1. 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 Deutsch H. 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-GdPTA-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 Days21-35 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 SE T1W-3DGE, FLASH, TE/TR/flip angle 2.5ms/15ms/40º Acquisition time 0.75-1.5 min Spatial resolution 0.156x0.156x1.2 mm 3 IV bolus injection: EPTA-Gd 0.03-0.075 mmol/kg TPTA-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 m 2= 0.003, min -1 m 2 =0.04, min -1 Fast clearance Slow clearance C b (t)= Dose(a 1 e -m 1 t + a 2 e -m 2 t ) Gd-complex Binding Affinity to ER Ki (μM) T1 Relaxivity 9.4T r1 (mM·s) -1 EPTA-Gd0. 97± 0.076.8±0.7 TPTA-Gd0.13 ± 0.014.7±0.1 PTA-Gd-3.0±0.1 Cellular Studies: Hormonal-Induced Activities mild-agonist agonist ERcMycERcMyc MCF 7 EPTA-GdTPTA-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-GdTPTA-GdPTA-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-GdTPTA-GdPTA-Gd 0.5 cm 0 1 3 2 EPTA-GdTPTA-GdPTA-Gd 1 st eigenvector projection coefficients N=9N=4 * N=9N=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. –Efficient 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 mice TPTA-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 17β- estradiol Green EPTA Blue Gd ER-LBD dimer bound to EPTA-Eu Li MJ, et al Med Chem. 2011, 54:3575-80. EPTA-GdTPTA-Gd PTA-Gd 17β-estradiol Tamoxifen Gunanathan C, et al, Bioconjug Chem. 2007, 18: 1361-5 r1 relaxivity of EPTA-Gd