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Purpose: To evaluate the utility of targeted photoacoustic imaging (PAI) in providing molecular information to complement intrinsic functional and anatomical details of the vasculature within prostate lesion. Experimental Design: We developed a PAI agent, AA3G-740, that targets gastrin-releasing peptide receptor (GRPR), found to be highly overexpressed in prostate cancer. The binding specificity of the agent was evaluated in human prostate cancer cell lines, PC3 and LNCaP, and antagonist properties determined by cell internalization and intracellular calcium mobilization studies. The imaging sensitivity was assessed for the agent itself and for the PC3 cells labeled with agent. The in vivo stability of the agent was determined in human plasma and in the blood of living mice. The in vivo binding of the agent was evaluated in PC3 prostate tumor models in mice, and was validated ex vivo by optical imaging. Results: AA3G-740 demonstrated strong and specific binding to GRPR. The sensitivity of detection in vitro indicated suitability of the agent to image very small lesions. In mice, the agent was able to bind to GRPR even in poorly vascularized tumors leading to nearly 2-fold difference in photoacoustic signal relative to the control agent. Conclusions: The ability to image both vasculature and molecular profile outside the blood vessels gives molecular PAI a unique advantage over currently used imaging techniques. The imaging method presented here can find application both in diagnosis and in image-guided biopsy. Clin Cancer Res; 20(14); 3721–9. ©2014 AACR.

Purpose: Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin αvβ6, a cell surface receptor being evaluated as a novel clinical biomarker. Experimental Design: To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3–6 nmol/L) to integrin αvβ6. The binders do not cross-react with related integrin αvβ5, integrin α5β1, or tumor-angiogenesis–associated integrin, αvβ3. Results: Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin αvβ6. Clinically relevant tumor-to-muscle ratios of 7.7 ± 2.4 to 11.3 ± 3.0 were achieved within 1 hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting αvβ6-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3). Conclusions: We have engineered highly stable cystine knot peptides with potent and specific integrin αvβ6-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging. Clin Cancer Res; 18(3); 839–49. ©2011 AACR.