MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Summary

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

Abstract

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

Introduction

Effective imaging of cancer related molecular targets is of great significance to improve the accuracy of earlier cancer detection and diagnosis. Magnetic resonance imaging (MRI) is a powerful clinical imaging modality with high spatial resolution and no ionization radiation¹. However, no targeted contrast agent is available for clinical MR cancer molecular imaging. Innovative design and development of targeted MRI contrast agents would greatly advance the application of MR cancer molecular imaging. Significant efforts have been made to develop targeted contrast agents for MR imaging of the biomarkers expressed on the surface of cancer cells. Due to relatively low sensitivity of MRI and low concentration of these biomarkers, it is a challenge to generate sufficient contrast enhancement for effective MR molecular imaging using small molecular targeted contrast agents^2,3. In order to obtain sufficient enhancement, various delivery systems such as liposomes, nanoparticles and polymer conjugates with a high payload of paramagnetic Gd(III) chelates have been prepared to increase local concentration of contrast agents at the target sites^4,5. Although these delivery systems were able to generate significant tumor enhancement in animal models, their large sizes resulted in slow and incomplete elimination from the body, resulting in prolonged accumulation of toxic Gd(III) ions, which may cause serious safety concerns⁶. Recently, some studies have shown that the limitations of MRI for molecular imaging can be overcome by selecting proper molecular biomarkers with high local expression in the lesions and using small molecular agents that can be readily excreted^7,8. The key feature of these agents is that they target molecular markers abundantly present in the diseased tissues with little presence in normal tissues. A high concentration of contrast agents can bind to these targets, resulting in sufficient contrast enhancement for effective MR molecular imaging. Since their size is smaller than the renal filtration threshold, unbound contrast agents can readily be excreted from the body with reduced background noise. We have selected a universal cancer-related biomarker, clotted plasma proteins, which abundantly exist in tumor stroma, and are rarely present in normal tissues⁹. We synthesized a targeted contrast agent containing a small targeting peptide CGLIIQKNEC (CLT1), which showed strong specific binding to the PC3 prostate tumor model¹⁰, and four Gd-DOTA monoamide chelates. Here, we provide a methodology for MR cancer molecular imaging to detect tumors in mice.

Protocol

Protocol adapted from a prior study11. 1. Conjugation of Gd-DOTA to CLT1 Peptide Using standard solid-phase peptide synthesis, synthesize CLT1 peptide (CGLIIQKNEC) from Fmoc-protected amino acids on a 2-chlorotrityl chloride resin (1.0 mmol). After adding final amino acid, cyclize the linear peptide on-resin with thallium(III) trifluoroacetate (1.09 g, 2.0 mmol, 2 equivalents) in DMF (20 ml) at 0 °C for 2 hr. Next, conjugate PEG and lysine sequ…

Representative Results

Figure 1 depicts synthesis of the targeted contrast agent CLT1-dL-(Gd-DOTA)4 and the overall scheme of the experiment. CLT1-dL-(Gd-DOTA)4 shows much higher relaxivity than clinical Gd-DOTA (Table 1). At 1.5 T, the T1 relaxivity per gadolinium of CLT1-dL-(Gd-DOTA)4 in PBS (pH 7.4) is approximately 3 times higher than that of Gd-DOTA10. Maestro imaging confirms the strong specific binding of Texas Red labeled CLT1 (CLT1-TR) to tumor with little binding…

Discussion

Critical Steps

Selection of Proper Biomarker and Targeting Small Peptide

To successfully develop a targeted contrast agent with small size, two key points need to be considered. First, it is important to select proper molecular biomarkers which are abundantly present in diseased tissues with little presence in normal tissues. Our selected cancer-related biomarker, clotted plasma proteins, meets this requirement. Second, the selected targeted small mol…

Materials

			REAGENTS
Fmoc protected amino acids	EMD Chemicals Inc
DOTA-tris(t-Bu)	TCI America
PyBOP, HOBt, HBTU	Nova Biochem
DIPEA, Thallium(III) trifluoroacetate, TIS	Sigma-Aldrich Corp.
Texas Red, succinimidyl ester, single isomer	Invitrogen	T20175
			EQUIPMENTS
Agilent 1100 HPLC system	Agilent
ZORBAX 300SB-C18 PrepHT column	Agilent
ICP-OES Optima 3100XL	Perkin-Elmer
MALDI-TOF mass spectrometer	Bruker	AutoflexTM Speed
Maestro FLEX In Vivo Imaging System	Cambridge Research & Instrumentation, Inc.
Biospec 7T MRI scanner	Bruker