Evaluation of Two Optical Probes for Imaging the Integrin αvß6- In Vitro and In Vivo in Tumor-Bearing Mice.

ABSTRACT

PURPOSE: The purpose of this study was to develop and evaluate two αvß6-targeted fluorescent imaging agents. The integrin subtype αvß6 is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. αvß6-targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH2-PEG28-A20FMDV2-K16R-PEG28 (1), a peptide that has high affinity and selectivity for the integrin αvß6 (a) 5-FAM-X-PEG28-A20FMDV2-K16R-PEG28 (2), and (b) IRDye800-PEG28-A20FMDV2-K16R-PEG28 (3). PROCEDURES Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for αvß6 was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroß6 (αvß6+) and DX3puro (αvß6-) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroß6/DX3puro and BxPC-3 (αvß6+) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model. RESULTS: Peptides were obtained in > 99% purity. IC50 values were 28 nM (2) and 39 nM (3). Rapid αvß6-selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the αvß6 positive subcutaneous and orthotopic tumors, with negligible uptake in the αvß6-negative tumor. Blocking studies with a known αvß6-targeting peptide demonstrated αvß6-specific binding of 3. CONCLUSION: Two fluorescence imaging agents were developed. The αvß6-specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the αvß6-positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.