Alpha 5 integrin dependent 3 D attachment appearance

Alpha 5 integrin dependent 3 D attachment, appearance, and migration (Not 2 D) (pliable) (cell-derived)

Some early studies in 3 -Dimensional Substrates: Bissell lab • HMT-3522 Breast Epithelial cells – S-1: nonmalignant – T 4 -2: tumorigenic • Grown on plastic: 2 cell lines appear similar • Grown in reconstituted basement membrane (r. BM): – S-1 cells form polarized, acinar structures in growth arrest – T 4 -2 cells form nonpolarized, proliferating amorphous structures

S-1 + anti-beta 4 T 4 -2 + anti-beta 1

Physiological relevance of blocking beta 1 function: Cells in suspension, treated with anti-B 1 antibody, injected SQ: Reduced tumor number and size

AIIB 2 = anti B 1 Tyrphosin = anti EGFR m. AB 225 = anti EGFR • Reduced b 1 levels • Reduced EGFR levels and activity Grown in Basement Membrane

Active FAK, ILK and MAP Kinase downstream of beta 1 and EGFR

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

Drug Induced Apoptosis ECM/Microenvironment (Laminin) Alpha 6 Beta 4 integrin (Hemidesmosome targeting domain) • Beta 4 integrin deletion (HTD) Polarity/ Basement Membrane • Tailless beta 4 integrin • Blocking Abs to alpha 6 NF-kappa. B translocation or beta 4 integrin • Mutant Ikappa. Balpha To nucleus • Proteosome inhibitor Apoptosis Survival

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

(uncleavable)

The Five Stages of Cell Migration

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

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Lymphocytes Neutrophils

The abrogation of beta 1 integrin function can generate single-cell dissemination. Insert movie with melanoma explants

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 Green = not treated Red = pretreated with PIs

• Elongated, spindle-shaped • Beta 1 integrin dependent • ECM-degrading enzymes colocalize with integrins • Reduced elongation • Increased morphodynamic flexibility • Loss of focal beta 1 integrin and MMP clustering at interactions with ECM • Less adhesion to collagens (lower B 1 & B 3 • More diffuse cortical actin distribution Lymphocytes Neutrophils

Role of Rho family of GTPases In Mesenchymal-Amoeboid Transition

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.

3 D in vitro matrigel Tumor Xenograft

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

Comparison of A 375 M 2 motility in 3 D vs. 2 D • Motility on 2 D substrate is not blocked by Y 27632 treatment Here insert movies 3 & 4

Green= beta 1 integrin BE ezrin A 375 M 2 BE M 2+ C 3 M 2+ Y 27… -/+ Ezrin Dominant Neg.

Rounded motility driven by Rho or ROCK does not require pericellular proteolysis WM 266. 4 Melanoma (mixed morphology) ? ?