Cell-cell interactions promotes metastasis The role of metastatic microenvironment

1. Cell-cell interactions promotes metastasisThe role of metastatic microenvironment Lubor Borsig Zürich Center for Integrative Human Physiology Cancer Network Zürich University of Zürich Bratislava, May 2008

2. Metastatic cells interact with host leukocytes and platelets when they enter the bloodstream Hematogenous Metastasis Clonal Expansion Growth Angiogenesis Invasion of basement membrane Passage through Extracellullar Matrix Intravasation Tumor Cell emboli Adhesion to endothelium Invasion of basement membrane Extravasation Growth and Angiogenesis

3. Metastatic tumor cells in circulation Integrins Selectins Ig CAM

4. L L P P P E E E-selectin L-selectin P-selectin Selectins and their ligands L-selectin Leukocyte P-selectin E-selectin Platelet Endothelial Cell E • - Sialylated fucosylated lactosamine oligosaccharides • sLeX based ligands (O-linked =mucins, N-linked) • - Glycosaminoglycans (e.g. heparin, heparan sulfate) • Glycolipids (e.g. Sulfatides) L P SELECTIN LIGANDS

5. Altered sugar chains CANCER Siaa2-3Galb1-4GlcNAcb1- 3 1 a Fuc Sialyl Lewisx (sLex) Normal and malignant epithelium NORMAL BASEMENT MEMBRANE Blood vessels

6. Prognostic significance of Sialyl Lewisx (sLex) expression in patients with colon adenocarcinoma from Nakamori et.al, 1997 Sialyl-Lex expression is associated with poor prognosis / disease progression in Adenocarcinomas of Breast, Lung, Pancreas, Bladder, Prostate, Biliary tree and Ovary

7. Possible interactions between carcinomas and selectins • P-dependent interactions promote metastasis • Leukocytes facilitate metastasis • Heparin as an inhibitor of metastasis Borsig L, NIPS 2004

8. Selectin deficiencies attenuate metastasis of colon carcinoma cells in syngeneic mice Borsig L, et al, PNAS 2002

9. P< 0.01 P-sel +/+ P-sel -/- Mice Psel+/+ Psel-/- Psel+/+ Psel+/+ OSGPase - - - + P-selectin deficiency attenuates metastasis formation • Human colon carcinoma cells intravenously injected in Rag-/- mouse background •Mice were euthanized after 6 weeks • PCR amplification of human specific Alu sequences, quantified by Dot-blot Borsig L, et al. PNAS 2001

10. Interaction of platelets with carcinoma cells is P-selectin dependent in vivo Calcein labeled LS180 cells were intravenously injected Green - tumor cells Red - CD41 stained platelets • P-selectin is required for optimal interactions of tumor cells with platelets Borsig L, et al. PNAS 2001

11. Interaction of carcinoma cells with platelets & monocytes is affected by P-selectin Green: tumor cells Blue: CD41+ platelets Red: Mac-1+ cells Loss of the “platelet coating” allows increased association of Mac-1-positive monocytes/macrophage precursors with tumor cells Borsig L, et al, PNAS 2001

12. Leukocytes facilitate metastasis in L-selectin dependent manner

13. * p< 0.05 Lsel+/+ Lsel -/- Leukocytes associate with tumor cells in L-selectin dependent manner

14. Temporal inhibition of L-selectin attenuates metastasis MEL-14 Fab injected at +6 h and +12 h after tumor cells Läubli et al. Cancer Res 2006

15. Early Heparin Late Heparin BEFORE TC AFTER TC Heparin as an inhibitor of metastasis

16. „Late“ Heparin treatment affects metastasis Heparin IV injected at +6 h and +12 h after tumor cells „Late“ Heparin attenuates metastasis in P-selectin independent manner Laubli et al. Cancer Res 2006

17. Diverse biological effects of heparin • Inhibition of the Clotting Cascade • Blockade of P- and L-selectin • Binding of Growth factors • Inhibition of Angiogenesis • Inhibition of Heparanases • Effects on Proteases and ECM components • Inhibition of the Clotting Cascade • Blockade of P- and L-selectin • Binding of Growth factors • Inhibition of Angiogenesis • Inhibition of Heparanases • Effects on Proteases and ECM components Heparin der. with a narrow biological activity

18. NA-H„selectin specific“ inhibitor RO.H selectin and heparanase inhibitor NA-RO.H heparanase specific inhibitor 58 G3496 G4146 100 G4000 Characterization of Heparin derivatives Hostettler N, et al. FASEB J 21:3562-72, 2007

19. GFP-quantitation Number of Foci Heparin treatment attenuates metastasis G3496 – „P-selectin specific“ inhibitor Heparin derivative with NO anticoagulant activity and low heparanase inhibitory activity attenuates metastasis Hostettler N, et al. FASEB J 21:3562-72, 2007

20. Heparinoids attenuate metastasis by P-selectin inhibition

21. Metastatic microenvironment PMN tumor cells platelets monocytes endothelium Activation of microvascular endothelial cells promotes metastasis

22. Co-culture system HT-29 and LS-180 tumor cell lines Microvascular endothelial cells platelets Monocytes PMN

23. Tumor cell interactions with platelets and leukocytes activate HMVEC LS180 HT-29 3.8 4.2 Tumor cells alone E-selectin 62.2 27.8 + Platelets, PMN and Monocytes CD105

24. Nuclear translocation of NF-κB HMVEC with PMN, monocytes and platelets - LS180 +LS180 DAPI p65 After 40 min of co-culture Colocalization of DAPI and p65 CD31

25. Leukocytes facilitate metastasis in L-selectin dependent manner

26. Leukocyte association with tumor cells wt mice Lsel-/- mice Pronounced association of neutrophils and monocytes/macrophages with metastasizing tumor cells is L-selectin dependent

27. How contribute Monocyte/Macrophages to metastasis?

28. TC extravasation is mediated by Monocytes Heart perfusion with Tomato lectin-TR + 24 h liposomes + 24 h clodronate

29. Conclusions and Outlook Cell-cell interactions and metastasis • Temporal inhibition of P-and L-selectin attenuate metastasis • Platelet and leukocyte interactions with TC facilitate metastasis • Endogenous selectin ligands potentiate metastasis • Heparin treatment reduces metastasis primarily through selectin inhibition • Selectin-specific heparins are equally good inhibitors of metastasis as heparin

30. Conclusions Acknowledgements University Zürich Ronzoni Institute Milano Heinz Läubli B. Casu Marie-A Boucabeille A. Naggi Nina Hostettler G. Torri Josep Garcia Claudia Ruedin B. Rappoport F of Medicine Eric BergerI. Vlodavsky IMCR University of Zürich R. Schwendener Supported by SNSF, Swiss Cancer League Zürich