Alpha5 integrin dependent 3D attachment, appearance, and migration

(Not 2D)

(cell-derived)

(pliable)

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

S-1 + anti-beta4

T4-2 + anti-beta1

Physiological relevance of blocking beta1 function:Cells in suspension, treated with anti-B1 antibody, injected SQ:

Reduced tumor number and size

AIIB2 = anti B1Tyrphosin = anti EGFRmAB225 = anti EGFR

•Reduced b1 levels•Reduced EGFR levels and activity

Grown in Basement Membrane

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

Bissell & Radisky 2001

Studies with HMT-3522 cell lines grown in a reconstituted BM linkIntegrin signalling, adherens junction assembly, growth factorSignalling, and tissue structure differentiation

ECM/Microenvironment(Laminin)

Alpha6Beta4 integrin(Hemidesmosome targeting domain)

NF-kappaB translocationTo nucleus

Survival

Polarity/ Basement Membrane

•Beta4 integrin deletion (HTD)•Tailless beta4 integrin•Blocking Abs to alpha6 or beta4 integrin•Mutant IkappaBalpha•Proteosome inhibitor

Apoptosis

Drug Induced Apoptosis

Membrane Type I Matrix Metalloproteinase Usurps Tumor GrowthControl 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 2D substrates: cells have fully mature focal contacts.In 3D substrates: clustered integrins couple to less-completely assembled focalinterations and a predominantly cortical actin cytoskeleton; stress fiber formation is rare.

x

x

x

LymphocytesNeutrophils

The abrogation of beta1 integrin function can generate single-cell dissemination.

Insert movie with melanoma explants

HT-1080 and MDA-MB-231 cells remain migratory afterpericellular proteolysis: compensation by mesenchymal-amoeboid transition.

First insert videos from this paper

Now insert dermis/ SQ video

Green = not treatedRed = pretreated with PIs

•Elongated, spindle-shaped•Beta1 integrin dependent •ECM-degrading enzymescolocalize with integrins

•Reduced elongation •Increased morphodynamic flexibility•Loss of focal beta1 integrin and MMP clustering at interactions with ECM•Less adhesion to collagens (lower 1 & B3•More diffuse cortical actin distribution

LymphocytesNeutrophils

Role of Rho family of GTPasesIn Mesenchymal-Amoeboid Transition

Effect of Rho and ROCK inhibitors on cells with a rounded vs. elongatedPhenotype - invasion into matrigel

Only rounded cells show a decrease in migration when treated.

3D in vitro matrigel

Tumor Xenograft

When grown in 2D, activation of Rho signalling completely inhibitsMotility of BE cells.

Effect of Rho levels on cellular phenotype

Comparison of A375M2 motility in 3D vs. 2D•Motility on 2D substrate is not blocked by Y27632 treatment

Here insert movies 3 & 4

Green= beta1 integrin ezrin

BE

M2

M2+ C3

M2+Y27…

A375M2 BE

-/+ Ezrin Dominant Neg.

WM266.4 Melanoma (mixed morphology) ??

Rounded motility driven by Rho or ROCK does not require pericellular proteolysis