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Alpha5 integrin dependent 3D attachment, appearance, and migration

Alpha5 integrin dependent 3D attachment, appearance, and migration. (Not 2D). (pliable). (cell-derived). Some early studies in 3-Dimensional Substrates: Bissell lab. HMT-3522 Breast Epithelial cells S-1: nonmalignant T4-2: tumorigenic Grown on plastic: 2 cell lines appear similar

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Alpha5 integrin dependent 3D attachment, appearance, and migration

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  1. Alpha5 integrin dependent 3D attachment, appearance, and migration (Not 2D) (pliable) (cell-derived)

  2. Some early studies in 3-Dimensional Substrates: Bissell lab • HMT-3522 Breast Epithelial cells • S-1: nonmalignant • T4-2: tumorigenic • Grown on plastic: 2 cell lines appear similar • Grown in reconstituted basement membrane (rBM): • S-1 cells form polarized, acinar structures in growth arrest • T4-2 cells form nonpolarized, proliferating amorphous structures

  3. T4-2 + anti-beta1 S-1 + anti-beta4

  4. Physiological relevance of blocking beta1 function: Cells in suspension, treated with anti-B1 antibody, injected SQ: Reduced tumor number and size

  5. AIIB2 = anti B1 Tyrphosin = anti EGFR mAB225 = anti EGFR • Reduced b1 levels • Reduced EGFR • levels and activity Grown in Basement Membrane

  6. Active FAK, ILK and MAP Kinase downstream of beta1 and EGFR

  7. Studies with HMT-3522 cell lines grown in a reconstituted BM link Integrin signalling, adherens junction assembly, growth factor Signalling, and tissue structure differentiation Bissell & Radisky 2001

  8. Drug Induced Apoptosis ECM/Microenvironment (Laminin) Alpha6Beta4 integrin (Hemidesmosome targeting domain) • Beta4 integrin • deletion (HTD) • Tailless beta4 integrin • Blocking Abs to alpha6 • or beta4 integrin • Mutant IkappaBalpha • Proteosome inhibitor Polarity/ Basement Membrane NF-kappaB translocation To nucleus Survival Apoptosis

  9. Membrane Type I Matrix Metalloproteinase Usurps Tumor Growth Control Imposed by the Three-Dimensional Extracellular Matrix Works in Collagen and Fibrin gels, not Matrigel; also in vivo

  10. (uncleavable)

  11. The Five Stages of Cell Migration

  12. On 2D substrates: cells have fully mature focal contacts. In 3D substrates: clustered integrins couple to less-completely assembled focal interations and a predominantly cortical actin cytoskeleton; stress fiber formation is rare.

  13. x x x

  14. Lymphocytes Neutrophils

  15. The abrogation of beta1 integrin function can generate single-cell dissemination. Insert movie with melanoma explants

  16. Green = not treated Red = pretreated with PIs HT-1080 and MDA-MB-231 cells remain migratory after pericellular proteolysis: compensation by mesenchymal-amoeboid transition. First insert videos from this paper Now insert dermis/ SQ video

  17. Reduced elongation • Increased morphodynamic flexibility • Loss of focal beta1 integrin and MMP • clustering at interactions with ECM • Less adhesion to collagens (lower B1 & B3 • More diffuse cortical actin distribution • Elongated, spindle-shaped • Beta1 integrin dependent • ECM-degrading enzymes • colocalize with integrins Lymphocytes Neutrophils

  18. Role of Rho family of GTPases In Mesenchymal-Amoeboid Transition

  19. Effect of Rho and ROCK inhibitors on cells with a rounded vs. elongated Phenotype - invasion into matrigel Only rounded cells show a decrease in migration when treated.

  20. 3D in vitro matrigel Tumor Xenograft

  21. Effect of Rho levels on cellular phenotype When grown in 2D, activation of Rho signalling completely inhibits Motility of BE cells.

  22. Comparison of A375M2 motility in 3D vs. 2D • Motility on 2D substrate is not blocked by Y27632 treatment Here insert movies 3 & 4

  23. Green= beta1 integrin ezrin BE M2 M2+ C3 M2+ Y27… A375M2 BE -/+ Ezrin Dominant Neg.

  24. Rounded motility driven by Rho or ROCK does not require pericellular proteolysis WM266.4 Melanoma (mixed morphology) ??

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