Fluorescence imaging of prostate cancer in living mice using a new mitochondrial-derived peptide

Objectives A new 16-amino-acid peptide (MOTS-c) encoded within the mitochondrial 12S rRNA has been identified. Cellular actions of MOTS-c inhibit the folate cycle and tether de novo purine biosynthesis, leading to AMPK activation. In this study, we developed a new fluorescent MOTS-c probe and evaluated its tumor-targeting efficacy and pharmacokinetics in athymic nude mouse model with a subcutaneous xenograft of PC3 prostate cancer cells.

Methods The N-terminal protected MOTS-c was conjugated with an alkyne-containing PEG unit followed by mixing with an azide-terminated Cy5.5 fluorophore (Cy5.5-N3). After deprotection, the Cy5.5-MOTS-c probe was obtained and subject to in vitro studies, and in vivo fluorescence imaging in subcutaneous PC-3 tumor mouse xenografts. A Cy5.5 filter set was used to acquire the fluorescence signals. Each animal bearing a PC-3 tumor received 1.5 nmol of Cy5.5-MOTS-c intravenously and imaged at various time points with or without a mixture of 20 mg/kg of unlabeled MOTS-c. The animals from non-blocking and blocking groups were euthanized at 4h pi. The tumors, tissues, and organs were dissected for ex vivo fluorescence imaging.

Results The click chemistry approach provided a rapid conjugation of MOTS-c with Cy5.5 in a quantitative yield. In vitro studies confirmed the fluorescence property of Cy5.5-MOTS-c. For in vivo fluorescence imaging, Cy5.5-MOTS-c showed rapid PC-3 tumor targeting at 0.5 h pi (T/M ratio: 1.87±0.33), and good tumor retention (T/M ratio: 2.16 ± 0.08 at 4 h pi). The specific PC-3 tumor accumulation of Cy5.5-MOTS-c was accomplished by a blocking study with unlabeled MOTS-c peptide in PC-3 tumor bearing mice. The tumor-to-muscle ratio of Cy5.5-MOTS-c at 2 h pi reached 2.14 ± 0.55 in non-blocking group vs. 1.53 ± 0.42 in blocking group. Ex vivo fluorescence imaging further confirmed the in vivo findings, demonstrating that Cy5.5-MOTS-c displayed an excellent PC-3 tumor uptake at 4h pi for the non-blocking group and significantly reduced PC-3 tumor uptake (p<0.05) for the blocking group.

Conclusions We have successfully developed a Cy5.5-MOTS-c peptide for highly sensitive and target-specific imaging of PC-3 tumors. Cy5.5-MOTS-c has the potential as a promising molecular probe for non-invasively localizing and characterizing prostate cancer.