VEGFR-3 Expression Is Restricted to Blood and Lymphatic Vessels in Solid Tumors
2008; Cell Press; Volume: 13; Issue: 6 Linguagem: Inglês
10.1016/j.ccr.2008.04.022
ISSN1878-3686
AutoresTatiana V. Petrova, Petri Bono, Wolfgang Holnthoner, Jessica Chesnes, Bronislaw Pytowski, Harri Sihto, Pirjo Laakkonen, Päivi Heikkilä, Heikki Joensuu, Kari Alitalo,
Tópico(s)Vascular Tumors and Angiosarcomas
ResumoThe mechanisms regulating growth of lymphatic vessels have recently emerged as a prominent area of cancer research because of the essential role of lymphatic vasculature in tumor metastasis (Alitalo and Carmeliet, 2002Alitalo K. Carmeliet P. Molecular mechanism of lymphangiogenesis in health and disease.Cancer Cell. 2002; 1: 219-227Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, Alitalo et al., 2005Alitalo K. Tammela T. Petrova T.V. Lymphangiogenesis in development and human disease.Nature. 2005; 438: 946-953Crossref PubMed Scopus (905) Google Scholar). VEGFR-3 is a receptor for the lymphangiogenic factors VEGF-C and VEGF-D (Achen et al., 1998Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. 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 (984) Google Scholar, Joukov et al., 1996Joukov 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 (1110) Google Scholar), which is expressed in all endothelia during early development but later becomes restricted to lymphatic endothelial cells (ECs) and certain fenestrated blood vascular ECs. VEGFR-3 has been used as a marker for lymphatic vessels along with LYVE-1, podoplanin, and PROX1 (Stacker et al., 2002Stacker S.A. Achen M.G. Jussila L. Baldwin M.E. Alitalo K. Lymphangiogenesis and cancer metastasis.Nat. Rev. Cancer. 2002; 2: 573-583Crossref PubMed Scopus (656) Google Scholar). However, VEGFR-3 is upregulated in blood vascular ECs in some tumors and chronic wounds (Paavonen et al., 2000Paavonen K. Puolakkainen P. Jussila L. Jahkola T. Alitalo K. Vascular endothelial growth factor receptor-3 in lymphangiogenesis in wound healing.Am. J. Pathol. 2000; 156: 1499-1504Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, Partanen et al., 1999Partanen T.A. Alitalo K. Miettinen M. Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors.Cancer. 1999; 86: 2406-2412Crossref PubMed Scopus (227) Google Scholar, Valtola et al., 1999Valtola R. Salven P. Heikkila P. Taipale J. Joensuu H. Rehn M. Pihlajaniemi T. Weich H. deWaal R. Alitalo K. VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer.Am. J. Pathol. 1999; 154: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). Furthermore, new data show that VEGFR-3 contributes to angiogenesis at least in certain tumors (Laakkonen et al., 2007Laakkonen P. Waltari M. Holopainen T. Pytowski B. Steiner P. Hicklin D. Persaud K. Tonra J.R. Witte L. Alitalo K. Vascular endothelial growth factor receptor-3 (VEGFR-3) is involved in tumor angiogenesis and growth.Cancer Res. 2007; 67: 593-599Crossref PubMed Scopus (189) Google Scholar). VEGFR-3 expression in tumor cells has recently been reported in several studies (Arinaga et al., 2003Arinaga M. Noguchi T. Takeno S. Chujo M. Miura T. Uchida Y. Clinical significance of vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 in patients with nonsmall cell lung cancer.Cancer. 2003; 97: 457-464Crossref PubMed Scopus (145) Google Scholar, Beierle et al., 2004Beierle E.A. Dai W. Langham Jr., M.R. Copeland 3rd, E.M. Chen M.K. Expression of VEGF receptors in cocultured neuroblastoma cells.J. Surg. Res. 2004; 119: 56-65Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, Kaushal et al., 2005Kaushal V. Mukunyadzi P. Dennis R.A. Siegel E. Johnson D.E. Kohli M. Stage-specific characterization of the vascular endothelial growth factor axis in prostate cancer: Expression of lymphangiogenic markers is associated with advanced-stage disease.Clin. Cancer Res. 2005; 11: 584-593PubMed Google Scholar, Leclers et al., 2006Leclers D. Durand K. Cook-Moreau J. Rabinovitch-Chable H. Sturtz F.G. Rigaud M. VEGFR-3, VEGF-C and VEGF-D mRNA quantification by RT-PCR in different human cell types.Anticancer Res. 2006; 26: 1885-1891PubMed Google Scholar, Li et al., 2003Li Q. Dong X. Gu W. Qiu X. Wang E. Clinical significance of co-expression of VEGF-C and VEGFR-3 in non-small cell lung cancer.Chin. Med. J. (Engl.). 2003; 116: 727-730PubMed Google Scholar, Witte et al., 2002Witte C. Thomas A. Ali N. Carlson N. Younes M. Expression of the vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligand VEGF-C in human colorectal adenocarcinoma.Anticancer Res. 2002; 22: 1463-1466PubMed Google Scholar), including one published in Cancer Cell (Su et al., 2006Su J.-L. Yang P.-C. Shih J.-Y. Yang C.-Y. Wei L.-H. Hsieh C.-Y. Chou C.-H. Jeng Y.-M. Wang M.-Y. Chang K.-J. et al.The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells.Cancer Cell. 2006; 9: 209-223Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). These data are at odds with previously published results, including ours (Valtola et al., 1999Valtola R. Salven P. Heikkila P. Taipale J. Joensuu H. Rehn M. Pihlajaniemi T. Weich H. deWaal R. Alitalo K. VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer.Am. J. Pathol. 1999; 154: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar), and call for a reassessment of the VEGFR-3 role in tumor metastasis. We have reinvestigated VEGFR-3 expression in human tumors and cell lines using immunohistochemical staining, FACS, RT-PCR, western, and northern blotting analyses. None of the cancer cell lines tested, including A549 and MDA-MB-231 cells previously reported to express VEGFR-3, produced VEGFR-3 mRNA, whereas high levels were present in human dermal microvascular endothelial cells (Figure 1A). In contrast, A549, LNM-35, and the MDA-MB-231 cells expressed mRNA encoding the VEGFR-3 ligand VEGF-C. Neither A549 nor MDA-MB-231 cells contained VEGFR-3 protein, as detected by western blotting or FACS analysis using monoclonal antibodies 9D9F9 or 2E11D11, which recognize the extracellular domain of VEGFR-3 (Figures 1B and 1C). MAB3491, which was used by Su et al., detected 125 kDa and 170 kDa bands in all cell lines tested but failed to detect VEGFR-3 in transfected 293T cells (Figure 1C). As the 293T, A549, and MDA-MB-231 cells lacked VEGFR-3 mRNA, these data indicate that MAB3491 recognizes a nonspecific antigen. RT-PCR using the same set of primers as reported by Su et al. (Figure 1D) or an alternative set of primers (data not shown) failed to demonstrate the presence of VEGFR-3 mRNA except in the human erythroleukemia cell line HEL and the human ovarian carcinoma cell line OVCAR8 (Laakkonen et al., 2007Laakkonen P. Waltari M. Holopainen T. Pytowski B. Steiner P. Hicklin D. Persaud K. Tonra J.R. Witte L. Alitalo K. Vascular endothelial growth factor receptor-3 (VEGFR-3) is involved in tumor angiogenesis and growth.Cancer Res. 2007; 67: 593-599Crossref PubMed Scopus (189) Google Scholar). Also, immunoprecipitation analyses failed to detect VEGFR-3 expression in MDA-MB-231 or A549 cells (Figure 1 E). Among 62 human cell lines tested, we have detected VEGFR-3 mRNA and protein in the erythroleukemia HEL, the ovarian carcinoma OVCAR8 (Figures 1D and 1E), the nephroblastoma SK-NEP-1, the retinoblastoma Y79, and the teratocarcinoma cell line Tera2 (Pajusola et al., 1992Pajusola K. Aprelikova O. Korhonen J. Kaipainen A. Pertovaara L. Alitalo R. Alitalo K. FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines.Cancer Res. 1992; 52: 5738-5743PubMed Google Scholar, Laakkonen et al., 2007Laakkonen P. Waltari M. Holopainen T. Pytowski B. Steiner P. Hicklin D. Persaud K. Tonra J.R. Witte L. Alitalo K. Vascular endothelial growth factor receptor-3 (VEGFR-3) is involved in tumor angiogenesis and growth.Cancer Res. 2007; 67: 593-599Crossref PubMed Scopus (189) Google Scholar). These results indicate that the induction of VEGFR-3 in cultured tumor cell lines is a rare event, and when observed it can be detected with the 9D9F9 antibody. To study whether VEGF-C can induce a cellular response in the absence of detectable VEGFR-3, we used a migration assay. As expected, VEGF-C induced migration of microvascular endothelial cells; however, both A549 and MDA-MB-231 cells failed to migrate in response to stimulation (Figure S1 available online). Finally, we stained 456 human neoplasms of 35 various histological types (Table S1), including the ones in which VEGFR-3 expression in tumor cells was reported (Arinaga et al., 2003Arinaga M. Noguchi T. Takeno S. Chujo M. Miura T. Uchida Y. Clinical significance of vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 in patients with nonsmall cell lung cancer.Cancer. 2003; 97: 457-464Crossref PubMed Scopus (145) Google Scholar, Li et al., 2003Li Q. Dong X. Gu W. Qiu X. Wang E. Clinical significance of co-expression of VEGF-C and VEGFR-3 in non-small cell lung cancer.Chin. Med. J. (Engl.). 2003; 116: 727-730PubMed Google Scholar, Shields et al., 2004Shields J.D. Borsetti M. Rigby H. Harper S.J. Mortimer P.S. Levick J.R. Orlando A. Bates D.O. Lymphatic density and metastatic spread in human malignant melanoma.Br. J. Cancer. 2004; 90: 693-700Crossref PubMed Scopus (134) Google Scholar, Su et al., 2006Su J.-L. Yang P.-C. Shih J.-Y. Yang C.-Y. Wei L.-H. Hsieh C.-Y. Chou C.-H. Jeng Y.-M. Wang M.-Y. Chang K.-J. et al.The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells.Cancer Cell. 2006; 9: 209-223Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, Witte et al., 2002Witte C. Thomas A. Ali N. Carlson N. Younes M. Expression of the vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligand VEGF-C in human colorectal adenocarcinoma.Anticancer Res. 2002; 22: 1463-1466PubMed Google Scholar). We detected VEGFR-3 expression only in blood or lymphatic vessels but not in tumor cells (Figure 1F). We have also re-examined 40 publications reporting VEGFR-3 expression in human tumors (Table S2) and found that all publications using 9D9F9 antibody report the expression in blood and/or lymphatic vessels but not tumor cells. Strikingly, 90% of studies using Santa Cruz Biotechnology antibodies reported VEGFR-3 expression in tumor cells. Indeed, the polyclonal antibody sc-321, used in at least five publications, produced strong staining of tumor cells (Figure S2A). However, detailed examination revealed that sc-321 stains nonspecifically both VEGFR-3-positive and -negative endothelial cells (Figure S2B), which may account for the nonspecific staining of tumor specimens. However, the sc-321 antibody recognized VEGFR-3 by western blotting (Figure S2C; Veikkola et al., 2003Veikkola T. Lohela M. Ikenberg K. Makinen T. Korff T. Saaristo A. Petrova T. Jeltsch M. Augustin H.G. Alitalo K. Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function.FASEB J. 2003; 17: 2006-2013Crossref PubMed Scopus (64) Google Scholar). Using a variety of retrieval procedures, we were not able to obtain reliable staining of paraffin sections with MAB3491, which also failed to recognize VEGFR-3 by western blotting (Figure 1C) or immunoprecipitation analysis (data not shown). Staining of frozen sections of human tumors, where the antigens are usually well preserved, revealed colocalization of the 9D9F9 signal and the blood vessel marker von Willebrand factor, as expected (Valtola et al., 1999Valtola R. Salven P. Heikkila P. Taipale J. Joensuu H. Rehn M. Pihlajaniemi T. Weich H. deWaal R. Alitalo K. VEGFR-3 and its ligand VEGF-C are associated with angiogenesis in breast cancer.Am. J. Pathol. 1999; 154: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). On the contrary, MAB3491 produced intense staining that did not colocalize with staining for von Willebrand factor, strongly suggesting nonspecific binding (Figure S3A). To rule out the possibility that VEGFR-3 is induced in cancer cells only in vivo, we also stained frozen sections of MDA-MB-231 tumor xenografts in nude mice using both 9D9F9 and MAB3491. However, no significant staining above the background level was observed (Figure S3B). Except for vascular tumors (Partanen et al., 1999Partanen T.A. Alitalo K. Miettinen M. Lack of lymphatic vascular specificity of vascular endothelial growth factor receptor 3 in 185 vascular tumors.Cancer. 1999; 86: 2406-2412Crossref PubMed Scopus (227) Google Scholar), the expression of VEGFR-3 is uncommon in solid tumors and instead limited to blood vascular and lymphatic endothelial cells. We therefore submit that the lymphatic and blood vessels, and not tumor cells, represent the major target of anti-VEGFR-3 therapy in cancer. Moreover, our results call for caution when using anti-VEGFR-3 antibodies that may not be optimal for immunohistochemistry of paraffin-embedded sections, as well as when interpreting the staining results, where signal to noise ratio should be taken into account when evaluating staining specificity. Our data are also a reminder that the thorough validation of antibodies should be regarded as an absolute requirement, especially when they produce unexpected results. Furthermore, RT-PCR results should be interpreted carefully, as even minimal numbers of transcripts, which do not translate into functional protein levels or cellular responses, can be easily detected. Based on the immunohistochemical stainings and analyses of tumor cell lines by flow cytometry, northern, RT-PCR, and western blotting, we conclude that VEGFR-3 expression by cultured cancer cells and neoplastic cells in most solid tumors is negligible when compared to the levels observed in endothelial cells. Supported by the Academy of Finland, Finnish Cancer Organizations, and Helsinki University Hospital Research Funds. Download .pdf (1.77 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Three Supplemental Figures, and Two Supplemental Tables Tumor Suppressor PTEN Mediates Sensing of Chemoattractant GradientsIijima et al.CellMay 31, 2002In BriefShallow gradients of chemoattractants, sensed by G protein-linked signaling pathways, elicit localized binding of PH domains specific for PI(3,4,5)P3 at sites on the membrane where rearrangements of the cytoskeleton and pseudopod extension occur. Disruption of the PI 3-phosphatase, PTEN, in Dictyostelium discoideum dramatically prolonged and broadened the PH domain relocation and actin polymerization responses, causing the cells lacking PTEN to follow a circuitous route toward the attractant. Exogenously expressed PTEN-GFP localized to the surface membrane at the rear of the cell. Full-Text PDF Open ArchiveFurther Evidence for Expression and Function of the VEGF-C/VEGFR-3 Axis in Cancer CellsSu et al.Cancer CellJune 10, 2008In BriefVEGFRs are primarily expressed by endothelial cells but are also expressed by nonendothelial cell types, including cancer cells (Witte et al., 2002; Li et al., 2003; Van Trappen et al., 2003; Wu et al., 2003; Su et al., 2007). VEGFR-3 influences the potential for a lymphangiogenic response (Alitalo and Carmeliet, 2002; Joukov et al., 1996; Skobe et al., 2001). Recent studies have indicated that VEGFR-3 is also expressed in a variety of human malignancies and that the VEGF-C/VEGFR-3 axis contributes to tumor progression (Dias et al., 2002; Marchio et al., 1999; Masood et al., 2003; Mylona et al., 2007; Van Trappen et al., 2003; Witte et al., 2002). Full-Text PDF Open Archive
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