, 14:235 | Cite as

Neutrophil granulocyte derived MMP-9 is a VEGF independent functional component of the angiogenic switch in pancreatic ductal adenocarcinoma

  • Dirk Bausch
  • Thomas Pausch
  • Tobias Krauss
  • Ulrich Theodor Hopt
  • Carlos Fernandez-del-Castillo
  • Andrew L. Warshaw
  • Sarah P. Thayer
  • Tobias KeckEmail author
Original Paper



Vascular endothelial growth factor (VEGF) that is secreted by tumor cells plays a key role in angiogenesis. Matrix metalloproteinase 9 (MMP-9) is produced by inflammatory cells, such as stromal granulocytes (PMN), remodels the extracellular matrix and is known to promote angiogenesis indirectly by interacting with VEGF. The aim of this study was to determine the role of PMN-derived MMP-9, its interaction with VEGF, and the efficacy of anti-angiogenic therapy targeting MMP-9 with oral Doxycycline and VEGF with Bevacizumab in pancreatic cancer (PDAC).

Methodology/principal findings

Inhibitors to MMP-9 (Doxycycline) and VEGF (Bevacizumab) were used alone or in combination in an in vitro angiogenesis assay to test their effect on angiogenesis caused by MMP-9, VEGF, PMN and PDAC cells. In an in vivo model of xenografted PDAC, treatment effects after 14 days under monotherapy with oral Doxycycline or Bevacizumab and a combination of both were evaluated.

In vitro, PMN-derived MMP-9 had a direct and strong proangiogenic effect that was independent and additive to PDAC-derived VEGF. Complete inhibition of angiogenesis required the inhibition of VEGF and MMP-9. In vivo, co-localization of MMP-9, PMN and vasculature was observed. MMP inhibition with oral Doxycycline alone resulted in a significant decrease in PDAC growth and mean vascular density comparable to VEGF inhibition alone.


PMN derived MMP-9 acts as a potent, direct and VEGF independent angiogenic factor in the context of PDAC. MMP-9 inhibition is as effective as VEGF inhibition. Targeting MMP-9 in addition to VEGF is therefore likely to be important for successful anti-angiogenic treatment in pancreatic cancer.


VEGF MMP-9 Neutrophil granulocyte Pancreatic cancer 



The authors would like to thank Nancy Neyhard, Amy Stirman, Silke Hempel and Bettina Waldvogel for their technical assistance and Andrew Liss for his assistance composing the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285(21):1182–1186PubMedCrossRefGoogle Scholar
  2. 2.
    Hanahan D, Folkman J (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86(3):353–364PubMedCrossRefGoogle Scholar
  3. 3.
    Ferrara N (2002) VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer 2(10):795–803PubMedCrossRefGoogle Scholar
  4. 4.
    Nagy JA, Vasile E, Feng D, Sundberg C, Brown LF, Manseau EJ, Dvorak AM, Dvorak HF (2002) VEGF-A induces angiogenesis, arteriogenesis, lymphangiogenesis, and vascular malformations. Cold Spring Harb Symp Quant Biol 67:227–237PubMedCrossRefGoogle Scholar
  5. 5.
    Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246(4935):1306–1309PubMedCrossRefGoogle Scholar
  6. 6.
    Roberts WG, Palade GE (1995) Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J Cell Sci 108(Pt 6):2369–2379PubMedGoogle Scholar
  7. 7.
    Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF (1983) Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219(4587):983–985PubMedCrossRefGoogle Scholar
  8. 8.
    Kalluri R (2003) Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer 3(6):422–433PubMedCrossRefGoogle Scholar
  9. 9.
    Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z, Hanahan D (2000) Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol 2(10):737–744PubMedCrossRefGoogle Scholar
  10. 10.
    Nozawa H, Chiu C, Hanahan D (2006) Infiltrating neutrophils mediate the initial angiogenic switch in a mouse model of multistage carcinogenesis. Proc Natl Acad Sci USA 103(33):12493–12498PubMedCrossRefGoogle Scholar
  11. 11.
    Ardi VC, Kupriyanova TA, Deryugina EI, Quigley JP (2007) Human neutrophils uniquely release TIMP-free MMP-9 to provide a potent catalytic stimulator of angiogenesis. Proc Natl Acad Sci USA 104(51):20262–20267. doi: 10.1073/pnas.0706438104 PubMedCrossRefGoogle Scholar
  12. 12.
    Hashimoto G, Inoki I, Fujii Y, Aoki T, Ikeda E, Okada Y (2002) Matrix metalloproteinases cleave connective tissue growth factor and reactivate angiogenic activity of vascular endothelial growth factor 165. J Biol Chem 277(39):36288–36295PubMedCrossRefGoogle Scholar
  13. 13.
    Hawinkels LJ, Zuidwijk K, Verspaget HW, de Jonge-Muller ES, van Duijn W, Ferreira V, Fontijn RD, David G, Hommes DW, Lamers CB, Sier CF (2008) VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis. Eur J Cancer 44(13):1904–1913. doi: 10.1016/j.ejca.2008.06.031 PubMedCrossRefGoogle Scholar
  14. 14.
    Ahn GO, Brown JM (2008) Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: role of bone marrow-derived myelomonocytic cells. Cancer Cell 13(3):193–205. doi: 10.1016/j.ccr.2007.11.032 PubMedCrossRefGoogle Scholar
  15. 15.
    Nakamura T, Kuwai T, Kim JS, Fan D, Kim SJ, Fidler IJ (2007) Stromal metalloproteinase-9 is essential to angiogenesis and progressive growth of orthotopic human pancreatic cancer in parabiont nude mice. Neoplasia 9(11):979–986PubMedCrossRefGoogle Scholar
  16. 16.
    Condeelis J, Pollard JW (2006) Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124(2):263–266. doi: 10.1016/j.cell.2006.01.007 PubMedCrossRefGoogle Scholar
  17. 17.
    Huang S, Van Arsdall M, Tedjarati S, McCarty M, Wu W, Langley R, Fidler IJ (2002) Contributions of stromal metalloproteinase-9 to angiogenesis and growth of human ovarian carcinoma in mice. J Natl Cancer Inst 94(15):1134–1142PubMedGoogle Scholar
  18. 18.
    Coussens LM, Tinkle CL, Hanahan D, Werb Z (2000) MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell 103(3):481–490PubMedCrossRefGoogle Scholar
  19. 19.
    Pandol S, Edderkaoui M, Gukovsky I, Lugea A, Gukovskaya A (2009) Desmoplasia of pancreatic ductal adenocarcinoma. Clin Gastroenterol Hepatol 7(11 Suppl):S44–S47PubMedCrossRefGoogle Scholar
  20. 20.
    Roland CL, Dineen SP, Toombs JE, Carbon JG, Smith CW, Brekken RA, Barnett CC Jr (2010) Tumor-derived intercellular adhesion molecule-1 mediates tumor-associated leukocyte infiltration in orthotopic pancreatic xenografts. Exp Biol Med(Maywood) 235(2):263–270CrossRefGoogle Scholar
  21. 21.
    Bloomston M, Zervos EE, Rosemurgy AS 2nd (2002) Matrix metalloproteinases and their role in pancreatic cancer: a review of preclinical studies and clinical trials. Ann Surg Oncol 9(7):668–674PubMedCrossRefGoogle Scholar
  22. 22.
    Hotz HG, Hines OJ, Hotz B, Foitzik T, Buhr HJ, Reber HA (2003) Evaluation of vascular endothelial growth factor blockade and matrix metalloproteinase inhibition as a combination therapy for experimental human pancreatic cancer. J Gastrointest Surg 7 (2): 220–227; discussion 227–228. doi: S1091255X02001579 Google Scholar
  23. 23.
    Bramhall SR, Schulz J, Nemunaitis J, Brown PD, Baillet M, Buckels JA (2002) A double-blind placebo-controlled, randomised study comparing gemcitabine and marimastat with gemcitabine and placebo as first line therapy in patients with advanced pancreatic cancer. Br J Cancer 87(2):161–167. doi: 10.1038/sj.bjc.6600446 PubMedCrossRefGoogle Scholar
  24. 24.
    Evans JD, Stark A, Johnson CD, Daniel F, Carmichael J, Buckels J, Imrie CW, Brown P, Neoptolemos JP (2001) A phase II trial of marimastat in advanced pancreatic cancer. Br J Cancer 85(12):1865–1870. doi: 10.1054/bjoc.2001.2168 PubMedCrossRefGoogle Scholar
  25. 25.
    Bramhall SR, Rosemurgy A, Brown PD, Bowry C, Buckels JA (2001) Marimastat as first-line therapy for patients with unresectable pancreatic cancer: a randomized trial. J Clin Oncol 19(15):3447–3455PubMedGoogle Scholar
  26. 26.
    Van Cutsem E, Vervenne WL, Bennouna J, Humblet Y, Gill S, Van Laethem JL, Verslype C, Scheithauer W, Shang A, Cosaert J, Moore MJ (2009) Phase III trial of bevacizumab in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer. J Clin Oncol 27(13):2231–2237PubMedCrossRefGoogle Scholar
  27. 27.
    Ko AH, Dito E, Schillinger B, Venook AP, Xu Z, Bergsland EK, Wong D, Scott J, Hwang J, Tempero MA (2008) A phase II study evaluating bevacizumab in combination with fixed-dose rate gemcitabine and low-dose cisplatin for metastatic pancreatic cancer: is an anti-VEGF strategy still applicable? Invest New Drugs 26(5):463–471PubMedCrossRefGoogle Scholar
  28. 28.
    Kindler HL, Friberg G, Singh DA, Locker G, Nattam S, Kozloff M, Taber DA, Karrison T, Dachman A, Stadler WM, Vokes EE (2005) Phase II trial of bevacizumab plus gemcitabine in patients with advanced pancreatic cancer. J Clin Oncol 23(31):8033–8040PubMedCrossRefGoogle Scholar
  29. 29.
    Golub LM, Lee HM, Ryan ME, Giannobile WV, Payne J, Sorsa T (1998) Tetracyclines inhibit connective tissue breakdown by multiple non-antimicrobial mechanisms. Adv Dent Res 12(2):12–26PubMedCrossRefGoogle Scholar
  30. 30.
    Hanemaaijer R, Visser H, Koolwijk P, Sorsa T, Salo T, Golub LM, van Hinsbergh VW (1998) Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells. Adv Dent Res 12(2):114–118PubMedCrossRefGoogle Scholar
  31. 31.
    Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52(11):2745–2756PubMedCrossRefGoogle Scholar
  32. 32.
    Boyum A (1968) Separation of leukocytes from blood and bone marrow. Introduction. Scand J Clin Lab Invest Suppl 97:7PubMedGoogle Scholar
  33. 33.
    Korff T, Augustin HG (1998) Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J Cell Biol 143(5):1341–1352PubMedCrossRefGoogle Scholar
  34. 34.
    Korff T, Augustin HG (1999) Tensional forces in fibrillar extracellular matrices control directional capillary sprouting. J Cell Sci 112(Pt 19):3249–3258PubMedGoogle Scholar
  35. 35.
    Nagase H, Visse R, Murphy G (2006) Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res 69(3):562–573PubMedCrossRefGoogle Scholar
  36. 36.
    Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17:463–516PubMedCrossRefGoogle Scholar
  37. 37.
    Aimes RT, Quigley JP (1995) Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J Biol Chem 270(11):5872–5876PubMedCrossRefGoogle Scholar
  38. 38.
    Patterson ML, Atkinson SJ, Knauper V, Murphy G (2001) Specific collagenolysis by gelatinase A, MMP-2, is determined by the hemopexin domain and not the fibronectin-like domain. FEBS Lett 503(2–3):158–162PubMedCrossRefGoogle Scholar
  39. 39.
    Akahane T, Akahane M, Shah A, Connor CM, Thorgeirsson UP (2004) TIMP-1 inhibits microvascular endothelial cell migration by MMP-dependent and MMP-independent mechanisms. Exp Cell Res 301(2):158–167. doi: 10.1016/j.yexcr.2004.08.002 PubMedCrossRefGoogle Scholar
  40. 40.
    Dufour A, Sampson NS, Zucker S, Cao J (2008) Role of the hemopexin domain of matrix metalloproteinases in cell migration. J Cell Physiol 217(3):643–651. doi: 10.1002/jcp.21535 PubMedCrossRefGoogle Scholar
  41. 41.
    Paez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Vinals F, Inoue M, Bergers G, Hanahan D, Casanovas O (2009) Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15(3):220–231PubMedCrossRefGoogle Scholar
  42. 42.
    Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS (2009) Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 15(3):232–239PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Dirk Bausch
    • 1
  • Thomas Pausch
    • 2
  • Tobias Krauss
    • 3
  • Ulrich Theodor Hopt
    • 1
  • Carlos Fernandez-del-Castillo
    • 4
  • Andrew L. Warshaw
    • 4
  • Sarah P. Thayer
    • 4
  • Tobias Keck
    • 1
    Email author
  1. 1.Department of General and Visceral SurgeryUniversity of FreiburgFreiburgGermany
  2. 2.Klinik für Allgemein, Viszeral- und TransplantationschirurgieUniversität HeidelbergHeidelbergGermany
  3. 3.Radiologische Universitätsklinik Freiburg i. Br.Freiburg i. Br.Germany
  4. 4.Department of SurgeryMassachusetts General Hospital and Harvard Medical SchoolBostonUSA

Personalised recommendations