Development of Follicle-Stimulating Hormone Receptor Binding Probes to Image Ovarian Xenografts

Abstract

The Follicle-Stimulating Hormone Receptor (FSHR) is used as an imaging biomarker for the detection of ovarian cancer (OC). FSHR is highly expressed on ovarian tumors and involved with cancer development and metastatic signaling pathways. A decapeptide specific to the FSHR extracellular domain is synthesized and conjugated to fluorescent dyes to image OC cells in vitro and tumors xenograft model in vivo. The in vitro binding curve and the average number of FSHR per cell are obtained for OVCAR-3 cells by a high resolution flow cytometer. For the decapeptide, the measured EC50 was 160 μM and the average number of receptors per cell was 1.7 × 10⁷. The decapeptide molecular imaging probe reached a maximum tumor to muscle ratio five hours after intravenous injection and a dose-dependent plateau after 24-48 hours. These results indicate the potential application of a small molecular weight imaging probe specific to ovarian cancer through binding to FSHR. Based on these results, multimeric constructs are being developed to optimize binding to ovarian cells and tumors.

Keywords: Decapeptide; Follicle-stimulating hormone receptor; Multimeric peptide; Near-infrared imaging; Ovarian cancer.

Figure 1

Dose Response Curve for BI-10^FAM. The average fluorescence intensity and its standard deviation (n=5) was plotted as a function of peptide concentration (1.0 μM, 10.0 μM, 50 μM, 250 μM, and 500 μM) and fitted to a sigmoidal curve (solid line). Note, the error bars of the first three points are small and covered by the size of markers.

Figure 2

Kinematics of BI-10^AF750 Molecular Imaging (MI) Probe. (Primary Axis) Plotted is the tumor-to-reference tissue (muscle) ratio (TRR) as a function of time for three mice (M1-M3) each injected with 120 μg (blue), 40 μg (green), and 15 μg (red) of the BI-10^AF750 MI probe. The peak TRR occurred within 4–8 hours post-injection. (Secondary Axis) Similarly, the average dose-normalized fluorescence signal is plotted as function of time for muscle (n=3) from which the BI-10^AF750 clearance rate was determined to be 4 hours.

Figure 3

The TRR for a Cohort of Ovarian Cancer Xenograft Mouse Model. The average TRR and its standard deviation (n=3) was calculated using NIR imaging (LB981 NightOWL) prior to and 6 hours post-injection of the BI-10^AF750 MI probe (80 μg).

Figure 4

In vivo NIRF Imaging of BI-10^AF750. Three mice were i.v. injected with 80 μg of the BI-10^AF750 MI probe, and NIR fluorescence images were acquired 6 hours post injection. A threshold was applied to better depict the tumors. Signals in the kidneys and bladder indicate renal filtration. No signal was observed in the liver at this time. An indication of tumor cell growth along the subcutaneous needle injection was observed in two mice (left and right).

Figure 5

NIR Optical Images of BI-10^AF750 Molecular Imaging (MI) Probe. (Left) Displayed is the bright field image of a mouse with an ovarian tumor on its flank, and (Right) the corresponding fluorescence image 6 hours post-injection of the BI-10^AF750 MI probe. The arrows depict the anatomical features of the mouse.