Supplementary Material for: Preclinical evaluation of 99mTc-labeled LHRH analog as cancer receptor imaging.

Introduction: Breast cancer is the main cause of cancer-related mortality in women in the developed world. In particular, receptors of Luteinizing Hormone-Releasing Hormone (LHRH or GnRH) are overexpressed in this malignant disease. The aim of this study was to develop a new molecular probe [99mTc]Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/Nicotinic Acid (NA) as a novel molecular imaging agent for breast cancer. Methods: HYNIC-GSG-LHRH(D-Lys6) was acquired and radiolabeled with [99mTc]Tc. Radiochemical purity and stability under different conditions were evaluated by Instant thin-layer chromatography (ITLC) and High-Performance Liquid Chromatography (HPLC). Lipophilicity was determined by the partition coefficient test. In vitro cell binding studies were performed in different human and mice breast cancer cell lines (MDA-MB-231, MDA-MB-435, MCF-7, BT474 and 4T1) as well as in normal murine fibroblasts (NIH-3T3) and CHO-K1 as a negative control. Biodistribution studies were performed in normal Balb/c mice and 4T1 tumor-bearing Balb/c mice up to 6 h post-injection (p.i.). SPECT/CT images were performed in 4T1 tumor-bearing Balb/c mice up to 5 h p.i. Results: [99mTc]Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA complex was labeled with a high radiochemical purity (>98%) and remained stable for up to 4 h. It exhibited good hydrophilicity (Log P = - 2.82± 0.04) and also demonstrated significant and specific binding across all evaluated breast cancer cell lines. Biodistributions studies showed a high renal clearance and low nonspecific binding (<2% Act/g) in most organs, as well as appreciable tumor uptake (5.8 ± 0.5 %ID/g 1 h p.i.) and high tumor-to-muscle ratio (maximum of 30.5 ± 11.2 at 1 h p.i.). SPECT/CT imaging of 4T1-tumor bearing Balb/c mice revealed results consistent with the biodistribution studies, showing a tumor-to-non-tumor ratio of greater than 3.5 at all evaluated times points. In vivo blocking studies confirmed specificity for the LHRH receptor. Conclusions: [99mTc]Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA complex has shown significant potential for in vivo visualization of LHRH receptors expression in breast cancer.