Background
The majority of cancer deaths are due to metastatic spread of tumor cells. The mortality rate among breast cancer patients is also largely the result of metastasis, the common sites being the lymph nodes, lung, liver and bone. Lymph node metastasis is one of the most important adverse prognostic factors for breast cancer [
1]. In principle, cancer cells spread through the body by different mechanisms, such as direct invasion of surrounding tissue, hematogenous metastasis and/or lymphatic metastasis. Thus, development of vascular supply is a key factor in the growth and metastatic spread of cancers. The ability to block the signaling system that enables the spread of cancer would be a major step forward in the prevention of tumor metastasis, and would consequently reduce both morbidity and mortality.
The vascular endothelial growth factor (VEGF) family of molecules is critical for vascular development and pathological sprouting. The growth of blood vessels (angiogenesis) is primarily initiated by activation of VEGFR-1 and VEGFR-2 by VEGF-A, whereas lymphangiogenesis is predominantly driven by VEGF-C, which activates VEGFR-2 and VEGFR-3 expressed in lymphatic endothelial cells. Recently, blockade of VEGFR-3 signaling by soluble VEGFR-3 (sVEGFR-3) or the blocking antibody inhibits lymph node metastasis in experimental animal cancer models and associated with reduction in lymphangiogenesis but not anginogenesis of the tumors [
2‐
6]. More recently, an endogenous soluble isoform of VEGFR-2 (esVEGFR-2) that sequesters VEGF-C was identified and shown to be the first endogenous specific inhibitor of lymphatic vessel growth [
7]. esVEGFR-2 is a truncated form of 230 kDa membrane-bound form of VEGFR-2 resulting from alternative splicing. In addition, tissue-specific loss of esVEGFR-2 in mice induces, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting angiogenesis. Treatment with esVEGFR-2 inhibits lymphangiogenesis but not angiogenesis induced by corneal suture injury or transplantation, enhances corneal allograft survival and suppressed lymphangioma cell proliferation [
7].
VEGF-C is the major lymphangiogenic factor highly expressed in a variety of malignant tumors including mammary cancer [
8]. Furthermore, over-expression of VEGF-C has been reported to be associated with a poor prognosis and lymph node metastasis in breast cancer patients [
9,
10]. A number of animal studies using cell lines [
2,
11,
12] and transgenic mice [
13] have been conducted in an attempt to demonstrate that VEGF-C over-expression is able to promote cancer metastasis. Thus, tumor cell-derived VEGF-C is thought to enhance lymph node metastasis. Moreover, VEGF-A is well-known to exert a crucial role in tumor angiogenesis [
14]. An adequate blood supply is required to sustain the uncontrolled cell proliferation characteristic of malignant tumors, and tumorigenesis and metastasis have been associated with angiogenesis in tumors [
14]. Therefore, lymphangiogenesis and angiogenesis in tumors have become potential targets for cancer therapy. The recent discovery of esVEGFR-2 [
7] and its selective inhibition of VEGF-C signaling, led to the interrogation of whether it would serve as a therapeutic tool for preventing cancer metastasis and dissecting the precise individual contribution of lymphangiogenesis and VEGF-C signaling in this milieu.
In the present study, we examined whether gene therapy with an alternative splicing variant esVEGFR-2 (an endogenous inhibitor of lympphangiogenesis) might lead to suppression of lymphatic metastasis in a mouse immunocompetent mammary cancer model. In addition, since endostatin is also a naturally occurring molecule and exerts both inhibitions of blood and lymphatic vessels, this protein served as a positive control [
15,
16].
Methods
Vectors
The open reading frame of e
sVegfr2 was cloned from mouse corneal cDNA and inserted into a pcDNA3.1 vector for
in vivo overexpression as previously described [
7]. Empty vector pcDNA3.1 was used as a control vector and referred to as pVec. The plasmid pBLAST-mEndo XVIII (InvivoGen, Inc. San Diego, CA, USA), which encodes murine endostatin with the addition of the IL-2 signal sequence for secretion, was used as a positive control [
15]. For simplicity in this manuscript, the vectors are referred to as pesVEGFR-2 and pEndo, respectively. All plasmid vectors were extracted from
Escherichia coli (DH5α strain) and purified by means of a modified alkaline lysis procedure using a Plasmid Maxi Kit (Qiagen Inc., Valencia, CA, USA) and further purified with centrifugal filters (Ultrafree-MC, Millipore Co., Bedford, MA, USA).
Cell line and animals
Mouse mammary tumor virus (MMTV), purified from medium in which Jyg-MC cells (established from mammary tumors of the Chinese wild mouse) were grown, was inoculated into the inguinal mammary glands of female BALB/c mice, resulting in the development of mammary carcinomas [
17]. The BJMC3879 mammary adenocarcinoma cell line was subsequently derived from a metastatic focus within a lymph node from one of the inoculated BALB/c mice and the cell line continues to show a high metastatic propensity, especially to lymph nodes and lungs, a trait retained through culture [
18‐
20]. This cell line and inoculated tumors expressed VEGF-C and VEGFR-3 [
21]. The BJMC3879luc2 mammary carcinoma cell line was generated by stable transfection with
luc2 gene (an improved
firefly luciferase gene) to parent cell line BJMC3879 cells [
22]. BJMC3879luc2 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum with streptomycin/penicillin in an incubator under 5% CO
2 at 37°C.
A total of 30 female, six-week-old BALB/c mice was used in this study (Japan SLC, Hamamatsu, Japan). The animals were housed five per plastic cage on wood chip bedding with free access to water and food under controlled temperature (21 ± 2°C), humidity (50 ± 10%), and lighting (12:12 hour light:dark cycle). All animals were held for a one-week acclimatization period before study commencement. All manipulations of mice were performed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals in Osaka Medical College, Japanese Government Animal Protection and Management Law (No.105) and Japanese Government Notification on Feeding and Safekeeping of Animals (No.6).
In vivoesVEGFR-2 gene therapy on mammary cancer model
BJMC3879luc2 cells (2.5 × 10
6 cells/0.3 ml in phosphate buffered saline) were inoculated into the right inguinal region of 30 female BALB/c mice. The animals were then randomly allocated into three groups of 10 mice each: the pVec (control), pesVEGFR-2, or pEndo groups. Two weeks post-inoculation, when the tumors had reached 0.4 to 0.5 cm in diameter, we injected pVec, pesVEGFR-2 or pEndo directly into the tumors and then immediately performed
in vivo gene electrotransfer by applying a conductive gel (Echo Jelly; Aloka Co., Ltd., Tokyo, Japan) topically to the unshaved skin over the injected tumor. The vectors were injected using a 27-gauge needle at a concentration of 0.5 μg/μl in sterile saline while the animals were under isoflurane anesthesia. A total volume of 150 μl was introduced into larger tumors, while smaller tumors of 0.6 to 0.7 cm were infused until we detected leakage of the vector solution. Electric pulses were delivered directly to the tumor via "forceps" platinum plate electrodes (CUY650-10; Nepa Gene Co. Ltd., Ichikawa, Japan) using a CUY21EDIT square-wave electropulser (Nepa Gene Co., Ltd.). Conditions for gene electrotransfer used in the present study were intratumoral injection of 50 to 75 μg plasmid (dependent on tumor size as mentioned above), eight pulses with a pulse length of 20 milliseconds at 100 volts. The parameters for optimal gene electrotransfer were previously determined [
15,
18,
20].
Using calipers, we measured the size of each treated mammary tumor weekly and calculated tumor volumes using the formula
maximum diameter ×
(minimum diameter)
2
×
0.4 [
23]. Individual body weights were also recorded at weekly intervals. All surviving animals were injected intraperitoneally with 50 mg/kg 5-bromo-2'-deoxyuridine (BrdU; Sigma Co., St. Louis, MO, USA) at one hour prior to sacrifice. After six weeks of treatment, all mice were euthanized under isoflurane anesthesia and the mammary tumors and certain lymph nodes (that is, nodes from axillary and femoral regions as well as any that appeared abnormal) were removed. We then immediately fixed a portion of each tissue sample in 10% phosphate-buffered formalin and processed through to paraffin embedding; an additional portion of each tumor was also immediately frozen in liquid nitrogen for molecular analysis. Lungs were routinely inflated with the fixative, excised and immersed back into the fixative. We subsequently trimmed and examined all lobes for metastatic foci before processing through histology, where they were cut into 4-μm-thick sections and stained with hematoxylin and eosin (H&E) for histopathological examination or remained unstained sections for immunohistochemistry.
Bioluminescence imaging in vivo
At Week 6, while under isoflurane inhalation using an SBH Scientific anesthesia system (SBH Designs Inc., Windsor, Ontario, Canada), a minimum of five mice from each group were injected intraperitoneally with D-luciferin potassium salts (Wako Pure Chemical Industries, Osaka, Japan) at 3 mg/mouse. Bioluminescence imaging with a Photon Imager (Biospace Lab, Paris, France) was performed. The bioluminescent signals received during the six-minute acquisition time were imaged and quantified using Photovision software (Biospace Lab).
p53 immunohistochemistry
The labeled streptavidin-biotin (LSAB) method (Dako Co., Glostrup, Denmark) was used for p53 immunohistochemistry. Unstained sections were immersed in distilled water and heated for antigen retrieval prior to incubation with a p53 mouse monoclonal antibody (Clone Pab240; Santa Cruz Biotechnology, Santa Cruz, CA, USA) that reacts to the mutant protein in fixed specimens.
Blood and lymphatic microvascular densities in mammary tumors
Immunohistochemistry was performed on samples using the blood and lymphatic vessel markers CD34 and Lyve-1 respectively to quantitatively assess the number of microvessels present in primary mammary carcinomas. Rat anti-CD34 (Hycult Biotech, Uden, The Netherlands) and rabbit anti-LYVE-1 (Acris Antibodies GmbH, Herford, Germany) were used as primary antibodies and were detected using goat anti-rat Alexa-594 and goat anti-rabbit Alexa-488 (Molecular Probes, Life Technol. Corp., Carlsbad, CA, USA). Nuclear staining was performed with Vectashield mounting medium with DAPI (Vector Labs, Inc., Burlingame, CA, USA). The probes were then visualized at high magnification (x200) using a laboratory microscope equipped with a high pressure mercury burner for fluorescence (Olympus Co., Tokyo, Japan). The mammary carcinoma tissues immunohistochemically stained were observed and digitally captured whole periphery of the tumors at high magnification (x200) under fluorescence with a 590 nm or 495 nm excitation filter. The corresponding three images (CD34, Lyve-1 and DAPI) were merged into a single image and the number of CD34+/Lyve-1- and the number of CD34-/Lyve-1+ vessels were counted.
Dilated lymphatic vessels with cancer cell invasion
Mammary tumor sections from paraffin-embedded tissues were immunohistochemically stained using the LSAB method (Dako Co.). A hamster anti-podoplanin monoclonal antibody (AngioBio Co., Del Mar, CA, USA), against a lymphatic endothelium marker was used. To quantitatively assess the number of lymphatic vessels having intraluminal tumor cells in whole periphery area of the primary mammary carcinomas, the slides were scanned at low-power (x100) magnification to identify podoplanin-positive lymphatic vessels, and were then confirmed whether the lymphatic vessel contain mammary cancer cells or not at higher (x200 to 400) magnification. The number of podoplanin-positive lymphatic vessels containing intraluminal tumor cells in whole periphery of the tumors was counted and expressed as the average ± SD.
Apoptosis and cell proliferation in mammary tumors
For the quantitative analyses of apoptotic cell death, sections from paraffin-embedded tumors were assayed using the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling (TUNEL) method in conjunction with an apoptosis in situ detection kit (Wako Pure Chemical Industries) with minor modifications to the manufacturer's protocol. TUNEL-positive cells (mainly regarded as apoptotic cells) were counted in viable regions peripheral to areas of necrosis in tumor sections. The slides were scanned at low-power (x100) magnification to identify those areas having the highest number of TUNEL-positive cells; four areas neighboring the highest area of TUNEL-positive cells were then selected and counted at higher (x200 to 400) magnification to obtain mean ± SD values. The numbers of TUNEL-positive cells were expressed as numbers per cm2.
The tumors from five animals from each treatment group were subsequently evaluated for cell proliferation rates (BrdU labeling indices) as inferred by BrdU incorporation. DNA was denatured in situ by incubating unstained paraffin-embedded tissue sections in 4 N HCl solution for 20 minutes at 37°C. The incorporated BrdU was visualized after exposure to an anti-BrdU mouse monoclonal antibody (Clone Bu20a, Dako Co.). The numbers of BrdU-positive S-phase cells per 250 mm2 were counted in four random high power (×200) fields of viable tissue, and the BrdU labeling indices were expressed as numbers per cm2.
Statistical analyses
Significant differences in the quantitative data between groups were analyzed using the Student's t-test via the Welch method which provides for insufficient homogeneity of variance. The differences in metastatic incidence were examined by Fisher's exact probability test, with P < 0.05 or P < 0.01 considered to represent a statistically significant difference.
Discussion
In the present study, gene therapy with vectors expressing esVEGFR-2 significantly suppressed the multiplicity of lymph node metastasis and lung metastatic nodules in an immunocompetent metastatic mammary cancer model, whereas pEndo (as a positive control) strongly inhibited overall metastasis. Survival rates tended to be prolonged in the pesVEGFR-2 and pEndo groups, although this tendency was not statistically significant. Tumor volume was significantly reduced in the pesVEGFR-2 and pEndo groups, and this reduction was associated with decreased cell proliferation as assessed by BrdU labeling indices. In addition, the antitumor effects in the pEndo group were significantly stronger than the antitumor effects in the pesVEGFR-2 group. The inhibition of metastasis in these groups may simply be a reflection of the suppressed tumor growth and cell proliferation. However, this therapeutic benefit is apparent because conventional therapies are often insufficient to eradicate metastatic breast cancer. When the diameter of malignant breast tumors reaches 4 cm or larger, the chance of tumor recurrence and/or metastasis increases dramatically [
24]. The prolonged survival, reduced tumor volume, and suppression of metastasis after pesVEGFR-2 therapy suggests that esVEGFR-2 may potentially represent a novel therapy for cancer treatment.
Tumor cell dissemination is mediated by a number of mechanisms, including direct invasion into local tissue, lymphatic spread, and hematogenous spread. The most common pathway of initial dissemination is via the afferent ducts of the lymphatics [
25]. The lymphatic capillaries present in tissues and tumors provide entrance into the lymphatic system, allowing cancer cell migration to the lymph nodes. VEGF-C expression correlates with lymph node metastasis in a variety of human cancers, including breast neoplasms [
8,
26]. In many animal models of cancer, VEGF-C enhances tumor lymphangiogenesis, the metastatic spread of tumor cells to lymph nodes and, in some cases, distant organ metastasis [
27]. Downregulation of VEGF-C with siRNA reduces lymph node metastasis in murine mammary cancer models [
20,
28]. In addition, VEGFR-3, the VEGF-C receptor, is predominantly expressed on lymphatic endothelial cells [
29], and VEGF-C-dependent activation of VEGFR-3 stimulates the growth of lymphatic endothelial cells and lymphatics [
30]. Blockade of VEGFR-3 signaling by sVEGFR-3 or blocking antibody inhibits lymph node metastasis in experimental animal cancer models and is associated with a reduction in lymphangiogenesis but not angiogenesis of tumors [
2‐
4]. In contrast, Laakkonen
et al. reported that VEGFR-3 blocking antibody therapy significantly suppresses both angiogenesis and lymphangiogenesis [
31]. In addition, Burton
et al. reported that sVEGFR-3 significantly inhibits lymphangiogenesis and slightly inhibits tumor blood vasculature. They speculated that the inhibition of tumor blood vasculature could likely be responsible for the delay in tumor growth
in vivo [
5].
We recently demonstrated that naturally occurring esVEGFR-2 is a VEGF-C antagonist that selectively inhibits lymphangiogenesis and is associated with normal alymphatic cornea [
7]. In fact, the present study shows that the multiplicity of lymph node metastasis and lung metastatic nodules was significantly reduced in the pesVEGFR-2 group and associated with a decreased number of lymphatic vessels but not blood vessels in mammary carcinomas. However, as shown in Table
1, pesVEGFR-2 did not decrease the number of unilateral or bilateral metastasis in the lungs, ovaries, kidneys and adrenals, which are types of hematogenous metastasis. Thus, treatment with pesVEGFR-2 that primarily inhibits lymphangiogenesis may be ineffective in this experimental setting. But, since pesVEGFR-2 significantly decreased the number of metastatic nodules in the lungs, some possibilities are raised. An initial pathway of lung metastasis may also be through the lymphatic pathway (thoracic duct); cancer cells then influx into the left subclavian vein, pass through the right ventricle of the heart and pulmonary artery, and then settle and grow in the lung tissue. In addition, cancer cells metastasize to lymph nodes and invade into blood microvessels within the lymph node and then hematogenously spread to the lungs. If so, the number of cancer cells that metastasize to the lungs may be decreased. Alternatively, the secreted esVEGFR-2 in blood may inhibit the survival of cancer cells circulating in the blood, or it may inhibit the settlement of cancer cells in the lungs. Further investigation is necessary to explore these possibilities. In addition, we observed a significant decrease in the number of lymphatic vessels with tumor cells in their lumina in the pesVEGFR-2 and pEndo groups as compared to the pVec control group. This finding indicates an inhibitory effect on migration into tumor lymphatic vessels that supports a significant reduction in lymph node metastasis in these groups. In addition, the number of CD8
+ T cells and dendritic cells is significantly increased in inoculated murine mammary tumor cells stably transfected with VEGF-C siRNA, suggesting that VEGF-C modulates the immune response [
28]. Therefore, the immune response may participate in the antimetastatic potential of pesVEGFR-2 in the immunocompetent mammary cancer model in the present experiment.
Previous studies have found that the systemic administration of the anti-VEGFR-3 blocking antibody inhibits lymph node metastasis and reduces lymphatic vessel density in orthotopic lung [
2] or gastric tumors [
3] in nude mice. However, there were no changes in angiogenesis or tumor weight. The results of the present study raise the question of why esVEGFR-2 suppresses tumor growth without suppressing tumor angiogenesis. VEGF-C induces tumor growth in orthotopic prostate tumors [
32] or gastric carcinomas [
33] in nude mice. Indeed, in the present study, esVEGFR-2 decreased cell proliferation as determined by BrdU-labeling indices. Since esVEGFR-2 is an antagonist of VEGF-C, it is possible that pesVEGFR-2 could inhibit tumor growth as well, as indicated by the present study. On a related note, sVEGFR-3 can not only bind VEGFR-3 but also acts as a trap for VEGF-C, which blocks VEGFR-3 signaling [
2,
5].
Endostatin is a 20-kDa C-terminal fragment of collagen XVIII that inhibits endothelial cell proliferation and tumor angiogenesis by several mechanisms. These mechanisms include: blocking the binding of VEGF
121 and VEGF
165 to the KDR/Flk-1 receptor, which mediates endothelial cell motility and proliferation; blocking phosphorylation of the tyrosine receptor; and blocking activation of the intracellular signaling kinases ERK, p38 MAPK, and p125 FAK [
34,
35]. Gene therapy with endostatin causes significant tumor growth arrest in various cancers in laboratory animals [
36,
37]. We previously showed that gene therapy with endostatin suppresses tumor growth and metastasis (lymph nodes and lungs) and is associated with the inhibition of blood vessels and lymphatic vessels in the mouse mammary cancer model [
15], which is consistent with the results of the present study. Brideau
et al. reported that J4 transgenic mice overexpressing endostatin (driven by the keratin K14 promoter) in epidermal basal cells exhibited inhibited angiogenesis and lymphangiogenesis in skin tumors induced by a carcinogen followed by a tumor promoter agent [
16]. The skin tumors in the J4 transgenic mice were less aggressive than tumors in wild-type mice [
16]. Thus, endostatin inhibits both blood vessels and lymphatic vessels. Endostatin is a naturally occurring molecule like pesVEGFR-2, not a recombinant protein; hence, we selected endostatin as a positive control. In the present study, pEndo strongly suppressed overall metastasis (lymphatic and hematogenous metastasis) and was associated with decreased angiogenesis and lymphangiogenesis in tumors. A possible reason why metastasis in the pesVEGFR-2 group was not strong as compared to the pEndo group is that pesVEGFR-2 inhibited tumor lymphangiogenesis but not angiogenesis.
Acknowledgements
This investigation was supported, in part, by a Grant-in-Aid for Science Research (C)(2) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No.21591682 to MA Shibata). JA was supported by grants from the U.S. National Institutes of Health, National Eye Institute, Doris Duke Charitable Foundation, Burroughs Wellcome Fund, and Research to Prevent Blindness (Senior Scientific Investigator & Unrestricted Awards), and the E. Vernon & Eloise C. Smith Endowment Fund. We thank Ms. Mika Yoshida and Yumi Namita for their excellent secretarial assistance.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MS and ES carried out all experiments. RA performed esVEGFR-2 vector construction. Transplantation was performed by JM. Immunofluorescence staining was conducted by MS in consultation with YI. MS wrote the manuscript in consultation with RA and JA. JA edited the manuscript and assisted in experimental design. YO is a head in the department. All authors have read and approved the final manuscript to be submitted.