1. Special approaches of tumor biology and chemotaxis Orsolya Láng 2012.

2. TUMOR CELLS AND MIGRATION METASTASISPRIMARY TUMOR Angiogenezis Adhesion

3. CELL and CELL CYCLE

4. Growth factors Adhesion molecules Chemokines Regulatory proteins Apoptosis

5. TUMOR CELL

6. CELL KINETICS

7. Doubling time of the tumor volume (Td) 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 / Td Tpd= *Ts/Li Tpd: Potential tumor volume doubling time Td: tumor volume doubling time Lymphoma 48 h Lung cancer 108 h Usually 15-125 h Lymphoma 4 weeks Colon adenoma 90 weeks Usually 18-200 days

8. Volume of the tumor tissue ~10 division =*1000 cell number increase (2 10 =1024) ~20 division= 10 6 cells = 1 mg= 1 mm 3 ~30 division= 10 9 cells = 1 g= 1 cm 3 ~40 division= 10 12 cells = 1 kg 1 tumor cell ~30-33,25 division =1-10 cm 3 Time of the clinical symptomes / diagnosis ~27 division = 0,1cm 3 Earliest time of diagnosis ~40 division = 10 12 cell Fatal

9. BIOLOGY OF THE TUMORPROGRESSION

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

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 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  OEDEMA, decresed blood flow

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. Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation Extravasationcirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE

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

17. Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation Extravasationcirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE

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 Ca 2+ dependent anchorage It binds Sialyl-Le x carbohydrates „ROLLING” ! Tumor cells express increased amount of sialil-Le x or -Le a

20. INTEGRINS Transmembrane receptors Form cell-ECM interaction 8 , 14  subunites ~20 heterodimer Ca 2+, Mg 2+ 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   D G R

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

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

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 Ca 2+ 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. Increases N-Cadherin B-Catenin SrC Ras Ihibits E-Cadherin aE-Catenin cMET FGFR PTEN Adhesion molecules Signal pathways Factors influencing the metastatic potential of the melanoma cells

28. Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation ExtravasationCirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE Integrins cadherins Selectins CAM

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

30. MATRIX METTALLOPROTEINASES (MMP) Zn 2+ dependent endopeptidase (MMP28) ECM degradation – tissue remodelling Interstitial collagenase (MMP2) Stromalysin Gelatinase (MMP9) Membrane type MMP Produces biologically active molecules

31. MOLECULAR STRUCTURE OF THE MATRIX METTALLOPROTEINASES SUBSTRATE OF TIMP

32. MMP/TIMP EXPRESSION IN BREAST CANCER

33. MMP – TUMORPROGRESSION ?!?

34. Angiogenesis Local invasion ECM Adhesion Proteolysis Migration Intravasation ExtravasationCirculation Metastasis Tumor cell Primary tumor Adhesion Proteolysis Migration Angiogenesis VEGF Angiogenin FGF spreading METASTATIC CASCADE Integrins cadherins Selectins CAM MMP/TIMP Cathepsin Plasminogen

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

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

42. EXTRAVASATION ? Attachment Migration

43. LOCALISATION OF THE METASTASIS

44. CHEMOKINES – TISSUE SPECIFIC LOCALISATION

46. adhesion motility ?