Ardi - Tsri 2008


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  • Ardi - Tsri 2008

    1. 1. The Yin and the Yang of Matrix Metalloproteinase 9: The contribution of stromal and tumor MMP-9 in tumor angiogenesis and dissemination. Veronica C. Ardi, PhD The Scripps Research Institute
    2. 2. Outline <ul><li>The role of MMP-9 derived from inflammatory neutrophils in: </li></ul><ul><ul><li>Physiologic angiogenesis </li></ul></ul><ul><ul><li>Tumor angiogenesis </li></ul></ul><ul><li>Tumor-derived MMP-9 contribution to: </li></ul><ul><ul><li>Physiologic angiogenesis </li></ul></ul><ul><ul><li>Tumor dissemination and metastasis </li></ul></ul>
    3. 3. Angiogenesis <ul><li>Tumor-induced angiogenesis has been linked to inflammation. </li></ul><ul><ul><li>Tumor cells recruit inflammatory cells at the site of primary tumor formation. </li></ul></ul><ul><ul><li>Inflammatory cells produce and release pro-angiogenic factors. </li></ul></ul><ul><ul><li>Angiogenesis requires growth factors (e.g. VEGF, bFGF, TGF), chemokines (e.g. IL-6, IL-8), and matrix metalloproteinases (e.g. MMP-9, MMP-13), </li></ul></ul><ul><ul><li>which are produced by inflammatory cells. </li></ul></ul><ul><ul><li>Positive association between presence/elevated levels of matrix metalloproteinases, especially MMP-9, and tumor angiogenesis. </li></ul></ul>
    4. 4. Matrix metalloproteinase-9: gelatinase B Aim: to study the role of MMP-9 derived from inflammatory neutrophils in physiologic angiogenesis and in tumor-induced angiogenesis
    5. 5. Avian Chorioallantoic Membrane (CAM) Angiogenesis Assay Angiogenic factors 3-D collagen onplants
    6. 7. Analyses of neutrophil contents and purified MMP-9 Neutrophil releasate Eluate (purified MMP-9) Releasate depleted of MMP-9 Zymograph MMP-9 dimer MMP-9/ lipocalin heterodimer MMP-9 monomer Silver stained gel MW (kDa) 250 - 150 - 100 - 75 - 50 - 37 - 25 - 20 - 15 - 10 - Eluate (purified MMP-9) MW markers Neutrophil releasate Releasate depleted of MMP-9 Western Blot 220 - 120 - 100 - 80 - 60 - 50 - MW (kDa) purified MMP-9 Neutrophil releasate
    7. 8. Neutrophils, their contents, and neutrophil-derived MMP-9 induce angiogenesis. *, p<0.001 Ardi et al., Proceedings of the National Academy of Sciences 2007. 104(51):20262-7
    8. 9. Activation of neutrophil-derived MMP-9 is necessary to induce angiogenesis Zymograph pro-form active form Non-blocking 8-3H Blocking 7-11C Activation of pro-MMP-9 mAb specific to human MMP-9 APMA: - + + + MMP-9 specific mAb: PBS PBS 7-11C 8-3H - + + + Purified MMP-9: Additional supplement: - - 7-11C 8-3H * * Angiogenesis, fold change
    9. 10. Catalytic activity of MMP-9 is required for angiogenesis Neutrophil pro MMP-9: - + + + Additional supplement: - - TIMP-1 TIMP-2 Angiogenesis, fold change * *
    10. 11. TIMP-free MMP-9 derived from neutrophils induces angiogenesis while MMP-9 derived from tumor or monocyte/macrophage (U937) cell lines does not induce angiogenesis
    11. 12. Isolation of MMP-9/TIMP-1 Stoichiometric complex Neutrophil MMP-9 complexed with TIMP-1 loses the ability to induce angiogenesis Gelatin Sepharose Affinity Chromatography MMP-9 proenzyme Excess TIMP-1 Unbound TIMP-1 (flow thru) Purified pro-MMP-9/ TIMP-1 complex pro-MMP-9/TIMP-1 complex unbound TIMP-1 TIMP-1 MMP-9 monomer MMP-9 dimer MMP-9/ NGAL NeutrophilMMP-9 Buffer MMP-9 +TIMP-1 Angiogenesis, fold change *
    12. 13. Neutrophil MMP-9 induces angiogenesis in the mouse model. * Mouse with Angiotubes pro MMP-9 Hemoglobin (mg/ml) control pro MMP-9/TIMP-1
    13. 14. Physiologic Angiogenesis Summary <ul><li>Human neutrophils and their released products induce physiologic angiogenesis. </li></ul><ul><li>Neutrophil-derived MMP-9 (which importantly is free of TIMP-1) functions as a pro-angiogenic enzyme at subnanogram levels. </li></ul><ul><li>TIMP-free neutrophil MMP-9 functions as a potent pro-angiogenic enzyme in both avian and mammalian models. </li></ul><ul><li>MMP-9 derived from tumor cells or monocytes/macrophages are TIMP-1 complexed and does not induce angiogenesis above control. </li></ul>
    14. 15. Aim: Role of neutrophils and their secreted MMP-9 in tumor angiogenesis
    15. 16. Role of Neutrophil MMP-9 in tumor angiogenesis <ul><li>Tumor angiogenesis induced by two HT-1080 and PC3 cell variants </li></ul><ul><ul><li>Low disseminating HT-1080 or PC3 ( lo-diss ) </li></ul></ul><ul><ul><li>High disseminating HT-1080 or PC3 ( hi-diss ) </li></ul></ul>Angiogenesis, fold change PC- lo/diss PC- hi/diss HeLa * Angiogenic index HT- lo/diss HT- hi/diss *
    16. 17. Tumor-induced angiogenesis is strongly linked with recruitment of host inflammatory cells. <ul><li>Influx of inflammatory cells to the site of primary tumor development. </li></ul>* Cells per square Density of MMP-9 positive inflammatory host cells CAM Tumor HT- lo/diss CAM Tumor HT- hi/diss
    17. 18. Tumor-induced angiogenesis is enhanced by neutrophil-derived MMP-9 Angiogenesis, fold change HeLa HT- hi/diss HT- lo/diss * * Neutrophil MMP-9: - - + -
    18. 19. Neutrophil releasate and neutrophil-derived MMP-9 rescues dampened tumor-induced angiogenesis Angiogenesis, % HT- hi/diss Supplemented with Ibuprofen * * Additional supplement: Neutrophil releasate Neutrophil MMP-9 - - Releasate depleted of MMP-9
    19. 20. Possible targets of tumor-induced angiogenesis Panomics: TranSignal Angiogenesis Array
    20. 21. HT- hi/diss -induced angiogenesis is partially sensitive to anti-VEGF while completely abrogated with anti-FGF=2 p< 0.01 Angiogenesis, % HT- hi/diss anti-IL-8 antibody: - +
    21. 22. Tumor Angiogenesis Summary <ul><li>Neutrophil MMP-9 facilitates tumor-induced angiogenesis. </li></ul><ul><li>Neutrophil MMP-9 enhances tumor angiogenesis at low nanogram levels. </li></ul><ul><li>Neutrophil MMP-9 rescues tumor angiogenesis inhibited by anti-inflammatory drugs. </li></ul><ul><li>Inhibiting FGF-2 pathway blocks tumor-induced angiogenesis. </li></ul><ul><li>TIMP-free MMP-9 derived from inflammatory neutrophils could make FGF-2 bioavailable. </li></ul>
    22. 23. Metastasis: Tumor Dissemination Cascade Angiogenesis Primary Tumor Possible metastatic subclone Invasion through extracellular matrix Intravasation Interaction with host blood cells Formation of tumor cell embolus Arrest in capillaries and adhesion to basement membrane Extravasation Metastatic deposit and growth in secondary organ Metastatic Tumor Transformed cell Clonal expansion, growth, diversification Adhesion to and invasion of basement membrane
    23. 24. Metastasis Studies in Chick Embryo Model Experimental metastasis: via Intravenous Spontaneous Metastasis: via CAM Tumors
    24. 25. Human Tumor-Chick Embryo Model of Intravasation and Metastasis Day 10: Making the window Day 10: Inoculating the tumor cells Day 16: Harvesting the primary tumor Day 16: Tumor on CAM Day 16: Harvested Lower CAM (repository of intravasated cells) Day 16: Lung and Liver Harvest (sites of early metastatic growth) Lung Liver
    25. 26. Quantifying Intravasation & Metastasis in Human Tumor-Chick Embryo Model Relative frequency of human cells in the analyzed tissues Primary tumor analysis Lungs Metastatic Foci Intravasated cells Lower CAM alu PCR analysis Quantitative Real-Time alu PCR alu Standard Curve Human cells spiked into chick tissue Human cells detected by Alu qPCR
    26. 27. HT-1080 Cell Variants: In Vivo Characteristics in the Chick Embryo
    27. 28. <ul><li>Matrix metalloproteinases (MMPs) has been strongly linked to tumor progression </li></ul><ul><li>Individual MMPs may have specific roles in tumor dissemination </li></ul><ul><li>In this study, we examined the role of specific MMPs and TIMPs (tissue inhibitors of metalloproteinases) in tumor dissemination </li></ul>
    28. 29. Downregulation of MMP-1, MMP-2, & MMP-9 increases tumor dissemination Modulation of HT- hi/diss tumor cells Specific MMPs may have a protective role and negatively regulate tumor progession.
    29. 30. Acknowledgements <ul><li>Quigley Lab </li></ul><ul><li>Jim Quigley </li></ul><ul><li>Elena Deryugina </li></ul><ul><li>Erin Conn </li></ul><ul><li>Juneth Partridge </li></ul><ul><li>Chenxing Li </li></ul>Funding - National Institute of Health (training grant)