Chidambaram Gunanathan Raanan Margalit Inbal Eti Biton Ady Yosepovich David Milstein Hadassa Degani In Vitro and In Vivo Molecular Imaging of the ID: 926601
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Slide1
Methods
Results
Adi
Pais
, Chidambaram
Gunanathan
,
Raanan
Margalit
,
Inbal
Eti
Biton
,
Ady
Yosepovich
,
David Milstein, Hadassa Degani
In Vitro and In Vivo Molecular Imaging of the Estrogen Receptor using Novel ER-Targeted MRI Contrast Agents
Conclusions
We thank Tamar
Kreizman, Drs Dalia Seger & Edna Furman-Haran, Minjun Li & Prof. Joel Sussman for contributing to the success of this project This work was supported by Israel Science Foundation grant 235/08 and National Institutes of Health Grant CA 42238, and by Ernst and Anni DeutschH. Degani is the incumbent of the Fred and Andrea Fallek Professorial Chair for Breast Cancer Research
Acknowledgement
Introduction
Estrogen
receptor
alpha
(ER) is a member of the nuclear receptor family and serves as a key regulator in the female reproductive organs, including the mammary gland. ER is over expressed in ~70% of breast cancers and is a
prominent prognostic
marker for breast cancer and serves to select patients for hormonal therapy. we describe a novel method for Imaging ER in vivo using targeted ER contrast agents.
EPTA-Gd
PTA-Gd
TPTA-Gd
17β-estradiol
tamoxifen
Anatomical image
ER+
Cell implantation:
mammary fat pad
,
Left:
WT MDA-MB-231 : ER-Right: Tet Inducible ER transfected MDA-MB-231 : ER+
ER induction: 0.2 mg/ml tetracycline in drinking water
Day 14
Days
21-35
Ovariectomy
Day 7
DCE-MRI
Day 0
ER-
ER
Immunostaining
Orthotopic
Human Breast Cancer
in Female SCID
In vivo MRI protocol
9.4T Biospec AVANCE II spectrometer (Bruker)T2W anatomical-RARE SET1W-3DGE, FLASH, TE/TR/flip angle 2.5ms/15ms/40º Acquisition time 0.75-1.5 minSpatial resolution 0.156x0.156x1.2 mm3IV bolus injection:
EPTA-Gd 0.03-0.075 mmol/kgTPTA-Gd 0.075 mmol/kg PTA-Gd 0.15 mmol/kg
post contrast
coronal image
EPTA-
Gd
TPTA-Gd
PTA-Gd
Blood
Kidney
m
2
=0.03 min
-1
m2=0.003, min-1
m2=0.04, min-1
Fast clearance
Fast clearance
Slow clearance
Cb(t)= Dose(a1e-m1t + a2e-m2t )
Gd-complexBinding Affinity to ERKi (μM)T1 Relaxivity 9.4Tr1 (mM·s)-1EPTA-Gd0. 97± 0.076.8±0.7TPTA-Gd0.13 ± 0.014.7±0.1PTA-Gd-3.0±0.1
Cellular Studies: Hormonal-Induced Activities
mild-agonist
agonist
ER
cMyc
ER
cMyc
MCF7
EPTA-
Gd
TPTA-Gd
T47D cell growth
MCF7 cell growth
MCF7
r1
app
(ER+)=28.5±0.1, n=2
r1
app
(ER-)=19.6
*
Pval
<0.035, paired t-test, n=9
perfused cells
Blood
0.5 cm
DCE-MRI: enhancement profiles
in ER+ and ER- tumors
EPTA-
Gd
TPTA-
Gd
PTA-Gd
ER-
ER+
ER-
ER+
20’ post
ER+
ER-
Principal Component Analysis (PCA)
Eyal
E. et al. JMRI, 2009, 30:989-998
Eigenvalues
1
st
Eigenvector
EPTA-
Gd
TPTA-Gd
PTA-Gd
0.5 cm
0
1
3
2
EPTA-Gd
TPTA-Gd
PTA-
Gd
1
st
eigenvector projection coefficients
N=9
N=4
N=4
*
N=9
N=4
N=4
*
DCE-MRI
Statistics in ER+ and ER- tumors
PCA analysis
p<0.008
Enhancement analysis
20 min post contrast
p<0.05
EPTA
-
Gd
in Solution:
Fast water exchange, efficient paramagnetic r1
relaxivity
and
micromolar
affinity to ER.
in cell cultures (in vitro
)
Binds ER in cells; induces E2-like activity.
E
fficient
ER targeted T1 contrast agent in ER+ vs. ER- cells, 5-fold increased r1
relaxivity in cells upon binding to ER.In orthotopic tumors (in vivo)Efficient ER targeted T1 contrast agent in ER+ tumors.
EPTA-Gd can serve as a targeted probe for molecular imaging of ER in orthotopic breast cancer tumors implanted in miceTPTA-Gd - Binds with high affinity to muscle in vivo and cannot serve as a probe of ER in breast cancers.
Structure
x-ray crystallography of EPTA-
Eu
bound to ER ligand binding domain
Binding site structure
Purple 17
β
- estradiol
Green EPTA
Blue
Gd
ER-LBD dimer bound to EPTA-
Eu
Li MJ, et al Med Chem. 2011, 54:3575-80.
EPTA-Gd
TPTA-Gd
PTA-
Gd
17
β
-estradiol
Tamoxifen
Gunanathan C, et al, Bioconjug Chem. 2007, 18: 1361-5
r1
relaxivity
of EPTA-
Gd