Clinical & Experimental Metastasis

, Volume 25, Issue 3, pp 253–264

Graded hypoxia modulates the invasive potential of HT1080 fibrosarcoma and MDA MB231 carcinoma cells

Research Paper

Abstract

Spatial and temporal oxygen heterogeneity exists in most solid tumour microenvironments due to an inadequate vascular network supplying a dense population of tumour cells. An imbalance between oxygen supply and demand leads to hypoxia within a significant proportion of a tumour, which has been correlated to the likelihood of metastatic dissemination in both rodent tumour models and human patients. Experimentally, it has been demonstrated that near-anoxic in vitro exposure results in transiently increased metastatic potential in some tumour cell lines. The purpose of this study was to examine the effect of graded low oxygen conditions on the invasive phenotype of human tumour cells using an in vitro model of basement membrane invasion, in which we measured oxygen availability directly at the invasion surface of the transwell chamber. Our results show a relationship between culture vessel geometry and time to achieve hypoxia which may affect the interpretation of low oxygen experiments. We exposed the human tumour cell lines, HT1080 and MDA MB231, to graded normobaric oxygen (5% O2–0.2% O2) either during or prior to in vitro basement membrane invasion to simulate conditions of intravasation and extravasation. A secondary aim was to investigate the potential regulation of matrix metalloproteinase activity by oxygen availability. We identified significant reductions in invasive ability under low oxygen conditions for the HT1080 cell line and an increase in invasion at intermediate oxygen conditions for the MDA MB231 cell line. There were differences in the absolute activity of the individual matrix metalloproteinases, MMP-2, -9, -14, between the two cell lines, however there were no significant changes following exposure to hypoxic conditions. This study demonstrates cell line specific effects of graded oxygen levels on invasive potential and suggests that intermediate levels of low oxygen may increase metastatic dissemination.

Keywords

Hypoxia Invasion Metastasis Matrix metalloproteinases Tumour microenvironment 

Abbreviations

HIF

Hypoxia inducible factor

ITS

Insulin, transferrin, sodium selenite

mmHg

Millimeters of mercurcy

MMP

Matrix metalloproteinase

MT-MMP

Membrane type matrix metalloproteinase

pO2

Partial pressure of oxygen

TIMP

Tissue inhibitor of MMP

VEGF

Vascular endothelial growth factor

Supplementary material

10585_2007_9139_MOESM1_ESM.eps (873 kb)
Plating efficiency following low oxygen exposure. ( a) MDA MB231 and (b) HT1080 tumour cells were exposed to various low oxygen conditions for 24 h and assayed for plating efficiency under normal atmospheric conditions. Four replicate plates at each of two different seeding densities were used to calculate average plating efficiency for each experiment. n = (#) shows the number of separate experiments at each low oxygen condition. (EPS 873 kb)

References

  1. 1.
    Vaupel P, Kelleher DK, Hockel M (2001) Oxygen status of malignant tumors: pathogenesis of hypoxia and significance for tumor therapy. Semin Oncol 28(2 Suppl 8):29–35PubMedCrossRefGoogle Scholar
  2. 2.
    Brurberg KG, Skogmo HK, Graff BA et al (2005) Fluctuations in pO2 in poorly and well-oxygenated spontaneous canine tumors before and during fractionated radiation therapy. Radiother Oncol 77(2):220–226PubMedGoogle Scholar
  3. 3.
    Cardenas-Navia LI, Yu D, Braun RD et al (2004) Tumor-dependent kinetics of partial pressure of oxygen fluctuations during air and oxygen breathing. Cancer Res 64(17):6010–6017PubMedCrossRefGoogle Scholar
  4. 4.
    Cardenas-Navia LI, Braun R, Lewis K et al (2003) Comparison of fluctuations of oxygen tension in FSA, 9L, and R3230AC tumors in rats. Adv Exp Med Biol 510:7–12PubMedGoogle Scholar
  5. 5.
    Lanzen J, Braun RD, Klitzman B et al (2006) Direct demonstration of instabilities in oxygen concentrations within the extravascular compartment of an experimental tumor. Cancer Res 66(4):2219–2223PubMedCrossRefGoogle Scholar
  6. 6.
    Brurberg KG, Thuen M, Ruud EB et al (2006) Fluctuations in pO2 in irradiated human melanoma xenografts. Radiat Res 165(1):16–25PubMedCrossRefGoogle Scholar
  7. 7.
    Semenza GL (2003) Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3(10):721–732PubMedCrossRefGoogle Scholar
  8. 8.
    Jiang BH, Semenza GL, Bauer C et al (1996) Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. Am J Physiol 271(4 Pt 1):C1172–C1180PubMedGoogle Scholar
  9. 9.
    Chiarotto JA, Hill RP (1999) A quantitative analysis of the reduction in oxygen levels required to induce up-regulation of vascular endothelial growth factor (VEGF) mRNA in cervical cancer cell lines. Br J Cancer 80(10):1518–1524PubMedCrossRefGoogle Scholar
  10. 10.
    Koong AC, Denko NC, Hudson KM et al (2000) Candidate genes for the hypoxic tumor phenotype. Cancer Res 60(4):883–887PubMedGoogle Scholar
  11. 11.
    Lal A, Peters H, St Croix B et al (2001) Transcriptional response to hypoxia in human tumors. J Natl Cancer Inst 93(17):1337–1343PubMedCrossRefGoogle Scholar
  12. 12.
    Scandurro AB, Weldon CW, Figueroa YG et al (2001) Gene microarray analysis reveals a novel hypoxia signal transduction pathway in human hepatocellular carcinoma cells. Int J Oncol 19(1):129–135PubMedGoogle Scholar
  13. 13.
    Yoon DY, Buchler P, Saarikoski ST et al (2001) Identification of genes differentially induced by hypoxia in pancreatic cancer cells. Biochem Biophys Res Commun 288(4):882–886PubMedCrossRefGoogle Scholar
  14. 14.
    Brizel DM, Scully SP, Harrelson JM et al (1996) Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res 56(5):941–943PubMedGoogle Scholar
  15. 15.
    Hockel M, Schlenger K, Aral B et al (1996) Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res 56(19):4509–4515PubMedGoogle Scholar
  16. 16.
    Fyles A, Milosevic M, Hedley D et al (2002) Tumor hypoxia has independent predictor impact only in patients with node-negative cervix cancer. J Clin Oncol 20(3):680–687PubMedCrossRefGoogle Scholar
  17. 17.
    Brizel DM, Dodge RK, Clough RW et al (1999) Oxygenation of head and neck cancer: changes during radiotherapy and impact on treatment outcome. Radiother Oncol 53(2):113–117PubMedCrossRefGoogle Scholar
  18. 18.
    Subarsky P, Hill RP (2003) The hypoxic tumour microenvironment and metastatic progression. Clin Exp Metastasis 20(3):237–250PubMedCrossRefGoogle Scholar
  19. 19.
    Milosevic M, Fyles A, Hedley D et al (2004) The human tumor microenvironment: invasive (needle) measurement of oxygen and interstitial fluid pressure. Semin Radiat Oncol 14(3):249–258PubMedCrossRefGoogle Scholar
  20. 20.
    Pitson G, Fyles A, Milosevic M et al (2001) Tumor size and oxygenation are independent predictors of nodal diseases in patients with cervix cancer. Int J Radiat Oncol Biol Phys 51(3):699–703PubMedGoogle Scholar
  21. 21.
    Sundfor K, Lyng H, Rofstad EK (1998) Tumour hypoxia and vascular density as predictors of metastasis in squamous cell carcinoma of the uterine cervix. Br J Cancer 78(6):822–7PubMedGoogle Scholar
  22. 22.
    Young SD, Marshall RS, Hill RP (1988) Hypoxia induces DNA overreplication and enhances metastatic potential of murine tumor cells. Proc Natl Acad Sci USA 85(24):9533–9537PubMedCrossRefGoogle Scholar
  23. 23.
    Young SD, Hill RP (1990) Effects of reoxygenation on cells from hypoxic regions of solid tumors: anticancer drug sensitivity and metastatic potential. J Natl Cancer Inst 82(5):371–380PubMedCrossRefGoogle Scholar
  24. 24.
    Rofstad EK, Danielsen T (1999) Hypoxia-induced metastasis of human melanoma cells: involvement of vascular endothelial growth factor-mediated angiogenesis. Br J Cancer 80(11):1697–1707PubMedCrossRefGoogle Scholar
  25. 25.
    Zhang L, Hill RP (2004) Hypoxia enhances metastatic efficiency by up-regulating Mdm2 in KHT cells and increasing resistance to apoptosis. Cancer Res 64(12):4180–4189PubMedCrossRefGoogle Scholar
  26. 26.
    Erler JT, Bennewith KL, Nicolau M et al (2006) Lysyl oxidase is essential for hypoxia-induced metastasis. Nature 440(7088):1222–1226PubMedCrossRefGoogle Scholar
  27. 27.
    Chambers AF, Groom AC, MacDonald IC (2002) Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer 2(8):563–572PubMedCrossRefGoogle Scholar
  28. 28.
    Deryugina EI, Quigley JP (2006) Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev 25(1):9–34PubMedCrossRefGoogle Scholar
  29. 29.
    Coussens LM, Werb Z (1996) Matrix metalloproteinases and the development of cancer. Chem Biol 3(11):895–904PubMedCrossRefGoogle Scholar
  30. 30.
    Canning MT, Postovit LM, Clarke SH et al (2001) Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells. Exp Cell Res 267(1):88–94PubMedCrossRefGoogle Scholar
  31. 31.
    Munoz-Najar UM, Neurath KM, Vumbaca F et al (2006) Hypoxia stimulates breast carcinoma cell invasion through MT1-MMP and MMP-2 activation. Oncogene 25(16):2379–2392PubMedCrossRefGoogle Scholar
  32. 32.
    Osinsky SP, Ganusevich II, Bubnovskaya LN et al (2005) Hypoxia level and matrix metalloproteinases-2 and -9 activity in Lewis lung carcinoma: correlation with metastasis. Exp Oncol 27(3):202–205PubMedGoogle Scholar
  33. 33.
    Petrella BL, Lohi J, Brinckerhoff CE (2005) Identification of membrane type-1 matrix metalloproteinase as a target of hypoxia-inducible factor-2 alpha in von Hippel-Lindau renal cell carcinoma. Oncogene 24(6):1043–1052PubMedCrossRefGoogle Scholar
  34. 34.
    Lohi J, Lehti K, Westermarck J et al (1996) Regulation of membrane-type matrix metalloproteinase-1 expression by growth factors and phorbol 12-myristate 13-acetate. Eur J Biochem 239(2):239–247PubMedCrossRefGoogle Scholar
  35. 35.
    Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 102(1):196–202PubMedCrossRefGoogle Scholar
  36. 36.
    Leber TM, Balkwill FR (1997) Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels. Anal Biochem 249(1):24–28PubMedCrossRefGoogle Scholar
  37. 37.
    Kleiner DE, Stetler-Stevenson WG (1994) Quantitative zymography: detection of picogram quantities of gelatinases. Anal Biochem 218(2):325–329PubMedCrossRefGoogle Scholar
  38. 38.
    Albini A, Iwamoto Y, Kleinman HK et al (1987) A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 47(12):3239–3245PubMedGoogle Scholar
  39. 39.
    Albini A, Benelli R, Noonan DM et al (2004) The “chemoinvasion assay”: a tool to study tumor and endothelial cell invasion of basement membranes. Int J Dev Biol 48(5–6):563–571PubMedCrossRefGoogle Scholar
  40. 40.
    Chambers AF, Matrisian LM (1997) Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 89(17):1260–1270PubMedCrossRefGoogle Scholar
  41. 41.
    Sato H, Takino T, Miyamori H (2005) Roles of membrane-type matrix metalloproteinase-1 in tumor invasion and metastasis. Cancer Sci 96(4):212–217PubMedCrossRefGoogle Scholar
  42. 42.
    Strongin AY, Collier I, Bannikov G et al (1995) Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem 270(10):5331–5338PubMedCrossRefGoogle Scholar
  43. 43.
    Lehti K, Lohi J, Valtanen H et al (1998) Proteolytic processing of membrane-type-1 matrix metalloproteinase is associated with gelatinase A activation at the cell surface. Biochem J 334(Pt 2):345–353PubMedGoogle Scholar
  44. 44.
    Koch CJ (1984) A thin-film culturing technique allowing rapid gas-liquid equilibration (6 sec) with no toxicity to mammalian cells. Radiat Res 97(2):434–442PubMedCrossRefGoogle Scholar
  45. 45.
    Boag JW (1969) Oxygen diffusion and oxygen depletion problems in radiobiology. In: Ebert M, Howard A (eds), Current topics in radiation research. North-Holland Publishing Co., Amsterdam, pp 141–195Google Scholar
  46. 46.
    Chapman JD, Sturrock J, Boag JW et al (1970) Factors affecting the oxygen tension around cells growing in plastic Petri dishes. Int J Radiat Biol Relat Stud Phys Chem Med 17(4):305–328PubMedCrossRefGoogle Scholar
  47. 47.
    Chapman JD, Sturrock J, Boag JW et al (1969) The oxygen tension around mammalian cells growing on plastic Petri dishes and its effect on cell survival curves. Br J Radiol 42(497):399PubMedCrossRefGoogle Scholar
  48. 48.
    Levy AP, Levy NS, Wegner S et al (1995) Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia. J Biol Chem 270(22):13333–13340PubMedCrossRefGoogle Scholar
  49. 49.
    Levy AP, Levy NS, Goldberg MA (1996) Hypoxia-inducible protein binding to vascular endothelial growth factor mRNA and its modulation by the von Hippel-Lindau protein. J Biol Chem 271(41):25492–25497PubMedCrossRefGoogle Scholar
  50. 50.
    Forsythe JA, Jiang BH, Iyer NV et al (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16(9):4604–4613PubMedGoogle Scholar
  51. 51.
    Allalunis-Turner MJ, Franko AJ, Parliament MB (1999) Modulation of oxygen consumption rate and vascular endothelial growth factor mRNA expression in human malignant glioma cells by hypoxia. Br J Cancer 80(1–2):104–109PubMedCrossRefGoogle Scholar
  52. 52.
    Turcotte ML, Parliament M, Franko A et al (2002) Variation in mitochondrial function in hypoxia-sensitive and hypoxia-tolerant human glioma cells. Br J Cancer 86(4):619–624PubMedCrossRefGoogle Scholar
  53. 53.
    Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2(3):161–174PubMedCrossRefGoogle Scholar
  54. 54.
    Himelstein BP, Koch CJ (1998) Studies of type IV collagenase regulation by hypoxia. Cancer Lett 124(2):127–133PubMedCrossRefGoogle Scholar
  55. 55.
    Saed GM, Zhang W, Diamond MP (2000) Effect of hypoxia on stimulatory effect of TGF-beta 1 on MMP-2 and MMP-9 activities in mouse fibroblasts. J Soc Gynecol Investig 7(6):348–354PubMedCrossRefGoogle Scholar
  56. 56.
    Chen PS, Zhai WR, Zhou XM et al (2001) Effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells. World J Gastroenterol 7(5):647–651PubMedGoogle Scholar
  57. 57.
    Zhang J, Salamonsen LA (2002) Expression of hypoxia-inducible factors in human endometrium and suppression of matrix metalloproteinases under hypoxic conditions do not support a major role for hypoxia in regulating tissue breakdown at menstruation. Hum Reprod 17(2):265–274PubMedCrossRefGoogle Scholar
  58. 58.
    Ben Yosef Y, Lahat N, Shapiro S et al (2002) Regulation of endothelial matrix metalloproteinase-2 by hypoxia/reoxygenation. Circ Res 90(7):784–791PubMedCrossRefGoogle Scholar
  59. 59.
    Fahling M, Perlewitz A, Doller A et al (2004) Regulation of collagen prolyl 4-hydroxylase and matrix metalloproteinases in fibrosarcoma cells by hypoxia. Comp Biochem Physiol C Toxicol Pharmacol 139(1–3):119–126PubMedCrossRefGoogle Scholar
  60. 60.
    Cairns RA, Kalliomaki T, Hill RP (2001) Acute (cyclic) hypoxia enhances spontaneous metastasis of KHT murine tumors. Cancer Res 61(24):8903–8908PubMedGoogle Scholar
  61. 61.
    Cairns RA, Hill RP (2004) Acute hypoxia enhances spontaneous lymph node metastasis in an orthotopic murine model of human cervical carcinoma. Cancer Res 64(6):2054–2061PubMedCrossRefGoogle Scholar
  62. 62.
    Rofstad EK, Galappathi K, Mathiesen B et al (2007) Fluctuating and Diffusion-Limited Hypoxia in Hypoxia-Induced Metastasis. Clin Cancer Res 13(7):1971–1978PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  1. 1.Applied Molecular Oncology Division, Ontario Cancer Institute/Princess Margaret HospitalUniversity Health NetworkTorontoCanada
  2. 2.Department of Medical BiophysicsUniversity of TorontoTorontoCanada
  3. 3.Department of Radiation OncologyUniversity of TorontoTorontoCanada

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