The overall goal of this procedure is to perform molecular imaging of prostate cancer in mice using a safe and effective peptide contrast agent with magnetic resonance or MR.Molecular imaging. First, the successful development and proper size of orthotopic prostate tumors are verified in the prostate tumor mouse model. Next, tumor binding specificity of the peptide is confirmed with fluorescence imaging and histology in the mouse orthotopic prostate cancer model.
Then the targeted or control agent is injected after pre injection baseline MR image acquisition. Finally, contrast enhanced MR Images are acquired at different time points for up to 30 minutes. Ultimately, results can be obtained that show that the CLT one peptide targeted RI contrast agent can be effective for specific detection of prostate tumors through Mr.Molecular imaging techniques.
The main advantage of molecular MRI with a targeted MI contrast agents over existing technology like MRI and a pet MRI with a clinical contrast agents is that this technology can provide effective, enhanced images of prostate tumors with high resolution as a single imaging modality. We will demonstrate here. Effective molecular MRI of prostate tumor can be achieved by selecting abundant biomarker in tumor extracellular metrics and using small molecular targeted mi contrast agents Good skills in animal handlings and cannulation of a tail and MRI are also critical for obtaining high quality MRR images of tumors Using standard solid phase peptide synthesis, synthesize, and then characterize the GD DOTA mono amide complexes as illustrated here after generating a PC 3G FP prostate tumor animal model, according to the text protocol, anesthetize mice with 2%iso fluorine in oxygen in an induction chamber and perform a tail pinch to verify sedation.
Then use a fluorescence imager to verify the presence of tumors in the mouse. Inject 10 mol per mouse of Texas red labeled peptides into the tumor bearing mice. Two hours later, after sacrificing the mice by cervical dislocation, collect the tumor and major organs and immediately image using a fluorescence imager.
Next, after fixing the tumor tissues with formalin and cryosectioning into five micrometer slices, use a drop of DPI containing mounting medium to mount the tissue. Then image the slides using confocal laser scanning microscopy Approximately four weeks after inoculation, the tumors should have grown from 0.3 to 0.6 centimeters in diameter. After anesthetizing the mouse with 2%iso fluorine in oxygen in an induction chamber, apply ophthalmic ointment to maintain moisture in the mouse's eyes and perform a tail pinch to verify sedation.
Next, insert a 30 gauge needle into a one meter long tubing filled with heparinized saline. Then place the self-made catheter into the mouse tail vein using a 70 MRI scanner with a volume radio frequency coil. Place the mouse into the magnet, then attach a nose cone and administer 1.5%isof fluorine in oxygen to monitor rate and depth of respiration.
Place a respiratory sensor attached to a monitoring system on the animal's abdomen. Maintain the animal's body temperature at 37 degrees Celsius with a constant supply of hot air into the magnet. Now, place the animal into the MRI to identify the tumor location.
Begin with sagittal section images using a localizing sequence. Next, use a two DT one weighted gradient fat suppression sequence to acquire 2D axial images for contrast enhanced or CE MRI. After acquiring baseline images of the tumor begin to inject the targeted agent or control agent at a dose of 0.03 millimoles of gadolinium per kilogram.
Continue to acquire CE MRI images at different time points for up to 30 minutes to completely span and acquire images of the entire tumor. Collect 12 slices after imaging is complete, euthanize the animal with carbon dioxide and analyze and process the images according to the text protocol as shown here, Maestro imaging confirms the strong specific binding of Texas red labeled CLT one to the GFP PC three tumor with little binding to normal organs and tissues. While the non-specific scrambled peptide shows very little tumor binding.
This figure shows typical pre and post-injection contrast enhanced T one 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 up to 30 minutes. The biodistribution study shows the agent has minimal gadolinium retention in the main organs and tissues two days after injection. Our preliminary shows that CLT one targeted MRI contrast agents can be specifically delivered to the tumor at a relatively low dose.
Yeah, we have demonstrated here effective molecular MRI of a prostate cancer can be achieved by selecting a proper abundant biomarker in tumor microenvironment and using a small molecular targeted MRI contrast agent. After watching this video, you should have a good understanding about how to develop a proper techniques for effective molecular MI or for small malignant tumors in animal models using a targeted MI contrast agent.
