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[Cancer Research 66, 3971-3977, April 15, 2006]
© 2006 American Association for Cancer Research

Priority Reports

Placenta Growth Factor Overexpression Inhibits Tumor Growth, Angiogenesis, and Metastasis by Depleting Vascular Endothelial Growth Factor Homodimers in Orthotopic Mouse Models

Lei Xu, David M. Cochran, Ricky T. Tong, Frank Winkler, Satoshi Kashiwagi, Rakesh K. Jain and Dai Fukumura

Edwin L. Steele Laboratory, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

Requests for reprints: Lei Xu, Department of Radiation Oncology, Massachusetts General Hospital, 100 Blossom Street, Cox-7, Boston, MA 02114. Phone: 617-726-8051; Fax: 617-726-1961; E-mail: lei{at}steele.mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of placenta growth factor (PlGF) in pathologic angiogenesis is controversial. The effects of PlGF on growth, angiogenesis, and metastasis from orthotopic tumors are not known. To this end, we stably transfected three human cancer cell lines (A549 lung, HCT116 colon, and U87-MG glioblastoma) with human plgf-2 full-length cDNA. Overexpression of PlGF did not affect tumor cell proliferation or migration in vitro. The growth of PlGF-overexpressing tumors grown orthotopically or ectopically was impaired in all three tumor models. This decrease in tumor growth correlated with a decrease in tumor angiogenesis. The PlGF-overexpressing tumors had decreased vessel density and increased vessel diameter, but vessel permeability was not different from the parental tumors. Tumors overexpressing PlGF exhibited higher levels of PlGF homodimers and PlGF/vascular endothelial growth factor (VEGF) heterodimers but decreased levels of VEGF homodimers. Our study shows that PlGF overexpression decreases VEGF homodimer formation and inhibits tumor progression. (Cancer Res 2006; 66(8): 3971-7)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The role of placenta growth factor (PlGF) in pathologic angiogenesis is controversial. Three seminal studies by Carmeliet et al. showed that PlGF enhances pathologic angiogenesis by initiating crosstalk between vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 (1–3). On the contrary, studies from Yihai Cao et al. showed the opposite (4, 5). The effects of PlGF on the growth, angiogenesis, and metastasis of orthotopic tumors are not known.

The present study was designed to study the effect of PlGF overexpression on orthotopic tumor growth, angiogenesis, and metastasis. To this end, we stably transfected three tumor cell lines with Plgf-2 cDNA and implanted them ectopically (s.c.) and orthotopically (A549 lung carcinoma cells into the tail vein, HCT116 colon carcinoma cells into the cecum wall, and U87-MG glioblastoma cells into the brain). We found that PlGF inhibits in vivo tumor growth, angiogenesis, and metastasis. We also found depletion of active VEGF homodimers in PlGF-overexpressing tumors to be a potential mechanism of the observed effect of PlGF overexpression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. The human tumor cell lines HCT116, A549, and U87-MG were obtained from the American Type Culture Collection (Manassas, VA).

Reagents. Recombinant human VEGF, PlGF, and PlGF/VEGF heterodimers and PlGF-specific antibodies were obtained from R&D Systems (Minneapolis, MN).

Plasmid construct and transfection. Full-length plgf-2 cDNA (Genbank accession no. NM_002632) was cloned into peak12 vector driven by EF-1{alpha} promoter (obtained from Dr. Brian Seed, Massachusetts General Hospital, Boston, MA). This expression vector was stably transfected into A549, HCT116, and U87 cells using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA) as instructed by the manufacturer. The transfected cells were selected with 0.5, 0.8, and 0.8 µg/mL puromycin, respectively.

Northern blot analysis. Northern blot analysis was done as described previously (6). Plgf and ß-actin cDNA probes were synthesized by PCR using primers for PlGF, 5'-TCGTCAGAGGTGGAAGTGGT-3'; reverse primer, 5'-CTTCATCTTCTCCCGCAGAG; and for ß-actin, 5'-TGTATGCCTCTGGTCGTACC-3'.

ELISA for the quantitative measurement of PlGF and VEGF homodimers and PlGF/VEGF heterodimers. PlGF and VEGF homodimers were quantified using an ELISA kit specific for human PlGF and VEGF (R&D Systems). For quantification of PlGF/VEGF heterodimers, a PlGF ELISA plate was used. However, instead of the horseradish peroxidase–conjugated anti-PlGF polyclonal antibody, an enzyme-conjugated anti-VEGF polyclonal antibody was used for detection. This ELISA assay is specific for detection of PlGF/VEGF heterodimers and shows no cross-reactivity with PlGF or VEGF homodimers.

Implantation of tumor. The mice were anesthetized with ketamine/xylazine (90:9 mg/kg body weight) in PBS. To grow s.c. tumors, mice were given injections at the s.c. site of 1 x 106 viable tumor cells suspended in 0.2 mL of HBSS. Tumor volume was calculated as: tumor volume = length x (width)2 / 2.

To grow pulmonary tumor colonies, mice were given injections of 5 x 105 viable tumor cells in 0.1 mL HBSS through the lateral tail vein.

To grow cecal tumors, severe combined immunodeficient (SCID) mice received 1 x 106 viable cells by procedures previously described in detail (7). The weight of the cecal tumors was determined at necropsy.

To grow brain tumors, mice with cranial windows were used (8). Viable cells (3 x 105) were injected 1.5 mm lateral to the superior sagital sinus of the anterior part of the brain, into a depth of 0.4 mm. The injection was done with a Hamilton syringe slowly over 10 minutes, and the injection angle was 53 degrees.

Immunohistochemistry. CD31 (1:800; BD Biosciences, San Jose, CA) staining was carried out in 8-µm frozen sections. Microvessel density was determined as described before (9). Immunofluorescence double staining for CD31, and terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) was done as described previously (10). To quantify CD31/TUNEL+ cells, the number of double-positive cells was counted in 10 random 1.335-mm2 fields at x100 magnification. Endothelial cell nuclei were counter stained with 4',6-diamidino-2-phenylindole. Three tumor samples per group were studied. Macrophages were stained with F4/80 antibody (1:10; Serotec, Raleigh, NC). The number of positive cells was counted in 10 random 0.329-mm2 field under x200 magnification.

Permeability assay. The effective vascular permeability was measured using tetramethylrhodamine-labeled bovine serum albumin (BSA; Molecular Probes, Eugene, OR) according to published method (11, 12). The extravasated fluorescent BSA signal at a given region in the tumor was measured and quantified every 2 minutes for 23 minutes immediately after the injection. A macro program was used to quantify the rate of extravasation of BSA normalized by the density of blood vessels in the same region.

In vivo microscopy using the cranial window model. The mouse cranial window model was used to study the angiogenesis in brain tumors. In vivo microscopy using epifluorescence and multiphoton techniques was done as described previously (13).

Statistical analysis. All data are expressed as mean ± SE. The significance of differences between two groups was analyzed using the Student's t test (two tailed) or Mann-Whitney U test (two tailed).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human lung, brain, and colon carcinoma cell lines express high levels of VEGF and low levels of the PlGF gene. We screened 12 tumor cell lines, including human lung, ovarian, colon, brain, and kidney tumors, for expression of PlGF by Northern blot analysis. BeWo, a trophoblast cell line, was used as positive control. All cells expressed low levels of PlGF and high levels of VEGF mRNA (data not shown). A549, U87-MG, and HCT116 cells were selected and used in the following study because of their moderate level of PlGF mRNA and protein expression (A549, 53 ± 13 pg/mL; HCT116, 64 ± 24 pg/mL; U87, 105 ± 22 pg/mL). All three cell lines express high levels of VEGF protein (A549, 2,130 ± 54 pg/mL; HCT116, 1,989 ± 240 pg/mL; U87, 868 ± 65 pg/mL).

Characteristics of tumor cells transfected with Plgf. To analyze the effect of PlGF on tumor growth and metastasis, we cloned the full-length human Plgf-2 cDNA (Genbank accession no. NM_002632) and transfected it into A549, HCT116, and U87-MG cells. To avoid clonal variations, three high expression clones were combined. High expression of PlGF was detected in Plgf-2-transfected cells but not in parental or mock-transfected cells by both Northern blot and ELISA (Fig. 1A and B ).


Figure 1
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  		Figure 1. PlGF overexpression inhibits tumor progression at subcutaneous site. Parental, mock-, and Plgf-transfected A549, HCT116, and U87-MG cells were cultured under confluent conditions. A, mRNA was extracted, and a Northern blot analysis was done. Representative of at least three independent experiments. B, protein extracts were analyzed by ELISA. High levels of PlGF were detected in the PlGF-transfected cells. SCID mice were injected s.c. with 1 x 106 (C) A549, (D) HCT116, and (E) U87 cells with or without the Plgf-2 gene. Tumor growth was measured every 3 days. Points, mean; bars, SE. *, P < 0.001 compared with the corresponding wild-type tumor values.

 
Transfection with vector alone or Plgf-2 cDNA had no effect on tumor cell proliferation in vitro as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and overexpression of PlGF did not change the expression of VEGF, VEGFR-1, and VEGFR-2 mRNA levels as determined by Northern blot analysis (data shown as Supplementary Fig. S1).

PlGF inhibits tumor growth in vivo. To determine whether overexpression of PlGF affects tumor growth, we injected A549, HCT116, and U87-MG cells with or without the exogenous Plgf-2 gene into the s.c. space of SCID mice. Both A549 and A549-PlGF cells produced slow-growing tumors in the s.c. space. Although A549-PlGF tumors grew significantly slower than the parental and mock-transfected A549 tumors at early time points (days 14-17), the A549-PlGF tumor growth eventually caught up by day 38 (Fig. 1C). Mice injected with parental and mock-transfected HCT116 cells formed larger tumors than mice injected with HCT116-PlGF cells (P < 0.001) at all time points (Fig. 1D). Mice injected with U87 and U87-PlGF cells showed similar tumor growth pattern until day 25, after which the U87 tumors grew significantly faster than PlGF-overexpressing tumors (Fig. 1E).

To study the effect of PlGF on tumor metastasis and angiogenesis, orthotopic tumor models were used. Ten of 10 mice i.v. injected with parental A549 cells developed lung nodules 5 weeks later, whereas only five of eight mice injected with A549-PlGF cells developed lung nodules (Table 1 ). Mice injected with A549 and mock-transfected cells produced larger numbers of lung lesions than mice injected with A549-PlGF cells. These results clearly show that PlGF overexpression decreased the incidence and number of lung metastases formed.


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  		Table 1. Effect of PlGF on A549, HCT116, and U87 cell grown orthotopically in mice

 
All mice injected orthotopically into cecum wall with either parental HCT116 or HCT116-PlGF cells developed tumors. Mice injected with parental and mock-transfected (data not shown) HCT116 cells developed larger tumors than mice injected with HCT116-PlGF cells (Table 1). In the group injected with parental HCT116 cells, two mice developed liver metastasis, and one mouse developed blood ascites.

Mice injected with parental, mock-, or PlGF-transfected U87 cells into the brain of nude mice were killed on day 21 after injection. At the time of autopsy, mice injected with parental U87 cells developed tumors 15.3 ± 9.2 mm2 in area as observed from the cranial window, whereas mice injected with U87-PlGF tumors developed larger tumors (19.6 ± 6.3 mm2 in area; P > 0.4).

PlGF expression levels in the A549, HCT116, and U87 tumor samples were confirmed by Northern blot and ELISA analysis. The expression of VEGFR-1 and VEGFR-2 was not detectable with immunoprecipitation analysis (data not shown).

Effect of PlGF overexpression on angiogenesis, macrophage infiltration, and vessel permeability in vivo. To investigate the effect of PlGF on tumor angiogenesis in vivo, we stained the s.c. A549 tumors for CD31 (data are shown as number of CD31-positive vessels per 1.335 mm2 area). A549-PlGF tumors had more large blood vessels in their periphery and larger central regions of necrosis and a lower microvessel density (22 ± 4) compared with parental and mock-transfected tumors (44 ± 2 and 46 ± 1.5, respectively; Fig. 2 ).


Figure 2
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  		Figure 2. PlGF overexpression inhibits tumor angiogenesis. CD31 staining was done on (A and B) parental A549 tumors, (E and F) A549-PlGF tumors, (C and D) parental HCT116 tumors, and (G and H) HCT116-PlGF tumors. Magnification, x4 objective. Bar, 100 µm. Magnification, x20 objective. Bar, 50 µm. U87 cells (I; n = 5) and U87-PlGF cells (J; n = 7) were injected directly into the brain of nude mice. When the tumor reached 4 x 4 mm in diameter, two-photon microscopy images were taken of the tumor blood vessels (x5). Blood vessels were contrast enhanced by i.v. injection of FITC-dextran. Images are 2,000 µm across. K to P, in all three tumor models, tumor macrophages were stained with F4/80 antibody. Bar, 50 µm. Q to V, immunofluorescence double staining for CD31 and TUNEL. Endothelial cells were identified by CD31 staining (red), and DNA fragmentation was detected by localized terminal dUTP nick-end labeling (green) within the nucleus of apoptotic cells. Fragmented DNA in apoptotic endothelial cells in the merged images became yellow (arrows). Bar, 50 µm.

 
HCT116 tumors grown in the cecum wall were collected at the time of autopsy and stained with anti-CD31 antibody. The morphology of vessels in tumors from PlGF-transfected cells seemed to be vastly different from that of parental tumors, with the former having a larger diameter and the blood vessels distributed more around the periphery of the tumors. HCT116-PlGF tumors had a lower microvessel density (27 ± 3) compared with parental and mock-transfected tumors (62 ± 5 and 56 ± 6, respectively; Fig. 2).

Angiogenic activity in brain tumors was measured by intravital microscopy. Parental and mock-transfected U87 tumors had extensive tortuous and irregular small vessels. In contrast, U87-PlGF tumors showed significantly lower vessel density and larger mean vessel diameter compared with the other two groups. Vascular hyperpermeability is a hallmark of tumor vessels. U87-PlGF tumors had a similar vascular permeability compared with parental and mock-transfected U87 tumors (Table 1).

Next, we evaluated endothelial cell apoptosis in the tumors. Immunofluorescence double labeling of the tumors for CD31 expression and TUNEL positivity revealed that overexpression of PlGF increased the percentage of TUNEL-positive endothelial cells in all three tumor models. In A549 s.c. tumors, percentages increased from 1 ± 1% in the parental group to 5 ± 2% in A549-PlGF tumors; in HCT116 colon tumors, percentages increased from 0.3 ± 0.57% in the parental group to 5.6 ± 1.5% in HCT116-PlGF group; and in U87 brain tumors, percentages increased from 0.6 ± 1.1% in the parental group to 6.3 ± 2.1% in U87-PlGF group (P < 0.05; Fig. 2).

PlGF has been shown to activate normal monocytes (14) and increase macrophage infiltration in atherosclerotic lesions (15). By immunohistochemical staining for F4/80 marker for macrophages, we did not observe changes in macrophage infiltration (shown as number of F4/80-positive cells per 0.329 mm2 area) in A549 s.c. tumors (32 ± 8 in parental tumors versus 30 ± 5 in A549-PlGF tumors), in HCT116 tumors grown in the cecum wall (27 ± 8 in parental tumors versus 22 ± 5 in HCT116-PlGF tumors), and in U87 tumor implanted in the brain (319 ± 47 in parental tumors versus 287 ± 24 in U87-PlGF tumors).

Formation of PlGF/VEGF heterodimers in tumors. Previous studies showed that PlGF forms heterodimers with VEGF intracellularly and acts as an antagonist of VEGF (4). We quantified by ELISA the amount of PlGF and VEGF homodimers and PlGF/VEGF heterodimers in tumors with or without the exogenous Plgf-2 gene. As expected, high levels of VEGF homodimers were detected in tumor lysate from parental A549, HCT116, and U87 cells; high levels of PlGF homodimers were detected in Plgf-2-transfected tumors (Fig. 3 ). The amount of homodimeric VEGF in tumor tissues that overexpressed PlGF decreased significantly compared with parental tumors, whereas PlGF/VEGF heterodimers increased significantly. This indicates that highly expressed PlGF depleted VEGF homodimers, and that the majority of VEGF produced by the PlGF-transfected cells was involved in heterodimerization with PlGF.


Figure 3
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  		Figure 3. Quantitative measurement of PlGF and VEGF homodimers and PlGF/VEGF heterodimers in tumor tissues. Tumor lysates derived from A549 (A), HCT116 (B), and U87 (C) tumors with or without PlGF were analyzed for the presence of PlGF homodimers, VEGF homodimers and PlGF/VEGF heterodimers using ELISA assay. *, P < 0.0001 compared with corresponding wild-type tumor values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we observed that overexpression of human Plgf depleted VEGF homodimers and inhibited angiogenesis and growth of human lung, colon, and brain tumor cells when grown s.c. and/or orthotopically into mice. We choose to overexpress Plgf-2 because the effects of PlGF-2 on the growth, angiogenesis, and metastasis from orthotopic tumors are not known.

We chose to use A549, HCT116, and U87-MG cells in our study for two reasons. First, they express moderate levels of PlGF and high levels of VEGF constitutively compared with very low PlGF levels in most of the 12 tumor cell lines that we screened. Second, the expression of VEGF has been correlated with prognosis in human lung and colon cancer and in glioblastomas (16, 17), but the function of PlGF in the progression of these tumors was not known.

Carmeliet et al. showed that loss of PlGF caused impaired tumor growth and angiogenesis in embryonic stem cell–derived tumors (1), and that recombinant PlGF amplified the endothelial response to VEGF through VEGFR-1 transphosphorylation of VEGFR-2. In addition, PlGF/VEGF stimulated the survival of Plgf–/– capillary endothelial cells and increased tube formation of porcine aortic endothelial cells transfected with VEGFR-1 and VEGFR-2 in vitro. Furthermore, when given into the mice, 1.5 µg recombinant PlGF/VEGF increased myocardial angiogenesis to the same extent as recombinant VEGF (2). These studies used recombinant PlGF and PlGF/VEGF without affecting the constitutively expressed VEGF homodimer level. Our study used three tumor models that highly express VEGF constitutively. When PlGF is up-regulated in these tumors, the effect of depleting VEGF homodimers from the tumor cells seems to be greater than the effect of PlGF augmentation of VEGF function. In our study, A549-PlGF tumors implanted s.c. grew slower than the parental group at early time points (days 14-17) but caught up with the growth of the parental tumors at day 38. ELISA analysis showed A549-PlGF tumor still expresses relatively high levels of VEGF homodimers. This could contribute to the late tumor growth seen in this model. Thus, it remains to be determined to what extent and under which conditions the VEGF homodimer depleting effect or the VEGFR2 signal augmentation effect of PlGF is dominant in tumor progression and angiogenesis.

Yihai Cao's study reported that PlGF/VEGF heterodimers inhibit the angiogenic effect of VEGF in the mouse cornea assay (4), and overexpression of PlGF-1 with a retention signal leads to VEGF/PlGF-1 heterodimerization and results in abrogated tumor growth in Lewis lung carcinoma (5). It has been shown that PlGF/VEGF heterodimers are naturally present in tissues when both factors are synthesized in the same cells (18). Our study showed that PlGF overexpression inhibited tumor growth and metastasis at orthotopic sites. After PlGF transfection, VEGF mRNA levels did not change in tumor cells in vitro. By Northern blot (shown as Supplementary Fig. S1), we did not observe any differences in VEGF, VEGFR-1, and VEGFR-2 expression, excluding a direct negative effect of PlGF on VEGF and VEGF receptor transcription. Instead, the effect is due to a posttranslational regulation that controls the levels of heterodimeric and homodimeric VEGF. When PlGF is overexpressed, PlGF/VEGF heterodimer formation increased, consistent with previously published results (18). In addition, we showed that VEGF homodimers were depleted in PlGF-overexpressing tumors in vivo, leading to decreased angiogenesis and tumor progression.

PlGF activates VEGFR-1. VEGFR-1 has been shown to be a negative regulator of physiologic vascular development in the embryo (19, 20) and a positive regulator under pathologic conditions when PlGF is abnormally highly expressed (21). Although some studies suggest that VEGFR-1 has a direct role in transducing angiogenic signals, others report that it may act as a decoy receptor for VEGF (21–23), or down-regulate VEGFR-2-mediated endothelial cell proliferation through PI3K pathways (23, 24). Recent studies showed that inhibition of VEGFR-1 function by neutralizing antibodies leads to reduced tumor growth and a decreased number of perivascular haematopoietic cells in the tumors (3, 25). In addition, recombinant PlGF enhanced VEGFR-2 phosphorylation in capillary endothelial cells derived from Plgf–/– mice, indicating the function of PlGF in the intramolecular and intermolecular crosstalk between VEGFR-1 and VEGFR-2 (2). However, in our study, because the majority of the tumor tissue was composed of tumor cells, which express only low levels of VEGF receptors, VEGFR-1 and VEGFR-2 expression levels were not detectable.

In our study, we observed that when implanted orthotopically, PlGF-overexpressing U87 tumors exhibited different vessel morphology and vessel density but exhibited no difference in tumor growth. One of the mechanisms that could contribute to the growth of the orthotopic brain tumors might be the intensive macrophage infiltration (Fig. 2). Macrophages have been shown to express multiple growth factors that promote tumor growth and metastasis (26). Because there was significantly higher macrophage infiltration in U87 compared with other tumor types, and there was no significant difference in the macrophage infiltration between the parental and PlGF-overexpressing U87 tumors, the effect of VEGF homodimer depletion on tumor growth by PlGF overexpression may have been masked.

VEGF has been shown to increase vascular permeability. Our study showed that the PlGF-overexpressing tumors has less detectable VEGF homodimer; however, PlGF-overexpressing tumors tend to have relatively lower vascular permeability compared with the mock tumors, but the difference was not statistically significant. It is possible that PlGF is compensating for a reduction in VEGF-induced permeability. Indeed, previous studies have shown that PlGF alone or in synergy with VEGF can induce vascular permeability (27, 28). Furthermore, there was no difference in perivascular cell or basement membrane coverage in tumor tissues with and without PlGF overexpression (Table 1). This is consistent with our in vitro studies, which we observed that PlGF have no effects on proliferation and migration of perivascular cell precursors (10T1/2 cells; data not shown).

This report is the first to show the effect of PlGF on the growth, angiogenesis, and metastasis of lung, colon, and brain tumors grown orthotopically. Additional studies are clearly needed to further understand the complex PlGF/VEGF system and to evaluate PlGF as a potential target in cancer therapy.


   Acknowledgments
 
Grant support: National Cancer Institute Program Project grant PO1-CA80124 (D. Fukumura and R.K. Jain).

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.

We thank Chelsea J. Swandal for her technical support and Jennifer Lobo for her assistance in immunohistochemistry staining and imaging.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 8/25/04. Revised 11/ 4/05. Accepted 1/10/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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