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.
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.
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 agents2,3. 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 sites4,5. 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 excreted7,8. 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 adapted from a prior study11.
1. Conjugation of Gd-DOTA to CLT1 Peptide
2. Characterization of Contrast Agents
3. Cell Culture and Development of Animal Model with Orthotopic PC3 Prostate Tumor
4. Confirmation of Tumor Binding Specificity of the Peptides with Fluorescence Imaging and Histology
5. Magnetic Resonance Imaging (MRI)
6. Image Processing and Analysis
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 to normal organs and tissues, while the non-specific scrambled peptide (sCLT1-TR) shows very little tumor binding (Figure 2). Figure 3 shows typical pre and post-injection contrast enhanced (CE) T1-weighted images. The targeted agent results in greater and longer enhancement in tumor tissue compared to non-targeted scrambled agents. Contrast enhancement in the urinary bladder gradually increases over time, indicating that the contrast agents were excreted via renal filtration. Quantitative signal analysis reveals that the targeted agent produced more significant signal enhancement in the tumor tissue than the control agent (p<0.05) up to 30 min. The biodistribution study shows the agent has minimal Gd retention in the main organs and tissues 2 days after injection. Our preliminary data shows that CLT1 targeted MRI contrast agents can be specifically delivered to the tumor at relatively low dose (one third of clinical dose).
r2 (mM-1.s-1) | r1 (mM-1.s-1) | Gd content (mmol-Gd/g) | |||
per Gd | per molecule | per Gd | per molecule | ||
CLT1-(Gd-DOTA)4 | 10.1 ± 1.0 | 40.4 ± 3.0 | 13.0 ± 3.1 | 52.0 ± 9.6 | 1.05 ± 0.10 |
sCLT1-(Gd-DOTA)4 | 11.5 ± 1.4 | 46.0 ± 5.0 | 12.4 ± 0.3 | 49.6 ± 1.3 | 0.97 ± 0.08 |
Gd-DOTA4 | 2.9* | 2.9* | 3.2* | 3.2* |
Table 1. Physicochemical properties of the targeted and scrambled MRI contrast agents at 1.5 T, 37 °C (*Relaxivities for Gd-DOTA in water at 37 °C are from reference 10).
Figure 1. Graphical depiction of the synthesis procedure and overall experiment. Click here to view larger figure.
Figure 2. Tumor binding of CLT1-TR. Targeted CLT1-TR (A) and non-targeted sCLT1-TR (B) were injected intravenously to athymic nude mice bearing orthotopic PC3-GFP prostate at a dose of 10 nmol/mouse. After 2 hr, tumors and various organs were collected and imaged. Green fluorescence images are from PC3-GFP tumor cells. Red fluorescence images are from CLT1-TR (A) and sCLT1-TR (B) probes. 1. tumor; 2. spleen; 3. heart; 4. kidney; 5. testicle; 6. liver; 7. lung; 8. muscle; 9. brain. Click here to view larger figure.
Figure 3. Representative T1-weighted axial 2D gradient images of orthotopic PC-3 human prostate tumor before and after intravenous injection of CLT1-dL-(Gd-DOTA)4 (A) and sCLT1-dL-(Gd-DOTA)4 (B) at 0.03 mmol Gd/kg in nu/nu mice. Tumor labeled with red circle and bladder labeled with green circle. Click here to view larger figure.
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 molecular agents should have high binding affinity for the biomarkers so that sufficient contrast agents can bind to these targets, resulting in enough contrast enhancement. In this study, we chose the clot-binding peptide CLT1 that shows strong specific binding to the clotted plasma proteins in the tumor11.
Contrast Agent Synthesis
The peptide synthesis involves oxidizing two thiol groups in the linear peptide molecule to form final cyclic CLT1 peptide. Whilst peptide cyclisation is usually conducted in solution at low concentration to minimize potential aggregation, dimerization and oligomerization, on-resin cyclization takes advantage of the pseudo-dilution phenomenon as well as the removal of reagents by simple washing and filtration13. In our synthesis procedure, we find that on-resin cyclization using thallium(III) trifluoroacetate works well with high yield and little dimerization, although thallium(III) trifluoroacetate is very toxic and care must be taken while handling it. A second approach uses 20% DMSO in solution at low peptide concentration. However more steps are needed to remove DMSO, and lyophilizing the large volume solution takes longer. By using either method, it is crucial to make sure of the complete formation of intramolecular disulfide bond. When complexing the peptide ligand with Gd, the free thiols can induce substantial aggregations because trace metals in the buffer catalyze air oxidation, resulting in the overoxidation of cysteine to form polymerization14. This usually dramatically decreases the yield and purity of final product. To make sure complete disulfide band formation, first we need to use mild oxidization conditions to avoid formation of intermolecular disulfide bond resulting in dimer or higher oligomer formation. Secondly, long enough reaction time should be maintained to make sure that all thiol groups are oxidized before the Gd conjugation step.
Animal Model and Cannulation of Mouse Tail Vein
Preparation of the animal tumor model and tail vein cannulation are also very important aspects of this procedure. During the surgery, the tools/bench must be kept clean and sterile to avoid potential infection which could injure or kill the mice. In order to avoid moving the mouse before and after injection, it is necessary to cannulate mouse tail vein. To prepare the catheter for cannulation, break a 30 gauge needle from the hub and insert the blunt end into a 1.0 m long tubing, and connect another end of the tubing to a syringe loaded with 1% heparinized saline. Push the syringe to fill up the tubing. Dilate the tail with warm water (~105 °F). Insert the needle into the vein. Blood backflow into the tubing indicates successful insertion of the catheter. To verify that the needle is in the vein, gently push the syringe and the saline can be smoothly injected, the vein is cleared with the injection of a small amount of saline. Fix the catheter in place with tape before moving the mouse to the scanner.
Limitations and Possible Modifications
There are several limitations in our current study. First, from the preliminary results we notice that CLT1 washes out very quickly from the targeting site although it shows excellent binding to the tumor. In vivo proteolytic degradation likely causes the instability of CLT1. Modification of CLT1 to protect against proteolytic degradation should improve its stability and prolong its wash-out time in vivo. Using D- type amino acids to replace natural L- type amino acids is a potential strategy to achieve this goal. Second, the current study doesn't clarify which exact components in the clotted plasma proteins the CLT1 peptide binds to. Finally, we synthesize the contrast agents mainly using solid phase synthesis with relatively low yield and high cost. Developing a liquid phase synthesis method should increase the yield and lower the cost.
Applications
We are developing techniques for accurate detection of small malignant tumors with contrast enhanced MRI. MRI with the targeted agent provides a more effective and safer alternative for cancer molecular imaging.
The authors have nothing to disclose.
This work is supported in part by the American Heart Association GRA Spring 09 Postdoctoral Fellowship (09POST2250268) and the NIH R01 CA097465. We highly appreciate Dr. Wen Li and Dr. Vikas Gulani for MRI protocol test and setup, and Ms. Yvonne Parker for her assistance on tumor implantation.
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 |