Research ArticleExperimental Studies

Therapy with siRNA for Vegf-c but Not for Vegf-d Suppresses Wide-spectrum Organ Metastasis in an Immunocompetent Xenograft Model of Metastatic Mammary Cancer

MASA-AKI SHIBATA, EIKO SHIBATA, JUNJI MORIMOTO and MARIKO HARADA-SHIBA
Anticancer Research October 2013, 33 (10) 4237-4247;

Abstract

Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. Vascular endothelial growth factors-C and D (VEGF-C and VEGF-D) are heavily involved in lymphangiogenesis. Using small interfering RNA (siRNA) against mouse Vegf-c, and Vegf-d, we sought to inhibit metastasis in a model of metastatic murine mammary cancer. BJMC3879Luc2 cell-induced mammary carcinomas received direct intratumoral injections in vivo of either plasmid VEGF-C/D siRNA (psiVEGF-C, psiVEGF-D) or a vector control followed by in vivo gene electrotransfer weekly for seven weeks. Treatment with psiVEGF-C and with psiVEGF-D resulted in lower tumor volumes as compared to the controls. Treatment with psiVEGF-C suppressed wide-spectrum organ metastasis involving lung and lymph nodes. Treatment with psiVEGF-C further reduced the number of dilated lymphatic vessels with invading cancer cells and inhibited tumor blood microvessel density. In contrast, although treatment with psiVEGF-D was not effective and gave equivocal results, it did induce some insignificant reduction in tumor volume increment, average numbers of lymph node metastases and average number of intratumoral dilated lymphatic vessels. In conclusion, specific silencing of the Vegf-c gene suppresses wide-spectrum organ metastasis, including the lung and lymph nodes. However, therapy with siRNA for Vegf-d was not adequately effective in this murine system.

Breast cancer is the most common malignant disease in women worldwide. According to the International Agency for Research on Cancer, an estimated 1,384,000 patients were diagnosed with breast cancer in 2008. In 2008, 458,000 women died of breast cancer worldwide (1). Perhaps more worrisome is the increasing incidence among younger women under 40 years of age (2-4). Breast cancer lethality is largely due to metastasis, preferentially to lymph nodes, lung and bone (5). In order to reduce both morbidity and mortality, less toxic, more effective chemopreventive and anti-metastatic treatments are desperately required.

Lymph node metastasis is one of the most important adverse prognostic factors for breast carcinoma (6). Members of the vascular endothelial growth factor (VEGF) family promote the formation of new blood and lymphatic vessels in tumor tissues, enabling the spread of tumor cells (7). Within the VEGF family, both VEGF-C and VEGF-D have been reported to induce lymphangiogenesis via activation of VEGF receptor-3 (VEGFR-3) expressed on lymphatic endothelial cells (8, 9). In animal models, VEGF-C and VEGF-D have also been shown to enhance lymphangiogenesis and associated lymphatic metastasis (10-16), while clinical studies showed an association of overexpression of either VEGF-C or VEGF-D with lymph node metastasis and poor prognosis in patients with breast cancer (17-20).

Using an immunocompetent mouse mammary cancer model, we previously demonstrated inhibition of Vegf-c and Vegf-a by gene silencing using vectors expressing short interfering RNA (siRNA) which led to suppression of lymphatic and/or hematogenous metastasis (21). Here we confirmed the anti-metastatic action of Vegf-c siRNA using a different siRNA sequence reported previously (21), and examined the effectiveness of silencing related Vegf-d by the same method.

Materials and Methods

Cell line. The BJMC3879 mammary adenocarcinoma cell line was derived from a metastatic focus within a lymph node of an inoculated BALB/c mouse in an earlier study. When inoculated into BALB/c mice, the line continues to retain a high metastatic propensity, especially to lymph nodes and lungs (21-23). In addition, BJMC3879 cells have been reported to carry the p53 mutation, as inferred by immunohistochemistry (24). The BJMC3879Luc2 mammary carcinoma cell line used in our investigations was generated by stable transfection of the luc2 gene (an improved firefly luciferase gene) into the parent BJMC3879 cell line (25). BJMC3879Luc2 cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum with streptomycin/penicillin at 37°C under 5% CO2.

Animals. Thirty six-week-old female BALB/c mice (Japan SLC, Hamamatsu, Japan) 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 h light-dark cycle) conditions. All animals were held for a one-week acclimatization period before study commencement. This animal experiment was approved by the Animal Experiment Committee of Osaka Medical College (approved no. 19017). Mice were treated in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals at Osaka Medical College, the Japanese Government Animal Protection and Management Law (No.105) and the Japanese Government Notification on Feeding and Safekeeping of Animals (No.6).

siRNA sequences for silencing mouse Vegf-c or Vegf-d. Four different sequences each of siRNA oligonucleotides targeting Vegf-c and Vegf-d, were synthesized using the Qiagen kit and protocol (Qiagen, GmbH, Hilden, Germany). The four siRNA sequences for mouse Vegf-c were as follows: Vegf-C1, 5’-GAAUGACUAUAUAAUUUAUtt-3’ and 5’-AUAAAUUAUAUAGUCAUUCtt-3’; Vegf-C2, 5’-CACAGAAGUGCUUCCUUAAtt-3’ and 5’-UUAAGGAAGCACUUCUGUGtg; Vegf-C3, GACGUUGUUUGAAAUUACAtt-3’ and 5’-UGUAA UUUCAAACAACGUCtt-3’; Vegf-C4, 5’-CAGGGAAUUUGAUG AGAAUtt-3’ and 5’-AUUCUCAUCAAAUUCCCUGtt-3’. The four siRNA sequences for mouse Vegf-d were as follows: Vegf-D1, 5’-GGGCAAUGCUCAUGAGUUAtt-3’ and 5’-UAACUCAUGAGCAUUGCCCtt-3’; Vegf-D2, 5’-CGCCAUCCUUACUCAAUUAtt-3’ and 5’-ggGCGGUAGGAAUGAGUUAAAU; Vegf-D3, 5’-CGAGUUAGUGCCUGUUAAAtt t-3’ and 5’-UUUAACAGGCACU AACUCGgg-3’; Vegf-D4, 5’-GGAUAACACCAAAUGUAAAtt-3’ and 5’-UUUACAUUUGGUGUUAUCCca-3’. The scrambled negative siRNA control was purchased from Qiagen.

Transfection of siRNAs into mammary cancer cells. BJMC3879Luc2 cells were plated in 24-well plates at 8×104 cells/well and transfected with 25 nM siRNA-Vegf-C1 to -C4, siRNA-Vegf-D1 to -D4 or 25 nM negative siRNA control using the Hyperfect transfection reagent (HyperFect; Qiagen). At 24 h post-transfection, cells were collected and total RNA extracted using an RNeasy Mini Kit (Qiagen). cDNAs were synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Manheim, Germany). Real-time reverse transcription-PCR (RT-PCR) was performed using a LightCycler (Roche Diagnostics) as follows: an initial step at 95°C for 10 min, followed by 40 cycles of 10 s at 95°C, 10 s at 60°C, and 10 s at 72°C. The primer sequences for mouse Vegf-c, Vegf-d and glyceraldehyde-3-phsophate dehydrogenase (Gapdh) are shown in Table I. The relative mRNA levels of Vegf-c or Vegf-d were calculated using a 2−ΔΔCt method (26) which is based on the fact that the difference in threshold cycles (ΔCt) between Vegf-c or Vegf-d and Gapdh internal control is proportional to the relative expression level of the gene of interest.

siRNA expression vectors. To construct short hairpin RNAs (shRNA) targeting mouse Vegf-c or Vegf-d, the following oligonucleotides were designed: Vegf-C1 siRNA, 5’-GATCCGAATGACTATATAATTTATtagtgctcctggttgATAAATTATATAGTCATTCTTTTTTA-3’; and Vegf-D1 siRNA, 5’-GATCCGGGCAATGCTCATGAGTTAtagtgctcctggttgTAACTCATGAGCATTGCCCTTTTTT A-3’. The lower case letters in each sequence above indicate the 15-nucleotide spacer separating the antisense segments. Each complementary oligonucleotide was annealed and ligated into a pBAsi-mU6PurDNAvector (Takara Bio, Inc., Otsu, Japan). This vector contains an RNA polymerase III promoter, the mouse U6 promoter, which can generate high amounts of shRNAs. A control siRNA vector was also purchased from Takara Bio, Inc. This control siRNA vector contains a scrambled sequence with no homology to any human or mouse mRNA. The control siRNA sequence was as follows: 5’-GATCCGTCTTAATCGCGTATAAGGCtagtgctcctggttgG CCTTATACGCGATTAAGACTTTTTTA-3’. For simplicity, in this article, the therapeutic vectors are referred to as psiVEGF-C, psiVEGF-D, and psiSCR (scrambled control).

In vivo siRNA Vegf-c or Vegf-d gene therapy. We inoculated BJMC3879Luc2 cells (5×106 cells/0.3 ml in PBS) subcutaneously into the right inguinal region of 30 female BALB/c mice. The animals were then randomly allocated into three groups of 10 mice each, as the psiVEGF-C, psiVEGF-D or psiSCR (control) groups. Two weeks post-inoculation, when the resulting tumors had reached 0.3-0.5 cm in diameter, we injected psiVEGF-C, psiVEGF-D or psiSCR (0.5 μg/μl saline) directly into the tumors using a 27-gauge needle while the animals were under isoflurane anesthesia. In vivo gene electrotransfer was performed immediately after by applying a conductive gel (Echo Jelly; Aloka Co., Ltd., Tokyo, Japan) topically to the unshaved skin over the tumor and to the surface of small platinum ‘forceps’ electrode plates. Electric pulses were then delivered directly into the tumor via these plate electrodes (CUY650-10; Nepa Gene Co., Ltd., Ichikawa, Japan) using a CUY21EDIT square-wave electropulser (Nepa Gene Co., Ltd.) generating 8 pulses with a pulse length of 20 milliseconds at 100 volts (22, 27-29). Vector injection and gene electrotransfer were performed once a week for seven weeks. As the tumors grew, a volume of ≤150 μl of vector solution was introduced into larger masses, while smaller tumors of 0.6-0.7 cm in diameter were infused until we detected leakage of the vector solution. We hoped to reliably administer of 50-75 μg plasmid/tumor, dependent on tumor size.

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 (30). Individual body weights were also recorded at weekly intervals. After seven weeks of treatment, all mice were euthanized and the mammary tumors and certain lymph nodes (i.e. 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 in the fixative. We subsequently trimmed and examined all lobes for metastatic foci before processing for histology, where they were cut into 4-μm slices and stained with hematoxylin and eosin (H&E) for histopathological examination.

View this table:
Table I.

Primer sequences for RT-PCR analysis.

In vivo bioluminescent imaging. At experimental week 7, we anesthetized four or five mice from each group using isoflurane inhalation administered via the SBH Scientific anesthesia system (SBH Designs, Inc., Windsor, Ontario, Canada). Each anesthetized mouse received an intraperitoneal injection of 3 mg of D-luciferin potassium salts (Wako Pure Chemical Industries, Osaka, Japan) for bioluminescent screening with a Photon Imager (Biospace Lab, Paris, France). We quantified the bioluminescent signals received during the 6-min acquisition time with the Photovision software (Biospace Lab).

Histopathological analysis. All mice were euthanized and necropsied at week 7. Mammary tumors, lungs, lymph nodes, and abnormal organs/tissues were removed from each mouse, fixed in 10% phosphate buffered formaldehyde solution, paraffin-embedded and cut at 4 μm. Mounted tissue sections were stained with either H&E for histopathological examination or left unstained for immunohistochemical analysis. We routinely removed lymph nodes from the axillary and femoral regions, as well as any lymph nodes that appeared abnormal. Lungs were inflated with formaldehyde solution prior to excision and fixation and each lung lobe examined individually for metastatic foci prior to paraffin embedding.

Microvascular density in mammary tumors. We quantitatively assessed lymphatic and blood microvessel density in primary mammary carcinomas by immunohistochemistry (IHC) using the avidin-biotin complex (ABC) method (LSAB kit; Dako, Glostrup, Denmark) with hamster anti-podoplanin monoclonal antibody (AngioBio Co., Del Mar, CA, USA), a lymphatic endothelium marker, and rabbit anti-CD31 polyclonal antibody (Lab Vision Co., CA, USA), a specific marker for blood vessel endothelium. Since histological confirmation of lymphatic vessel invasion by tumor cells has prognostic value in various malignancies, we counted the number of podoplanin-positive lymphatic vessels containing intraluminal tumor cells and expressed them as the mean±SD. We additionally counted the number of CD31-positive blood microvessels by first scanning the slides at low magnification (×100) to locate areas of highest vessel density and then selecting five sites within those areas to count at higher magnification (×200-400). Values obtained were expressed as the mean±SD (31).

Figure 1.
Figure 1.

Effectiveness of siRNA sequences targeted to vascular endothelial growth factor-C (Vegf-c) or Vegf-d. We transfected BJMC3879Luc2 mammary carcinoma cultures with four different sequences of siRNA oligonucleotides and measured the relative mRNA levels of Vegf-c (A) and Vegf-d (B) in mouse mammary carcinoma cells. As compared to the negative control siRNA, constructed from scrambled sequences, siRNA-Vegf-C1 reduced Vegf-c mRNA levels to 66% that of the control. The other siRNA constructs C2, C3, and C4 were less effective, lowering Vegf-c mRNA by 57%, 56%, and 53%, respectively siRNA-Vegf-d-D1 reduced Vegf-d mRNA levels to 67% to those of the control.

Statistical analyses. Significant quantitative differences in intergroup data were analyzed using Welch's Student's t-test, which provides for insufficient homogeneity of variance. The variations 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.

Results

Selection of functional siRNA sequence for mouse Vegf-c or Vegf-d. Figures 1A and B illustrate the knockdown ratios of each Vegf-c and Vegf-d siRNA oligonucleotides tested in BJMC3879Luc2 cells. Out of the siRNAs targeted to these growth factors, the strongest silencing was achieved with oligonucleotides Vegf-C1 and Vegf-D1 (66% and 67% respectively).

Figure 2.
Figure 2.

Comparison of body weights and mammary tumor volumes in BALB/c female mice receiving intratumoral treatment with control psiSCR, psiVEGF-C or psiVEGF-D. A: Body weights were similar in all groups until the end of the study at seven weeks when mortality, which was greatest in psiSCR- and psiVEGF-D-treated mice, may have contributed to a slight decrease in those groups. B: From weeks 3-6, gains in tumor volumes were significantly suppressed in psiVEGF-C-transfected mice vs. psiSCR-treated controls. psiVEGF-D treatment tended to depress tumor volume increase throughout the study, but not to a statistically significant degree. Data represent the mean±SD. Although initial animal number was ten in each group, but survival number of animals at the end of the experiment was five in the psiSCR-treated group, nine in the psiVEGF-C-treated group and seven in the psiVEGF-D-treated group. All cause of death was due to cancer metastasis. *p<0.05; **p<0.01 compared to psiSCR-treated controls.

Body weights and tumor growth of animals under Vegf-c or Vegf-d siRNA treatment. At seven weeks, one animal from the psiVEGF-C group, and three animals from the psiVEGF-D group died due to metastasis vs. five animals in the control psiSCR group. As shown in Figure 2A, body weights did not differ statistically between all mice throughout the experiment until week 7, when there was a decrease in the weights of control and psiVEGF-D-treated animals (psiVEGF-C: 20.9±2.0 g; psiVEGF-D: 19.8±2.4 g; psiSCR: 19.4±1.9 g).

Figure 3.
Figure 3.

Bioluminescent imaging at week 7 in four representative mice receiving control or targeted vascular endothelial growth factor-C (Vegf-c)/Vegf-d siRNA. Mammary tumors were generated by inoculating BMJC3879Luc2 cells, which were stably transfected with a luciferase gene, into the inguinal mammary pads. Bioluminescent signals were lowest in mice treated with psiVEGF-C, indicating markedly reduced metastasis, vs. mice receiving either control psiSCR or psiVEGF-D. However, there was also some signal reduction in the psiVEGF-D-treated mice compared to the controls.

Tumor volumes are presented in Figure 2B. As compared to the psiSCR-treated mice, tumor volume increase in the psiVEGF-C-treated group significantly slowed from weeks 2 through 6. Increases in volumes of psiVEGF-D-treated tumors also tended to be lower in relation to controls, but the differences were not significant.

Bioluminescent imaging of transfected mice. Bioluminescent imaging revealed signals indicative of metastatic growth in mandibular, axillary, and inguinal lymphatic regions in all groups; however, less metastatic expansion occurred in mice from both the psiVEGF-C- and -D-treated groups as compared to control animals (Figure 3), with a more pronounced reduction in VEGF-C-treated mice.

Histopathological analysis of mammary tumors. Mammary tumors induced by BJMC3879Luc2 cell inoculations uniformly proved to be moderately-differentiated adenocarcinomas; we found no histopathological differences among groups. However, we did note treatment-induced variations in metastatic incidence and spread. Figure 4 summarizes the incidence and average number of metastatic lesions counted across multiple organs, and Figure 5 illustrates the histopathological presentation. Lymph node metastasis occurred in 100% of mice in the control psiSCR- and psiVEGF-D-treated groups, but in only 67% of mice treated with psiVEGF-C (Figure 4A; Figure 5A-C); furthermore, treatment with psiVEGF-C caused a significant reduction in the average number of cancerous nodes per mouse (Figure 4B). Some insignificant reduction also occurred in the psiVEGF-D-treated group as compared to the psiSCR-treated group.

Figure 4.
Figure 4.

Quantitative analysis of metastasis in lymph nodes, lung and other organs. A: Lymph node metastasis occurred in 100% of psiSCR- and psiVEGF-D-treated mice, while metastasis was reduced to 67% that of the control with psiVEGF-C treatment. B: The average number of metastatic lymph nodes per mouse was significantly lower in mice receiving psiVEGF-C, as compared to those receiving psiSCR. A tendency for decreased nodal involvement was seen in psiVEGF-D-treated mice, but this was not statistically significant. C: psiVEGF-C-treated mice also exhibited decreased incidence of lung metastasis, while treatment with psiVEGF-D had no effect. D: Development of larger metastatic lung nodules >250 μm was significantly reduced only in the psiVEGF-C-treated group. E: psiVEGF-C treatment appeared to reduce the total overall metastatic burden; Silencing Vegf-d had minimal effect. F: The average number of organs with metastases per mouse was significantly reduced only in the psiVEGF-C-treated group. Data represent the mean±SD. Number of animals examined was nine in the psiSCR-treated group, nine in the psiVEGF-C-treated group and ten in the psiVEGF-D-treated group. *p<0.05; **p<0.01 compared with the psiSCR-treated control.

Figure 5.
Figure 5.
Figure 5.

Mammary tumor metastases as demonstrated by hematoxylin and eosin (H&E) histopathology. A: In a lymph node of a control mouse, metastatic carcinoma cells fill the sinusoidal space. B: Metastatic carcinoma cells are more localized to the subcapsular space in a lymph node from a mouse receiving psiVEGF-C. C: In a lymph node from a mouse receiving psiVEGF-D, metastatic carcinoma cells were observed in both the subcapsular and sinusoidal spaces. D: Metastatic foci in the lungs of a psiSCR-treated control mouse, showing many small-to-large foci and nodules. E: Metastatic lung lesions were much smaller in the lungs of mice treated with psiVEGF-C than in the control mice. F: Metastatic foci in the lungs of mice given psiVEGF-D were similar to those found in psiSCR mice in terms of number and size. G: Kidney metastasis in a control mouse receiving psiSCR. H: Metastasis to the adrenal of a control mouse. I: Ovarian metastasis in a mouse receiving psiVEGF-D. J: Uterine metastasis in a mouse receiving psiVEGF-C. A-C and G-J, original magnification ×100; D-F, original magnification ×40.

Metastasis to the lungs followed a similar pattern, as illustrated in Figures 4C-D and 5D-F. Lung metastasis developed in ~89% of mice in both the psiSCR- and psiVEGF-D-treated groups, while treatment with psiVEGF-C reduced the incidence to 44% (Figure 4C). This degree of reduction was not statistically significant. However, down-regulation of Vegf-c did significantly reduce the number of large (>250 μm) metastatic nodules (Figure 5E) as compared to the control (Figure 4D; Figure 5D and F).

Metastatic foci were also observed in kidneys, adrenals, ovaries and uterus (Figure 5G-J). With respect to bilateral organs, metastasis to the only unilateral organ was counted as one, and metastases to the bilateral organs were counted as two. The multiplicity of overall metastasis is presented in Figure 4E and F. The total number of organs affected per group tended to be much lower in mice treated with psiVEGF-C as compared to mice in both the control and the psiVEGF-D treatment groups (Figure 4E); similarly, the average number of all organs with metastasis per mouse was significantly lower in the psiVEGF-C-treated group as compared to the psiSCR-treated group (Figure 4F).

Lymphatic and blood microvessel density in treated mammary tumors. Intratumoral podoplanin-positive lymphatic microvessels are demonstrated in Figure 6A-C. Lymphatic vessels were well-developed in the outer, superficial layers of the mammary tumors in all groups, and tumor cells were frequently observed within the lumina of dilated vessels in both control (Figure 6A) and treated animals (Figure 6B and C). As shown in Figure 7A, significantly fewer vessels containing cancer cells were detected in psiVEGF-C-treated vs. psiSCR-treated tumors, suggesting suppression of lymphatic migration. Fewer vessels harboring migrating cells were also found in psiVEGF-D-treated vs. control tumors, but the difference was significant.

Blood microvessel density, as determined by immunohistochemical analysis for the blood vessel endothelial cell marker CD31 (Figure 6D-F), was significantly lower in the group given siRNA therapy targeting Vegf-c compared to the psiSCR-treated group (Figure 7B).

Discussion

As the presence and degree of metastasis is a significant prognostic indicator for survival in most types of cancer, anti-metastatic therapies are of paramount importance. Dissemination of cancer cells can occur in different ways e.g. local tissue invasion and/or migration via blood and lymphatic vessels. Malignant cells also move into the bloodstream to reach distant sites, and lymph/blood crossover occurs; lung metastasis may initially occur through the lymphatics, but cancer cells can influx into the thoracic duct, the left subclavian vein, through the right ventricle and settle into lung tissue. The most common pathway of initial dissemination of many solid malignancies is via the lymphatics, with varying patterns of spread via afferent ducts (32). Lymphatic and blood capillaries present in tumors and surrounding tissues provide migrating cells entrance to the system. Involvement of the nearby sentinel nodes precedes involvement of more distal nodes and subsequent seeding of distant organs.

Figure 6.
Figure 6.

Immunohistochemical (IHC) analysis of lymphangiogenesis and angiogenesis in mammary tumors transfected with psiSCR, psiVEGF-C, or psiVEGF-D. Lymphatic vessels were often dilated and frequently contained migrating tumor cells (arrows) within the lumina. A: Podoplanin-positive lymphatic endothelium in a control mouse with multiple intraluminal cells. B: Notice fewer dilated lymphatic vessels with migrating cancer cells in a mouse treated with psiVEGF-C. C: Intratumoral dilated lymphatic vessels of a mouse treated with psiVEGF-D. D: Representative section of a tumor transfected with psiSCR, showing a high density of well-developed and CD31-positive blood microvessels. E: CD31-positive blood microvessels tended to be fewer in tumors transfected with psiVEGF-C than with control or psiVEGF-D. F: Tumors treated with psiVEGF-D appeared similar to those in the psiSCR group. A-C: Anti-podoplanin IHC, original magnification ×200. D-F: anti-CD31 IHC, original magnification ×100.

Figure 7.
Figure 7.

Quantitative analysis of lymphatic cellular migration and blood microvessel density. A: Compared to the control, the average number of lymphatic vessels containing intraluminal cancer cells was lower in both psiVEGF-C and psiVEGF-D-treated mice, but the difference was only statistically significant with psiVEGF-C treatment. B: Blood microvessel density in psiVEGF-C-treated tumors was also significantly reduced vs. psiSCR-treated control. Data represent the mean±SD. *p<0.05 compared with the psiSCR-treated controls. In both vascular analyses, the number of examined animals was four or five in the psiSCR-treated group, nine in the psiVEGF-C-treated group and seven in the psiVEGF-D-treated group.

Expression of VEGF-C and VEGF-D correlates with lymph node metastasis in a variety of human cancer types, including breast neoplasms (19, 33, 34), although, in contrast to VEGF-C, the role of VEGF-D is more equivocal. In at least one study, no association between VEGF-D and lymph node metastasis was found in human breast cancer (35), while another group of investigators linked high expression of VEGF-A and VEGF-C, but not VEGF-D, with poor prognosis (36). More recently, a clinical study demonstrated that only tumor-derived VEGF-C induced pre-metastatic sentinel node lymphangionenesis in primary breast cancer (37).

In many animal models of cancer, overexpression of both VEGF-C and VEGF-D evidently enhanced tumor lymphangiogenesis and nodal/distant organ metastasis (10-16), and siRNA down-regulation of VEGF-C reduced lymph node and lung metastases in murine mammary models (21, 38). An endogenous soluble isoform of the VEGF-C receptor, sVEGFR-2 was identified and shown to be a specific inhibitor of lymphatic vessel growth (39); a subsequent study showed sVEGFR-2 suppressed tumor growth and lymph node metastasis in a mouse mammary model specifically through inhibition of lymphangiogenesis (23). VEGFR-3, the co-receptor for both VEGF-C and VEGF-D, is predominantly expressed on lymphatic endothelial cells (40), and VEGF-C/D-dependent receptor activation stimulates both lymph endothelial cells and lymphatic vessel development (8, 9). Using soluble VEGFR-3 to sequester VEGF-C and VEGF-D effectively blocked VEGFR-3 signaling and inhibited lymphangiogenesis and lymph node metastasis in animal models (41-43), while an antibody to VEGF-D suppressed VEGF-D-induced lymphatic spread (14).

We attempted to refine Vegf-c/d down-regulation by first isolating the most effective silencing siRNA sequences and then directly treating highly metastatic mammary tumors in vivo with those sequences in a transfectable plasmid vector. Although intratumoral transfection with psiVEGF-C or psiVEGF-D reduced the average number of metastatic lymph nodes, statistically significant reduction was obtained only with psiVEGF-C transfection. Silencing Vegf-c also resulted in significant reductions in treated tumor volumes, in the average number of metastatic nodules of the lungs, and in the average number of other organs affected by metastasis. In contrast, siRNA therapy targeting Vegf-d gave equivocal results; it was ineffective in modulating overall organ metastasis, but did show some reduction of tumor volume increment, average number of lymph node metastases and average number of dilated lymphatic vessels with invading cancer cells. Carter et al. reported that once human breast carcinomas reach 4 cm or larger, the chance of tumor recurrence and metastasis increases dramatically (44). The reduction in tumor volume and size with psiVEGF-C could thus have clinical significance. A similar pattern emerged when analyzing blood microvessel density and lymphatic vessel invasion; transfection with psiVEGF-D tended to reduce both parameters, but psiVEGF-C treatment was significantly more effective. This was a much stronger anti-metastatic effect than we had previously obtained (21), due, we believe, to the selection and use of a more effective Vegf-c siRNA sequence.

In conclusion, treatment with psiVEGF-C, but not psiVEGF-D, significantly suppressed wide-spectrum organ metastasis and several parameters of tumor metastasis in a mouse model with prognostic significance in human cancer, suggesting a potential clinical therapeutic option in the treatment of human metastatic disease.

Acknowledgements

This investigation was supported by a Grant-in-Aid for Scientific Research (C)(2) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (no. 17591360 to M.A. Shibata). We thank Ms. Naomi Nakano (Osaka Health Science University) and Ms. Akiko Yoshida (National Cerebral and Cardiovascular Center Research Institute) for their excellent secretarial assistance.

  • Received July 26, 2013.
  • Revision received September 11, 2013.
  • Accepted September 12, 2013.

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Therapy with siRNA for Vegf-c but Not for Vegf-d Suppresses Wide-spectrum Organ Metastasis in an Immunocompetent Xenograft Model of Metastatic Mammary Cancer
MASA-AKI SHIBATA, EIKO SHIBATA, JUNJI MORIMOTO, MARIKO HARADA-SHIBA
Anticancer Research Oct 2013, 33 (10) 4237-4247;
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