In Vivo Distribution and Therapeutic Efficacy of Radioiodine-Labeled pH-Low Insertion Peptide Variant 3 in a Mouse Model of Breast Cancer

PURPOSE: Extracellular acidity is a marker of highly aggressive breast cancer (BC). pH-low insertion peptides (pHLIPs) target the acidic tumor microenvironment. This study evaluates the distribution and therapeutic efficacy of radioiodine-labeled pHLIP variant 3 (Var3) in a mouse model of BC. METHODS: The binding of fluorescein isothiocyanate (FITC)- or radioiodine-125 ((125)I) labeled Var3-pHLIP to MDA-MB-231, 4T1, and SK-BR-3 BC cell lines under different pH values was evaluated in vitro. The distribution of (125)I-labeled Var3-pHLIP and wild-type- (WT-) pHLIP in tumor-bearing mice was analyzed in vivo using micro-SPECT/CT imaging. The therapeutic efficacy of radioiodine-131 ((131)I)-labeled Var3-pHLIP in MDA-MB-231 xenografts was evaluated by relative tumor volume measurement and immunohistochemical analysis. RESULTS: The binding ability of FITC- or (125)I-labeled Var3-pHLIP to tumor cells increased with the decrease in pH. The tumor-to-background ratio of (125)I-Var3-pHLIP in BC xenografts showed the best imaging contrast at 24 h or 48 h postinjection. The uptake of (125)I-Var3-pHLIP in MDA-MB-231 xenografts at 2 h postinjection was significantly higher than that of (125)I-WT-pHLIP (3.76 ± 0.37 vs. 2.87 ± 0.60%ID/g, p = 0.046). The relative tumor volume in MDA-MB-231 xenografts was significantly lower in the (131)I-Var3-pHLIP-treated group than in the groups treated with Var3-pHLIP (p = 0.027), (131)I (p = 0.001), and saline (p < 0.001). The (131)I-Var 3-pHLIP group presented a lower expression of Ki67 and a higher expression of caspase 3. CONCLUSION: Radioiodine-labeled Var3-pHLIP effectively targeted BC cells in an acidic environment and inhibited the growth of MDA-MB-231 xenografts by ionizing radiation.