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.

Protocol adapted from a prior study11.
1. Conjugation of Gd-DOTA to CLT1 Peptide
<ol>
	<li>Using standard solid-phase peptide synthesis, synthesize CLT1 peptide (CGLIIQKNEC) from Fmoc-protected amino acids on a 2-chlorotrityl chloride resin (1.0 mmol).</li>
	<li>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 &#176;C for 2 hr.</li>
	<li>Next, conjugate PEG and lysine sequentially to the N-terminus of CLT1 peptide (1.0 mmol) on resin by reacting sequentially with Fmoc-NH-PEG-COOH (3.0 mmol), Fmoc-Lys(Fmoc)-OH (3.0 mmol), and a second batch of Fmoc-Lys(Fmoc)-OH (6 mmol).</li>
	<li>Remove Fmoc with piperidine. Add DOTA-tris(t-Bu) (4.58 g, 8.0 mmol, 2 equivalents to each amino group) to react with the four free amines from the lysine dendrimer on the resin for 2 hr.</li>
	<li>Finally, cleave and deprotect the product from the resin by treating with a cocktail of trifluoroacetic acid, water, and triisobutylsilane (TIS) (20 ml, 95/2.5/2.5) for 8 hr at RT. Remove the resin by filtration followed by washing with trifluoroacetic acid (TFA). Add the combined filtrates dropwise to cold ethyl ether (200 ml), centrifuge, and wash with ethyl ether 4x. Dry under vacuum to give a colorless solid (1.99 g, 61% yield).</li>
	<li>Purify the crude product with preparative HPLC using a gradient of 0-40% solvent B (0.1% TFA in acetonitrile) in solvent A (0.1% TFA aqueous solution) for 20 min and 40-90% solvent B in solvent A for 10 min.</li>
	<li>Dissolve CLT1-dL-(DOTA)4 (250 mg, 0.077 mmol) in DI water (15 ml) and adjust the pH to 6 using 1 M NaOH. Add Gd(OAc)3.4H2O (472 mg, 0.462 mmol, 1.5 equivalents to DOTA monoamide) in portions to the solution, while maintaining the pH at 6 using 1 M NaOH. Stir the reaction solution at RT for 48 hr.</li>
	<li>Complex the residual Gd(III) with ethylenediaminetetraacetic acid (EDTA) (90 mg, 0.31 mmol), and purify the crude product with a size exclusion column to give the final product CLT1-dL-(Gd-DOTA)4 (169 mg, 57%).</li>
</ol>
2. Characterization of Contrast Agents
<ol>
	<li>Measure Gd(III) content with inductively coupled plasma-optical emission spectroscopy.</li>
	<li>Acquire matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra in linear mode with 2,5-dihydroxybenzoic acid (2,5-DHB) as a matrix.</li>
	<li>Measure relaxation times of the aqueous solution of the contrast agents of CLT1-dL-(Gd-DOTA)4 and a nontargeted control agent sCLT1-dL-(Gd-DOTA)4 with different concentrations at 60 MHz (1.5 T) with a relaxometer at 37 &#176;C. Use an inversion-recovery pulse sequence to measure T1 and a Carr-Purcell-Meiboom-Gill sequence with 500 echoes to measure T2. Calculate T1 and T2 relaxivities of the agents from the slopes of the plots of 1/T1 and 1/T2 versus the Gd concentrations.</li>
</ol>
3. Cell Culture and Development of Animal Model with Orthotopic PC3 Prostate Tumor
<ol>
	<li>Culture PC3 prostate cancer cells with constitutive expression of green fluorescence protein (GFP) in RPMI medium supplemented with 5% fetal bovine serum and penicillin/streptomycin/fungizone, harvest by trypsinization and resuspend at density of 2.5 x 104 cells/&#956;l PBS.</li>
	<li>Maintain male NIH athymic nude mice (4-5 weeks old) at the Athymic Animal Core Facility, Case Western Reserve University (CWRU) according to an animal protocol approved by the CWRU Institutional Animal Care and Use Committee.</li>
	<li>The surgical surfaces were sprayed with 2% chlorhexidine solution and then covered with absorbent drapes. Sterile surgical gloves were worn during the procedure. Before animal surgery, i.p. inject Avertin (250 mg/kg) to anesthetize the mice. Alternatively, ketamine/xylazine /acepromazine can be used at a concentration of 50/5/1 mg/kg. Ophthalmic ointment was applied to the mouse to maintain adequate moisture during the anesthesia.</li>
	<li>Start the surgery by cutting a small incision through the skin and peritoneum along the lower midline at a length of about 1 cm using a sterile scalpel. A sterile surgical drape was covered around the incision site during survival surgeries.</li>
	<li>Gently exteriorize and stabilize the prostate dorsal lobes to expose the prostate. Inject the suspension of PC3-GFP cells in PBS (20 &#956;l) into the prostate with a 30 gauge needle. Finally close the incision with wound autoclip12.</li>
	<li>For post-procedure monitoring, monitor animals twice per day for 1 week. Signs of illness include lack of eating, dark urine collecting around urethra, and unsteady gait which could indicate infection or occlusion of the bladder in the staples.</li>
	<li>After 10 days, remove the staples using a staple remover. Monitor the animals daily to assess tumor size and any evidence of pain such as weight loss, behavioral deviations, and motor defects. Euthanize the animals which showed a tumor size exceeding the ethically allowed limit or evidence of pain during tumor growth.</li>
</ol>
4. Confirmation of Tumor Binding Specificity of the Peptides with Fluorescence Imaging and Histology
<ol>
	<li>After tumor cell inoculation, allow approximately 4 weeks of tumor growth before beginning with the imaging. Prior to injection of the peptide probes, acquire GFP fluorescence images with live mice on a fluorescence imager to verify the presence of tumor.</li>
	<li>I.v. inject Texas Red labeled peptides to the tumor bearing mice at a dose of 10 nmol/mouse.</li>
	<li>Sacrifice the mice 2 hr later by cervical dislocation, collect the tumor and major organs, and immediately image on a fluorescence imager. Use green light filters for GFP (excitation: 444-490 nm; emission: 515 nm long-pass filter; acquisition settings: 500-720 in 10 nm steps) and red light filters for Texas Red (excitation: 576-621 nm; emission: 635 nm long-pass filter; acquisition settings: 630-800 in 10 nm steps). 10 msec exposure time for GFP and 150 msec for Texas Red.</li>
	<li>Immediately after measurement of whole tissue fluorescence, collect tumor tissues, fix with formalin, and cryosection into 5-&#956;m slices.</li>
	<li>After rinsing with PBS, mount with 1 drop of mounting medium containing 4&#39;,6-diamidino-2-phenylindole (DAPI), image immediately on a confocal laser scanning microscope.</li>
</ol>
5. Magnetic Resonance Imaging (MRI)
<ol>
	<li>Approximately 4 weeks after tumor cell inoculation, allow the tumors to grow up to 0.3-0.6 cm in diameter. Use a 7 T MRI scanner with a volume radio frequency (RF) coil for MRI study.</li>
	<li>Anesthetize the mouse with a 2% isoflurane-oxygen mixture in an isoflurane induction chamber. Ophthalmic ointment was applied to the mouse to maintain adequate moisture during the anesthesia.</li>
	<li>Insert a 30 gauge needle into a 1.0 m long tubing filled with heparinized saline. Place the self-made catheter in the mouse tail vein.</li>
	<li>Place the mouse into the magnet and keep under inhalation anesthesia with 1.5% isoflurane-oxygen via a nose cone.</li>
	<li>Place a respiratory sensor connected to a monitoring system on the abdomen to monitor rate and depth of respiration. Maintain the body temperature at 37 &#176;C by blowing hot air into the magnet through a feedback control system.</li>
	<li>Begin with sagittal section images using a localizing sequence to identify the tumor location (TR/TE = 200/3.7 msec, FOV = 3.0 cm, slice thickness = 2.2 mm, slice number = 16, average = 2, flip angle = 45&#176;, matrix = 128 x 128).</li>
	<li>Use a 2D T1-weighted gradient fat suppression sequence to acquire 2D axial images for CE-MRI (TR/TE = 151.2/1.9 msec, FOV = 3.0 cm x 3.0 cm, slice thickness = 1.2 mm, slice number = 12, average = 1, flip angle = 80&#176;, matrix = 128 x 128).</li>
	<li>After pre-injection baseline MR image acquisition, begin to inject the targeted agent or control agent at a dose of 0.03 mmol Gd/kg by flushing with 200 &#956;l of saline.</li>
	<li>Continue to acquire CE-MRI images at different time points for up to 30 min.</li>
</ol>
6. Image Processing and Analysis
<ol>
	<li>Use imaging software for image analysis.</li>
	<li>Draw the regions of interest (ROIs) over the whole tumor and the kidneys in the two-dimensional imaging plane and measure average signal intensity.</li>
	<li>To quantify the CE-MRI data, measure tumor or kidney contrast enhancement (&#916;SNR) by calculation of the increase in post-contrast SNR over pre-contrast SNR using the following equation: &#916;SNR = (St/&#963;t) - (S0/&#963;0), where S0 and St denote the signal in the tumor or the kidneys before and after contrast, and &#963;0 and &#963;t are the standard deviation of noise measured from the background air before and after contrast.</li>
	<li>Calculate the p values using the student&#39;s two-tailed t-test, assuming statistical significance at p&#60;0.05.</li>
</ol>

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Imaging Biol. 13, 653-662 (2011).</li><li>Schmieder, A. H., Winter, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+MR+imaging+of+melanoma+angiogenesis+with+alphanubeta3-targeted+paramagnetic+nanoparticles.">Molecular MR imaging of melanoma angiogenesis with alphanubeta3-targeted paramagnetic nanoparticles.</a> Magn. Reson. Med. 53, 621-627 (2005).</li><li>Mulder, W. J., Strijkers, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+molecular+imaging+and+fluorescence+microscopy+for+identification+of+activated+tumor+endothelium+using+a+bimodal+lipidic+nanoparticle.">MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle.</a> FASEB J. 19, 2008-2010 (2005).</li><li>Wang, S. 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No conflicts of interest declared.

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...

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.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>Fmoc protected amino acids</td><td>EMD Chemicals Inc</td><td></td><td></td></tr><tr><td>DOTA-tris(&lt;em&gt;t&lt;/em&gt;-Bu)</td><td>TCI America</td><td></td><td></td></tr><tr><td>PyBOP, HOBt, HBTU</td><td>Nova Biochem</td><td></td><td></td></tr><tr><td>DIPEA, Thallium(III) trifluoroacetate, TIS</td><td>Sigma-Aldrich Corp.</td><td></td><td></td></tr><tr><td>Texas Red, succinimidyl ester, single isomer</td><td>Invitrogen</td><td>T20175</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENTS&lt;/strong&gt;</td></tr><tr><td>Agilent 1100 HPLC system</td><td>Agilent</td><td></td><td></td></tr><tr><td>ZORBAX 300SB-C18 PrepHT column</td><td>Agilent</td><td></td><td></td></tr><tr><td>ICP-&amp;#8211;S Optima 3100XL</td><td>Perkin-Elmer</td><td></td><td></td></tr><tr><td>MALDI-TOF mass spectrometer</td><td>Bruker</td><td>AutoflexTM Speed</td><td></td></tr><tr><td>Maestro FLEX In Vivo Imaging System</td><td>Cambridge Research Instrumentation, Inc.</td><td></td><td></td></tr><tr><td>Biospec 7T MRI scanner</td><td>Bruker</td><td></td><td></td></tr></tbody></table>

mr molecular imaging of prostate cancer with a small molecular clt1 peptide targeted contrast agent

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.

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...

Watch this Scientific Journal Video about MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent at JoVE.com

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

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 ...

mr-molecular-imaging-prostate-cancer-with-small-molecular-clt1

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11.1K Views.  Case Western Reserve University. 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.

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

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

Cancer Research

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bioengineering

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Chemistry

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Use of MRI-ultrasound Fusion to Achieve Targeted Prostate Biopsy

Here, we present a protocol to perform targeted prostate biopsy using a magnetic resonance imaging-ultrasound (MRI/US) fusion system. Prostate cancer has traditionally been diagnosed via transrectal ultrasound (TRUS) biopsy. Though considered the gold standard, TRUS is unable to visualize most prostate cancer lesions and therefore requires sampling of the entire prostate. This biopsy method often undergrades prostate cancer and fails to detect up to 35% of cancers on initial biopsy. Prostate MRI has been shown to have excellent sensitivity in the detection of cancerous lesions, and advancements in MRI technology during the last decade have led to the development of targeted biopsy. In targeted biopsy, a software platform overlays MRI data onto live TRUS images to create a fused MRI/US three-dimensional model of the prostate. Regions suspicious for malignancy on MRI are contoured by a radiologist, uploaded into the fusion system, and then displayed within the live MRI/US fused model. The urologist is then able to directly biopsy these targets. When compared to conventional TRUS biopsy, MRI/US fusion technology has been demonstrated to improve the detection of clinically significant cancer while reducing insignificant cancer detection. This technology, therefore, has the potential to diagnose prostate cancer primarily in men who would benefit from treatment.

Presented herein is a protocol to perform targeted biopsy of the prostate using an MRI-ultrasound fusion system.

Here, we present a protocol to perform targeted prostate biopsy using a magnetic resonance imaging-ultrasound (MRI/US) fusion system. Prostate cancer has ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Biology

A Cognitive Fusion-guided Prostate Biopsy Using Multiparametric Magnetic Resonance Imaging and Transrectal Ultrasound

Traditional transrectal ultrasound (TRUS)-guided prostate biopsy has limited sensitivity and specificity, particularly for detecting early-stage prostate cancer, due to a lack of precise lesion targeting. An improved cognitive fusion-guided prostate biopsy method has been developed to enhance lesion targeting by integrating three parameters of prostate multiparametric MR (mpMRI) images into TRUS images. Prostate mpMRI measurement is initially performed to obtain three key parameters: the rotation angle (&#945;), the distance from the rectal wall (X), and the distance from the prostate apex (Y). These parameters are then cognitively applied in real-time, TRUS-guided prostate needle biopsy to detect target lesions. This improved transperineal cognitive fusion biopsy method enhances diagnostic accuracy, improves reproducibility, and reduces reliance on operator experience. Clinical application in 423 patients demonstrated a prostate cancer detection rate of 73.5%, with 62.9% classified as clinically significant cancers. Compared with equipment-intensive methods such as MRI-ultrasound fusion biopsy, this approach is cost-effective, practical, and well-suited for broader clinical adoption. Additionally, the method&#39;s flexibility supports integration with other imaging techniques, such as 68Ga-PSMA PET/CT, further improving detection rates for patients with high-risk prostate cancer.

Prostate biopsy is the gold standard diagnostic method for prostate cancer. Cognitive fusion-guided prostate biopsy, which combines transrectal ultrasound with pre-measured MRI parameters, improves biopsy accuracy and enhances the detection rate of clinically significant prostate cancer.

Traditional transrectal ultrasound (TRUS)-guided prostate biopsy has limited sensitivity and specificity, particularly for detecting early-stage prostate ...

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

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