Name: hyperpolarized 13c metabolic magnetic resonance spectroscopy and imaging
Uploaded: 2016-12-30T13:00:00
Description: Watch this Scientific Journal Video about Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging at JoVE.com

E.K. gratefully acknowledges the support of the Graduate School of Bioengineering (GSB) at Technische Universit&#228;t M&#252;nchen. This work was supported by the German Research Foundation (DFG) within the SFB Collaborative Research Center 824, "Imaging for Selection, Monitoring, and Individualization of Cancer Therapies."

1. Sample Stock Solution Preparation
<ol>
	<li>Add gadoterate meglumine (GadM, 0.5 mol/L) to concentrated [1-13C]pyruvic acid to give a final concentration of 1-mmol/L GadM. Add trityl radical tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2-4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063) to this mixture to give a final concentration of 15 mmol/L. Vortex until complete dissolution. 
	NOTE: This stock solution preparation is designed for usage with a 3.35-T preclinical DNP hyperpolarizer....

13CMRSI with hyperpolarized probes is a promising method to monitor metabolism in real time in vitro and in vivo. One very important aspect when employing this experimental process is the proper standardization, especially regarding in vitro experiments. First, the preparation of the sample needs to be done properly and consistently to achieve the same concentration of hyperpolarized material in each experiment. This requires a precise weighing of both the sample to be hyperpolarized...

Presently, the most widely used clinical method for tumor staging, restaging, treatment response monitoring, and recurrence detection of a wide variety of cancers is [18F]-FDG PET.1 However, recently, several novel and alternative approaches have emerged. One of those methods is 13CMRSI. This technique involves the introduction of the 13C-molecule into a biological sample, followed by minimally invasive MRI to assess the metabolism in vitro or in vivo in real time. Nevertheless, the biggest challenge of 13CMRSI, compared to the other methods such as [18F]-FDG PET or computed tomography, is its low signal-to-noise ratio.
The NMR signal is directly proportional to the level of polarization, a ratio of the spin &#189; nuclei population difference in two energy states to the total population (Figure 1A). The polarization is a product of the gyromagnetic ratio (&#947;) of the nuclei and the applied magnetic field strength over the temperature. A typical polarization of 1H nuclei is in the order of 0.001% to 0.005% at 3 T, which gives a relatively poor signal-to-noise ratio. Today&#39;s state-of-the-art MRI has been a successful imaging method only due to the high abundance of 1H in biological samples and the high gyromagnetic ratio of 1H (&#947;1H = 42.576 MHz/T). However, observing other nuclei, such as carbon, is more demanding. The only stable, magnetically active carbon isotope, 13C, makes up only 1.1% of all carbon atoms. In addition, the gyromagnetic ratio of 13C (&#947;13C = 10.705 MHz/T) is four times lower than that of 1H, leading to a lower detection efficiency. In summary, the low 13C abundance and low &#947;13C cause thermal 13C measurements to achieve 0.0176% of the sensitivity of a 1H-NMR measurement in vivo.
Dynamic Nuclear Polarization
A method to overcome the relatively poor sensitivity of 13C measurements is DNP. It was originally described for metals in 1953 by Albert W. Overhauser. In his article, he stated: &#34;It is shown that if the electron spin resonance of the conduction electrons is saturated, the nuclei will be polarized to the same degree they would be if their gyromagnetic ratio were that of the electron spin.&#34;2 Later that year, Carver and Slichter experimentally confirmed Overhauser&#39;s hypothesis3. In 1958, Abragam and Proctor described this effect for electrons in liquids and named it the &#34;solid effect.&#34; At temperatures below 4 K, electron-spin polarization reaches nearly 100% and is more than three orders of magnitude higher than the nuclear-spin polarization (Figure 1B)4. This occurs because the gyromagnetic ratio of the electron (&#947;e = 28024.944 MHz/T) is three orders of magnitude higher than the nuclear gyromagnetic ratios. The weak interactions between electrons and nuclei, such as the Overhauser effect, the solid effect, the cross effect, and the thermal mixing effect, allow the transfer of polarization from electron spins to nuclear spins using microwave irradiation with a frequency close to the corresponding electron paramagnetic resonance (EPR) frequency5,6. DNP theory has been further developed to involve more electrons and thermal mixing. Nevertheless, to date, no unified quantitative theoretical description of DNP has been published7,8.
<img alt="Figure 1" src="/files/ftp_upload/54751/54751fig1.jpg" /> 
Figure 1: Understanding Dynamic Nuclear Polarization and Hyperpolarization. A) A schematic comparison of the spin population in the thermal equilibrium polarization state and the hyperpolarized state. B) The polarization is dependent upon temperature. The polarization of an electron (e-) reaches 100% below 1.4 K. The DNP allows the transfer of the polarization from the e- to the 13C nuclei, which increases their polarization up to 105-fold. <a href="http://cloudflare.jove.com/files/ftp_upload/54751/54751fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To introduce DNP in studies of biological systems using 13C NMR, subsequent rapid sample dissolution had to be developed. 50 years after Overhauser&#39;s hypothesis, Jan H. Ardenkjaer-Larsen et al. solved the technically challenging issue of bringing the hyperpolarized frozen sample into the liquid state with minimal hyperpolarization loss6. Dissolution DNP opened a new field of research called 13CMRSI, providing a new method to investigate and characterize various disease states9,10. As stable carriers of an unpaired electron, a trityl radical tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2-4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063) or (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO) is usually used. These are mixed with the desired 13C-labeled molecule and exposed to microwave irradiation with a frequency close to the corresponding EPR frequency. Using this technique, the polarization of 13C nuclei can be increased up to 37%11. This results in a 105-fold polarization enhancement compared to the thermal equilibrium polarization11,12. However, as soon as the microwave irradiation is stopped and/or the 13C-molecule is transferred to the liquid state, the polarization decays with the longitudinal relaxation time (T1) of the 13C nucleus that was polarized. Thus, the invention of fast dissolution techniques or any subsequent technique shortening the time before experimental measurement (i.e., injection) is crucial for biological applications13.
There are three major requirements that the candidate molecule needs to fulfill for successful 13CMRSI studies. First, the 13C nucleus of interest has to have a sufficiently long T1 (&#62; 10 s). The choice of the 13C-label is crucial. The best candidate nuclei are carbons with no direct contact with 1H-nuclei via a bond. It also needs to be rapidly metabolized within 2 - 3 T1 times, resulting in a downstream metabolic product with a significantly different chemical shift from the original substance. The sample mixture must also form an amorphous glass when in a solid state so that the spatial distribution decreases the distance between the electron and 13C, allowing the transfer of polarization. If the candidate molecule does not form amorphous glass naturally, it needs to be highly soluble in a glassing agent, such as glycerol or dimethyl sulfoxide14. These requirements result in a relatively small number of candidate molecules. However, even after the successful discovery of a suitable molecule, developing a working protocol for hyperpolarization can be technically challenging9,14,15.
In recent years, several substrates have been successfully polarized, such as [1-13C]pyruvate12,16-36, [2-13C]pyruvate37, [1-13C]ethyl pyruvate38, [1-13C]lactate39, [1-13C]fumarate40-43, 13C-bicarbonate36,44,45, [1-13C]sodium acetate43,46-49, 13C-urea6,36,50,51, [5-13C]glutamine15,52,53, [1-13C]glutamate53,54, [1-13C]2-oxoglutarate55, [1-13C]alanine, and others14,56. A particularly interesting and commonly used substrate for hyperpolarization is [1-13C]pyruvate. It is widely used in preclinical studies to investigate the cellular energy-metabolism in various diseases14,17,22. [1-13C]pyruvate meets all the requirements for successful hyperpolarization, including a relatively long T1 and rapid transport across the cell membrane before subsequently being metabolized. Preclinical studies with [1-13C]pyruvate are currently being translated into the clinic57.
Metabolism of Pyruvate
It is well known that there is a direct link between mutations in a cancer cells&#39; DNA and changes in their metabolic pathways. Already in the 1920s, Otto Warburg discovered that there is an increased metabolism of glucose and production of lactate in tumors compared to healthy tissue58-60. Subsequently, various alternations in other metabolic pathways, such as the pentose-phosphate pathway, the tricarboxylic acid cycle, oxidative phosphorylation, and the synthesis of nucleotides and lipids, have been described.
Pyruvate is the final product of glycolysis. In the tumor, it undergoes anaerobic glycolysis catalyzed by LDH61 and reacts with the reduced form of the coenzyme nicotinamide adenine dinucleotide (NADH), resulting in lactate and the oxidized form of the coenzyme (NAD+). Alternatively, pyruvate undergoes a transamination reaction with glutamate to form alanine, catalyzed by alanine transaminase (ALT). Both reactions are readily reversible. Pyruvate also undergoes decarboxylation catalyzed by pyruvate dehydrogenase (PDH) to carbon dioxide and acetyl-CoA, representing an irreversible reaction at this step. Alternations in these reaction rates can be linked to tumor metabolism17,21,22,25,62. The metabolic pathways are summarized in Figure 2.
<img alt="Figure 2" src="/files/ftp_upload/54751/54751fig1.jpg" /> 
Figure 2: Diagram of the major metabolic reaction of pyruvate. Pyruvate/lactate conversion is catalyzed by LDH, and pyruvate/alanine conversion is catalyzed by ALT. Pyruvate is irreversibly converted to acetyl-CoA and CO2 by PDH, and CO2 is in a pH-dependent equilibrium with bicarbonate80. <a href="http://cloudflare.jove.com/files/ftp_upload/54751/54751fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The detection of hyperpolarized [1-13C]pyruvate and its metabolites has been previously demonstrated in the rat heart37,63-65, liver66, muscle, and kidney62,67. One study demonstrated significant differences in the lactate-to-alanine ratio between the normal and fasted rat liver66 and demonstrated a highly elevated and hyperpolarized [1-13C]lactate level in liver cancer68,69. There is evidence that the tumor grade can be identified in a transgenic adenocarcinoma of mouse prostate (TRAMP) using hyperpolarized [1-13C]pyruvate22, with the hyperpolarized lactate levels showing a high correlation with the histological grade of the excised tumors. The alanine catalyzed from pyruvate by ALT has also been suggested as a useful marker in rat hepatocellular carcinoma23.
Measuring the pyruvate-lactate metabolic flux has been used for monitoring ischemia63,65,70 and as a response to treatment with cytotoxic chemotherapy17,40, targeted drugs24,25,41, or radiotherapy26 in animal models. It has also been used for the detection of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 response in glioblastoma and breast cancer mouse models25. Changes in pyruvate metabolism in brain tumors26 and prostate cancer24,71 have also been observed after treatment.
Prostate Carcinoma
Prostate carcinoma is the predominant cancer in elderly men and the second leading cancer related to death in men worldwide72. To date, no reliable, non-invasive methods are available for an early diagnosis and characterization of prostate cancer73,74, emphasizing the urgent need for novel metabolic imaging techniques to enable stringent detection and staging of patients. Prostate carcinoma was used as a model to demonstrate the possibilities of dissolution DNP combined with 13CMRSI in patients57. This work was continued in a first clinical trial employing [1-13C]pyruvate and 13CMRSI for the imaging of prostate cancer, and it has just recently has been completed (NCT01229618).
The motivation behind this work was to illustrate in more detail and for a wider audience the application of the 13CMRSI method in a preclinical setting with cells. Measuring the LDH-catalyzed metabolism of [1-13C]pyruvate to [1-13C]lactate in vitro in the PC3 prostate carcinoma cell line, we demonstrate the possible application of dissolution DNP in in vitro studies and address the crucial steps and challenges during experiments.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HyperSense DNP Polariser</td>
			<td style="width:375px;">Oxford Instruments</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">3.35 T preclinical DNP hyperpolarizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GE/Agilent MR901</td>
			<td style="width:375px;">GE Healthcare/Agilent Technologies</td>
			<td style="width:136px;"></td>
			<td>7 T preclinical MRI scanner, with small bore designed for experiments onrodent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Spinsolve Carbon</td>
			<td style="width:375px;">Magritek</td>
			<td style="width:136px;"></td>
			<td>1 T NMR spectrometer with permanent magnet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Deuterium Oxide</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">7789-20-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium phosphate dibasic</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium phosphate monbasic</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">7558-80-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sodium hydroxide</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">1310-73-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Disodium edetate</td>
			<td style="width:375px;">Sigma Aldrich</td>
			<td style="width:136px;">6381-92-6</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:247px;">Pyruvic acid - 13C1</td>
			<td style="width:375px;">Cambridge Isotopes Laboratories</td>
			<td style="width:136px;">CLM-8077-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Dotarem (0.5 mmol/L)</td>
			<td style="width:375px;">Guerbet</td>
			<td style="width:136px;"></td>
			<td>gadoterate meglumine&#160;&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">tris (8-carboxy-2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2&#8211;4,5]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (OX063)</td>
			<td style="width:375px;">GE Healthcare</td>
			<td style="width:136px;"></td>
			<td>trityl radical used as a sourse of free electron</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PC3 cell line</td>
			<td style="width:375px;">ATCC</td>
			<td style="width:136px;">CRL1435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F-12K medium&#160;</td>
			<td style="width:375px;">ATCC</td>
			<td style="width:136px;">30-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:375px;">ATCC</td>
			<td style="width:136px;">SCRR-30-2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsine-EDTA Solution, 1X</td>
			<td style="width:375px;">ATCC</td>
			<td style="width:136px;">30-2101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sample plastic cup</td>
			<td style="width:375px;">Oxford Instruments</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Trypan blue</td>
			<td style="width:375px;">Bio-Rad</td>
			<td>145-0013-MSDS</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The results of the &#8220;microwave sweep&#8221; are illustrated in Figure 3. It shows that the optimal microwave frequency for the [1-13C]pyruvate sample is at 94.156&#160;GHz for the local 3.35-T hyperpolarizer. All following hyperpolarization experiment (n = 14) were performed using this microwave frequency with a power of 100&#160;mW. The microwave irradiation was applied for 60 to 80 min, leading to a solid-state hyperpolarization higher than 90%. The results are presented in Figu...

Watch this Scientific Journal Video about Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging at JoVE.com

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.

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 ...

The overall goal of this procedure is to hyperpolarize carbon 13-labelled molecules using dynamic nuclear polarization, and to measure their enyzmatic conversion using carbon 13 magnetic resonance, retroscopic imaging, in vitro. This method increases the resonant signal of carbon by 10, 000 to 100, 000 fold, allowing the layered compounds to be distinguished from their metabolic products, and to be localized in the body by imaging. The main advantage of this method, compared to say, the VR techniques, is that it provides a view of the metabolism of naturally occurring substances in living organisms in real time.Also this method can provide insight into the metabolism of cancer and as it is in vitro. It can also be applied to animal models and human patients. The application of this technique exceeds tumor localization.It can be used to find and distinguish metabolic tumor sub-types, and to monitor treatment alongside state-of-the-art techniques. To begin the procedure, first prepare a one millimolar solution of gadoterate meglumine, and concentrated one-carbon 13 pyruvate. Add trityl radical OX063 to the mixture to achieve a 15 millimolar final concentration, and dissolve it completely.In the DNP Polarizer software, click Cool Down'to decrease the temperature of the variable temperature insert, or VTI, below 1.4 kelvin. Once the VTI has cooled, place about eight microliters of the carbon 13-labelled sample solution in a plastic sample cup. Attach the cup to the sample insertion rod.In the software, click Insert Sample'Select Normal Sample'and then Next'This raises the sample holder and opens the inlet valves to the VTI, allowing sample insertion. At the prompt, use the insertion rod to push the sample cup into the VTI. Detach the sample cup and carefully withdraw the insertion rod.Confirm that the sample was detached from the rod and was not pushed out of the VTI during the closing procedure. Click Next'to close the valves and lower the sample cup into the liquid helium, and then click Finish'To find an optimal polarization frequency, first start the RINMR software. In the configuration screen, click Select Sample'and Do Micro Sweep'Create the file where the data will be stored.Switch back to the DNP Polarizer software. Select the Calibrate'tab, set the microwave sweep parameters, and then start the microwave sweep process. Once the microwave sweep is finished, note the frequency with a maximal measured signal amplitude.Return to the RINMR software, and select Solid Build Up'Then, in the DNP Polarizer software, click Polarization'Enter the optimal microwave frequency and the power. Click Next'Enable Polarization Buildup Monitoring'and click Finish'Polarize the sample for a sufficient length of time to achieve over a 95%polarization. During polarization, connect a collection flask to the hyperpolarizer sample exhaust tube, and place it near the spectrometer.Set up a one tesla NMR spectrometer with the 13 carbon scan series of 110 degree RF pulses at three second intervals. During pyruvate polarization, begin cell culture preparation. Remove the cell medium from cell culture flasks.Wash the cells with about 10 milliliters of phosphate-buffered saline. Add five milliliters of trypsin to the culture flask. Incubate the culture for three to five minutes at 37 degrees celsius, and then deactivate the trypsin with about five milliliters of F12K medium containing 10%fetal calf serum by volume.Then, transfer the volume of cell suspension containing the desired number of cells to a plastic centrifuge tube. Centrifuge the cell sample at 1200 G for five minutes. Discard the supernatant and re-suspend the pallet in sufficient F12K medium with 10%FCS to achieve a final volume of 800 microliters.Transfer the suspension to a 1.5 milliliter microcentrifuge tube, and place the tube in a plastic container filled with warm water. To begin data collection via carbon 13 magnetic resonance spectroscopy, once the cell sample is prepared and the pyruvate polarization is greater than 95 percent, click Run Dissolution'in the DNP Polarizer software. Load the meted volume of dissolution agent via the top valve of the dissolution chamber.Move the dissolution stick into the active position. Confirm by clicking Next'Once the cell sample is ready for the experiment, click Finish'in the DNP Polarizer software to initialize the dissolution process. Wait until the dissolution process is finished.Add 200 microliters of the hyperpolarized pyruvate solution to 800 microliters of the cell sample. Mix well with a pipette, and then place about 600 microliters of the mixture in a five millimeter NMR tube. Load the NMR tube into the spectrometer and click Run'to start the measurement.Then, move the dissolution stick with the attached sample cup to the cleaning position and click Finish'in the DNP Polarizer software. To begin carbon 13 magnetic resonance imaging, after starting the hyperpolarization process, transfer 800 microliters of re-suspended cell sample with a cell number of at least 10 to the eighth into a three milliliter syringe. Connect the syringe to a long catheter and place the syringe on an MRI scanning bed.Make sure that the catheter is long enough to reach outside of the MRI scanner. Place a calibration phantom, containing ten molar carbon 13 urea with 10%gadoterate meglumine next to the syringe. Place a carbon 13-tuned RF receiver coil on the syringe.Insert the scanning bed into the scanner so that the object of interest is in the isocenter of the MRI scanner. Connect the connection flask to the hyperpolarizer sample exhaust tube and place it close to the end of the catheter. Run a standard three plane localization sequence and adjust the syringe position if necessary.Then, set up and run a proton T2-weighted anatomical sequence covering the syringe localization. From the resulting anatomical images, select five continuous slices of the region of interest, including the carbon 13 phantom. Set up a carbon 13 spectroscopic calibration acquisition, and select the 2D block siegert calibration sequence from the pulse sequence library.Download the pulse sequence to the scanner, and run a spectroscopic pre-scan. In the spectrum magnitude plot, set the carbon 13 calibration phantom peak to the center of the scanner frequency. Set the receiver gains to maximum and click Scan'to run the calibration sequence.Record the reported transmit gain and centric frequency. Then, set up a carbon 13 chemical shift imaging acquisition for the chosen anatomical slices. Choose the 2D echo-planar spectroscopic imaging sequence and download the pulse sequence to the scanner.Run the spectroscopic pre-scan, and adjust the centric frequency and transmit gain based on the calibration values to finish setting up the MRI spectrometer. Once the pyruvate reaches 95%polarization, click Run Dissolution'in the DNP Polarizer software and load the dissolution agent. Move the dissolution stick into the active position.Click Next'and then Finish'When the dissolved hyperpolarized sample reaches the collection flask, draw about 1 milliliter of the hyperpolarized solution into a syringe, and inject it into the catheter so that 200 microliters reaches the cell sample. Click Scan'to start the acquisition of the experimental data. Once data collection is finished, move the dissolution stick with the attached sample cup to the cleaning position, and click Finish'in the DNP NMR software.The metabolic exchange rate of pyruvate to lactate was studied in prostate carcinoma PC3 cells by monitoring the carbon 13 NMR spectroscopy signal intensities of hyperpolarized lactate and pyruvate with respect to frequency and time. The lactate kinetic values were evaluated by a simple ratio model and by a two-site exchange differential model, and normalized to the number of cells. In both models, a trend of decreasing lactate production with an increasing number of cells was observed.This is attributed to higher cell concentrations and greater viscosity of the sample. Samples with cell counts below two times ten to to the seven show a very small lactate signal to noise ratio and therefore large standard deviation values indicating that samples should contain at least that number of cells to obtain informative data. Spatial localization was achieved with MRI with a good spectral resolution allowing carbon 13 images to be acquired for specific frequency regions.The spatial distribution was determined from the raw data. The carbon 13 image could also be co-registered with the previously acquired proton image. Once mastered and properly performed this experiment can be done in less than 60 minutes.After watching this video, you should have a good understanding of how to perform a hyperpolarization of a desired molecule using the dynamic nuclear polarization technique and how to do simple NMR spectroscopy and cell imaging experiments. While attempting this procedure, it's important to be very fast. After the hyperpolarized sample leaves the polarizer, remember that you lose all your gained NMR signal in less than two minutes.In conclusion, dissolution DNP can be used for a variety of applications. This includes studying diseases such as diabetes, or looking at metabolic changes of various types of cancer, but also measuring tissue pH.

hyperpolarized-13c-metabolic-magnetic-resonance-spectroscopy

Research

JoVE Journal

Cancer Research

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...