A high-affinity, high-stability photoacoustic agent for imaging gastrin-releasing peptide receptor in prostate cancer

Abstract

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.

Figure 1. Design and spectral characterization of the GRPR imaging agent

A) The highest photoacoustic signal among the small molecule dyes tested was observed for IRDyeQC-1, ATTO 740, RD831 and ICG. B) Among the dyes that provided a high photoacoustic signal, ATTO-740 showed superior photostability not changing the appearance after being exposed to light for 7 days. C) The absorbance of ATTO740 did not change during a 30 minute irradiation with 740 nm laser light. D) GRPR agent, AA3G-740 consists of antagonist binding moiety, triple glycine linker and ATTO740 as a signaling dye. The control agent, CAA3G-740, has the same signaling dye and linker but a scrambled binding sequence. E) AA3G-740 provides the highest photoacoustic signal at 750 nm and highest fluorescence at 760 nm.

Figure 2. Binding specificity

A) AA3G-740 efficiently labeled PC3 cells that have high level of GRPR (black filled area). Co-incubation with bombesin prevented the binding, resulting in minimal cell fluorescence (gray filled area). B) Low GRPR expressing cells, LNCaP, showed minimal increase in fluorescence (grey filled area). C) Control agent, CAA3G-740 containing a scrambled binding sequence was not able to label PC3 cells (grey filled area). Black outline represent unstained cells in all three panels.

Figure 3. Functional properties of AA3G-740

A) The agent was found to be primarily surface bound. The internalized portion increased over time but it represented a smaller portion of the total amount. B) AA3G-740 binding did not cause calcium internalization at any of the concentrations tested. Both results suggest antagonistic properties of the agent.

Figure 4. Sensitivity of detection

A) The photoacoustic signal was linearly dependent on the amount of the agent. The lowest amount that could reliably be detected above the background was found to be 100 pmol. B) The lowest detectable number of PC3 cells labeled with AA3G-740 was determined to be 0.5 million.

Figure 5. Agent stability

The stability of the agent was determined in human male plasma (A) and in mouse blood (B). There was no evidence of decomposition within one hour of incubation in human plasma (grey line 30 min incubation, dotted line 60 min incubation). In mouse blood, at 30 minutes post injection, the major peak corresponded to the intact agent with minute amount of possible peptide adducts also visible (arrow). The three spectra in B correspond to data from three mice. There was no indication of proteolysis occurring in the blood.

Figure 6. Imaging of GRPR in PC3 tumors

PC3 tumors implanted in nude mice were imaged before and after the injection of 10nmol active or control agent. A) While the injection of AA3G-740 led to close to a 2 fold increase in photoacoustic signal, CAA3G-740 injection caused a minimal change over the background, pre-injection, signal. The scale bar represents 0.5 mm. B) Different accumulation of the two agents was confirmed ex vivo by optical imaging. C) The quantitation of the photoacoustic signals revealed significant difference between tumors injected with the two agents at both time points. The signal in tumors injected with AA3G-740 (n=6) increased over time, while in the ones injected with CAA3G-740 (n=4) it remained largely constant. The errors bars represent standard deviation. *p<0.05.

Figure 7. AA3G-740 excretion route

Agent (10 nmol) was injected in mice (n=3) and major organs imaged ex vivo 30 minutes post injection. The highest signal was observed for gallbladder and liver, followed by intestine, kidneys and spleen. H-heart, L- lung, P- pancreas, S-spleen, K- kidneys, Li- liver, G-gallbladder, I-intestine, M-muscle, B-bone.