Mechanisms of Cell Invasion

1. Mechanisms of Cell Invasion • Cancer Biology Course • Feb 26, 2008

2. Outline • Background Material on Invasion & Metastasis • Human tumor histopathology • Culture Models • Molecular Mechanisms • Stromal / Tumor Cell Interactions • Orthotopic Model for Skin Cancer • Sahai paper • Invadopodia Meeting

3. Reading Material • Cell Biology Text - Molecular Cell Biology, Lodish 6th Edition: Chaps 17, 18 & 19. • Cancer Biology Text - The Biology of Cancer, Weinberg, 2007. Chap 14. • Original Article for Discussion • Gaggioli et al. 2007. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol. 9:1392-1400.

4. Online Material • Bob Weinberg / MIT. “How Cancer Begins” - Streaming Podcast Lecture • http://mitworld.mit.edu/play/149/ • Cell Migration Consortium • Lists of reagents, background info, literature • http://www.cellmigration.org/resource/ • Invadopodia/Podosome Consortium - New • http://www.invadosomes.org/

5. Invasion and Metastasis

6. Adenoma in Intestinal Epithelium

7. Adenoma in Intestinal Epithelium

8. Invasion

9. Breast Cancer Slide Link Link - Click Here

10. Metastasis via Lymphatics Lymphatic Drainage of the Breast

11. Metastasis via Lymphatics Identification of “Sentinel” Lymph Node with Dye

12. Metastasis via Lymphatics H&E Staining: Breast Ca in Lymph Node

13. Metastasis via Lymphatics Keratin Staining: Breast Ca in Lymph Node

14. Culture Models of Invasion • Simple Criteria for Transformation • Loss of contact inhibition of growth • Growth in soft agar • Tumors in nude mice • Cells on Fluorescent Matrix • Cells in 3-D Matrix • Orthotopic Models - Sahai paper

15. Cells on Fluorescent Matrix Actin Matrix Smooth Muscle Cell Matrix Actin Macrophage

16. “Invadopodia”

17. MMP Degradation of Matrix

18. Monolayer vs Gel

19. Cells in 3D Culture

20. Breast Cancer Cells MCF-10Awith ErbB2 Inducible Dimer

21. Induced Receptor Dimerization w/ AP1510

22. Screening cDNAs associated w/breast cancer

23. Screening cDNAs associated w/breast cancer

24. Screening cDNAs associated w/breast cancer

25. Expression Profiles for Invasive Cells

26. Expression Profiles for Invasive Cells

27. Lamellipodia induced by HER2/Neu

28. Growth Factor Pathways & Oncogenes • Receptor Tyr Kinases • EGFR, PDGFR, VEGFR • Cytoplasmic Tyr Kinases • Src, Syk-ZAP70, Abl • Small G-proteins • Ras

29. Connections from Integrins & Srcto Cell Migration • Integrins activate Src & FAK • Tyrosine kinases • Focal contacts / adhesions • Paxillin & Vinculin

30. Ras-like Protein Signaling

31. Connections from Integrins & Srcto Cell Migration • Integrins activate Src & FAK • Tyrosine kinases • Focal contacts / adhesions • Paxillin & Vinculin • Cortactin - Src Substrate • Stabilizes Arp2/3 Networks • Signaling Scaffold

32. Other Rho’s: RhoC and Metastasis

33. Connections from Ras-like Proteinsto Cell Migration • Rac - Lamellipodia • WAVE family of Arp2/3 activators • Cdc42 • WASp family of Arp2/3 activators • RhoA • Myosin-II Activation for Contraction • Others • Formins - mDia • CARMIL?

34. Tumor / Stroma:Paracrine Scenario a: Tumor cells activate surrounding stromal cells, i.e., fibroblasts, macrophages, and blood vessels. b: Fibroblasts respond by undergoing smooth muscle differentiation, blood vessels by angiogenesis, and both myofibroblasts and macrophages secrete additional cytoklnes. c: Modulated extracellular matrix production derives primarily from activated myofibroblasts, recruited vascular cells, and leaky blood vessels. d: Tumor cells, myofibroblasts, and activated macrophages cooperate in proteolytic action at invasive front. e: In the tumor center, angiogenesis is inhibited, tissue undergoes extensive fibrosis, and tumor cells may become necrotic.

35. Stromal Changes in Breast Cancer:Fibroblasts

36. Macrophages in Invasion and Intravasation

37. Macrophages Degrade Matrix

38. Sahai paper - Orthotopic model of cancer cell invasion • Gaggioli et al. 2007. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol. 9:1392-1400.

39. Confocal Microscopy Pinhole Focal Plane

40. Two-Photon Microscopy Thin Region of Illumination Two Photons Absorbed

41. Cells Crawling on Collagen Fibers Time-lapsed movie of high metastatic CFP-labeled cells (white) and low metastatic GFP-labeled cells (green) crawling on collagen fibers (purple) in vivo.

42. Lamellipodia / Ruffles in situ Time-lapsed movie of a CFP labeled cell with a ruffling lamella on a field of GFP labeled cells in a living tumor.

43. Ameboid Movements of Cells High metastatic CFP-labeled cells (white) and low- metastatic GFP labeled cells (green) crawling on collagen fibers (purple).

44. Orthotopic Model for Skin Cancer Invasion • SCC12 - Squamous cell carcinoma cells • Stromal fibroblasts - from cancers • Matrix - dense fibrillar collagen w/ laminin & collagen IV

45. Epidermal Cancer Cells Figure 1. Fibroblasts promote and lead collective SCC invasion. (a) H&E-stained sections of SCC12 cells cultured in an organotypic system in the absence or presence of stromal fibroblasts. Mesench Cancer Cells Untransf Keratinocytes

46. Movie 1 SCC cells invade as collective strands. Sequential confocal sections of SCC12 and A431 cells invading in co-cultures with tumour fibroblasts (stained with phalloidin): note how all cells are incorporated into invading chains.

47. Invasion Proportional to Fibroblast #

48. Cells, not Conditioned Media

49. Contact between Cells Needed

50. Fibroblasts lead SCC cells (Upside-down images) Figure 1. Fibroblasts promote and lead collective SCC invasion. (f) Representative images of SCC12 cells (green) and stromal fibroblasts (red) invading into naïve matrix.