ELLA

SEDSSAY

ERVICES

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AngiogenesisAngiogenesis, the formation of new blood vessels from pre-existing ones, is a physiological process duringgrowth and development. Beyond that angiogenesis is mandatory for tumor growth and is involved in otherpathological disorders (e.g. psoriasis, macular degeneration). The angiogenic cascade can be divided in at leastfive stages: matrix degradation, endothelial cell (EC) migration, EC proliferation, lumen formation and vesselmaturation. The cellular angiogenesis assay mimics the in vivo situation and the first stages of the angiogeniccascade are involved. Despite its complexity the cellular angiogenesis assay demonstrates a high degree ofreproducibility and robustness.

Assay Procedure

Study Example

Cellular Angiogenesis Assay(Spheroid-Based)

Figure 1: Assay procedure. Human EC spheroids (fibroblasts and smooth muscle cell spheroids may also be used) aremixed in a collagen or fibrin matrix and transferred into 24 well plates. After polymerization the test compounds and/orstimulation factors (VEGF-A, FGF-2, Deferoxamine, HB-EGF, etc. ) are added. The assay is incubated at 37 °C for 24 h andsubsequently fixed. EC sprouting is quantified by measuring the sprout length and the cumulative sprout length (CSL) isdetermined.

Figure 3: Study example.The angiogenesis inhibitors PTK787 andSunitinib were tested for inhibition of ECsprouting (VEGF-A and Deferoxamineinduced) and fibroblast scattering. Theselective inhibitor PTK787 is active on ECsprouting but does not affect fibroblastscattering. In contrast Sunitinib which has abroader kinase inhibition profile thanPTK787 inhibits both EC sprouting andfibroblast scattering.

Assay Features

Figure 2: Assay features. Unstimulated EC spheroids show limited sprouting into the surrounding collagen matrix (left). ECsprouting can be induced by pro-angiogenic factors like VEGF-A (right). The number and length of the sprouts corresponds tothe angiogenic activity. Pro-angiogenic compounds induce 3D EC sprouting originating from the spheroid whereas angiogenicinhibitors prevent sprout formation induced by pro-angiogenic factors (e.g. VEGF-A, FGF-2).

-9 -8 -7 -6

0

50

100

IC50 [M] =Hill Slope =

R2 =-1.22.6e-007

0.90

PTK787 [log M]

EC s

prou

ting

[% o

f Def

erox

amin

e co

ntro

l]

-9 -8 -7 -6

0

50

100 -0.829.9e-008IC50 [M] =

Hill Slope =R2 = 0.92

PTK787 [log M]

EC s

prou

ting

[% o

f VEG

F-A

cont

rol]

-8 -7 -6

0

50

100

-2.31.3e-006IC50 [M] =

Hill Slope =R2 = 0.78

PTK787 [log M]

Fibr

obla

st s

catt

erin

g[%

of c

ontr

ol]

-8 -7 -6 -5

0

50

100

-3.51.0e-006IC50 [M] =

Hill Slope =R2 = 0.88

Sunitinib [log M]

EC s

prou

ting

[% o

f Def

erox

amin

e co

ntro

l]

-8 -7 -6 -5

0

50

100

-1.41.6e-007IC50 [M] =

Hill Slope =R2 = 0.79

Sunitinib [log M]

EC s

prou

ting

[% o

f VEG

F-A

cont

rol]

-8 -7 -6 -5

0

50

100

-0.766.4e-007IC50 [M] =

Hill Slope =R2 = 0.95

Sunitinib [log M]

Fibr

obla

st s

catt

erin

g[%

of c

ontr

ol]

EC sprouting

VEGF-A stimulated Deferoxamine stimulated

Fibroblast scattering

Sunitinib

PTK787

-9 -8 -7 -6

0

50

100

IC50 [M] =Hill Slope =

R2 =-1.22.6e-007

0.90

PTK787 [log M]

EC s

prou

ting

[% o

f Def

erox

amin

e co

ntro

l]

-9 -8 -7 -6

0

50

100 -0.829.9e-008IC50 [M] =

Hill Slope =R2 = 0.92

PTK787 [log M]

EC s

prou

ting

[% o

f VEG

F-A

cont

rol]

-8 -7 -6

0

50

100

-2.31.3e-006IC50 [M] =

Hill Slope =R2 = 0.78

PTK787 [log M]

Fibr

obla

st s

catt

erin

g[%

of c

ontr

ol]

-8 -7 -6 -5

0

50

100

-3.51.0e-006IC50 [M] =

Hill Slope =R2 = 0.88

Sunitinib [log M]

EC s

prou

ting

[% o

f Def

erox

amin

e co

ntro

l]

-8 -7 -6 -5

0

50

100

-1.41.6e-007IC50 [M] =

Hill Slope =R2 = 0.79

Sunitinib [log M]

EC s

prou

ting

[% o

f VEG

F-A

cont

rol]

-8 -7 -6 -5

0

50

100

-0.766.4e-007IC50 [M] =

Hill Slope =R2 = 0.95

Sunitinib [log M]

Fibr

obla

st s

catt

erin

g[%

of c

ontr

ol]

EC sprouting

VEGF-A stimulated Deferoxamine stimulated

Fibroblast scattering

Sunitinib

PTK787

spheroid formationplacing spheroids

in matrixsprout length quantification

-9 -8 -7 -6

0

50

100 -0.829.9e-008IC50 [M] =

Hill Slope =R2 = 0.92

PTK787 [log M]

EC s

prou

ting

[% o

f VEG

F-A

cont

rol]

-9 -8 -7 -6

0

50

100 -0.829.9e-008IC50 [M] =

Hill Slope =R2 = 0.92

PTK787 [log M]

EC s

prou

ting

[% o

f VEG

F-A

cont

rol]