VEGFR-3 and Its Ligand VEGF-C Are Associated with Angiogenesis in Breast Cancer
1999; Elsevier BV; Volume: 154; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65392-8
ISSN1525-2191
AutoresReija Valtola, Petri Salvén, Päivi Heikkilä, Jussi Taipale, Heikki Joensuu, Marko Rehn, Taina Pihlajaniemi, Herbert A. Weich, Robert M. deWaal, Kari Alitalo,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoRecently, monoclonal antibodies against the human vascular endothelial growth factor receptor VEGFR-3 were shown to provide a specific antigenic marker for lymphatic endothelium in various normal tissues. In this study we have investigated the expression of VEGFR-3 and its ligand VEGF-C in normal breast tissue and in breast tumors by immunohistochemistry. VEGFR-3 was weakly expressed in capillaries of normal breast tissue and in fibroadenomas. In intraductal breast carcinomas, VEGFR-3 was prominent in the “necklace” vessels adjacent to the basal lamina of the tumor-filled ducts. VEGF receptor 1 and 2 as well as blood vessel endothelial and basal lamina markers were colocalized with VEGFR-3 in many of these vessels. Antibodies against smooth muscle α-actin gave a weak staining of the necklace vessels, suggesting that they were incompletely covered by pericytes/smooth muscle cells. A highly elevated number of VEGFR-3 positive vessels was found in invasive breast cancer in comparison with histologically normal breast tissue (P < 0.0001, the Mann-Whitney test). VEGF-C was located in the cytoplasm of intraductal and invasive cancer cells. The results demonstrate that the expression of VEGFR-3 becomes up-regulated in the endothelium of angiogenic blood vessels in breast cancer. The results also suggest that VEGF-C secreted by the intraductal carcinoma cells acts predominantly as an angiogenic growth factor for blood vessels, although this paracrine signaling network between the cancer cells and the endothelium may also be involved in modifying the permeabilities of both blood and lymphatic vessels and metastasis formation. Recently, monoclonal antibodies against the human vascular endothelial growth factor receptor VEGFR-3 were shown to provide a specific antigenic marker for lymphatic endothelium in various normal tissues. In this study we have investigated the expression of VEGFR-3 and its ligand VEGF-C in normal breast tissue and in breast tumors by immunohistochemistry. VEGFR-3 was weakly expressed in capillaries of normal breast tissue and in fibroadenomas. In intraductal breast carcinomas, VEGFR-3 was prominent in the “necklace” vessels adjacent to the basal lamina of the tumor-filled ducts. VEGF receptor 1 and 2 as well as blood vessel endothelial and basal lamina markers were colocalized with VEGFR-3 in many of these vessels. Antibodies against smooth muscle α-actin gave a weak staining of the necklace vessels, suggesting that they were incompletely covered by pericytes/smooth muscle cells. A highly elevated number of VEGFR-3 positive vessels was found in invasive breast cancer in comparison with histologically normal breast tissue (P < 0.0001, the Mann-Whitney test). VEGF-C was located in the cytoplasm of intraductal and invasive cancer cells. The results demonstrate that the expression of VEGFR-3 becomes up-regulated in the endothelium of angiogenic blood vessels in breast cancer. The results also suggest that VEGF-C secreted by the intraductal carcinoma cells acts predominantly as an angiogenic growth factor for blood vessels, although this paracrine signaling network between the cancer cells and the endothelium may also be involved in modifying the permeabilities of both blood and lymphatic vessels and metastasis formation. Vascular endothelial growth factor (VEGF) is a well-known hypoxia-induced stimulator of endothelial cell growth and angiogenesis,1Ferrara N Davis-Smyth T The biology of vascular endothelial growth factor.Endocr Rev. 1997; 18: 4-25Crossref PubMed Scopus (3668) Google Scholar, 2Shibuya M Role of VEGF-FLT receptor system in normal and tumor angiogenesis.Adv Cancer Res. 1995; 67: 281-316Crossref PubMed Scopus (276) Google Scholar which is also up-regulated by various hormones and cytokines, such as transforming growth factor-β.3Ferrara N: The role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int 1999, In pressGoogle Scholar VEGF is a ligand for two tyrosine kinase receptors named VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 is mainly expressed in endothelial cells, whereas VEGFR-1 can also be found in monocytes.4Clauss M Weich H Breier G Knies U Rockl W Waltenberger J Risau W The vascular endothelial growth factor receptor Flt-1 mediates biological activities: Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis.J Biol Chem. 1996; 271: 17629-17634Crossref PubMed Scopus (751) Google Scholar The inhibition of VEGF activity by specific monoclonal antibodies has been reported to reduce the growth of experimental tumors and their blood vessel density.5Kim KJ Li B Winer J Armanini M Gillett N Phillips HS Ferrara N Inhibition of vascular endothelial growth factor induced angiogenesis suppresses tumour growth in vivo.Nature. 1993; 362: 841-844Crossref PubMed Scopus (3325) Google Scholar Primary breast cancers are known to express several angiogenic polypeptides of which VEGF was the most abundant.6Relf M LeJeune S Scott P Fox S Smith K Leek R Moghaddam A Whitehouse R Bicknell R Harris A Expression of the angiogenic factors vascular endothelial growth factor, acidic and basic fibroblast growth factor, tumor growth factor β-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis.Cancer Res. 1997; 57: 963-969PubMed Google Scholar, 7Bicknell R Lewis C Ferrara N Tumour Angiogenesis. Oxford University Press, Oxford1997Google Scholar Tumor cells contained high levels of VEGF mRNA in both invasive and noninvasive, ductal (in situ) breast carcinomas.8Brown LF Berse B Jackman RW Tognazzi K Guidi AJ Dvorak HF Senger DR Connolly JL Schnitt SJ Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer.Hum Pathol. 1995; 26: 86-91Abstract Full Text PDF PubMed Scopus (606) Google Scholar The endothelial cells adjacent to the in situ carcinomas expressed VEGFR-1 and VEGFR-2 mRNA in a continuous pattern. Thus, VEGF and its receptors may contribute to the angiogenic progression of malignant breast tumors because correlations have been found between tumor vascular density and the prognosis of the disease.9Weidner N Folkman J Pozza F Bevilacqua P Allred EN Moore DH Meli S Gasparini G Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma.J Natl Cancer Inst. 1992; 84: 1875-1887Crossref PubMed Scopus (1811) Google ScholarVEGFR-3 is a receptor tyrosine kinase that is similar to the two VEGF receptors in structure but does not bind VEGF,10Pajusola K Aprelikova O Pelicci G Weich H Claesson-Welsh L Alitalo K Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors.Oncogene. 1994; 9: 3545-3555PubMed Google Scholar placenta growth factor (PlGF),11Sawano A Takahashi T Yamaguchi S Aonuma M Shibuya M Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for placenta growth factor, which is related to vascular endothelial growth factor.Cell Growth Differ. 1996; 7: 213-221PubMed Google Scholar or VEGF-B.12Olofsson B Korpelainen E Mandriota S Pepper MS Aase K Kumar V Gunji Y Jeltsch MM Shibuya M Alitalo K Eriksson U VEGF-B binds to VEGFR-1, and regulates plasminogen activator activity in endothelial cells.Proc Natl Acad Sci USA. 1998; 95: 11709-11714Crossref PubMed Scopus (443) Google Scholar VEGFR-3 is initially expressed in all embryonic endothelia, but its expression in the blood vessel endothelium decreases during development, and it becomes largely restricted to the lymphatic endothelium in adult tissues.13Kaipainen A Korhonen J Mustonen T van Hinsbergh VM Fang G-H Dumont D Breitman M Alitalo K Expression of the fms-like tyrosine kinase FLT4 gene becomes restricted to endothelium of lymphatic vessels during development.Proc Natl Acad Sci USA. 1995; 92: 3566-3570Crossref PubMed Scopus (1174) Google Scholar In the early embryos, VEGFR-3 plays an important role in blood vessel development.14Dumont DJ Jussila L Taipale J Lymboussaki A Mustonen T Pajusola K Breitman M Alitalo K Cardiovascular failure in mouse embryos deficient in VEGF receptor-3.Science. 1998; 282: 946-949Crossref PubMed Scopus (689) Google Scholar We have shown that monoclonal antibodies against VEGFR-3 provide the first specific antigenic marker for lymphatic endothelial cells in several normal tissues.15Jussila L Valtola R Partanen T Salven P Heikkilä P Matikainen M-T Renkonen R Kaipainen A Detmar M Tschachler E Alitalo R Alitalo K Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3.Cancer Res. 1998; 58: 1599-1604PubMed Google Scholar In adults, very little or no staining was observed in most blood vessel endothelia, whereas increased expression was found in Kaposi's sarcoma spindle cells and in vascular skin tumors.15Jussila L Valtola R Partanen T Salven P Heikkilä P Matikainen M-T Renkonen R Kaipainen A Detmar M Tschachler E Alitalo R Alitalo K Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3.Cancer Res. 1998; 58: 1599-1604PubMed Google Scholar, 16Lymboussaki A Partanen TA Olofsson B Thomas-Crusells J Fletcher C deWaal R Kaipainen A Alitalo K Expression of the vascular endothelial growth factor receptor VEGFR-3 in lymphatic endothelium of the skin and in vascular tumors.Am J Pathol. 1998; 153: 395-403Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar In addition, increased VEGFR-3 mRNA has been found in metastatic lymph nodes and in lymphangiomas.13Kaipainen A Korhonen J Mustonen T van Hinsbergh VM Fang G-H Dumont D Breitman M Alitalo K Expression of the fms-like tyrosine kinase FLT4 gene becomes restricted to endothelium of lymphatic vessels during development.Proc Natl Acad Sci USA. 1995; 92: 3566-3570Crossref PubMed Scopus (1174) Google ScholarThe two known ligands of VEGFR-3 have a high degree of homology to VEGF. Thus, they have been named as VEGF-C17Joukov V Pajusola K Kaipainen A Chilov D Lahtinen I Kukk E Saksela O Kalkkinen N Alitalo K A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar and VEGF-D18Achen M Jeltsch M Kukk E Mäkinen T Vitali A Wilks A Alitalo K Stacker S Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4).Proc Natl Acad Sci USA. 1998; 95: 548-553Crossref PubMed Scopus (1011) Google Scholar and consist of proteolytically processed polypeptides, which form disulfide-linked dimers.18Achen M Jeltsch M Kukk E Mäkinen T Vitali A Wilks A Alitalo K Stacker S Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4).Proc Natl Acad Sci USA. 1998; 95: 548-553Crossref PubMed Scopus (1011) Google Scholar, 19Joukov V Sorsa T Kumar V Jeltsch M Claesson-Welsh L Cao Y Saksela O Kalkkinen N Alitalo K Proteolytic processing regulates receptor specificity and activity of VEGF-C.EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (638) Google Scholar Experiments in transgenic mice have shown that VEGF-C is a growth factor for the developing lymphatic vessels,20Jeltsch M Kaipainen A Joukov V Meng X Lakso M Rauvala H Swartz M Fukumura D Jain RK Alitalo K Hyperplasia of lymphatic vessels in VEGF-C transgenic mice.Science. 1997; 276: 1423-1425Crossref PubMed Scopus (1103) Google Scholar, 21Oh S-J Jeltsch MM Birkenhager R McCarthy JE Weich HA Christ B Alitalo K Wilting J VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane.Dev Biol. 1997; 188: 96-109Crossref PubMed Scopus (430) Google Scholar although it can also bind to VEGFR-2 expressed in blood vessel endothelia and induce capillary endothelial cell migration in culture17Joukov V Pajusola K Kaipainen A Chilov D Lahtinen I Kukk E Saksela O Kalkkinen N Alitalo K A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar, 19Joukov V Sorsa T Kumar V Jeltsch M Claesson-Welsh L Cao Y Saksela O Kalkkinen N Alitalo K Proteolytic processing regulates receptor specificity and activity of VEGF-C.EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (638) Google Scholar and angiogenesis ischemic rabbit hindlimb22Witzenbichler B Asahara T Murohara T Silver M Spyridopoulos I Magner M Principe N Kearney M Hu J-H Isner JM Vascular endothelial growth factor-C (VEGF-C/VEGFR-2) promotes angiogenesis in the setting of tissue ischemia.Am J Pathol. 1998; 153: 381-394Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar and in mouse cornea.23Cao Y Linden P Farnebo J Cao R Eriksson A Kumar V Qi J-H Claesson-Welsh L Alitalo K Vascular endothelial growth factor induces angiogenesis in vivo.Proc Natl Acad Sci USA. 1998; 95: 14389-14394Crossref PubMed Scopus (492) Google Scholar Expression of VEGF-C mRNA has been detected also in malignant human tumors, including nearly half of the breast cancers analyzed.24Salven P Lymboussaki A Heikkilä P Joensuu H Alitalo K Vascular endothelial growth factors VEGF-B and VEGF-C are expressed in human tumors.Am J Pathol. 1998; 153: 103-108Abstract Full Text Full Text PDF PubMed Scopus (292) Google ScholarWhile active angiogenesis is known to be a prerequisite for tumor growth beyond a few mm3 in size,25Folkman J Angiogenesis in cancer, vascular, rheumatoid and other disease.Nat Med. 1995; 1: 27-31Crossref PubMed Scopus (7176) Google Scholar lymphangiogenesis in normal or pathological adult tissues, including malignant tumors has not been reported.25Folkman J Angiogenesis in cancer, vascular, rheumatoid and other disease.Nat Med. 1995; 1: 27-31Crossref PubMed Scopus (7176) Google Scholar, 26Jain R Delivery of novel therapeutic agents in tumors: physiological barriers and strategies.J Natl Cancer Inst. 1989; 81: 570-576Crossref PubMed Scopus (511) Google Scholar, 27de Waal R van Altena M Erhard H Lack of lymphangiogenesis in human primary cutaneous melanoma: consequences for the mechanism of lymphatic dissemination.Am J Pathol. 1997; 150: 1951-1957PubMed Google Scholar It has been suggested that solid tumors may compress the nearby lymphatic vessels, which cannot penetrate the tumor mass because of an elevated interstitial fluid pressure within the tumor.28Baish J Netti P Jain R Transmural coupling of fluid flow in microcirculatory network and interstitium in tumors.Microvasc Res. 1997; 53: 128-141Crossref PubMed Scopus (124) Google Scholar If lymphangiogenesis takes place during cancer progression, cancers with active lymphangiogenesis could be predisposed to metastatic spread via the lymphatic system and thus to poor survival. We wanted to use VEGFR-3 as a marker to study the lymphatic vasculature of breast carcinomas. To our surprise, we found that in normal breast tissue, VEGFR-3 was very weakly expressed in blood capillary endothelium in addition to lymphatic endothelia. In breast carcinomas, VEGFR-3 was also expressed in the lymphatic endothelium, but more detailed analysis showed that the expression of this receptor becomes up-regulated in angiogenic capillaries and that some of the nearby tumor cells express the VEGF-C protein.Materials and MethodsFreshly Frozen Tissue SamplesFreshly frozen breast tissue samples were retrieved from the files of the Department of Pathology, University of Helsinki. The samples consisted of ductal carcinoma (n = 6), lobular carcinoma (n = 6), intraductal carcinoma (n = 8), fibroadenoma (n = 4), and histologically normal breast tissue (n = 12). Two of the ductal carcinomas were grade I, three were grade II, and one was grade III. All samples had been frozen immediately after surgical excision in liquid nitrogen and stored at −70°C.ImmunohistochemistryMouse monoclonal antibodies (mAbs) against human VEGFR-3 were produced as described earlier.15Jussila L Valtola R Partanen T Salven P Heikkilä P Matikainen M-T Renkonen R Kaipainen A Detmar M Tschachler E Alitalo R Alitalo K Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3.Cancer Res. 1998; 58: 1599-1604PubMed Google Scholar The VEGFR-3 extracellular protein domain (VEGFR-3EC) was expressed via a recombinant baculovirus in insect cells and purified from the culture medium. Mouse monoclonal antibodies against VEGFR-3EC were then produced using standard methods, and the immunoglobulin fraction was purified by protein A affinity chromatography from hybridoma ascites fluid or Tecnomouse® culture supernatants.Five-μm cryosections of the tissues were air-dried and fixed in cold acetone for 10 minutes. The sections were rehydrated in phosphate-buffered saline (PBS) and incubated for 30 minutes in 5% normal horse serum at room temperature. The sections were then incubated for 2 hours in a humid atmosphere at room temperature with the 9D9 mAbs at the concentration of 1.0 μg/ml. Other anti-VEGFR-3 mAb against distinct epitopes of the VEGFR-3EC were also studied; clones 2E11 and 7B8 were used at the concentrations of 9.5 and 8.5 μg/ml, respectively. A subsequent incubation for 30 minutes in biotinylated anti-mouse serum was followed by a 60-minute incubation using reagents of the Vectastain Elite Mouse IgG ABC kit (Vector Laboratories, Burlingame, CA). Peroxidase activity was developed with 3-amino-9-ethyl carbazole (AEC, Sigma, St. Louis, MO) for 10 minutes. Finally, the sections were stained with hematoxylin for 20 seconds. Negative controls were done by omitting the primary antibody or by using irrelevant primary antibodies of the same isotype. The purified baculoviral immunogen15Jussila L Valtola R Partanen T Salven P Heikkilä P Matikainen M-T Renkonen R Kaipainen A Detmar M Tschachler E Alitalo R Alitalo K Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3.Cancer Res. 1998; 58: 1599-1604PubMed Google Scholar was used to block the binding of the 9D9 antibodies as another negative control. In these experiments the antibodies were incubated overnight with a 10-fold molar exess of the VEGFR-3EC protein in PBS. After centrifugation for 4 minutes at 4000 rpm, 4°C, the supernatant was carefully collected and then used as primary antibody. The 5-μm cryosections adjacent to the ones stained with the anti-VEGFR-3 antibodies were immunostained for PAL-E (0.15 μg/ml, Monosan, Uden, The Netherlands), laminin (1:4000 dilution of the supernatant of clone LAM-89, Sigma), collagen XVIII (1.9 μg/ml), α-smooth muscle actin (SMA, 0.5 μg/ml, clone 1A4, Sigma), VEGFR-1 (1:200 dilution of the supernatant of clone 19), or VEGFR-2 (dilution 1:100).29Simon M Röckl W Hornig C Gröne EF Theis H Weich HA Fuchs E Yayon A Gröne H-J Receptors of vescular endothelial growth factor/vascular permeability factor (VEGF/VPF) in fetal and adult human kidney: localization and (125 I) VEGF binding sites.J Am Soc Nephrol. 1998; 9: 1032-1044PubMed Google ScholarFollowing the staining procedures, all samples were examined by a trained pathologist (P.H.). The blood vascular densities were obtained from the slides stained for PAL-E,27de Waal R van Altena M Erhard H Lack of lymphangiogenesis in human primary cutaneous melanoma: consequences for the mechanism of lymphatic dissemination.Am J Pathol. 1997; 150: 1951-1957PubMed Google Scholar following the guidelines recommended by Gasparini and Harris.30Gasparini G Harris A Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool.J Clin Oncol. 1995; 13: 765-782Crossref PubMed Scopus (454) Google Scholar The VEGFR-3 positive vessel densities were studied in the same way. A slide was first scanned at low magnification, and intratumoral vessel density was then assessed by counting the number of stained vessels per a ×400-magnification high power field (hpf) in the areas with the highest vascular density (“vascular hotspots”) or in the areas with highest VEGFR-3 positive vessel density. A minimum of five fields was counted per slide, after which the three highest counts were averaged.Double-Staining ProceduresVEGFR-3 and PAL-E Double StainingDouble staining with PAL-E and VEGFR-3 mAbs was used to differentiate immunohistochemical staining of lymphatic and blood vessels in two intraductal carcinomas. Briefly, acetone-fixed 5-μm cryosections were incubated for 1 hour with anti-PAL-E antibodies with biotinylated horse anti-mouse antibody (Vectastain Elite Mouse IgG ABC kit, Vector Laboratories) for 30 minutes with ABC-peroxidase (Vectastain, 1:100) for 45 minutes and developed finally with 3-amino-9-ethyl carbazole (AEC) for 10 minutes. For the second step, the sections were incubated with anti-VEGFR-3 antibodies for 1 hour (0.14 μg/ml), followed by biotinylated anti-mouse antibody for 30 minutes (1:200 dilution of the supernatant of clone), ABC-peroxidase for 30 minutes (1:100), biotinylated tyramin solution (1:2.000) containing 0.01% peroxide for 5 minutes, ABC-alkaline phosphatase (1:100) for 20 minutes, and developed with Fast Blue (Sigma) for 20 minutes, according to a procedure previously described for in situ hybridization signal enhancement.31Kerstens H Poddighe P Hanselaar A A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine.J Histochem Cytochem. 1995; 43: 347-352Crossref PubMed Scopus (283) Google Scholar Five-μm cryosections adjacent to the double stained ones were also immunostained with VEGFR-3 antibodies only, as described above.VEGFR-3 and Ki-67 Double StainingTo study if the endothelial cells in VEGFR-3 positive necklace vessels in intraductal carcinoma were undergoing angiogenesis, two intraductal samples were chosen for double staining for the nuclear proliferation marker Ki-67 (0.5 mg/ml, Dako Immunoglobulins, Glostrup, Denmark) and VEGFR-3. Five-μm frozen sections were rehydrated and incubated for 20 minutes in 5% normal goat serum at room temperature. The sections were incubated for 1 hour at room temperature with the rabbit polyclonal Ki-67 antibody at a concentration of 5 μg/ml. A subsequent incubation for 30 minutes in biotinylated anti-rabbit serum was followed by a 30-minute incubation using reagents of the Vectastain Elite ABC anti-rabbit kit (Vector Laboratories). Peroxidase activity was developed with 3-amino-9-ethyl carbazole (Sigma) for 10 minutes. Subsequently, the sections were rehydrated and incubated for 20 minutes in 5% in normal horse serum at room temperature. The sections were incubated for 1 hour with the anti-VEGFR-3 monoclonal antibodies at the concentration of 2.6 μg/ml. A subsequent incubation for 30 minutes in biotinylated anti-mouse serum was followed by a 30-minute incubation using reagents of the Vectastain Elite ABC anti-mouse IgG kit (Vector Laboratories). Peroxidase activity was developed with 3,3′-diaminobenzidine (Sigma) for 10 minutes. Finally, the sections were stained with hematoxylin. Negative controls were performed by omitting the primary antibodies or by using irrelevant primary antibodies.Production, Analysis, and Use of Polyclonal Anti-VEGF-C AntibodiesPolyclonal antibodies were produced in rabbits against a synthetic peptide corresponding to the amino acid residues 2–18 of the N-terminus of mature secreted human VEGF-C (residues 104–120 of the VEGF-C prepropeptide) as described earlier.17Joukov V Pajusola K Kaipainen A Chilov D Lahtinen I Kukk E Saksela O Kalkkinen N Alitalo K A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.EMBO J. 1996; 15: 290-298Crossref PubMed Scopus (1143) Google Scholar, 19Joukov V Sorsa T Kumar V Jeltsch M Claesson-Welsh L Cao Y Saksela O Kalkkinen N Alitalo K Proteolytic processing regulates receptor specificity and activity of VEGF-C.EMBO J. 1997; 16: 3898-3911Crossref PubMed Scopus (638) Google Scholar The antisera were affinity-purifed using the immunogenic polypeptide coupled to an epoxy-activated sepharose-6B column and tested for specific staining of VEGF-C using cells infected with an adenoviral vector expressing VEGF-C or control β-galactosidase (unpublished data of Michael Jeltsch, Seppo Ylä-Herttuala and the authors). The possibility of a cross-reaction between VEGF-C and VEGF-D was studied by immunoprecipitation analysis. 293T cells grown in Dulbecco's modified Eagle's medium − 10% fetal calf serum were transfected using the calcium phosphate precipitation method with equivalent amounts of plasmids pcDNA3.1-VEGF-C, pcDNA3.1-VEGF-D, CMV-βgal, pIg-βgal, pIg-VEGFR-3(1–3 loop), and pIg-VEGFR-1(1–5 loop). When producing the receptor bodies, serum-free culture medium containing 0.2% bovine serum albumin was changed to the cells 48 hours after transfection, and after an additional 30 hours the medium was collected, clarified by centrifugation, and used in the experiments. Metabolic labeling of VEGF-C and VEGF-D produced by the transfected cells was carried out by addition of 100 μCi/ml of Pro-Mix L-[35S] in vitro cell labeling mix (Amersham) to culture medium devoid of cysteine and methionine. After 6 hours, the medium was collected and immunoprecipitated using affinity purifed rabbit antibodies against VEGF-C or rabbit anti-VEGF-D antisera (a generous gift from Drs. Marc Achen and Steven Stacker).The eight intraductal carcinomas and all of the invasive carcinomas analyzed for VEGFR-3 were chosen for further analyses of the expression of VEGF-C. Five-μm cryosections adjacent to the sections stained with the anti-VEGFR-3 antibodies were air-dried and fixed in cold acetone for 10 minutes. The sections were rehydrated in PBS and incubated for 30 minutes in 5% normal goat serum and then for 2 hours in a humid atmosphere at room temperature with the rabbit polyclonal antibodies against human VEGF-C diluted 1:200 in PBS. A subsequent incubation for 30 minutes in biotinylated anti-rabbit serum was followed by a 60-minute incubation using reagents of the Vectastain Elite Rabbit IgG ABC kit (Vector Laboratories). The sections were further processed as described above. As a negative control, the purified immunogen was used to block the binding of the VEGF-C antibodies. In these experiments, VEGF-C antibodies were incubated overnight with a 10-fold molar excess of the VEGF-C protein in PBS. After centrifugation for 4 minutes at 4000 rpm, 4°C, the supernatant was carefully collected and used in the immunostainings.For comparison, two intraductal carcinomas were immunostained with commercially available anti-VEGF-C polyclonal antibodies (c = 200 μg/ml, Santa Cruz Biotechnology, Santa Cruz, California) raised against the amino terminus of the human VEGF-C precursor. After acetone fixation sections were incubated with 5% normal rabbit serum and with 4.0 μg/ml anti-VEGF-C antibodies for 2 hours at room temperature, a subsequent incubation for 30 minutes in biotinylated anti-goat serum was done and was followed by a 60-minute incubation using reagents of the Vectastain Elite Goat IgG ABC kit (Vector Laboratories).Production of Collagen Type XVIII Monoclonal AntibodiesMonoclonal antibodies to human type XVIII collagen were generated by DiaBor Ltd. (Oulu, Finland) by immunization of mice with the recombinant polypeptide QH48.18,32Saarela J Ylikärppä R Rehn M Purmonen S Pihlajaniemi T Complete primary structure of two variant forms of human type XVIII collagen and tissue-specific differences in the expression of the corresponding transcripts.Matrix Biol. 1998; 16: 319-328Crossref PubMed Scopus (113) Google Scholar corresponding to the common region of the N-terminal NC1 domain of human type XVIII collagen. The clones were screened by enzyme-linked immunosorbent assay and Western analysis using the polypeptide QH48.18 and also by immunofluorescence staining of frozen human tissue sections (data not shown). The screening of the hybridoma clones resulted in three monoclonal antibodies, which were positive in all three assays mentioned (enzyme-linked immunosorbent assay, Western, immunofluorescence staining). One of the antibodies that gave the strongest signals, DB144-N2, was used in subsequent experiments. It gave an identical staining pattern to that of the polyclonal anti-all hu(XVIII), eg, in adult human skin and kidney samples (data not shown).33Saarela J Rehn M Oikarinen A Autio-Harmainen H Pihlajaniemi T The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans.Am J Pathol. 1998; 153: 611-626Abstract Full Text Full Text PDF PubMed Scopus (205) Google ScholarResultsVEGFR-3 in Histologically Normal Breast Tissue and in Benign FibroadenomasImmunohistochemical staining of VEGFR-3 in normal breast tissue showed a very weak staining in capillaries of the interductal stroma. These vessels did not form any specific pattern but were scattered throughout the stroma (Figure 1A). The density of the VEGFR-3 positive vessels in the normal breast tissue samples ranged from 6 to 17 per hpf, median 9 (n = 12). Most of such vessels were strongly stained for the blood vascular endothelial marker PAL-E and for the basal lamina component, collagen XVIII (black arrows in Figure 1), suggesting that VEGFR-3 was expressed weakly in the blood vessels of normal breast tissue. However, some thin vessels in the stroma, which were clearly stained for VEGFR-3 were negative for PAL-E and only weakly positive for the collagen type XVIII, suggesting that they were lym
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