Estrogen Receptor-Targeted Contrast Agents for Molecular Magnetic Resonance Imaging of Breast Cancer Hormonal Status

Abstract

The estrogen receptor (ER) α is overexpressed in most breast cancers, and its level serves as a major prognostic factor. It is important to develop quantitative molecular imaging methods that specifically detect ER in vivo and assess its function throughout the entire primary breast cancer and in metastatic breast cancer lesions. This study presents the biochemical and molecular features, as well as the magnetic resonance imaging (MRI) effects of two novel ER-targeted contrast agents (CAs), based on pyridine-tetra-acetate-Gd(III) chelate conjugated to 17β-estradiol (EPTA-Gd) or to tamoxifen (TPTA-Gd). The experiments were conducted in solution, in human breast cancer cells, and in severe combined immunodeficient mice implanted with transfected ER-positive and ER-negative MDA-MB-231 human breast cancer xenografts. Binding studies with ER in solution and in human breast cancer cells indicated affinities in the micromolar range of both CAs. Biochemical and molecular studies in breast cancer cell cultures showed that both CAs exhibit estrogen-like agonistic activity, enhancing cell proliferation, as well as upregulating cMyc oncogene and downregulating ER expression levels. The MRI longitudinal relaxivity was significantly augmented by EPTA-Gd in ER-positive cells as compared to ER-negative cells. Dynamic contrast-enhanced studies with EPTA-Gd in vivo indicated specific augmentation of the MRI water signal in the ER-positive versus ER-negative xenografts, confirming EPTA-Gd-specific interaction with ER. In contrast, TPTA-Gd did not show increased enhancement in ER-positive tumors and did not appear to interact in vivo with the tumors' ER. However, TPTA-Gd was found to interact strongly with muscle tissue, enhancing muscle signal intensity in a mechanism independent of the presence of ER. The specificity of EPTA-Gd interaction with ER in vivo was further verified by acute and chronic competition with tamoxifen. The chronic tamoxifen treatment also revealed that this drug increases the microvascular permeability of breast cancer xenograft in an ER-independent manner. In conclusion, EPTA-Gd has been shown to serve as an efficient molecular imaging probe for specific assessment of breast cancer ER in vivo.

Keywords: breast cancer; contrast agents for MRI; estrogen receptor; estrogen receptor-targeted probes; molecular imaging.

Figure 1

The chemical structure of 17β-estradiol pyridine-tetra-acetate-Gd (EPTA-Gd) and tamoxifen pyridine-tetra-acetate-Gd (TPTA-Gd) (A) and their competitive displacement curves by tritiated 17β-estradiol (³HE2) on a human recombinant ERα in reference to tamoxifen competition (B).

Figure 2

T1 and T2 measurements with EPTA-Gd and TPTA-Gd in perfused ER-positive and ER-negative MDA-MB-231 human breast cancer cells. ΔR1 and ΔR2 are defined as the difference between R1 (or R2) of cells perfused with medium containing the contrast agent and R1 (or R2) of these cells perfused with contrast-free medium. (A) T1 relaxivity of EPTA-Gd in ER-positive and negative cells: r1 (ER-positive) = 28.5 ± 0.1 mM⁻¹s⁻¹ (n = 2) and r1 (ER-negative) = 19.6 mM⁻¹s⁻¹. (B) T1 relaxivity measurements of TPTA-Gd in ER-positive and negative cells: r1 (ER-positive) = 42.1 mM⁻¹s⁻¹ and r1 (ER-negative) = 36 mM⁻¹s⁻¹. The cells were perfused in the NMR tube and treated with increasing concentrations of each contrast agent. T1-relaxation time of water protons was determined at 9.4 T using inversion recovery pulse sequence. Different symbols for ER-positive cells in (A) represent two independent experiments. r1 relaxivities were calculated as the slope of the linear fit to the data as explained in the text. (C) Change in T1-relaxation rates, ΔR1, in the perfused cells after washing out EPTA-Gd (7.5 μM) or TPTA-Gd (7.5 μM) from the perfusion system with fresh medium. (D) Change in T2-relaxation rates, ΔR2, in the perfused cells as in (C). Data presented in (C,D) are mean ± SD of three to six measurements recorded 30–60 min after the beginning of the washout process.

Figure 3

Binding affinity of EPTA-Eu to ER in human breast cancer cells. ER-positive and ER-negative MDA-MB-231 human breast cancer cells, grown on microspheres, were incubated in the presence of the indicated concentrations of EPTA-Eu for 1 h at 37°C and subjected to DELFIA assay. (A) Total binding (red) was determined in ER-positive cells and non-specific binding (black) was determined in ER-negative cells. Specific binding (blue) was calculated by subtracting non-specific from total binding. (B) Saturation-binding curve. Data points of specific binding were fitted to one-site binding equation yielding Kd = 0.56 μM, BMAX = 60.9 pmole, and R2 = 0.97. Data of OD scale were converted to molar units by comparing to OD values of known EPTA-Eu concentrations.

Figure 4

ERα expression in various human breast cancer cell lines. (A) Western blots of recombinant hERα and of ERα in human breast cancer cell extracts. (B) Quantification of western blot experiments (n = 2).

Figure 5

Effect of EPTA-Gd and TPTA-Gd on the proliferation rates of human breast cancer cells. (A) Dose-dependent effect of EPTA-Gd on the proliferation rate of ER-positive T47D human breast cancer cells. (B) Dose-dependent effect of TPTA-Gd and of TPTA-Gd + E2 on the proliferation rate of ER-positive MCF7 human breast cancer cells. (C) Dose-dependent effect of EPTA-Gd on the proliferation rate of ER-negative WT MDA-MB-231 human breast cancer cells. Cell proliferation was measured by the cell viability MTT assay, and each data point represents mean ± SD of six-replicate wells. Control: cells cultivated in E2-free medium.

Figure 6

Changes in the expression level of ERα (left) and cMyc (right) in MCF7 cells induced by EPTA-Gd (A) and TPTA-Gd (B) at a concentration of 5 μM. In each panel, a representative blot is at the upper raw and the α-tubulin for normalization is at the bottom raw. The curve shows the fold change in expression levels, n = 2.

Figure 7

Immunohistology staining of ERα (A) and MRI signal enhancement patterns induced by EPTA-Gd (B,D) and by TPTA-Gd (C,E) in ER-positive and ER-negative MDA-MB-231 human breast cancer xenografts. The injected dose of EPTA-Gd and TPTA-Gd was 0.075 mmol/kg. The curves in (B,D) show mean ± SD of EPTA-Gd-induced enhancement (n = 5). The curves in (C,E) show mean ± SD of TPTA-Gd-induced enhancement (n = 4).

Figure 8

Enhancement maps in muscle tissue 20 min after bolus injection of EPTA-Gd (A) and TPTA-Gd (B) into the tail vein of the mice and mean ± SD enhancement curves induced by EPTA-Gd [(C), n = 5] and TPTA-Gd [(D), n = 4]. The injected dose of EPTA-Gd and TPTA-Gd was 0.075 mmol/kg. The enhancement maps are overlaid on the corresponding T1-weighted images.

Figure 9

EPTA-Gd induced enhancement patterns (A,B) and enhancement maps at 40 min (C,D) in ER-positive and ER-negative MDA-MB-231 xenografts treated with acute (1 h) and chronic (3 days) tamoxifen. The anatomy of the xengorafts is demonstrated in the T2-weighted images (C,D). The enhancement maps are overlaid on the corresponding T1-weighted images.