Special approaches of tumor biology and chemotaxis

1. Special approaches of tumor biology and chemotaxis OrsolyaLáng 2012.

2. TUMOR CELLS AND MIGRATION PRIMARY TUMOR METASTASIS Angiogenezis Adhesion

3. CELL and CELL CYCLE

4. Apoptosis Growth factors Chemokines Regulatory proteins Adhesion molecules

5. TUMOR CELL

6. CELL KINETICS

7. Doubling time of the tumor volume (Td) Lymphoma 4 weeks Colon adenoma 90 weeks Usually 18-200 days • Time of the Cell cycle (Tc): Tc= Ts / Li Ts: S phase Li: labeling index (proportoin of cells in S phase) • Growth fraction(GF): GF=P / (P+Q’) P: number of the mitotic cells Q: number of the cells in interphase • Rate of the cell loss (): = 1-Tpd /TdTpd= *Ts/Li Tpd: Potential tumor volume doubling time Td: tumor volume doubling time Lymphoma 48 h Lung cancer 108 h Usually 15-125 h

8. 1 tumor cell ~27 division = 0,1cm3 Earliest time of diagnosis ~30-33,25 division =1-10 cm3 Time of the clinical symptomes / diagnosis ~40 division = 1012cell Fatal Volume of the tumor tissue ~20 division= 106 cells = 1 mg= 1 mm3 ~30 division= 109 cells = 1 g= 1 cm3 ~40 division= 1012 cells = 1 kg ~10 division =*1000 cell number increase (210 =1024)

9. BIOLOGY OF THE TUMORPROGRESSION

10. Tumorigenesis Exogen and endogen factors Genom instability Activation of the oncogene Inactivation of tumorsuppressors Growth rate De-Differentation Invazivity Ectopic survival capacity Epithelial cell Hiperplastic adenoma Displatic Carcinoma in situ Invasive and metastasis carcinoma Local and systemic factors inhibition acceleration

11. Important steps of tumor progression • Transformation of the microenvironment: • stromal cells, • ECM components, • proteolytic degradation • Induction of the angiogenesis • Escaping from immune-mediated rejection • Formation of metastasis

12. MICROENVIRONMENT – STROMAL CELLS • Cell types: fibroblasts, myofibroblasts, endothelial cells, lymphocytes, macrophages • Function: host defence ! MALT - B cell helps to maintain lymphomas ! Growth factors are released by the stromal cells (VEGF- angiogenesis)

13. ANGIOGENESIS  OEDEMA, decresed blood flow • Hypoxia  formation of new vessels, proliferation of the endothelial cells • Types: vessels arteriovenous shunts „dead end” /lack of smooth muscle , weak vessel wall, irregular shape(insuficient endothelial cell and basement membrane layers)/ sinuses /wall is formed by tumor cells/ Venous circulation • VEGF  induces angiogensis increases permeability • Lack of lymphatic vessels

14. Strategies that tumors use to escape from immune-mediated rejection are: • To decrease the antigen expression • To inhibit the immune-reactive cells: degrade the chemoattractans decrease their cell adhesion inhibite their phagocytotic activity

15. METASTATIC CASCADE VEGFAngiogeninFGF Tumor cell Metastasis Angiogenesis Primary tumor Angiogenesis Local invasion Adhesion Proteolysis Migration Adhesion Proteolysis Migration ECM circulation Extravasation Intravasation spreading

16. INVASION In situ carcinoma DECREASED CELL ADHESION, INCREASED MOTILITY ECM proteolysis

17. METASTATIC CASCADE VEGFAngiogeninFGF Tumor cell Metastasis Angiogenesis Primary tumor Angiogenesis Local invasion Adhesion Proteolysis Migration Adhesion Proteolysis Migration ECM circulation Extravasation Intravasation spreading

18. CELL ADHESION • Significant change in cell-cell and cell-ECM interactions • Molecules: selectins integrins immunoglobulin superfamily cadherins catenins

19. SELECTINS • Cell-cel junctions • Types: E- endothelial cells P- trombocytes L- leukocytes • Extracellular C-lectin domain • Ca2+ dependent anchorage • It binds Sialyl-Lex carbohydrates • „ROLLING” ! Tumor cells express increased amount of sialil-Lex or -Lea

20.   INTEGRINS D R G • Transmembrane receptors • Form cell-ECM interaction • 8 , 14  subunites ~20 heterodimer • Ca2+, Mg2+dependent anchorage • „RGD” sequence is the specific substrate • Signalling: outside-in – signalling • inside-out – adhesion • Increased expression of integrins promotes angiogenesis and helps to bind MMPs at the cell surface • EXTRAVASATION, ATTACHMENT

21. ECM integrin PTEN SHC GRB2/SOS FAK PI(3)K ILK RAC CDC42 RAS RAF MEK MAPK PKB/AKT GSK3 BAD -catenin Ciklin D1 apoptosis cellcycle gene expression motility cellproliferation Integrin or celladhesion regulated signalling pathways

22. integrin PTEN PI(3)K RAC CDC42 Integrin or celladhesion regulated signalling pathways ECM SHC GRB2/SOS FAK ILK RAS RAF MEK MAPK PKB/AKT GSK3 BAD -katenin Ciklin D1 apoptsis cellcycle gene expression motility cellproliferation

23. Molecular partners of the integrins • Cytoskeletal components: actinin, talin,F- actin, filamin • Adaptors: rack 1, ICAP-1 • Calcium binding proteins: CIB, calreticulin • Protein kinases: pp125FAK, p59 ILK • Membrane proteins: CD9, CD16,CD47… caveolin, urokinase-plazminogen-activator receptor • Ligands in ECM: collagen, laminin, fibronectin, fibrinogen, von Willebrand factor, osteopontin, elastin

24. IMMUNGLOBULIN SUPERFAMILY • has 5 Ig-like domains at the extracellular region • forms cell-cell junction • interacts with integrins VCAM - 41, PECAM - v3 • takes essential part in extravasation • ! Over expression of ICAM-1, MUC18 increased inavsion • ! Down-regulation of VCAM-1  increased metastatic potential(faster detachment)

25. CADHERIN • Is a transmembrane glycoprotein • Forms homophyl cell-cell junctions • Ca2+ dependent anchorage • Classical types: E- epithelial P- placenta N- neural, Intracellular part interacts with catenins to connect aktin filaments ! Increased expression  invasion

26. CATENIN • Is an intracellular molecule • Fixes cadherins to F-actin ! Catenin expression is often decreased in carcinomas !-catenin binds to the az APC gén termékéhez

27. Factors influencing the metastatic potential of the melanoma cells Ihibits E-Cadherin aE-Catenin cMET FGFR PTEN Increases N-Cadherin B-Catenin SrC Ras Adhesionmolecules Signal pathways

28. METASTATIC CASCADE VEGFAngiogeninFGF Tumor cell Metastasis Angiogenesis Integrins cadherins Selectins CAM Primary tumor Angiogenesis Local invasion Adhesion Proteolysis Migration Adhesion Proteolysis Migration ECM Circulation Extravasation Intravasation spreading

29. INVASION PROTEOLYSIS • Components of the basement membrane(BM) andECM: IV collagen, laminin, proteoglycanes • Tumorcells (stromal cells) secrete proteases • Cathepsin • Matrix metalloproteinase (MMP) • Plazmin, tPA ,Urokinase (plasminogen activator inhibitor 1&2) • TIMP Tissue inhibitor of metalloproteinases

30. MATRIX METTALLOPROTEINASES (MMP) • Zn2+ dependent endopeptidase (MMP28) • ECM degradation – tissue remodelling • Interstitial collagenase (MMP2) • Stromalysin • Gelatinase (MMP9) • Membrane type MMP • Produces biologically active molecules

31. SUBSTRATE OF TIMP MOLECULAR STRUCTURE OF THE MATRIX METTALLOPROTEINASES

32. MMP/TIMP EXPRESSION IN BREAST CANCER

33. ?!? MMP – TUMORPROGRESSION

34. METASTATIC CASCADE VEGFAngiogeninFGF Tumor cell Metastasis Angiogenesis Integrins cadherins Selectins CAM Primary tumor Angiogenesis Local invasion MMP/TIMP Cathepsin Plasminogen Adhesion Proteolysis Migration Adhesion Proteolysis Migration ECM Circulation Extravasation Intravasation spreading

35. MIGRATORY MECHANISMS IN TUMOR Small-cell lung cancer

36. FORMS OF MIGRATORY ADAPTATION

37. 2D –3D MIGRATIONS

38. STEPS OF 3D MIGRATION 1. Pseudopod protrusion 2. Formation of focal contact 3. Focal ECM proteolysis 4. Actomyosin contraction 5. Detachment

39. Cell-cell interactions visualized in tumorigenesis

40. METASTATIC CASCADE VEGFAngiogeninFGF Tumor cell Metastasis Angiogenesis Integrins cadherins Selectins CAM Primary tumor Angiogenesis Local invasion MMP/TIMP Cathepsin Plasminogen Adhesion Proteolysis Migration Adhesion Proteolysis Migration ECM AMF/gp78 Autotaxin HGF/c-MET Circulation Extravasation Intravasation spreading

41. HEMATOGENIC DISSEMINATION !! Tumor markers e.g. cytokeratin, mucin

42. ? EXTRAVASATION Attachment Migration

43. LOCALISATION OF THE METASTASIS

44. CHEMOKINES– TISSUE SPECIFIC LOCALISATION

46. ? motility adhesion