Experimental study of [⁶⁸Ga]Ga-HER2-RGD, a dual-target molecular probe, for breast cancer PET/CT imaging

Radiolabeled heterodimeric peptides have emerged as a highly promising targeting strategy for PET imaging due to their superior binding properties. The Human epidermal growth factor receptor 2 (HER2)- and Arg-Gly-Asp (RGD)-targeting moieties bind to HER2 and integrin αvβ3, respectively, receptors that are both overexpressed in many different types of tumors. This study focuses on the synthesis and evaluation of the heterodimeric radioligand [⁶⁸Ga]Ga-DOTA-HER2-RGD for PET imaging of the breast cancers-bearing mouse models that overexpress HER2 and/or integrin αvβ3.

The heterodimeric radiopeptide was successfully synthesized and radiolabeled with ⁶⁸Ga, achieving a radiochemical purity of > 95%. The receptor-binding properties and tumor-targeting efficacy were evaluated in vitro and in vivo using three breast cancer cell lines-MDA-MB-435 S (high HER2, high αvβ3), MDA-MB-231 (high HER2, low αvβ3), and MCF-7 (low HER2, low αvβ3)-and their respective xenograft models. Flow cytometry verified the cell-surface expression profiles of HER2 and integrin αvβ3 in all three cell types. In vitro cell studies showed that the MDA-MB-435 S cell line exhibited the highest uptake of [⁶⁸Ga]Ga-HER2-RGD, followed by MDA-MB-231 and MCF-7. High specificity was demonstrated in blocking studies using excess unlabeled HER2-RGD. Competitive binding assays revealed that [⁶⁸Ga]Ga-HER2-RGD exhibited high binding affinity, with an IC50 value of 12.9 nM. Micro-PET/CT imaging revealed a significant accumulation of [⁶⁸Ga]Ga-HER2-RGD in MDA-MB-435 S xenografts, with no or very low uptake in nontarget organs and tissues. Consistent with the in vitro findings, the tumor-to-nontumor ratios across the three xenograft models followed the order: MDA-MB-435 S > MDA-MB-231 > MCF-7. A significant decrease in [⁶⁸Ga]Ga-HER2-RGD uptake in the MDA-MB-435 S xenografts was observed after the administration of blocking amount of HER2-RGD.

[⁶⁸Ga]Ga-HER2-RGD demonstrates specific dual-receptor-targeting properties towards HER2 and integrin αvβ3, as evidenced by in vitro and in vivo studies. This radiopeptide shows promise as a novel PET agent for the noninvasive visualization of HER2 and/or integrin αvβ3 expression in breast cancer, and is capable of semi-quantitatively evaluating the expression levels of HER2 and/or integrin αvβ3 to a certain extent.