Selective Visualization of ROR1-Positive TNBC of the Mesenchymal-Like Subtype Using Peptide-Based 68 Ga-PET Radiotracers

Triple-negative breast cancer (TNBC) lacks effective molecular targets for imaging. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is overexpressed in the mesenchymal-like (claudin-low) subtype of TNBC. Here, we developed two optimized linear peptide radiotracers, DR1 and DR2, derived from the complementarity-determining regions (CDR) of an anti-ROR1 antibody, conjugated with DOTA and labeled with 68 Ga. Both peptides showed nanomolar binding affinities ( K D = 93.4 ± 8.2 nM for DR1 and 38.0 ± 4.6 nM for DR2) and favorable target-binding properties. [ 68 Ga]Ga-DOTA-DR2 exhibited excellent in vitro stability (≥95% radiochemical purity after 4 h in human serum), rapid in vivo clearance ( t 1/2β = 0.66 ± 0.11 h), and specific cellular uptake. Micro-PET/CT imaging in MDA-MB-231 xenograft mice revealed rapid tumor visualization, with a peak tumor uptake of 2.75 ± 0.61 %ID/g at 1 h and a tumor-to-muscle ratio of 7.98 at 2 h. Blocking studies confirmed ROR1-specific targeting. Collectively, [ 68 Ga]Ga-DOTA-DR2 may represent a promising PET tracer for imaging ROR1-positive mesenchymal-like TNBC, providing a foundation for further optimization.