Regular Article

British Journal of Cancer (2001) 84, 558–564. doi:10.1054/bjoc.2000.1641 www.bjcancer.com
Published online 13 February 2001

Human bladder cancer invasion model using rat bladder in vitro and its use to test mechanisms and therapeutic inhibitors of invasion

C Fujiyama1,3,*, A Jones1,2,3,*, S Fuggle3, R Bicknell1, D Cranston2 and A L Harris1

  1. 1Molecular Oncology Unit, ICRF, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS
  2. 2Department of Urology, Churchill Hospital, Oxford, OX3 7LJ, UK
  3. 3Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospita, Oxford, OX3 9DU, UK

*These authors contributed equally to this paper.

Received 21 July 2000; Revised 16 November 2000; Accepted 17 November 2000.

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Abstract

As well as being a passive support, the extracellular matrix also regulates key biological processes such as invasion, differentiation and angiogenesis. We have therefore developed an in vitro model of bladder cancer invasion using de-epithelialized rat bladder to allow for tumour cell–extracellular matrix interactions. Onto this we have seeded a panel of human bladder cancer cell lines (RT4, RT112, 253J and EJ28 (T24)) representing progression from well to poorly differentiated phenotypes and used as models of superficial to invasive bladder cancer. The better differentiated cell lines RT4 and RT112 reproducibly grew as stratified epithelium, whereas poorly differentiated EJ28 cells invaded across a broad front. Invasion was not simply related to proliferation rate, measured either as doubling time on plastic (non-invasive 253J and invasive EJ28 having the same doubling time) or by Ki-67 proliferation index within the model. We used the model to test the ability of 4 compounds that interfere with tumour cell–extracellular matrix interactions (suramin, N-acetylcysteine and the urokinase plasminogen activator pathway antagonists Å5 compound and monoclonal antibody Mab 3936) to inhibit invasion. At non-toxic concentrations, all significantly inhibited invasion (P< 0.05), although to varying degrees, suramin and Å5 almost completely and N-acetylcysteine the least. In conclusion, this model shows the urokinase system is important for bladder invasion and can be used to investigate other mechanisms of bladder cancer invasion and also for the testing of intravesical drugs. © 2001 Cancer Research Campaign

Keywords:

invasion, in vitro assay, bladder cancer, suramin, N-acetylcysteine

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References

  1. Albini A, D'Agostini F, Giunciuglio D, Paglieri I, Balansky R and De Flora S (1995) Inhibition of invasion, gelatinase activity, tumour take and metastasis of malignant cells by N-acetylcysteine. Int J Cancer 61: 121–129 | Article | PubMed | ISI | ChemPort |
  2. Bajour K, Noel A, Gerard RD, Masson V, Brunner N, Holst-Hansen C, Skobe M, Fusenig NE, Carmeliet P, Collen D and Foidart JM (1998) Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularisation. Nature Med 4: 923–928 | Article |
  3. Behrendt N, Ronne E and Dano K (1993) Binding of the urokinase-type plasminogen activator to its cell surface receptor is inhibited by low doses of suramin. J Biol Chem 268: 5985–5989 | PubMed | ISI | ChemPort |
  4. Bhat GJ, Gunaje JJ and Idell S (1999) Urokinase-type plasminogen activator induces tyrosine phosphorylation of a 78-kDa protein in H-157 cells. Am J Physiol 277: L301–L309
  5. Booth C, Harnden P, Trejdosiewicz LK, Scriven S, Selby PJ and Southgate J (1997) Stromal and vascular invasion in an human in vitro bladder cancer model. Lab Invest 76: 843–857 | PubMed | ISI | ChemPort |
  6. Borgstrom P, Bourdon MA, Hillan KJ, Sriramarao P and Ferrara N (1998) Neutralising anti vascular endothelial growth factor antibody completely inhibits angiogenesis and growth of human prostate carcinoma microtumours in vivo. Prostate 35: 1–10 | Article | PubMed | ISI | ChemPort |
  7. Brooks PC, Stromblad S, Klemke R, Visscher D, Sarkar FH and Cheresh DA (1995) Antiintegrin alphavbeta3 blocks human breast cancer growth. J Clin Invest 96: 1815–1822 | Article | PubMed | ISI | ChemPort |
  8. Chern H-D, Becich MJ, Persad RA, Romkes M, Smith P, Collins C, Li Y-H and Branch RA (1996) Clonal analysis of human recurrent superficial bladder cancer by immunohistochemistry of P53 and retinoblastoma proteins. J Urol 156: 1846–1849 | Article | PubMed | ISI |
  9. Conese M and Blasi F (1995) The urokinase/urokinase-receptor system and cancer invasion. Baillières Clin Haematol 8: 365–389
  10. Cook GP and Hampton JA (1997) Effects of ibuprofen on the in vitro invasiveness of a human transitional cell carcinoma. Anticancer Res 17: 365–368
  11. Crew JP, O'Brien T, Bradburn M, Fuggle S, Cranston D and Harris AL (1997) Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer. Cancer Res 57: 5281–5285 | PubMed | ISI | ChemPort |
  12. Davies G, Jiang WG and Mason MD (1999) Cell–cell adhesion molecules and their associated proteins in bladder cancer cells and their role in mitogen induced cell–cell dissociation and invasion. Anticancer Res 19: 547–552 | PubMed | ChemPort |
  13. Dickinson AJ, Savage PB, Newcomb PV, Lodge R and Sibley GN (1995) The expression of urokinase, its receptor and plasminogen activator inhibitor-1 in bladder cancer (abstract). J Urol 153: 406A
  14. Eisenberger M, Reyno LM, Jodrell DI, Sinibaldi VJ, Tkaczuk KH, Sridhara R, Zuhowski EG, Lowitt MH, Jacobs SC and Egorin MJ (1993) Suramin, an active drug for prostate cancer: interim observations in a phase I trial. J Natl Cancer Inst 85: 611–621
  15. Elliot AY, Cleveland P, Cervenka J, Castro AE, Stein N, Hakala TR and Fraley EE (1974) Characterisation of a cell line from human transitional cell cancer of the urinary tract. J Natl Cancer Inst 53: 1341–1349 | PubMed |
  16. Fadl-Elmula I, Gorunova L, Mandahl N, Elfving P, Lundgren R, Mitelman F and Heim S (1999) Cytogenetic monoclonality in multifocal uroepithelial carcinomas: evidence of intraluminal tumour seeding. Br J Cancer 81: 6–12 | Article |
  17. Folkman J (1990) What is the evidence that tumours are angiogenesis dependent?. J Natl Cancer Inst 82: 4–6 | Article | PubMed | ChemPort |
  18. Fujiyama C, Masaki Z and Sugihara H (1995) Reconstruction of the urinary bladder mucosa in three dimensional collagen gel culture: Fibroblast-extracellular matrix interaction of transitional epithelial cells. J Urol 153: 2060–2067 | Article | PubMed | ISI | ChemPort |
  19. Gilbert HA, Logan SL, Kagan AR, Friedman HA, Cove JK, Fox M, Muldoon TM, Lonni YW, Rowe JH, Cooper JF, Nussbaum H, Chan P, Rao A and Starr A (1978) The natural history of papillary transitional cell carcinoma of the bladder and its treatment in an unselected population on the basis of histologic grading. J Urol 119: 488–492
  20. Grondhal-Hansen J, Christensen IJ, Briand P, Pappot H, Mouridsen HT, Blichert-Toft M, Dano K and Brunner N (1997) Plasminogen activator inhibitor type 1 in cytosolic tumour extracts predicts prognosis in low-risk breast cancer patients. Clin Cancer Res 3: 233–239 | ChemPort |
  21. Habuchi T, Takahashi R, Yamada H, Kakehi Y, Sugiyama T and Yoshida O (1993) Matachronous multifocal development of urothelial cancers by intraluminial seeding. Lancet 342: 1087–1088 | Article | PubMed | ISI | ChemPort |
  22. Harris AL and Neal DE (1992) Bladder cancer-field versus clonal origin. N Engl J Med 326: 759–761 | PubMed | ISI | ChemPort |
  23. Hasui Y, Marutsuka K, Nishi S, Kitada S, Osada Y and Sumiyoshi A (1994) The content of urokinase-type plasminogen activator and tumour recurrence in superficial bladder cancer. J Urol 151: 16–20
  24. Hudson ML and McReynolds LM (1997) Urokinase and the urokinase receptor: Association with in vitro invasiveness of human bladder cancer cell lines. J Natl Cancer Inst 89: 709–717 | Article | PubMed | ChemPort |
  25. Irani K, Xia Y, Zweier JL, Sollott SJ, Der CJ, Fearon ER, Sundaresan M, Finkel T and Goldschmidt-Clermont PJ (1997) Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science 275: 1649–1652 | Article | PubMed | ISI | ChemPort |
  26. Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS and Ferrara N (1993) Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 362: 841–844 | Article | PubMed | ISI | ChemPort |
  27. Lamm DL and Griffith JG (1992) Intravesical therapy: Does it affect the natural history of superficial bladder cancer?. Semin Urol 10: 39–44 | PubMed |
  28. Liotta LA, Rao CN and Barsku SH (1983) Tumour invasion and the extracellular matrix. Lab Invest 49: 636–649 | PubMed | ISI | ChemPort |
  29. Ma D, Gerard RD, Li XY, Alizadeh H and Niederkorn JY (1997) Inhibition of metastasis of intraocular melanomas by adenovirus-mediated gene transfer of plasminogen activator inhibitor type 1 (PAI-1) in an athymic mouse model. Blood 90: 2738–2746 | PubMed | ChemPort |
  30. Miyake H, Yoshimura K, Hara I, Eto H, Arakawa S and Kamidono S (1997) Basic fibroblast growth factor regulates matrix metalloproteinases production and in vitro invasiveness in human bladder cancer cell lines. J Urol 157: 2351–2355 | Article | PubMed | ISI | ChemPort |
  31. O'Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR and Folkman J (1997) Endostatin: An endogenous inhibitor of angiogenesis and tumour growth. Cell 88: 277–285 | Article | PubMed | ISI | ChemPort |
  32. Oshinsky GS, Chen Y, Jarett T, Anderson AE and Weiss GH (1995) A model of bladder tumour xenografts in the nude rat. J Urol 154: 1925–1929 | PubMed | ChemPort |
  33. Ossowski L (1992) Invasion of connective tissue by human carcinoma cell lines: requirement for urokinase receptor, and interstitial collagenase. Cancer Res 52: 6754–6760 | PubMed | ISI | ChemPort |
  34. Ossowki L, Russo-Payne H and Wilson EL (1991) Inhibition of urokinase-type plasminogen activator by antibodies: the effect on dissemination of a human tumour in the nude mouse. Cancer Res 51: 274–281 | PubMed | ISI | ChemPort |
  35. Parada LF, Tabin CJ, Shih C and Weinber RA (1982) Human EJ bladder carcinoma oncogene is homolgue of Harvey sarcoma virus ras gene. Nature 297: 474–478 | Article | PubMed | ISI | ChemPort |
  36. Pedersen H, Brunner N, Francis D, Osterlind K, Ronne E, Hansen HH, Dano K and Grondahl-Hansen J (1994) Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue. Cancer Res 54: 4671–4675 | PubMed | ISI | ChemPort |
  37. Pesenti E, Sola F, Mongelli N, Grandi M and Spreafico F (1992) Suramin prevents neovascularisation and tumour growth through blocking of basic fibroblast growth factor activity. Br J Cancer 66: 367–372 | PubMed | ChemPort |
  38. Pyke C, Kristensen P, Ralfkiaer E, Grondahl-Hansen J, Eriksen J, Blasi F and Dano K (1991) Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas. Am J Pathol 138: 1059–1067 | PubMed | ISI | ChemPort |
  39. Raghavan D, Shipley WU, Garnick MB, Russell PJ and Richie JP (1990) Biology and management of bladder cancer. N Engl J Med 322: 1129–1138 | PubMed | ISI | ChemPort |
  40. Rebel JM, Thijssen C, Vermey M, Delouvee A, Zwarthoff EC and Van der Kwast TH (1994) E-cadherin expression determines the mode of replacement of normal urothelium by human bladder carcinoma cells. Cancer Res 54: 5488–5492
  41. Redwood SM, Liu BC, Weiss RE, Hodge DE and Droller MJ (1992) Abrogation of the invasion of human bladder tumour cells by using proetease inhibitor(s). Cancer 69: 1212–1219 | PubMed | ISI | ChemPort |
  42. Scriven S, Booth C, Thomas DFM, Trejdosiewicz LK and Southgate J (1997) Reconstitution of human urothelium from monolayer cultures. J Urol 158: 1147–1152 | Article | PubMed | ISI | ChemPort |
  43. See WA, Miller JS and Williams RD (1989) Pathophysiology of transitional tumour cell adherence to sites of urothelial injury in rats: mechanisms mediating intravesical recurrence due to implantation. Cancer Res 49: 5414–5418
  44. Sidransky D, Frost P, Von Eschenbach A, Oyasu R, Preisinger AC and Vogestein B (1992) Clonal origin of bladder cancer. N Engl J Med 326: 737–740 | PubMed | ISI | ChemPort |
  45. Silverman DT, Rothman N and Devesa SS (1999) Epidemiology of bladder cancer. Bladder Cancer: Biology, Diagnosis and Management, Syrigos KN, Skinner DG (eds), pp 11–55, Oxford University Press: Oxford
  46. Skobe M, Rockwell P, Goldstein N, Vosseler S and Fusenig NE (1997) Halting angiogenesis suppresses carcinoma cell invasion. Nature Med 3: 1222–1227 | Article |
  47. Soloway MS and Masters S (1980) Urothelial susceptibility to tumour cell implantation – influence of cauterization. Cancer 46: 1158–1163
  48. Southgate J, Hutton KAR, Thomas DFM and Trejdosiewicz LK (1994) Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab Invest 71: 583–594 | PubMed | ISI | ChemPort |
  49. Theodorescu D, Cornil I, Fernandez BJ and Kerbel RS (1990) Overexpression of normal and mutated forms of HRAS induces orthopic bladder invasion in human transitional cell carcinoma. Proc Natl Acad Sci USA 87: 9047–9051 | PubMed | ChemPort |
  50. Theodorescu D, Laderoute KR and Gulding KM (1998) Epidermal growth factor receptor-regulated human bladder cancer motility is in part a phosphatidylinositol 3-kinase-mediated process. Cell Growth Differ 9: 919–928 | PubMed | ISI | ChemPort |
  51. Vladovski I, Miao H-Q, Benezra M, Lider O, Bar-Shavit R, Schmidt A and Peretz T (1997) Involvement of the extracellular matrix, heparan sulphate proteoglycans, and heparan sulphate degrading enzymes in angiogenesis and metastatis. Tumour angiogenesis, Bicknell R, Lewis CE, Ferrara N (eds), pp 125–140, Oxford University Press: Oxford
  52. Wallace DMA, Smith JHF, Billington S, Smith MR, Stemplewski HE and Tipton PW (1984) Promotion of bladder tumours by endoscopic procedures in an animal model. Br J Urol 56: 658–662
  53. Walther MM, Figg WD and Lineham WM (1996) Intravesical suramin: a novel agent for the treatment of superficial transitional-cell carcinoma of the bladder. World J Urology 14: S8–S11
  54. Yip L, Dart RC and Hurlbut KM (1998) Intravenous administration of oral N-acetylcysteine. Crit Care Med 26: 40–43