Increased Melanoma Growth and Metastasis Spreading in Mice Overexpressing Placenta Growth Factor
2006; Elsevier BV; Volume: 169; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.051041
ISSN1525-2191
AutoresMarcella Marcellini, Naomi De Luca, Teresa Riccioni, Alessandro Ciucci, Angela Orecchia, Pedro Miguel Lacal, Federica Ruffini, Maurizio Pesce, Francesca Cianfarani, Giovanna Zambruno, Augusto Orlandi, Cristina Maria Failla,
Tópico(s)Cancer Cells and Metastasis
ResumoPlacenta growth factor (PlGF), a member of the vascular endothelial growth factor family, plays an important role in adult pathological angiogenesis. To further investigate PlGF functions in tumor growth and metastasis formation, we used transgenic mice overexpressing PlGF in the skin under the control of the keratin 14 promoter. These animals showed a hypervascularized phenotype of the skin and increased levels of circulating PlGF with respect to their wild-type littermates. Transgenic mice and controls were inoculated intradermally with B16-BL6 melanoma cells. The tumor growth rate was fivefold increased in transgenic animals compared to wild-type mice, in the presence of a similar percentage of tumor necrotic tissue. Tumor vessel area was increased in transgenic mice as compared to controls. Augmented mobilization of endothelial and hematopoietic stem cells from the bone marrow was observed in transgenic animals, possibly contributing to tumor vascularization. The number and size of pulmonary metastases were significantly higher in transgenic mice compared to wild-type littermates. Finally, PlGF promoted tumor cell invasion of the extracellular matrix and increased the activity of selected matrix metalloproteinases. These findings indicate that PlGF, in addition to enhancing tumor angiogenesis and favoring tumor growth, may directly influence melanoma dissemination. Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family, plays an important role in adult pathological angiogenesis. To further investigate PlGF functions in tumor growth and metastasis formation, we used transgenic mice overexpressing PlGF in the skin under the control of the keratin 14 promoter. These animals showed a hypervascularized phenotype of the skin and increased levels of circulating PlGF with respect to their wild-type littermates. Transgenic mice and controls were inoculated intradermally with B16-BL6 melanoma cells. The tumor growth rate was fivefold increased in transgenic animals compared to wild-type mice, in the presence of a similar percentage of tumor necrotic tissue. Tumor vessel area was increased in transgenic mice as compared to controls. Augmented mobilization of endothelial and hematopoietic stem cells from the bone marrow was observed in transgenic animals, possibly contributing to tumor vascularization. The number and size of pulmonary metastases were significantly higher in transgenic mice compared to wild-type littermates. Finally, PlGF promoted tumor cell invasion of the extracellular matrix and increased the activity of selected matrix metalloproteinases. These findings indicate that PlGF, in addition to enhancing tumor angiogenesis and favoring tumor growth, may directly influence melanoma dissemination. Tumor growth and metastasis spreading depend on the formation of new blood vessels that provide oxygen and nutrients.1Folkman J Angiogenesis in cancer, vascular, rheumatoid and other disease.Nat Med. 1995; 1: 27-31Crossref PubMed Scopus (7218) Google Scholar To promote new vessel development, tumor cells secrete angiogenic growth factors that act on the neighboring endothelial cells, inducing proliferation and migration. Recent studies demonstrate that tumor vascularization does not exclusively rely on sprouting of pre-existing vessels but also depends on bone marrow-derived progenitor cells.2Lyden D Hattori K Dias S Costa C Blaikie P Butros L Chadburn A Hessing B Marks W Witte L Wu Y Hicklin D Zhu Z Hackett NR Crystal RG Moore MAS Hajjar KA Manova K Benezra R Rafii S Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.Nat Med. 2001; 7: 1194-1201Crossref PubMed Scopus (1693) Google Scholar Among the growth factors implicated in tumor angiogenesis, a major contribution has been ascribed to vascular endothelial growth factor (VEGF).3Neufeld G Tessler S Gitay-Goren H Cohen T Levi B-Z Vascular endothelial growth factor and its receptors.Prog Growth Factor Res. 1994; 5: 89-97Abstract Full Text PDF PubMed Scopus (212) Google Scholar In addition to the proliferative and anti-apoptotic action on endothelial cells, VEGF directly promotes proliferation and/or migration of VEGF receptor-expressing tumor cells.4Graeven U Fiedler W Karpinski S Melanoma-associated expression of vascular endothelial growth factor and its receptors Flt-1 and KDR.J Cancer Res Clin Oncol. 1999; 125: 621-629Crossref PubMed Scopus (81) Google Scholar, 5Gitay-Goren H Halaban R Neufeld G Human melanoma cells but not normal melanocytes express vascular endothelial growth factor receptors.Biochem Biophys Res Commun. 1993; 190: 702-709Crossref PubMed Scopus (137) Google Scholar, 6Lacal P Failla CM Pagani E Odorisio T Schietroma C Cianfarani F Falcinelli S Zambruno G D'Atri S Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor.J Invest Dermatol. 2000; 115: 1000-1007Crossref PubMed Scopus (157) Google Scholar VEGF and its receptors are also involved in mobilization of endothelial and hematopoietic stem cells (HSCs) from the bone marrow and their recruitment at the tumor site where they contribute to tumor angiogenesis.2Lyden D Hattori K Dias S Costa C Blaikie P Butros L Chadburn A Hessing B Marks W Witte L Wu Y Hicklin D Zhu Z Hackett NR Crystal RG Moore MAS Hajjar KA Manova K Benezra R Rafii S Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.Nat Med. 2001; 7: 1194-1201Crossref PubMed Scopus (1693) Google Scholar, 7Grunewald M Avraham I Dor Y Bachar-Lustig E Itin A Yung S Chimenti S Landsman L Abramovitch R Keshet E VEGF-induced adult neovascularization: recruitment, retention, and role of accessory cells.Cell. 2006; 124: 175-189Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar Although VEGF and its receptor-2 (VEGFR-2) were previously considered major targets for the therapeutic inhibition of angiogenesis, a significant role of VEGF receptor-1 (VEGFR-1) in modulation of angiogenesis has been reported.8Luttun A Tjwa M Moons L Wu Y Angelillo-Scherrer A Liao F Nagy JA Hooper A Priller J De Klerck B Compernolle V Daci E Bohlen P Dewerchin M Herbert J Fava R Matthys P Carmeliet G Collen D Dvorak HF Hicklin DJ Carmeliet P Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt-1.Nat Med. 2002; 8: 831-840Crossref PubMed Scopus (947) Google Scholar VEGFR-1 has been shown to be involved in tissue-specific localization and growth of tumor metastases.9Hiratsuka S Nakamura K Iwai S Murakami M Itoh T Kijima H Shipley JM Senior RM Shibuya M MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis.Cancer Cell. 2002; 2: 289-300Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 10Kaplan RN Riba RD Zacharoulis S Bramley AH Vincent L Costa C MacDonald DD Jin DK Shido K Kerns SA Zhu Z Hicklin D Wu Y Port JL Altorki N Port ER Ruggero D Shmelkov SV Jensen KK Rafii S Lyden D VEGFR-1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.Nature. 2005; 438: 820-827Crossref PubMed Scopus (2488) Google Scholar Besides VEGF, other members of the VEGF family, in particular placenta growth factor (PlGF) and VEGF-B, bind to VEGFR-1, but their relative role in VEGFR-1 activation is still undefined. PlGF plays an important function in adult pathological angiogenesis, whereas it is redundant for the development of the embryonic vasculature.11Carmeliet P Moons L Luttun A Vincenti V Compernolle V De Mol M Wu Y Bono F Devy L Beck H Scholz D Acker T DiPalma T Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.Nat Med. 2001; 7: 575-583Crossref PubMed Scopus (1396) Google Scholar PlGF is chemotactic for monocytes12Clauss M Weich H Breier G Knies U Rockl W Waltenberger J Risau W The vascular endothelial growth factor receptor Flt-1 mediates biological activities.J Biol Chem. 1996; 271: 17629-17634Crossref PubMed Scopus (754) Google Scholar and can restore early and late phases of hematopoiesis after bone marrow suppression through chemotaxis of progenitor cells.13Hattori K Heissig B Wu Y Dias S Tejada R Ferris B Hicklin DJ Zhu Z Bohlen P Witte L Hendrikx J Hackett NR Crystal RG Moore MAS Werb Z Lyden D Rafii S Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1+ stem cells from bone-marrow microenvironment.Nat Med. 2002; 8: 841-849Crossref PubMed Scopus (560) Google Scholar PlGF also acts on VEGFR-1-expressing smooth muscle cells and pericytes, thus promoting vessel maturation. In fact, PlGF-deficient mice display less mature vessels compared to wild-type (WT) controls.11Carmeliet P Moons L Luttun A Vincenti V Compernolle V De Mol M Wu Y Bono F Devy L Beck H Scholz D Acker T DiPalma T Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.Nat Med. 2001; 7: 575-583Crossref PubMed Scopus (1396) Google Scholar Besides directly activating its tyrosine kinase receptor, PlGF sustains VEGF activity, probably through increased phosphorylation of VEGFR-2.14Autiero M Waltenberger J Communi D Kranz A Moons L Lambrechts D Kroll J Plaisance S De Mol M Bone F Kliche S Fellbrich G Ballmer-Hofer K Maglione D Mayr-Beyrle U Dewerchin M Dombrowski S Stanimirovic D Van Hummelen P Dehio C Hicklin DJ Persico G Herbert JM Communi D Shibuya M Collen D Conway EM Carmeliet P Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt-1 and Flk-1.Nat Med. 2003; 9: 936-943Crossref PubMed Scopus (655) Google Scholar Regarding the contribution to tumor angiogenesis, PlGF expression has been detected in human tumor cells both in vitro and in vivo and is up-regulated during tumor progression.6Lacal P Failla CM Pagani E Odorisio T Schietroma C Cianfarani F Falcinelli S Zambruno G D'Atri S Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor.J Invest Dermatol. 2000; 115: 1000-1007Crossref PubMed Scopus (157) Google Scholar, 15Kodama J Seki N Tokumo K Nakanishi Y Yoshinouchi M Kudo T Placenta growth factor is abundantly expressed in human cervical squamous cell carcinoma.Eur J Gynaecol Oncol. 1997; 18: 508-510PubMed Google Scholar, 16Donnini S Machein MR Plate KH Weich HA Expression and localization of placenta growth factor and PlGF receptors in human meningiomas.J Pathol. 1999; 189: 66-71Crossref PubMed Scopus (71) Google Scholar Moreover, tumor growth and angiogenesis are markedly reduced in PlGF-deficient mice,11Carmeliet P Moons L Luttun A Vincenti V Compernolle V De Mol M Wu Y Bono F Devy L Beck H Scholz D Acker T DiPalma T Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.Nat Med. 2001; 7: 575-583Crossref PubMed Scopus (1396) Google Scholar and overexpression of PlGF in glioma cells leads to increased tumor growth and endothelial cell survival.17Adini A Kornaga T Firoozbakht F Benjamin LE Placental growth factor is a survival factor for tumor endothelial cells and macrophages.Cancer Res. 2002; 62: 2749-2752PubMed Google Scholar On the other hand, PlGF could play a negative role in tumor angiogenesis through the formation of PlGF/VEGF heterodimers. These heterodimers are naturally produced by normal and tumor cells in culture,18DiSalvo J Bayne M Conn G Kwok P Trivedi P Soderman D Palisi T Sullivan K Thomas K Purification and characterization of a naturally occurring vascular endothelial growth factor/placenta growth factor heterodimer.J Biol Chem. 1995; 270: 7717-7723Crossref PubMed Scopus (245) Google Scholar, 19Cao Y Linden P Shima D Browne F Folkman J In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor.J Clin Invest. 1996; 98: 2507-2511Crossref PubMed Scopus (184) Google Scholar, 20Failla CM Odorisio T Cianfarani F Schietroma C Puddu P Zambruno G Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000; 115: 388-395Crossref PubMed Scopus (94) Google Scholar binding and activating VEGFR-2, although with reduced affinity compared to VEGF homodimers. Therefore, some authors suggest that the expression of PlGF in tumor tissue could result in inhibition of VEGF-mediated tumor angiogenesis because of the augmented formation of less active PlGF/VEGF heterodimers and the depletion of VEGF homodimers.21Eriksson A Cao R Pawliuk R Berg S Tsang M Zhou D Fleet C Tritsaris K Dissing S Leboulch P Cao Y Placenta growth factor-1 antagonizes VEGF-induced angiogenesis and tumor growth by the formation of functionally inactive PlGF-1/VEGF heterodimers.Cancer Cell. 2002; 1: 99-108Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar We have previously shown that PlGF overexpression in the skin, under the control of a keratin 14 promoter cassette (K14-PlGF mice), results in a substantial increase in number, branching, and size of dermal blood vessels.22Odorisio T Schietroma C Zaccaria ML Cianfarani F Tiveron C Tatangelo L Failla CM Zambruno G Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability.J Cell Sci. 2002; 115: 2559-2567PubMed Google Scholar Mature smooth muscle-coated vessels are abundant in the K14-PlGF mice. High levels of PlGF homodimers are produced by keratinocytes of transgenic mice whereas homodimeric VEGF is significantly reduced compared with control littermates. Therefore, this murine model represents a valuable tool to investigate the effect of PlGF in the biology of tumor growth and tumor-host interactions. In this study, we injected syngeneic murine melanoma cells in K14-PlGF transgenic mice and matched controls. Using this approach, we could demonstrate that PlGF transgenic animals substantially differ from WT littermates in melanoma growth rate, tumor vascularization, and metastasis formation. B16-BL6 murine melanoma cell line was maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 2 mmol/L glutamine, 10% fetal calf serum (Hyclone Laboratories, Logan, UT), and antibiotics. Human umbilical vein endothelial cells (HUVECs) were isolated from freshly delivered umbilical cords as previously described23Gimbrone MA Culture of vascular endothelium.Prog Hemost Thromb. 1976; 3: 1-28PubMed Google Scholar and cultured in Endothelial Cell Growth Medium-2 kit from Clonetics (BioWhittaker Inc., Walkersville, MD). The human melanoma cell line M146Lacal P Failla CM Pagani E Odorisio T Schietroma C Cianfarani F Falcinelli S Zambruno G D'Atri S Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor.J Invest Dermatol. 2000; 115: 1000-1007Crossref PubMed Scopus (157) Google Scholar was maintained in RPMI 1640 medium supplemented with glutamine, fetal calf serum, and antibiotics as described for the B16-BL6 cells. Human fibroblasts were isolated as previously described24Wirtz MK Glanville RW Steinmann B Rao VH Hollister DW Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2 (I) chain.J Biol Chem. 1987; 262: 16376-16385Abstract Full Text PDF PubMed Google Scholar and cultured in Dulbecco's modified Eagle's medium. The human fibroblast-conditioned medium was obtained from subconfluent cultures incubated for 24 hours in medium without serum, supplemented with 0.1% bovine serum albumin. Recombinant mouse PlGF-2 (rmPlGF) and VEGF (rmVEGF) were purchased from R&D Systems (Minneapolis, MN). Transgenic lines were established on a B6D2F1 background as previously described.22Odorisio T Schietroma C Zaccaria ML Cianfarani F Tiveron C Tatangelo L Failla CM Zambruno G Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability.J Cell Sci. 2002; 115: 2559-2567PubMed Google Scholar All mice were treated in accordance with the institutional guidelines for the care of experimental animals. For tumor implantation, mice were anesthetized by intraperitoneal injection of 15 μl/g 2.5% 2,2,2-tribromoethyl alcohol (Sigma-Aldrich, Milwaukee, WI). B16-BL6 cells (1 × 106) in 50 μl of Ca2+/Mg2+-free Hanks' balanced salt solution (ICN Biomedicals Inc., Aurora, OH) were inoculated intradermally in the right flanks of mice by using a 100-μl Hamilton syringe. Tumor attachment rate was 100%; tumor growth was followed daily and measured by a digital caliper. Tumor volume (V) was calculated as V = π/6xab2, where a is the longer and b is the shorter of two perpendicular tumor diameters. Mice were sacrificed 25 days after cell inoculation. Animals were necropsied, and organs were examined for the presence of macroscopic metastases. Tumors and lungs were then processed for histological analysis. For lung colonization assay, 1.5 × 104 B16-BL6 cells in 100 μl of Hanks' balanced salt solution were injected into the lateral tail vein. The animals were sacrificed 15 days after inoculation. The lungs were removed and processed for histological analysis. Four-μm-thick paraformaldehyde-fixed, paraffin-embedded tumor or lung sections were used, and specimens were deparaffinized, rehydrated, and processed as previously described.20Failla CM Odorisio T Cianfarani F Schietroma C Puddu P Zambruno G Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000; 115: 388-395Crossref PubMed Scopus (94) Google Scholar Hematoxylin and eosin (H&E) staining was performed by standard procedures. The antibodies used for blood vessel immunohistochemistry were an anti-mouse PECAM/CD31 polyclonal antibody (M-20; Santa Cruz Biotechnology, Santa Cruz, CA) used at a concentration of 2 μg/ml for 2 hours at 37°C and an anti-α-smooth muscle actin (α-SMA) monoclonal antibody (clone 144, Sigma-Aldrich) diluted 1:30, for 1 hour at room temperature. Negative controls were done by omitting the primary antibody. To detect SDF-1 in tumor sections, the polyclonal antibody (no. 22140192 APX; ImmunoKontact, Oxon, UK) was diluted 1:250 and incubated overnight at room temperature. The signal was amplified with the TSA biotin system (Perkin-Elmer Life Science, Boston, MA) following the manufacturer's instructions. Necrosis, total tumor area, and number of lung metastases were measured on serial H&E-stained sections at ×20 magnification using a Hamamatsu camera connected to a Nikon microscope and analyzed by Scion Image software (Scion Corp., Frederick, MD). CD31- and α-SMA-stained tumor and lung sections were evaluated with the AxioCam digital camera attached to the Axioplan 2 microscope (Carl Zeiss AG, Oberkochen, Germany). Calibration of each image and the number and area of blood vessels in the tumor stroma (tumor interior, distant from normal tissue more than 100 μm, ×200 magnification), in the tumor periphery (tumor area less than 100 μm distant from the interface with normal tissue, ×400 magnification), in the normal tissue/tumor interface (normal tissue comprised within 100 μm from the tumor edge, ×400 magnification), and in the pulmonary tissue (×200 magnification) were calculated using the KS300, version 3.0 software (Carl Zeiss AG). Quantification was performed on 10 fields per animal by two independent observers. For tumor section analysis, necrosis and hemorrhage areas were excluded. Results are presented as mean ± SEM. Values were evaluated by the nonparametric Mann-Whitney test; P ≤ 0.05 was considered significant. B16-BL6 melanoma cells in culture were maintained in basal medium containing 0.1% bovine serum albumin for 18 hours and then treated with 100 ng/ml of rmPlGF for 4, 12, and 24 hours. Cell pellets were resuspended in hypotonic buffer (10 mmol/L Tris-HCl, 1 mmol/L ethylenediaminetetraacetic acid, pH 7.4, with protease inhibitor cocktail tablets; Complete, Roche Diagnostics, Basel, Switzerland). An aliquot of extract was saved for determination of protein concentration and the remainder was boiled in the sodium dodecyl sulfate loading buffer. Eighty μg of protein per sample were separated by 6% gel electrophoresis and transferred to a nitrocellulose filter (Amersham Life Science, Buckinghamshire, UK). Protein detection was performed by using a polyclonal antibody against VEGFR-1 (C17, Santa Cruz Biotechnology) that recognizes both the murine and the human form of the receptor, at a concentration of 2 μg/ml, and with a monoclonal antibody against β-actin diluted 1:1000 (AC-40, Sigma-Aldrich). Detection was performed through a chemiluminescence assay (ECL; Amersham Life Science). B16-BL6 cells and HUVECs were seeded in 96-well tissue culture dishes at a density of 3 × 103 and 5 × 103cells/well, respectively, in the presence of complete medium. After 24 hours, culture medium was substituted with serum-free medium containing 10, 50, and 100 ng/ml of rmPlGF alone or in combination with 10 ng/ml of rmVEGF. The growth factors were then added every 24 hours. Three days later, the plates were stained with rhodamine B as described25Skehan P Stoneng R Scudiero D Monks A McMahon J Vistica D Warren JT Bokesch H Kenney S Boyd MR New colorimetric cytotoxicity assay for anticancer-drug screening.J Natl Cancer Inst. 1990; 82: 1107-1112Crossref PubMed Scopus (8881) Google Scholar and the absorbance read at 540 nm in a spectrophotometer (Victor; EG&G Wallac, Turku, Finland). Statistical analysis was performed by the unpaired Student's t-test; P ≤ 0.05 was considered significant. Blood was collected and white blood cell counts determined by using a hemocytometer. Plasma samples were obtained and murine PlGF, stem cell factor (SCF), and CXCL12/stromal cell-derived factor 1α (SDF-1α) were measured using the Quantikine Kit ELISA (R&D Systems, Eugene, OR). Peripheral blood mononuclear cells were isolated from heparinized blood after centrifugation over a discontinuous gradient using Lymphoprep (Axis-Shield PoC As, Oslo, Norway). Peripheral blood mononuclear cells (6 × 104) were plated in triplicate in a methylcellulose-based colony assay (StemCell Technologies, Vancouver, Canada) containing interleukin-6 and −3 and SCF. Colonies containing granulocyte/macrophage (CFU-GM), granulocyte (CFU-G), and macrophage cells (CFU-M) were scored after 10 days. Values were evaluated by the nonparametric Mann-Whitney test; P ≤ 0.05 was considered significant. Eight-μm polycarbonate filters (Nuclepore; Whatman Inc., Clifton, NJ) were coated with 10 μg of Matrigel (BD Biosciences, Bedford, MA). B16-BL6 cells (2 × 105) were introduced in the upper chamber of the Boyden chamber, and graded concentrations of rmPlGF or human fibroblast-conditioned medium were used as stimuli in the lower chamber. After a 3-hour incubation at 37°C, filters were fixed in 4% paraformaldehyde/phosphate-buffered saline and stained in 0.5% crystal violet. Cells from the upper surface were removed by wiping with a cotton swab, and the number of migrating cells attached to the lower surface of the filter was counted in 12 randomly selected microscopic fields (×200 magnification). The invasion index was calculated as the ratio between the number of cells per microscopic field in the experimental condition analyzed and the number of cells per microscopic field in the basal condition (ie, in the absence of any stimulus). Invasion index in the basal condition corresponds to 1. Specificity of rmPlGF-induced stimulation was analyzed by neutralization of the growth factor with a specific antibody (10 μg/ml monoclonal antibody [mAb] 465; R&D Systems) for 45 minutes at room temperature before the invasion test. As a control, an unrelated antibody was used in the same conditions (anti-human VEGF-D mAb 286; R&D Systems). Receptor involvement was analyzed by preincubating the cells with 10 μg/ml of neutralizing polyclonal antibodies anti-mouse VEGFR-1 (AF471; R&D Systems) or anti-mouse VEGFR-2 (AF644; R&D Systems). The invasion assay was performed in the presence of the antibodies. Statistical analysis was performed by the Student's t-test; P ≤ 0.05 was considered significant. B16-BL6 melanoma cells in culture were exposed to basal medium containing 0.1% bovine serum albumin for 18 hours and then treated with 100 ng/ml of rmPlGF for 3 hours. Total RNA was prepared using an RNeasy Midi kit (Qiagen, Chatsworth, CA) and reverse-transcribed for 50 minutes at 42°C using an oligo (dT)12-18 primer (Invitrogen) and the Superscript II enzyme (Invitrogen). Quantitative real-time PCR was performed using the SYBR Green PCR core reagent mix (Applied Biosystems, Foster City, CA) and gene-specific primers designed using the Primer Express software (Applied Biosystems). The primers used were: VEGFR-1 forward primer 5′-AGCCTACCTCACCGTGCAAG-3′ and reverse primer 5′-AAAAGAGGGTCGCAGCCAC-3′; MMP1 forward primer 5′-GGAGACCGGCAAAATGTGG-3′ and reverse primer 5′-TGCCCAAGTTGTAGTAGTTTTCCAG-3′; MMP2 forward primer 5′-ATCGCTCAGATCCGTGGTG-3′ and reverse primer 5′-TGTCACGTGGTGTCACTGTCC-3′; MMP3 forward primer 5′-GGGATGATGATGCTGGTATGG-3′ and reverse primer 5′-GCTTCACATCTTTTGCAAGGC-3′; MMP9 forward primer 5′-AAAACCTCCAACCTCACGGA-3′ and reverse primer 5′-GCGGTACAAGTATGCCTCTGC-3′; MMP10 forward primer 5′-GAGA-AATGGACACTTGCACCC-3′ and reverse primer 5′-AGGGAGTGGCCAAGTTCATG-3′; MMP13 forward primer 5′-CAAATGGTCCCAAACGAACTTAAC-3′ and reverse primer 5′-CCACTTCAGAATGGGACATATC-AG-3′. The reaction conditions were as follows: 2 minutes at 50°C (one cycle), 10 minutes at 95°C (one cycle), 15 seconds at 95°C, and 1 minute at 60°C (40 cycles). Gene-specific PCR products were continuously measured by means of the ABI Prism 5700 detection system (Perkin-Elmer, Norwalk, CT) and quantified with a gene-specific standard curve. Value normalization was performed by using a GAPDH standard curve (forward primer 5′-GTATGACTCCACTCACGGCAAA-3′ and reverse primer 5′-TTCCCATTCTCGGCCTTG-3′). Results are expressed as the relative fold increase of the PlGF-stimulated over the control group, which was used as a calibrator. B16-BL6 cells were seeded in 100-mm dishes at a density of 2.5 × 103 cells/cm2 in the presence of complete medium. After 24 hours, culture medium was substituted with either serum-free alone or serum-free medium containing 20 and 100 ng/ml of PlGF, and cells were maintained at 37°C for a further 24 hours. Conditioned media were collected and concentrated using Centricon YM-10 concentrators (Amicon Bioseparations; Millipore Corp., Bedford, MA). Protein content was determined by the Bradford assay (Bio-Rad, Hercules, CA). Thirty-five-μl aliquots were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 8% polyacrylamide gels containing 0.1% gelatin under nonreducing conditions, washed first in renaturing buffer (2.7% Triton X-100) and then in a developing buffer (50 mmol/L Tris-HCl, 200 mmol/L NaCl, 10 mmol/L CaCl2, pH 7.5) for 30 minutes, and left in the same buffer overnight at 37°C. Gels were stained with Coomassie brilliant blue R-250 (Bio-Rad) and destained with 5% methanol and 7% acetic acid. A semiquantitative analysis of MMP-2 and MMP-9 activity was performed in triplicate by densitometric methods using Fluor S Max Multi Imager (Bio-Rad), and intensity for each band was expressed as uncalibrated optical density (OD). To establish the role of PlGF in promoting tumor growth in vivo, B16-BL6 melanoma cells were injected intradermally into the flank of K14-PlGF transgenic mice and WT littermates (n = 9). Tumor size was measured daily starting from day 15 after injection, when the tumor mass began to be detectable in both transgenic and control animals. Mice were sacrificed 25 days after cell injection. As shown in Figure 1A, in K14-PlGF mice tumors grew exponentially throughout time with dramatic growth acceleration as compared with tumors in WT animals. A significant difference was already detectable at day 15 after cell injection. At day 25, there was a more than fivefold increase in tumor volume in K14-PlGF mice compared to WT littermates. Macroscopic analysis of tumors dissected from the transgenic animals revealed that the melanoma mass was surrounded by an extended area of prominent vascularization (Figure 1B). The B16-BL6 cell line showed a constitutive production of PlGF both in vitro, as assessed by immunoassay of the culture medium, and in vivo, as evaluated by immunohistochemistry. No difference in PlGF expression was observed in tumors grown in transgenic compared to WT mice (data not shown). Thus, to investigate whether the higher amount of PlGF secreted by transgenic mouse keratinocytes could directly stimulate melanoma cell growth, we first analyzed the expression of the PlGF tyrosine kinase receptor VEGFR-1 on B16-BL6 cells. Total RNA was extracted from cells treated or not with 100 ng/ml of rmPlGF for 3 hours, reverse-transcribed, and analyzed by real-time PCR. VEGFR-1 mRNA coding for the transmembrane form of the receptor was detected in melanoma cells and appeared slightly increased on cell stimulation with the growth factor (Figure 2A). At the protein level, VEGFR-1 production was analyzed through Western blotting of B16-BL6 cell extract using an antibody specific for the C-terminal portion of the receptor (Figure 2B). HUVEC extract was used as a positive control and showed two bands corresponding to glycosylated and unglycosylated forms of VEGFR-1.26Orecchia A Lacal PM Schietroma C Morea V Zambruno G Failla CM Vascular endothelial growth factor receptor-1 is deposited in the extracellular matrix by endothelial cells and is a ligand for the α5β1 integrin.J Cell Sci. 2003; 116: 3479-3489Crossref PubMed Scopus (84) Google Scholar B
Referência(s)