Abstract
Rat KMT‐17 fibrosarcoma‐derived endothelial cells were isolated by Percoll gradient centrifugation with an attaching‐speed separation technique, and their properties in culture were examined. The primary cultured tumor‐derived endothelial cells (TEC) showed angiotensin‐converting enzyme activity, positivity for Factor VIII‐related antigen staining, and typical capillary‐like formation on Matrigel. The primary cultured TEC monolayer showed greater permeability than normal tissuederived endothelial cell (aorta, vena cava and epididymal fat capillary) monolayers on FITC‐dextran diffusion (molecular weight 70,000). Leukocyte adhesion to TEC was reduced compared to that to fat‐derived capillary endothelial cells. These characteristics resembled those of tumor vascular endothelium, and were observed both in the primary and first‐passage cell cultures, but not in the fourth‐passage cell cultures. Our findings indicate that primary or subcultured TEC are applicable for studies of the physiological characteristics of tumor endothelial cells.
Keywords: Endothelial cell, Primary culture, Tumor, KMT‐17
Full Text
The Full Text of this article is available as a PDF (1.6 MB).
REFERENCES
- 1).Follkman , J.What is the evidence that tumors are angiogenesis dependent ? J. Natl. Cancer Inst. , 82 , 4 – 6 ( 1991. ). [DOI] [PubMed] [Google Scholar]
- 2).Ingber , D. , Fujita , T. , Kishimoto , S. , Sudo , K. , Kanamaru , T. , Brem , H. and Folkman , J.Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth . Nature , 348 , 555 – 557 ( 1990. ). [DOI] [PubMed] [Google Scholar]
- 3).Mahadevan , V. and Hart , I. R.Tumor angiogenesis and metastasis . Eur. J. Cancer , 27 , 679 – 680 ( 1991. ). [DOI] [PubMed] [Google Scholar]
- 4).Kim , K. J. , Li , B. , Winer , J. , Armanini , M. , Gillett , N. , Phillips , H. S. and Ferrara , N.Inhibition of vascular endothelial growth factor‐induced angiogenesis suppresses tumour growth in vivo . Nature , 362 , 841 – 844 ( 1993. ). [DOI] [PubMed] [Google Scholar]
- 5).Kondo , S. , Asano , M. and Suzuki , H.Significance of vascular endothelial growth factor/vascular permeability factor for solid tumor growth, and its inhibition by the antibody . Biochem. Biophys. Res. Commun. , 194 , 1234 – 1241 ( 1993. ). [DOI] [PubMed] [Google Scholar]
- 6).Hori , A. , Sasada , R. , Matsutani , E. , Naito , K. , Sakura , Y. , Fujita , T. and Kozai , Y.Suppression of solid tumor growth by immunoneutralizing monoclonal antibody against human basic fibroblast growth factor . Cancer Res. , 51 , 6180 – 6184 ( 1991. ). [PubMed] [Google Scholar]
- 7).Fotsis , T. , Zhang , Y. , Pepper , M. S. , Adlercreutz , H. , Montesano , R. , Nawroth , P. P. and Schweigerer , L.The endogenous oestrogen metabolite 2‐methoxyoestradiol inhibits angiogenesis and suppresses tumor growth . Nature , 368 , 237 – 239 ( 1994. ). [DOI] [PubMed] [Google Scholar]
- 8).Millauer , B. , Shawver , L. K. , Plate , K. H. , Risau , W. and Ullrich , A.Glioblastoma growth inhibited in vivo, by a dominant‐negative F1k‐1 mutant . Nature , 367 , 576 – 579 ( 1994. ). [DOI] [PubMed] [Google Scholar]
- 9).Montesano , R. , Vassalli , J. D. , Baird , A. , Guillemin , R. and Orci , L.Basic fibroblast growth factor induces angiogenesis in vitro . Proc. Natl. Acad. Sci. USA , 83 , 7297 – 7301 ( 1986. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10).Frater‐Schroder , M. , Risau , W. , Hallmann , R. , Gautschi , P. and Bohlen , P.Tumor necrosis factor type α, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo . Proc. Natl. Acad. Sci. USA , 84 , 5277 – 5281 ( 1987. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11).Roberts , A. B. , Sporn , M. B. , Assoian , R. K. , Smith , J. M. , Roche , N. S. , Wakefield , L. M. , Heine , U. I. , Liotta , L. A. , Falanga , V. , Kehrl , J. H. and Fauci , A. S.Transforming growth factor type β: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro . Proc, Natl. Acad. Sci. USA , 83 , 4167 – 4171 ( 1986. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12).Reese , T. S. and Karnovsky , M. J.Fine structural localization of a blood‐brain barrier to exogeneous peroxidase . J. Cell Biol. , 34 , 207 – 217 ( 1967. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13).Joo , F.The blood‐brain barrier in vitro: ten years of research on microvessels isolated from the brain . Neuro chem. Int. , 7 , 1 – 25 ( 1985. ). [DOI] [PubMed] [Google Scholar]
- 14).Luft , J. and Hechter , O.An electron microscopic correlation of structure with function in the isolated perfused cow adrenal, preliminary observations . J. Biophys. Biochem, Cytol. , 3 , 615 – 620 ( 1957. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15).Motta , P. , Muto , M. and Fujita , T.Three dimensional organization of mammalian adrenal cortex . Cell Tissue Res. , 196 , 23 – 38 ( 1979. ). [DOI] [PubMed] [Google Scholar]
- 16).Vaupel , P. , Kallinowski , F. and Okunieff , P.Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors . Cancer Res. , 49 , 6449 – 6465 ( 1989. ). [PubMed] [Google Scholar]
- 17).Matsumura , Y. and Maeda , H.A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs . Cancer Res , 46 , 6387 – 6392 ( 1986. ). [PubMed] [Google Scholar]
- 18).Hori , K. , Suzuki , M. , Abe , I. , Saito , S. and Sato , H.Increase in tumor vascular area due to increased blood flow by angiotensin II in rats . J. Natl. Cancer Inst. , 74 , 453 – 459 ( 1985. ). [PubMed] [Google Scholar]
- 19).Skinner , S. A. , Tutton , P. J. and O'Brien , P. E.Microvascular architecture of experimental colon tumors in the rat . Cancer Res. , 50 , 2411 – 2417 ( 1990. ). [PubMed] [Google Scholar]
- 20).Song , C. W. and Levitt , S. H.Quantitative study of vascularity in Walker carcinoma 256 . Cancer Res. , 31 , 587 – 589 ( 1971. ). [PubMed] [Google Scholar]
- 21).O'Connor , S. W. and Bale , W. F.Accessibility of circulating immunoglobulin G to the extravascular compartment of solid rat tumors . Cancer Res. , 44 , 3719 – 3723 ( 1984. ). [PubMed] [Google Scholar]
- 22).Underwood , J. C. E. and Carr , I.The ultrastructure and permeability characteristics of the blood vessels of a transplantable rat sarcoma . J. Pathol. , 107 , 157 – 166 ( 1972. ). [DOI] [PubMed] [Google Scholar]
- 23).Heuser , L. S. and Miller , F. N.Differential macromolecular leakage from the vasculature of tumors . Cancer , 57 , 461 – 464 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 24).Dvorak , H. F. , Nagy , J. A. , Dvorak , J. T. and Dvorak , A. M.Identification and characterization of the blood vessels of solid tumors that are leakey to circulating macromolecules . Am. J. Pathol. , 133 , 95 – 109 ( 1988. ). [PMC free article] [PubMed] [Google Scholar]
- 25).Dvorak , H. F. , Harvey , V. S. and McDonagh , J.Quantitation of fibrnogen influx and fibrin deposition and turnover in line 1 and line 10 guinea pig carcinomas . Cancer Res. , 44 , 3348 – 3354 ( 1984. ). [PubMed] [Google Scholar]
- 26).Jaffe , E. A. , Nachman , R. L. , Becker , C. G. and Minick , C. R.Culture of human endothelial cells derived from umbilical veins . J. Clin. Invest. , 52 , 2745 – 2756 ( 1973. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27).Fawcett , J. , Harris , A. L. and Bicknell , R.Isolation and properties in culture of human adrenal capillary endothelial cells . Biochem. Biophys. Res. Commun. , 174 , 903 – 908 ( 1991. ). [DOI] [PubMed] [Google Scholar]
- 28).Diglio , C. A. , Grammas , P. , Giacomelli , F. and Wiener , J.Primary culture of rat cerebral microvascular endothelial cells. Isolation, growth, and characterization . Lab. Invest. , 46 , 554 – 563 ( 1982. ). [PubMed] [Google Scholar]
- 29).Bowman , P. D. , Betz , A. L. and Goldstein , G. W.Primary culture of microvascular endothelial cells from bovine retina . In Vitro , 18 , 626 – 632 ( 1982. ). [DOI] [PubMed] [Google Scholar]
- 30).Kasai , M. , Yamaguchi , H. , Hosokawa , M. , Mizushima , Y. and Kobayashi , H.Increased sensitivity of murine leukemia virus‐infected tumor cells to lymphocyte‐mediated cytotoxicity . J. Natl. Cancer Inst. , 67 , 417 – 422 ( 1981. ). [PubMed] [Google Scholar]
- 31).Minami , A. , Mizushima , Y. , Takeichi , N. , Hosokawa , M. and Kobayashi , H.Dissociation of anti‐tumor immune responses in rats immunized with solubilized tumor associated antigens from a methylcholanthrene‐induced fibrosarcoma . Int. J. Cancer , 23 , 358 – 365 ( 1979. ). [DOI] [PubMed] [Google Scholar]
- 32).Chiba , I. , Oikawa , T. , Naiki , M. , Takimoto , M. , Miyoshi , I. , Mizuno , S. , Yamashita , K. , Yamagiwa , S. and Kobayashi , H.Enhanced immunogenicity of cultured rat fibrosarcoma KMT‐17 by cultivation in a low concentration of fetal calf serum . Cancer Res. , 47 , 1815 – 1819 ( 1987. ). [PubMed] [Google Scholar]
- 33).Friedland , J. and Silverstein , E.A sensitive fluorimetric assay for serum angiotensin‐converting enzyme . Am. J. Clin. Pathol. , 66 , 416 – 424 ( 1976. ). [DOI] [PubMed] [Google Scholar]
- 34).Kubota , Y. , Kleinman , H. K. , Martin , G. R. and Lawley , T. J.Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary‐like structures . J. Cell Biol. , 107 , 1589 – 1598 ( 1988. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35).Cole , O. F. , Fan , T. P. D. and Lewis , G. P.Isolation, characterization, growth and culture of endothelial cells from the rat aorta . Cell Biol. Int. Rep. , 10 , 399 – 405 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 36).Madri , J. A. and Williams , S. K.Capillary endothelial cell cultures: phenotypic modulation by matrix components . J. Cell Biol. , 97 , 153 – 165 ( 1983. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37).McGuire , P. G. and Orkin , R. W.Isolation of rat aortic endothelial cells by primary explant techniques and their phenotypic modulation by defined substrata . Lab. Invest. , 57 , 94 – 105 ( 1987. ). [PubMed] [Google Scholar]
- 38).Hashida , R. , Anamizu , C. , Yagyu‐Mizuno , Y. , Ohkuma , S. and Takano , T.Transcellular transport of fluorescein dextran through an arterial endothelial cell monolayer . Cell Struct. Funct. , 11 , 343 – 349 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 39).Mizuguchi , H. , Hashioka , Y. , Utoguchi , N. , Kubo , K. , Nakagawa , S. and Mayumi , T.A comparison of drug transport through cultured monolayers of bovine brain capillary and bovine aortic endothelial cells . Biol. Pharm. Bull. , 17 , 1385 – 1390 ( 1994. ). [DOI] [PubMed] [Google Scholar]
- 40).Siflinger‐Birnboim , A. , Del Vecchio , P. J. , Cooper , J. A. , Blumenstock , F. A. , Shepard , J. M. and Malik , A. B.Molecular sieving characteristics of the cultured endothelial monolayer . J. Cell. Physiol. , 132 , 111 – 117 ( 1987. ). [DOI] [PubMed] [Google Scholar]
- 41).Luscinskas , F. W. , Brock , A. F. , Arnaout , M. A. and Gimbrone , M. A. , Jr.Endothelial‐leukocyte adhesion molecule‐1‐dependent and leukocyte (CD11/CD18)‐dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine‐activated human vascular endothelium . J. Immunol. , 142 , 2257 – 2263 ( 1989. ). [PubMed] [Google Scholar]
- 42).Pauli , B. U. and Lee , C.Organ preference of metastasis: the role of organ‐specifically modulated endothelial cells . Lab. Invest. , 58 , 379 – 397 ( 1989. ). [PubMed] [Google Scholar]
- 43).Ryan , U. S.Isolation and culture of pulmonary endothelial cells . Environ. Health Perspect. , 56 , 103 – 114 ( 1984. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44).Diglio , C. A. , Grammas , P. , Giacomelli , F. and Wiener , J.Rat heart‐derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization . Tissue Cell , 20 , 477 – 492 ( 1988. ). [DOI] [PubMed] [Google Scholar]
- 45).Wu , N. Z. , Klitzman , B. , Dodge , R. and Dewhirst , M. W.Diminished leukocyte‐endothelium interaction in tumor microvessels . Cancer Res. , 52 , 4265 – 4268 ( 1992. ). [PubMed] [Google Scholar]
- 46).Rotrosen , D. and Gallin , J. I.Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F‐actin, and promotes albumin diffusion across cultured endothelial monolayers . J. Cell Biol. , 103 , 2379 – 2387 ( 1986. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 47).Carson , M. R. , Shasby , S. S. and Shasby , D. M.Histamine and inositol phosphate accumulation in endothelium: cAMP and a G protein . Am. J. Physiol. , 257 , L259 – L264 ( 1989. ). [DOI] [PubMed] [Google Scholar]
- 48).Hekimian , G. , Caste , S. , Van‐Sande , J. and Boeynaems , J. M.H2 receptor‐mediated responses of aortic endothelial cells to histamine . Am. J. Physiol. , 262 , H220 – H224 ( 1992. ). [DOI] [PubMed] [Google Scholar]
- 49).Meyer , J. , Mischeck , U. , Veyhl , M. , Henzel , K. and Galla , H. J.Blood‐brain barrier characteristic enzymatic properties in cultured brain capillary endothelial cells . Brain Res. , 514 , 305 – 309 ( 1990. ). [DOI] [PubMed] [Google Scholar]
- 50).Senger , D. R. , Galli , S. J. , Dvorak , A. M. , Perruzzi , C. A. , Harvey , V. S. and Dvorak , H. F.Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid . Science , 219 , 983 – 985 ( 1983. ). [DOI] [PubMed] [Google Scholar]
- 51).Senger , D. R. , Perruzzi , C. A. , Feder , J. and Dvorak , H. F.A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines . Cancer Res. , 46 , 5629 – 5632 ( 1986. ). [PubMed] [Google Scholar]
- 52).Brock , T. A. , Dvorak , H. F. and Senger , D. R.Tumorsecreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells . Am. J. Pathol. , 138 , 213 – 221 ( 1991. ). [PMC free article] [PubMed] [Google Scholar]
- 53).Maeda , H. , Noguchi , Y. , Sato , K. and Akaike , T.Enhanced vascular permeability in solid tumor is mediated by nitric oxide and inhibited by both new nitric oxide scavenger and nitric oxide synthase inhibitor . Jpn. J. Cancer Res. , 85 , 331 – 334 ( 1994. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54).Maeda , H. , Matsumura , Y. and Kato , H.Purification and identification of [hydroxyprolyl3]bradykinin in ascitic fluid from a patient with gastric cancer . J. Biol. Chem. , 263 , 16051 – 16054 ( 1988. ). [PubMed] [Google Scholar]
- 55).Matsumura , Y. , Kimura , M. , Yamamoto , T. and Maeda , H.Involvement of the kinin‐generating cascade in enhanced vascular permeability in tumor tissue . Jpn. J. Cancer Res. , 79 , 1327 – 1334 ( 1988. ). [DOI] [PMC free article] [PubMed] [Google Scholar]