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Tumor Metastasis
1
Invasion
A
Separation of tumor cells from the primary tumor mass.
dop
TKS5
PKC
PI3K
odi
a
ROS
Migration towards blood vessels.
5
P
Fascin
Actin
Cortactin
P
NKC1.2
TKS5
PIP2
Entry of tumor cells into a blood vessel and their activation by
platelet-activating factor.
P
P
Dynamin
N-WASP
WIP
ECM
TKS4
MT1 MMP
MT1 MMP
MMP9
MMP2
5
Intravasation
A
B
D
C
Attachment of activated tumor cells to the vessel wall,
Extravasation
Endothelial cells begin to proliferate and migrate towards
the tumor mass.
A
B
D
C
Tumor Mass
Hypoxia
HIF-1
9
VEGF
STOP
Secondary
Growth
Li
Rep mitl
e
Pot licat ss
en ive
tia
l
m
fro trol
n
pe
ca Co
Es une
m
Hallmarks
of Cancer
I
ATP
Tumor-associated
Macrophages
11
PLCγ
KDR/Flt-1
PI3K
Cox-2
PIP2
e-NOS
Akt
PGI2
ECM
12
DAG+IP3
•NO
mTOR
ENDOTHELIAL CELL
Tum
o
Infl r-pr
am om
ma ot
tio
Inh
Cell Comb™ Scratch Assay was used to create cell monolayers with multiple
wounds applied two directions with the Cell Comb™ was viewed at 2X
magnification (top) and 10X (phase contrast image, bottom).
Evading
Apoptosis
Genome
Instability
d
an Mutation
n
In Vitro Vascular Permeability Assays
(96-well, 24-well & Imaging Formats)
(Cat. Nos. ECM642, ECM644 & 17-10398)
The endothelial cell lining inside the vasculature defines
a semi-permeable barrier between the blood and the
interstitial spaces of the body. In vitro vascular permeability
studies involve an intact, confluent endothelial cell
monolayer cultured on semi-permeable membranes or
clear chamber slides. Disruptions of the barrier integrity
can be measured by multi-well plate-based assays or visual
detection.
E
to
ty
ivi
n
sit tio
ls
en era gna
ns olif g Si
Pr itin
ib
g
in
Extravasation
VEGF signaling in angiogenesis
7
10
Self-su
ffic
Prolife iency
in
ra
Signa tion
ls
USE
In Vitro Vascular Permeability Imaging Assay was used to visualize changes in
microvascular hyperpermeability.
New blood supply is established.
on
Invasi is
sue
tas
Tis Metas
and
TO STUDY
The Cell Comb™ Scratch Assay has been optimized to apply
a high density field of scratches to create a high proportion
of migrating cells to quiescent monolayer cells, that
permits sensitive detection of the biochemical events in the
migrating cell population.
8
• A
s the vessel extends, the tissue is remolded around the vessel and the sprouting endothelial cells
roll up to form a blood vessel tube.
USE
Vascular Permeability Assay
The basic scratch assay creates physical wounds in a cell
monolayer for observing cell migration.
Circulation
ECM
• MMPs degrade the basement membrane and extracellular matrix. Adhesion molecules
(such as integrins) help the forming blood vessel to grow towards the tumor.
12
Scratch Assay
Cell Migration Cell Comb™ Scratch Assay
(Cat. No. 17-10191)
Wound
Healing
6
• A
ngiogenic growth factors bind to specific receptors located on the endothelial cells and
stimulate their proliferation and migration.
11
QCM™ ECMatrix™ Cell Invasion Assay, colorimetric was used to demonstrate lack
of invasion by non-invasive NIH3T3 cells (top panel), and active invasion by HT1080 cells (bottom panel).
TO STUDY
Sustained Angiogenesis
Hypoxia in a growing tumor mass results in the production of
HIF, which stimulates the production and release of growth
factors and proteolytic enzymes.
D
B
Tumor cell divides; this is the beginning of a secondary
growth.
10
The most widely published method for measuring
extracellular matrix degradation due to “invadopodia” or
“podosomes” involves plating cells over a culture surface
coated with a thin layer of fluorescently labeled matrix, and
visualizing regions where the cell has degraded the matrix to
create an area devoid of fluorescence.
QCM™ Gelatin Invadopodia Assay (Green) was used to study cell migration across
a fluorescently-conjugated matrix. Dark spots are evidence of invadopodia.
umor cells reach distant sites via circulation in the
T
blood vessel.
Tumor-associated macrophages secrete growth factors.
Cell Migration QCM™ Gelatin Invadopodia Assays (Green
& Red) (Cat. Nos. ECM670 & ECM671)
Invasion
Intravasation
MMP9
Invadopodia formation and tissue invasion
9
USE
4
glycosylation of integrin, CD44, and cadherin, and exit from
blood vessel.
8
ECM Degradation
ECM
GRB2
MMP2
Increased expression of P-selectin and the formation of tumor
cell-platelet aggregates.
3
C
TO STUDY
USE
Primary Tumor
Extravasation
7
Boyden Chamber
The classic Boyden Chamber system places cells in a
extracellular matrix protein-coated porous membrane insert
over a chemoattractant-containing well. Invading cells
migrate through the pores, are stained and measured.
PAK
Cofilin
Circulation
6
ERK
SRC
TKS5
Intravasation
4
PKC
P
Invasion of tumor cells through surrounding tissue.
• Proteolytic destruction of the basement membrane and extracellular matrix (ECM).
3
A
D
Cell Migration QCM™ ECMatrix Cell Invasion Assay,
Cell Invasion Colorimetric (Cat. No. ECM550)
P
Inva
2
AFAP110
Tissue Invasion
2
C
Techniques & Assays
TO STUDY
Key Steps in Tumor Metastasis
1
B
Tumor-associated
Macrophages
Tube Formation Assay
TO STUDY
USE
Angiogenesis
In Vitro Angiogenesis Assay
(Cat. No. ECM625)
Fibrin In Vitro Angiogenesis Assay
(Cat. No. ECM630)
Millicell® μ-Angiogenesis Inhibition
Assay Kit (Cat. No. MMA125)
Millicell® μ-Angiogenesis Activation
Assay Kit (Cat. No. MMA130)
The tube formation assay has been typically employed to
demonstrate the angiogenic activity of vascular endothelial
cells in vitro. This assay can serve as a powerful method
to screen the vascular potential of a variety of cell types
including vascular cells, tumor cells, as well as other cells.
For a complete listing of
Cancer Products, visit:
www.emdmillipore.com/cancer
Im
d
taine
Sus genesis
gio
An
Me
t
a
Reprog bolic
ram
min
g
Fibrin In Vitro Angiogenesis Assay was used to study tube formation in HUVEC
(32x magnification).
EMD Millipore is a division of
Merck KGaA, Darmstadt, Germany
EMD Millipore, QCM, Cell Comb, and the M mark are trademarks and Millicell is a registered trademark of Merck KGaA, Darmstadt, Germany.
Lit. No. PS5747EN00 03/2013 Job No. BS-GEN-13-07931 Printed in the U.S.A. ©2013 EMD Millipore Corporation, Billerica, MA, USA.
Angiogenesis
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PKC Activation
p70S6K
Survival, Vascular Permeability, Migration, Proliferation
www.emdmillipore.com