Tracking mesenchymal stromal cell-derived extracellular vesicles (EVs) homing to breast cancer sites via Cu-64-NODAGA-duramycin radiolabeling and PET imaging

Small extracellular vesicles (sEVs) are lipid-membrane-enclosed nanoparticles (30–120 nm) secreted by virtually all cell types into the extracellular environment. sEVs play a critical role in intercellular communication by transferring bioactive molecules such as proteins, lipids, RNA, and DNA between cells. Mesenchymal stem cells (MSCs) naturally home to tumors and metastases while evading the host immune response. It is hypothesized that MSC derived sEVs (MSC-sEVs) also possess tumor-homing and immune-evading capacities and therefore could provide a novel targeted delivery vehicle for treatment of cancer. To support the clinical translation of MSC-sEVs, it is imperative to elucidate MSC-sEVs migratory itinerary in vivo to support translation to the clinical setting. MSC-sEVs are encircled by a phospholipid bilayer, and externalization of phosphatidylethanolamine (PE) to the outer leaflet of the plasma membrane is a hallmark of EV formation. Duramycin (Dur), a peptide-based molecular probe, specifically binds to PE, providing a unique approach to label EVs. This study aimed to radiolabel MSC-sEVs with 64Cu-Dur to produce 64Cu-Dur-MSC-sEVs and investigate their tumor-homing properties. PET imaging was used to visualize MSC-sEV migration to tumor sites in mice bearing breast cancer (BC) xenografts.

Methods:
sEVs were isolated from wild-type MSCs stably expressing red fluorescent protein (RFP, via lentiviral transduction) using a combination of differential centrifugation, microfiltration, and ultracentrifugation. Isolated MSC-sEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Biotin-linked Dur was used to label MSC-sEVs, and Streptavidin Protein-Alexa Fluor™ 488 was employed to visualize biotinylated Dur-MSC-sEVs within human MDA-MB-231-luc BC cells after incubation for 3 hours. Cells were washed with cold PBS, and Alexa Fluor™ 488-labeled EV uptake was imaged using a confocal microscope. Radiolabeling of MSC-sEVs was achieved in two steps: NODAGA-Dur was conjugated with 64CuCl₂ (185 MBq in 0.1M HCl) at 80°C for 30 minutes to produce 64Cu-NODAGA-Dur, which was then incubated with MSC-sEVs at room temperature for 15 minutes. Unbound 64Cu-Dur was removed using exosome spin columns. Purified 64Cu-Dur-MSC-sEVs (>96% radiochemical purity) were intravenously injected (3.7 MBq) into nude mice bearing MDA-MB-231 BC xenografts. PET/MR imaging was conducted at multiple time points up to 42 hours post-injection.

Results:
Confocal microscopy confirmed that biotinylated Dur-MSC-sEVs were effectively taken up by MDA-MB-231 BC cells, with strong visualization up to 3 hours post-incubation following PBS washes. PET/MR imaging revealed that 64Cu-Dur-MSC-sEVs migrated and localized in tumor lesions over time. Initial scans at 1 hour post-injection showed no detectable tumor accumulation, with prominent radioactivity observed in the liver and spleen, likely due to reticuloendothelial system uptake. By 18 hours post-injection, tumor radioactivity became visible, and a significant increase in tumor uptake was observed by 42 hours. Minimal radioactivity was detected in soft tissues and kidneys, indicating limited release of free 64Cu-Duramycin.

Conclusion:
This study demonstrates that Duramycin binds MSC-sEVs with high affinity, leveraging the abundant expression of PE on mammalian membranes. Radiolabeling with 64Cu-Dur provides a reliable method for tracking MSC-sEV migration to tumors using PET imaging. The 64Cu-Dur-sEV tracking method represents a promising, noninvasive tool for evaluating MSC-sEVs as novel delivery vehicles for targeted cancer therapies. Strategies to enhance MSC-sEV circulation time, improve tumor targeting, and minimize sequestration by the liver and spleen are warranted.