphatic endothelial cells and lymphatic vessels via activating VEGFR-3 (Flt4) or VEGFR-2 (KDR) on the cell membrane of endothelial cells (21) . However, the activity in proliferating endothelial cells via VEGF-C and VEGFR-2 loop is considered to be decreased in adult tissue. Accordingly, the main receptor of VEGF-C in adult tissue is considered Flt4. Oh et al. (22) reported that VEGF-C can induce proliferation of lymphatic endothelial cells and development of new lymphatic sinuses on the avian chorioallantoic membrane. It has been considered that the mechanism of the neogenesis of the lymphatic channel is activation of VEGFR-3 by VEGF-C, which is abundant in the proliferating endothelial cells of vascular sprouts and branching vessels of embryonic tissues (23) .
Neogenesis and dilation of the lymphatic vessels are often found in the stroma of gastric cancer tissue, and intravasation of cancer cells in the lymphatic vessels is known as lymphatic invasion. Thus far, the mechanism of the dilation of lymphatic vessels in gastric cancer tissue has been speculated to be the obstruction of lymphatic channel by the cancer cell emboli. However, the mechanisms of the lymphoangiogenesis in cancer tissue have remained unclear.
It is generally accepted that the cancer cell invasion into the lymphatic vessels is established by the destruction of stroma around the lymphatic vessels via activation of matrix-digesting enzymes, which are produced by cancer cells or fibroblasts (24 , 25) , and by the enhanced motility activity of cancer cells via motility factor and their receptor, such as AMF/AMFR (26) or c-met/HGF (9 , 27) .
This study clearly demonstrated the close relationship between VEGF-C expression and lymphatic invasion and lymph node metastasis. Tumors with high expression of VEGF-C had more remote lymph node involvement than did those with low VEGF-C expression. Considering that VEGF-C is selectively expressed in cancer cells but not in normal gastric mucosa or stromal elements, VEGF-C produced from gastric cancer may induce lymphovascular neogenesis and dilation of lymphatic vessel in the stroma of primary tumor (23) . Immunohistological study has shown that the VEGF-C receptor flt4 was expressed on the lymphatic endothelial cells in the stroma of primary tumors and normal mucosa. Furthermore, there was a close correlation between flt4 and VEGF-C mRNA expression in the primary tumors, and immunohistochemical examination showed that cancer cells in the lymphatic vessels frequently expressed VEGF-C. In contrast, flt4 mRNA was not detected in eight examined gastric cancer cell lines, and this finding was in accordance with immunohistological evidence showing that almost all gastric cancer cells did not express Flt4. Furthermore, Flt4 immunoreactivity was only found on the lymphatic endothelial cells in the stroma of primary tumors and stomach wall. These results strong suggest the possibility that VEGF-C produced by cancer cells stimulate the lymphoangiogenesis and dilation of lymphatic vessels by activating Flt4, which is expressed on the lymphatic endothelial cells. Joukov et al. (28) reported that mature VEGF-C (Mr 21,000) also increases the permeability of lymphatic vessels as well as the migration and proliferation of endothelial cells. Our Western blot analysis in the clinical specimens showed that the mature VEGF-C was found in the cancer cells. Accordingly, VEGF-C may enhance the intravasation of cancer cells via loosening the mutual attachment of lymphatic endothelial cells (28) . These results strongly suggest that cancer cells producing VEGF-C may have a higher potential for the intravasation into the lymphatic vessel, resulting in the lymph node involvement.
This study also demonstrated that the patients with VEGF-C-positive tumor had a poorer prognosis than did those with VEGF-C-negative tumor. Furthermore, VEGF-C tissue status was confirmed to be a significantly independent factor for poor prognosis. It has been speculated that the reason is the prevalent progression by the higher lymphatic spread and more infiltrating spread in VEGF-C-positive tumors than in the VEGF-C-negative tumors. Further studies of the proliferative and motility activity of VEGF-C-producing tumors should be performed.
In summary, this study demonstrates the close relationship between VEGF-C tissue status and lymphatic spread and postoperative prognosis in gastric cancer. VEGF-C-producing cancer cells may induce the proliferation and dilation of the lymphatic vessel in the stroma around them. In these circumstances, cancer cells can easily invade the lymphatic vessel, even if these cells have low production of matrix-digesting enzymes and scatter factors. The preoperative determination of VEGF-C tissue status by RT-PCR or immunohistochemistry using endoscopically biopsied materials may be useful in deciding the extent of surgical lymph node resection and postoperative chemotherapy.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom requests for reprints should be addressed, at Second Department of Surgery, School of Medicine, Kanazawa Unversity, Takara-Machi 13-1, Kanazawa 920, Japan. Phone: 81 762 65 2000; Fax: 81 762 34 4260; E-mail: email@example.com.
Received for publication 12/ 7/98. Revision received 4/ 7/99. Accepted for publication 4/13/99.
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