1. No category

VEGF-targeting by Aflibercept directly
attenuates the migration of colorectal
cancer cells
Anaïs Bouygues, Paul Mésange, Meriam Ayadi, Virginie Poindessous,
Thierry André, Aimery de Gramont and Annette K. Larsen
Laboratory of Cancer Biology and Therapeutics
Centre de Recherche Saint-Antoine
INSERM U938 and Université Pierre et Marie Curie
Paris, FRANCE
INTRODUCTION (1)
Ø  Colorectal cancer cells and tumors express VEGF
and VEGF receptor 1 (Flt-1) and, to a lesser
degree, VEGF receptor 2 (Flk-1/KDR) thereby
promoting autocrine VEGF signaling
Ø  Tumor-associated VEGF-signaling stimulates cell
migration and enhance survival after environmental
stress including hypoxia and 5-FU
INTRODUCTION (2)
Ø  VEGFR1 is activated by VEGF-A, VEGF-B and PlGF
Ø  Aflibercept is a recombinant protein that binds and
neutralizes VEGF-A, VEGF-B and PlGF
Ø  Aflibercept is approved for treatment of metastatic CRC
OBJECTIVES
Ø  Determine the influence of Aflibercept on the
migration of CRC cells
Ø  Establish which VEGF ligands and receptors
influence cellular motility
Ø  Explore if VEGF signaling is upregulated in CRC
cells with acquired 5-FU resistance and if yes, if this
can be attenuated by Aflibercept
Migration of human CRC cells
10000
Cell number
8000
Normoxia
Hypoxia
6000
4000
2000
0
Boyden chamber migration assay
Aflibercept attenuates CRC cell migration
** p < 0.01
*** p < 0.001
NORMOXIA
10000
Cell number
Cell number
8000
6000
4000
***
*** *** ***
2000
0
10000
**
Control
Aflibercept
8000
HYPOXIA
**
** ***
***
Control
**
Aflibercept
6000
***
***
4000
***
2000
0
*** *** ***
**
***
***
Influence of VEGFR2 on CRC cell migration
PIGF ** p < 0.01
*** p < 0.001
VEGF-­‐A VEGF-­‐B VEGF-­‐E HCT-116 cellular model
P P Control
VEGF-A
P P VEGFR-­‐
VEGF-E
1 P
P
VEGF
R-­‐2 NORMOXIA
6000
HYPOXIA
6000
***
***
3000
2000
1000
***
***
5000
Cell number
Cell number
5000
4000
P
P
4000
3000
2000
1000
0
Control
Anti-VEGFR1
Anti-VEGFR2
0
Control
Anti-VEGFR1
Anti-VEGFR2
Influence of VEGFR1 on CRC cell migration
PIGF ** p < 0.01
*** p < 0.001
VEGF-­‐A HCT-116 cellular model
P P Control
VEGF-B
P P VEGFR-­‐
PlGF
VEGF-­‐B 1 VEGF-­‐E P
P
P
P
VEGF
R-­‐2 NORMOXIA
HYPOXIA
6000
6000
4000
5000
**
***
Cell number
Cell number
5000
3000
2000
4000
3000
2000
1000
1000
0
0
Control
anti-VEGFR1
anti-VEGFR2
***
***
Control
anti-VEGFR1
anti-VEGFR2
Influence of Aflibercept on 5-FU toxicity
HCT-116
HCT-116 + Aflibercept
HCT-116/5-FU
HCT-116/5-FU + Aflibercept
MTT Viability assay
NORMOXIA
HYPOXIA
100
Cell viability
(% of control)
Cell viability
(% of control)
100
50
0
0,01
0,1
1
10
100
Concentration 5-FU (µM)
1000
50
0
0,01
0,1
1
10
100
Concentration 5-FU (µM)
1000
Acquired 5-FU resistance is accompanied by
increased CRC cell migration
*** p < 0.001
HCT-116
Cell number
5000
4000
3000
***
HCT-116/5-FU
***
2000
1000
0
Normoxia
Hypoxia
Boyden chamber migration assay
Aflibercept attenuates the migration of parental and
5-FU resistant cells under normoxia and hypoxia
Untreated
*** p < 0.001
5000
5000
4000
4000
***
3000
2000
1000
Aflibercept
HYPOXIA
***
Cell number
Cell number
NORMOXIA
***
3000
2000
***
1000
0
0
HCT-116
HCT-116/5-FU
HCT-116
HCT-116/5-FU
Boyden chamber migration assay
CONCLUSIONS (1)
Ø  The migration of CRC cells is highly cell type dependent
Ø  Aflibercept decreases the migration of CRC cells under
both normoxia and hypoxia
Ø  Migration is stimulated by VEGF-A, VEGF-B and PlGF, but
not VEGF-E, and is mediated by VEGFR1
CONCLUSIONS (2)
Ø  Aflibercept alone had no influence on the viability of HCT-116
parental and 5-FU resistant cells under normoxia or hypoxia
Ø  Aflibercept could attenuate the resistance of HCT-116/5-FU
cells to 5-FU under hypoxia
Ø  5-FU resistance was accompanied by increased migration
that could be attenuated by Aflibercept under both normoxia
and hypoxia
Thank you for your attention