Postdoctoral Projects

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

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. Avian Chorioallantoic Membrane (CAM) Angiogenesis Assay Angiogenic factors 3-D collagen onplants

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

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

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

10. Catalytic activity of MMP-9 is required for angiogenesis Neutrophil pro MMP-9: - + + + Additional supplement: - - TIMP-1 TIMP-2 Angiogenesis, fold change * *

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

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 *

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

15. Aim: Role of neutrophils and their secreted MMP-9 in tumor angiogenesis

18. Tumor-induced angiogenesis is enhanced by neutrophil-derived MMP-9 Angiogenesis, fold change HeLa HT- hi/diss HT- lo/diss * * Neutrophil MMP-9: - - + -

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

20. Possible targets of tumor-induced angiogenesis Panomics: TranSignal Angiogenesis Array

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: - +

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

24. Metastasis Studies in Chick Embryo Model Experimental metastasis: via Intravenous Spontaneous Metastasis: via CAM Tumors

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

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

27. HT-1080 Cell Variants: In Vivo Characteristics in the Chick Embryo

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.