This work was supported by the NSFC (No. U1532107) and Shanghai Jiao Tong University Biomedical Engineering project (No. YG2014MS53). The authors would like to acknowledge Jianying Li and Yan Shen for their helpful comments and technical support efforts in developing the DECT and PET/CT imaging method.

This work was performed in strict accordance with the standards established by the Guidelines for the Care and Use of Laboratory Animals of Shanghai Jiao Tong University and was approved by the laboratory Animal Ethics Committee of Ruijin Hospital.
1. Gastric Cancer Peritoneal Metastasis Animal Model
<ol>
	<li>Divide a moderately differentiated SGC-7901 human gastric cancer cell line into an SGC-7901/sFRP1 group and an SGC-7901/vector group. Culture the two groups of cells separately in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL streptomycin, and 100 &#181;g/mL penicillin at 37 &#176;C in a humidified atmosphere with 5% CO2.</li>
	<li>Use 4-6 week-old female athymic BALB/c nude mice with body weights of 25 to 30 g. Place animals under specific pathogen-free conditions in an animal facility.</li>
	<li>Divide mice randomly into the sFPR1 overexpression group and the sFPR1 empty loading group. 
	NOTE: Each group had ten nude mice; twenty mice were randomly divided into TGF-&#946;1 treatment group and TGF-&#946;1 control group, with up to 5 nude mice per animal cage.</li>
	<li>Establish the sFPR1-overexpression peritoneal metastasis xenograft models group by administering 150 &#181;L (2 x 106 cells/mL) suspensions of SGC-7901/sFRP1 cells via the abdominal cavity; SGC-7901/vector cells are administered to establish the empty loading group. 
	NOTE: Use a hemocytometer counting method to determine the concentration of cells20.</li>
	<li>Establish the peritoneal metastasis xenograft models group by administering 150 &#181;L (2 x 106 cells/mL) suspensions of SGC-7901 cells via the abdominal cavity. Administer the target therapy of TGF-&#946;1 inhibitor SB431542 after a period of two weeks of growth by intraperitoneal injection at a dose of 100 &#181;L/10 g of body weight every other day to mice in the treatment group.
	<ol>
		<li>Administer normal saline at the same dose to mice in the control group.</li>
	</ol>
	</li>
	<li>Perform the DECT and PET/CT scanning 1 day before treatment and 1 day, 7 days, 14 days, and 21 days after treatment.</li>
</ol>
2. DECT for Peritoneal Metastasis Animal Model
NOTE: The animal imaging experiment was achieved on the dual energy CT scanner (see Table of Materials). We created the related DECT imaging protocol according to the previous studies.
<ol>
	<li>Setup for DECT imaging protocol
	<ol>
		<li>On the imaging console computer, select the &#34;protocol management&#34; icon to enter the next interface, then click the &#34;protocol management&#34; option to view the protocol management screen.</li>
		<li>In the &#39;User protocol&#39; interface, select the abdomen area to enter the abdomen protocols list.</li>
		<li>Click the blank space in the protocol lists and select the &#34;New&#34; button to type the name of a new protocol: &#34;Animal DECT Scan&#34;. Press the &#34;Enter&#34; key on the keyboard and select the &#34;Scout&#34; button in the pop-up window, click &#34;OK&#34; to setup the scout series (the first series).</li>
		<li>Select the &#34;XY&#34; mode for &#39;Anatomical Reference Point&#39; and the &#34;head-first supine position&#34; for &#39;Patient Orientation&#39;. Click on the &#34;auto transfer&#34; option and select the workstation location where the image series will be uploaded. Name &#34;Scout phase&#34; in Series Description.</li>
		<li>In the &#39;View Edit&#39; screen, make sure the relevant scan parameters are set as the following: &#34;Start location&#34; and &#34;End Location&#34; options are set at &#34;S50&#34; and &#34;I50&#34; respectively, &#34;KV&#34; at &#34;100&#34;, &#34;mA&#34; at &#34;80&#34;, &#34;90&#176;&#34; for &#34;Lateral scout position&#34;, &#34;0&#176;&#34; for &#34;AP scout position&#34;, and the &#34;scout WW/WL&#34; at &#34;400/40&#34;.</li>
		<li>Next, create the second series for non-enhanced scanning. Click &#34;Create New Series&#34;, in the pop-up window to select the &#34;Axial&#34; and &#34;Create After&#34; icons.</li>
		<li>Name the series as &#34;-C phase&#34; in the Series Description and turn &#34;Show localizer&#34; on. In the Scan type interface, select the &#34;helical&#34; scan type and 0.5 s for &#34;Rotation Time&#34;, click &#34;Thick Speed&#34; option to set the parameters (Detector Coverage at 40 mm, Helical Thickness at 0.625 mm, Pitch and speed at 0.516:1/20.62, Rotation Time at 0.5 s) in the pop-up window, Interval at 0.625 mm, Gantry Tit at 0, SFOV select Small Body, kV at 100, click &#34;mA&#34; then type 600 for Manual mA.</li>
		<li>Click the &#34;Recon Parameters&#34; icon and open the &#34;Recon Option&#34; pop-up window. Select &#34;Plus&#34; in Recon Mode; click the &#34;Slice&#34; icon to select &#34;ss50 slice 50%&#34; mode in the &#39;ASiR Setup screen&#39;. Set the remaining parameters as follows: DFOV at 25 cm, R/L and A/P Center at 0 cm, Recon type select &#34;Stnd&#34;, Matrix Size at 512.</li>
		<li>Create the third scan series for enhanced scanning by repeating the step 2.1.8, name it as &#34;+C QC phase&#34; and turn &#34;Show localizer&#34; on; the first group is the setup of arterial phase series.</li>
		<li>In the &#39;Scan type&#39; interface, click on &#34;GSI (Gemstone Spectral Imaging)&#34; and select &#34;helical&#34; scan type, select the protocol &#34;GSI-52&#34; in the &#39;Abdomen GSI Preset Selection&#39; window. Set start Location and end Location according to the non-enhanced scanning.
		<ol>
			<li>Click the &#34;Recon Parameters&#34; icon and open the &#34;Recon Option&#34; pop-up window. In Recon Mode, select &#34;Plus&#34; and in &#39;GSI option&#39; click &#34;QC&#34;; the remaining parameters are the same as in step 2.1.8.</li>
		</ol>
		</li>
		<li>Click the &#34;R2&#34; icon and select &#34;YES&#34; in the &#34;Recon enabled&#34; tab. Select &#34;Thickness&#34; at 0.625 and type 0.625 for &#34;Interval&#34;. Open the &#34;Recon Option&#34; pop-up window, in Recon Mode select &#34;Plus&#34;; in GSI options, click &#34;Mono&#34; and set the keV to 70 keV; in the ASiR Setup window, select the &#34;GS40 40%&#34; mode for the GSI ASiR setup. The remaining parameters are set with step 2.1.8. Name this step as &#34;+C 70keV phase&#34;.</li>
		<li>Click the &#34;R3&#34; icon and select &#34;YES&#34; in the &#34;Recon enabled&#34; tab. Set &#34;Thickness&#34; at 1.25 and type 0.625 for &#34;Interval&#34;. Open the &#34;Recon Option&#34; pop-up window, in Recon Mode select &#34;Plus&#34; and &#34;IQ enhanced&#34;; in the GSI options, click &#34;Mono&#34;, set the keV to 70 keV and click &#34;GSI Data File&#34;; ensure the GSI ASiR setup is in accordance with step 13. The remaining parameters are set as in step 2.1.8. Name this as &#34;+C Mono phase&#34;.</li>
		<li>Click &#34;Add group&#34; to create two scan groups to represent the portal phase and the delay phase, respectively. Make sure that the &#34;Start Location&#34; and &#34;End Location&#34; ranges of each scan phase are consistent and the remaining parameters are the same as the arterial phase. Type the delay time in &#34;Preparation Group&#34;: the first group (arterial phase) at 0 s, the second group (portal phase) at 8 s, and the third group (delay phase) at 16 s.</li>
		<li>Click the &#34;Accept&#34; option to save the protocol after all the settings are done.</li>
	</ol>
	</li>
	<li>DECT imaging process
	<ol>
		<li>Select the nude mice randomly from the treatment and control groups before each scan. Place the selected animals in new cages and mark them separately.</li>
		<li>Fast the mice for 4 h with water but without food or bedding.</li>
		<li>Remove the experimental mice from the animal experiment center 1 h before the scan, and make sure the mice are placed in a new warm environment until the scan begins.</li>
		<li>Anesthetize all the experimental mice with an intraperitoneal injection of 2.5% pentobarbital sodium (1.0 mL/kg body weight) before DECT scan imaging, and confirm the depth of anesthesia by the toe pinch reflex. Use ointment on the eyes to prevent dryness while under anesthesia. 
		NOTE: Ensure that the head of each nude mouse is in the lower position when injecting the drugs, thus reducing damage to the internal organs. Pay attention to the injection site and the depth of injection. Place the tip of the syringe at a 45&#176; angle to the inside of the right/left lower abdomen, and ensure that the needle depth is such that injection into the bowel and other organs is avoided.</li>
		<li>Click the &#34;New Patient&#34; icon, input the basic information about the mouse including its patient ID and name. In the &#39;User protocol&#39;, click the &#34;abdomen protocol&#34; and select the &#34;Animal DECT Scan&#34; protocol to enter the operation interface.</li>
		<li>Once anesthesia is induced, move each mouse onto an animal fixture platform in the supine position, and fix its tail with tape to make sure it does not bend. Sterilize the tail with alcohol for subsequent contrast agent injection into the tail-vein.</li>
		<li>Move the CT scan bed so that the external positioning line laser is over the lower abdomen of the animal. Click the &#34;reset&#34; button when the positioning is completed. 
		NOTE: The placement of external positioning lines over the lower abdomen of the animal ensures that the animals are located as far as possible on the outside of the machine for easy contrast agent administration into the tail vein.</li>
		<li>Click the &#34;Confirm&#34; icon and follow the flashing order of the buttons on the keyboard to complete the scout scanning. Select the &#34;next series&#34; icon after the scout scanning is completed, and enter the interface for non-enhanced scanning.</li>
		<li>In the right screen, set the &#34;Start Location&#34; and &#34;End Location&#34; on the scout views to define the scan range. Maintain the same range in &#39;Lateral scout&#39; and &#39;AP scout&#39; and cover the entire body volume of the animal.</li>
		<li>Click the &#34;Confirm&#34; icon and follow the flashing order of the buttons on the keyboard to complete the scout scanning.</li>
		<li>Inject each mouse with iopamidol at a dosage of 0.2 mL/100 g through the tail vein. 
		NOTE: We chose to administer the contrast medium manually and keep the injection rate as stable as possible. It is most conducive to capture the early enhancement of the tumor during imaging.</li>
		<li>Click &#34;next series&#34; to perform enhanced scanning. Set &#34;Start Location&#34; and &#34;End Location&#34; as per the non-enhanced scan. Click the &#34;confirm&#34; icon and follow the flashing order of the buttons on the keyboard to complete the dynamic enhanced scans, includingarterial phase, portal phase, and delayed phase. 
		NOTE: Click the &#34;Confirm&#34; icon immediately to start scanning after the contrast agent is injected. This is essential and crucial for enhanced scanning to ensure the best image of the arterial phase is captured. However, some delay time after clicking the &#34;confirm&#34; icon can ensure that the experimental staff has withdrawn safely from the scanning room.</li>
		<li>Click &#34;End exam&#34; to exit the scanning interface after the scanning is complete; the image series will be automatically uploaded to the workstation.</li>
		<li>Place the animal into an empty cage after completion of the scan with all mice, and observe them until they have regained consciousness.Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Then transfer the mice into a clean animal room.</li>
	</ol>
	</li>
	<li>Post DECT imaging analysis
	<ol>
		<li>Locate the mice series on the DECT workstationinterface (see Table of Materials) and select the &#34;+C Mono phase&#34; series lists. Open &#34;GSI Volume Viewer&#34; and select &#34;GSI VV General&#34; protocol from the &#39;GSI Protocol Manager&#39; interface.</li>
		<li>Click the &#34;View Type&#34; active annotation at the upper-left corner of the image viewports and select &#34;coronal&#34; orientation from the drop-down menu.</li>
		<li>For one image viewport, click the &#34;Volume 1&#34; active annotation at the upper-left corner and select &#34;Mono&#34; volumes from the drop-down menu. Similarly, in another image viewport, select &#34;Iodine (Water)&#34; volumes. Click and hold the left mouse button, drag the image from &#34;Iodine (Water)&#34; viewport to &#34;Mono&#34; and check the &#34;mix the views&#34; box to get the color fused images.</li>
		<li>Click and drag from the center of the &#34;Image Scroll&#34; icon to observe images. Save the images which show positive results as color fused images.</li>
	</ol>
	</li>
</ol>
3. PET/CT for Peritoneal Metastasis Animal Model
NOTE: See the table of materials for the PET/CT imager used. We created the related PET/CT imaging protocol according to this article21.
<ol>
	<li>Setup Micro-PET/CT Imaging Protocol
	<ol>
		<li>For a whole-body CT scan, set current at 500 &#181;A, voltage at 80 kV, exposure time at 200 ms, and 240 steps for 240&#176; rotation. For X-ray detector, select resolution at &#34;low system magnification&#34; with 78 mm axial imaging field and single bed mode. Use the &#34;Common Cone-Beam Reconstruction&#34; method and select the &#34;real time reconstruction&#34; option, so that the host PC can connect with the dedicated real time reconstruction computer (Cobra) to initiate the task.</li>
		<li>For PET acquisition, in the &#34;acquire by time&#34; option set &#34;fixed scan time&#34; to 600 s (10 min). Set &#34;study isotope&#34; to F-18 and &#34;energy level&#34; to 350-650 keV.</li>
		<li>To produce the PET Histogram, set the &#34;dynamic frame&#34; as &#34;black&#34; to process data as one frame for the entire duration to achieve static scan. Set histogram type to &#34;3D&#34; and select the &#34;no scatter correction&#34; option.</li>
		<li>For PET reconstruction, reconstruct images using an OSEM3D algorithm followed by MAP or Fast MAP22 provided by PET/CT workstationsoftware (see Table of Materials).</li>
	</ol>
	</li>
	<li>Preparation before PET/CT imaging
	<ol>
		<li>Fast the mice which have undergone DECT experiments for 4 h and transfer the mice to new animal cages 30 min before imaging.</li>
		<li>Weigh the mice and record their weight.</li>
		<li>Follow the institute&#39;s safety procedures to acquire and carry the package containing radioactive materials (RAM). Use a protective shield to carry the 18F-FDG (5 mCi), and measure the radioactivity of the total 18F-FDG with a dose calibrator.</li>
		<li>Dilute the 18F-FDG with normal saline to the appropriate radioactivity of mice injection. 
		NOTE: The diluted activity of 18F-FDG should be available at 100-200 &#181;Ci/100 &#181;L for each mouse.
		<ol>
			<li>Draw 200 &#181;L 18F-FDG solution into a 1 mL syringe. Measure the radioactivity of the whole syringe with a dose calibrator and record the 18F-FDG preparation time.</li>
		</ol>
		</li>
		<li>Inject each mouse with 200 &#181;L 18F-FDG solution via the tail intravenous injection route and record the 18F-FDG injection time. After the injection of all mice, measure the residual radioactivity of the syringe with the dose calibrator immediately and record the time that the measurements were taken after completion of the injection.</li>
		<li>Calculate the injected 18F-FDG activity for each mouse by the following formula: Injected activity (&#181;Ci) = activity in syringe before injection - activity in syringe after injection.</li>
	</ol>
	</li>
	<li>PET/CT Imaging Process
	<ol>
		<li>Put the animal into an anesthesia induction chamber; anesthetize mouse using inhaled 3% Isoflurane in oxygen after the completion of 18F-FDG injection. 
		NOTE: Follow all animal welfare guidelines appropriate for operation; keep the mice warm by using the heating pad. Use ointment on eyes to prevent dryness while under anesthesia.</li>
		<li>Once anesthesia is induced, move the mouse onto the micro-CT scanning bed while maintaining continuous anesthesia and warming. Position the head of the mouse within a cone face mask that continuously delivers Isoflurane (2%) in oxygen at a flow rate of 2 L/min. Place the mouse in supine position to ensure the posture is consistent with that in DECT scans.</li>
		<li>Move the animal to the entrance of the PET/CT scanner, click the &#34;laser&#34; icon from the toolbar viewer, and use the touchpad control interface to move the bed so that the abdomen of the mouse is located at the center of the PET and CT field-of-view (FOV) during scanning. In the &#34;Laser Align&#34; window, select &#34;first scan type&#34; as &#34;CT scan&#34;, and &#34;PET acquisition included in workflow&#34; as the option.</li>
		<li>Open the &#34;Scout View&#34; window and acquire a scout view X-ray radiograph. Adjust the position of the animal bed so that the center field of view of the CT is located at the center of the mouse body.</li>
		<li>Select the protocol established previously (in step 3.1). Input the number of mice to be imaged (successively) in the pop-up window and click the &#34;Setup&#34; option, then enter the weight. Then click the &#34;Setup&#34; option again and follow the pop-up window instructions to complete the setup.</li>
		<li>Click the &#34;Start Workflow&#34; icon to start scanning.</li>
		<li>Evaluate the quality of the acquired CT and PET images after all the scans are completed. Transfer the data through the network to the post imaging analysis for further study. 
		NOTE: Adjust the window width and window level of the image to ensure that the contrast of the organs is displayed properly. Check the resolution of the organs in the images to confirm the imaging quality.</li>
		<li>Remove the animal from the imager and immediately euthanize by cervical dislocation. Use the imaging system for the next animal successively.</li>
	</ol>
	</li>
	<li>Post PET/CT imaging analysis
	<ol>
		<li>Open the PET/CT workstation software, import the CT and PET image series data into the software. In the &#34;Registration&#34; window, click the &#34;General analysis&#34; option to register CT and PET images together, and choose the &#34;Sky&#34; model under the &#34;Review&#34; window to show a perfect alignment between CT and PET images.</li>
		<li>Identify the peritoneal nodules with references provided by the co-registered images in the &#34;Region of Interest (ROI) Quantification&#34; window.</li>
		<li>In the &#34;Region of Interest (ROI) Quantification&#34; window, draw ROI with tools over the fused images, edit the size and shape of ROI, record the SUVmax value, then output and save the selected merged images.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Ferlay, 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=Cancer+incidence+and+mortality+worldwide:+sources,+methods+and+major+patterns+in+GLOBOCAN+2012.">Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.</a> Int J Cancer. 136 (5), 359-386 (2015).</li><li>Kobayashi, D., Kodera, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+chemotherapy+for+gastric+cancer+with+peritoneal+metastasis.">Intraperitoneal chemotherapy for gastric cancer with peritoneal metastasis.</a> Gastric Cancer. 20, 111-121 (2017).</li><li>Gu, W., Li, X., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-139+regulates+the+proliferation+and+invasion+of+hepatocellular+carcinoma+through+the+WNT/TCF-4+pathway.">miR-139 regulates the proliferation and invasion of hepatocellular carcinoma through the WNT/TCF-4 pathway.</a> Oncol Rep. 31 (1), 397-404 (2014).</li><li>Sugai, T., 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+analysis+of+gastric+differentiated-type+intramucosal+and+submucosal+cancers.">Molecular analysis of gastric differentiated-type intramucosal and submucosal cancers.</a> Int J Cancer. 127 (11), 2500-2509 (2010).</li><li>Shi, Y., He, B., You, L., Jablons, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+secreted+frizzled-related+proteins+in+cancer.">Roles of secreted frizzled-related proteins in cancer.</a> Acta Pharmacol Sin. 28 (9), 1499-1504 (2007).</li><li>Amin, N., Vincan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+signaling+pathways+and+cell+adhesion.">The Wnt signaling pathways and cell adhesion.</a> Front Biosci (Landmark Ed). 17, 784-804 (2012).</li><li>Jones, S. E., Jomary, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+Frizzled-related+proteins:+searching+for+relationships+and+patterns.">Secreted Frizzled-related proteins: searching for relationships and patterns.</a> Bioessays. 24 (9), 811-820 (2002).</li><li>Qu, Y., 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=High+levels+of+secreted+frizzled-related+protein+1+correlate+with+poor+prognosis+and+promote+tumourigenesis+in+gastric+cancer.">High levels of secreted frizzled-related protein 1 correlate with poor prognosis and promote tumourigenesis in gastric cancer.</a> Eur J Cancer. 49 (17), 3718-3728 (2013).</li><li>Pan, Z., 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=Determining+gastric+cancer+resectability+by+dynamic+MDCT.">Determining gastric cancer resectability by dynamic MDCT.</a> Eur Radiol. 20 (3), 613-620 (2010).</li><li>Pan, Z., 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=Gastric+cancer+staging+with+dual+energy+spectral+CT+imaging.">Gastric cancer staging with dual energy spectral CT imaging.</a> PLoS One. 8 (2), 53651 (2013).</li><li>Kim, M. J., Hong, J. H., Park, E. S., Byun, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+metastasis+from+primary+lung+adenocarcinoma+mimicking+primary+gastric+cancer.">Gastric metastasis from primary lung adenocarcinoma mimicking primary gastric cancer.</a> World J Gastrointest Oncol. 7 (3), 12-16 (2015).</li><li>Maeda, H., Kobayashi, M., Sakamoto, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+treatment+of+malignant+ascites+secondary+to+gastric+cancer.">Evaluation and treatment of malignant ascites secondary to gastric cancer.</a> World J Gastroenterol. 21 (39), 10936-10947 (2015).</li><li>Bensinger, S. J., Christofk, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+aspects+of+the+Warburg+effect+in+cancer+cell+biology.">New aspects of the Warburg effect in cancer cell biology.</a> Semin Cell Dev Biol. 23 (4), 352-361 (2012).</li><li>Smyth, E., 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=A+prospective+evaluation+of+the+utility+of+2-deoxy-2-[(18)+F]fluoro-D-glucose+positron+emission+tomography+and+computed+tomography+in+staging+locally+advanced+gastric+cancer.">A prospective evaluation of the utility of 2-deoxy-2-[(18) F]fluoro-D-glucose positron emission tomography and computed tomography in staging locally advanced gastric cancer.</a> Cancer. 118 (22), 5481-5488 (2012).</li><li>Oka, S., Uramoto, H., Shimokawa, H., Iwanami, T., Tanaka, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+Ki-67,+but+not+proliferating+cell+nuclear+antigen,+predicts+poor+disease+free+survival+in+patients+with+adenocarcinoma+of+the+lung.">The expression of Ki-67, but not proliferating cell nuclear antigen, predicts poor disease free survival in patients with adenocarcinoma of the lung.</a> Anticancer Res. 31 (12), 4277-4282 (2011).</li><li>Zhao, C. H., Bu, X. M., Zhang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypermethylation+and+aberrant+expression+of+Wnt+antagonist+secreted+frizzled-related+protein+1+in+gastric+cancer.">Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer.</a> World J Gastroenterol. 13 (15), 2214-2217 (2007).</li><li>Cheson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+functional+imaging+in+the+management+of+lymphoma.">Role of functional imaging in the management of lymphoma.</a> J Clin Oncol. 29 (14), 1844-1854 (2011).</li><li>Fuster, D., 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=Preoperative+staging+of+large+primary+breast+cancer+with+[18F]fluorodeoxyglucose+positron+emission+tomography/computed+tomography+compared+with+conventional+imaging+procedures.">Preoperative staging of large primary breast cancer with [18F]fluorodeoxyglucose positron emission tomography/computed tomography compared with conventional imaging procedures.</a> J Clin Oncol. 26 (29), 4746-4751 (2008).</li><li>Lin, H., 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=Secreted+frizzled-related+protein+1+overexpression+in+gastric+cancer:+Relationship+with+radiological+findings+of+dual-energy+spectral+CT+and+PET-CT.">Secreted frizzled-related protein 1 overexpression in gastric cancer: Relationship with radiological findings of dual-energy spectral CT and PET-CT.</a> Scientific Reports. 7, 42020 (2017).</li><li>Cadena-Herrera, D., 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=Validation+of+three+viable-cell+counting+methods:+Manual,+semi-automated,+andautomated.">Validation of three viable-cell counting methods: Manual, semi-automated, andautomated.</a> Biotechnol Rep (Amst). 7, 9-16 (2015).</li><li>Wang, X., Minze, L. J., Shi, Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+brown+fat+in+mice+with+18F-FDG+micro-PET/CT.">Functional imaging of brown fat in mice with 18F-FDG micro-PET/CT.</a> J Vis Exp. (69), (2012).</li><li>Grootjans, W., 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=Performance+of+3DOSEM+and+MAP+algorithms+for+reconstructing+low+count+SPECT+acquisitions.">Performance of 3DOSEM and MAP algorithms for reconstructing low count SPECT acquisitions.</a> Z Med Phys. 26 (4), 311-322 (2016).</li><li>Chang, H. R., 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=Improving+gastric+cancer+preclinical+studies+using+diverse+in+vitro+and+in+vivo+model+systems.">Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems.</a> BMC Cancer. 16, 200 (2016).</li><li>Chang, H. R., 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=HNF4alpha+is+a+therapeutic+target+that+links+AMPK+to+WNT+signalling+in+early-stage+gastric+cancer.">HNF4alpha is a therapeutic target that links AMPK to WNT signalling in early-stage gastric cancer.</a> Gut. 65 (1), 19-32 (2016).</li><li>Zheng, H. C., 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=BTG1+expression+correlates+with+pathogenesis,+aggressive+behaviors+and+prognosis+of+gastric+cancer:+a+potential+target+for+gene+therapy.">BTG1 expression correlates with pathogenesis, aggressive behaviors and prognosis of gastric cancer: a potential target for gene therapy.</a> Oncotarget. 6 (23), 19685-19705 (2015).</li><li>Yamada, A., Oguchi, K., Fukushima, M., Imai, Y., Kadoya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+2-deoxy-2-[18F]fluoro-D-glucose+positron+emission+tomography+in+gastric+carcinoma:+relation+to+histological+subtypes+depth+of+tumor+invasion,+and+glucose+transporter-1+expression.">Evaluation of 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography in gastric carcinoma: relation to histological subtypes depth of tumor invasion, and glucose transporter-1 expression.</a> Ann Nucl Med. 20 (9), 597-604 (2006).</li><li>Hirose, Y., 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=Relationship+between+2-deoxy-2-[(18)F]-fluoro-d-glucose+uptake+and+clinicopathological+factors+in+patients+with+diffuse+large+B-cell+lymphoma.">Relationship between 2-deoxy-2-[(18)F]-fluoro-d-glucose uptake and clinicopathological factors in patients with diffuse large B-cell lymphoma.</a> Leuk Lymphoma. 55 (3), 520-525 (2014).</li><li>Tchou, 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=Degree+of+tumor+FDG+uptake+correlates+with+proliferation+index+in+triple+negative+breast+cancer.">Degree of tumor FDG uptake correlates with proliferation index in triple negative breast cancer.</a> Mol Imaging Biol. 12 (6), 657-662 (2010).</li><li>Coleman, R. E., 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=Concurrent+PET/CT+with+an+integrated+imaging+system:+intersociety+dialogue+from+the+Joint+Working+Group+of+the+American+College+of+Radiology+the+Society+of+Nuclear+Medicine,+and+the+Society+of+Computed+Body+Tomography+and+Magnetic+Resonance.">Concurrent PET/CT with an integrated imaging system: intersociety dialogue from the Joint Working Group of the American College of Radiology the Society of Nuclear Medicine, and the Society of Computed Body Tomography and Magnetic Resonance.</a> J Am Coll Radiol. 2 (7), 568-584 (2005).</li><li>Brepoels, L., 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=Effect+of+corticosteroids+on+18F-FDG+uptake+in+tumor+lesions+after+chemotherapy.">Effect of corticosteroids on 18F-FDG uptake in tumor lesions after chemotherapy.</a> J Nucl Med. 48 (3), 390-397 (2007).</li><li>Spaepen, K., 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=[18)F]FDG+PET+monitoring+of+tumour+response+to+chemotherapy:+does+[(18)F]FDG+uptake+correlate+with+the+viable+tumour+cell+fraction.">[18)F]FDG PET monitoring of tumour response to chemotherapy: does [(18)F]FDG uptake correlate with the viable tumour cell fraction.</a> Eur J Nucl Med Mol Imaging. 30 (5), 682-688 (2003).</li></ol>

The authors have no conflicts of interest to declare.

Animal models have been used widely in the study of molecular mechanisms underlying gastric cancer, and to experiment with various therapeutic strategies23,24,25. In this study, we have described a detailed protocol for gastric cancer peritoneal metastasis nude mice modeling, using DECT and PET/CT to image gastric tumors for identifying tumor cell proliferation in real-time, and monitoring peritoneal metastasis and responses to ...

Gastric cancer (GC) remains the fourth most common malignancy and the second-leading cause of cancer mortality worldwide1. Although the accuracy in diagnosis and treatment of gastric cancer has been greatly improved, peritoneal metastasis is the most key point of gastric cancer prognosis or recurrence and is a definitive determinant of postoperative death2. It is generally accepted that peritoneal dissemination is a life-threatening mode of metastasis, wherein the disease becomes uncontrollable and the prognosis of the patient is poor once peritoneal dissemination is established. Therefore, the detection and therapeutic effect evaluation of gastric cancer peritoneal metastasis is crucial for clinical practice.
The increasing incidence and mortality of gastric cancer had spurred researchers to identify its molecular mechanisms. The high expression of genes such as secreted frizzled-related protein 1 (sFRP1) might lead to activation of the signal pathway in the early stages of gastric cancer, promoting the process of tumor growth, proliferation, differentiation, and apoptosis3,4,5,6,7. sFRP1-overexpression cells showed an increase in the expression of TGF&#946;, its downstream targets, and TGF&#946;-mediated EMT8. Previous studies have demonstrated that the TGF-&#946;1 level is correlated with peritoneal metastasis and the TNM stages of gastric cancer. We have described the changes in cancer cell proliferation regulated by sFRP1 overexpression and TGF-&#946;1 inhibition, and established animal models for peritoneal metastasis to show the performance of tumor imaging under the effects of gene regulation.
Animal models for gastric cancer are indispensable tools for researching tumor development and experimenting with various therapeutic strategies without having to sacrifice animals. Animal models have proven useful in studying the formation mechanisms of tumors and cells of origin, determining the presence of cancer stem cells, and examining various novel therapeutic strategies. Therefore, a real-time non-invasive technique can provide an accurate description of the development of gastric tumors and tumor response to treatments, which can identify the development of peritoneal metastasis nodules in nude mice and monitor the changes of a tumor in response to various experimental and therapeutic interventions.
Currently, multi-detector CT (MDCT) plays an important role in the TNM staging of gastric cancers and is useful for predicting tumor resectability preoperatively9. However, radiological studies of patients with histologically proven gastric carcinoma have mainly been based on morphology. DECT imaging extends the parameters to reflect functional information by providing monochromatic images and may be helpful for improving the N staging accuracy for gastric cancers. Furthermore, this technique will enable the acquisition of material-decomposition images, which may be useful to differentiate between differentiated and undifferentiated gastric carcinoma, and between metastatic and non-metastatic lymph nodes10. With the introduction of DECT, the functional imaging aspect of CT has also been added to clinical applications, contributing to evaluations of therapeutic efficacy and predicting patient prognoses11,12,13. PET/CT is a useful imaging technique for the detection and staging of gastric cancer and can evaluate the recurrence of the tumor effectively14. Tumor cell proliferation and angiogenesis were both considered to be necessary in the development of a detectable tumor15, tumor nodules showed a positive performance with higher SUVmax on PET/CT. Based on their preference for aerobic glycolysis, 18F-FDG, a glucose analog, has been exploited as a promising tracer in the diagnosis of malignancies, combined with PET/CT16. This method relies on the rapid glucose consumption of tumor tissue and has broad clinical applications, including assisting in the detection, staging, and evaluation of the prognosis of tumors, as well as monitoring the tumors&#39; response to therapy17,18. As non-invasive methods, DECT and PET/CT have been utilized to diagnose malignant tumors and to assess tumor response to various therapies.
Our group has been using this non-invasive imaging method with DECT and PET/CT scanners to detect and monitor the process of tumor growth and metastasis in living mice19. We explored imaging findings induced by the sFRP1-overexpression in gastric cancer cells in vivo using nude mice, with DECT and PET/CT, and described the changes of the SUVmax value following targeted therapy by the TGF-&#946;1 inhibitor to confirm the development of tumor nodules in the peritoneum after gene induction, and also studied the changes in tumor nodules in response to experimental treatments. In this paper, we present detailed procedures for modeling gastric tumor peritoneal metastasis in mice, and its detection and monitoring with DECT and PET/CT.

<table><tbody><tr><td>Iohexol</td><td>BEJING BEILU PHARMACEUTICAL CO,LTD</td><td>NMPN:H20053800</td><td>non-ionic contrast medium for DECT scan</td></tr><tr><td>normal saline</td><td>HUNAN KELUN PHARMACEUTICAL CO,LTD</td><td>NMPN:H43020455</td><td>placebo of control group</td></tr><tr><td>BALB/c nude mice&amp;nbsp;</td><td>SLAC LABORATORY ANIMAL</td><td>BALB/cASlac-nu</td><td>animal model</td></tr><tr><td>SGC-7901&amp;nbsp; cells</td><td>Library of typical culture of Chinese academy of sciences</td><td>TCHu 46</td><td>gastric cancer cell&amp;nbsp;</td></tr><tr><td>SB431542</td><td>Selleck</td><td>No.S1067</td><td>TGF-&amp;beta;1 inhibitor</td></tr><tr><td>GE Discovery CT750 HD</td><td>GE Healthcare</td><td></td><td>dual-energy spectral CT scanner&amp;nbsp;</td></tr><tr><td>AW Volumeshare5</td><td>GE Healthcare</td><td></td><td>dual-energy spectral CT workstation</td></tr><tr><td>Siemens Inveon micro-PET/CT</td><td>Siemens Preclinical Solution</td><td></td><td>positron emission tomography/&lt;br/&gt; computed tomography scanner&amp;nbsp;</td></tr><tr><td>Inveon Acquisition Workplace</td><td>Siemens Preclinical Solution</td><td></td><td>PET-CT workstation</td></tr></tbody></table>

gene regulation and targeted therapy in gastric cancer peritoneal metastasis: radiological findings from dual energy ct and pet/ct

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of metastasis that accompanies unresectable gastric cancer and is a definitive determinant of prognosis. Preventing and controlling the development of peritoneal metastasis could play a role in helping to prolong the survival of gastric cancer patients. A non-invasive and efficient imaging technique will help us to identify the invasion and metastasis process of peritoneal metastasis and to monitor the changes in tumor nodules in response to treatments. This will enable us to obtain an accurate description of the development process and molecular mechanisms of gastric cancer. We have recently described experiment using dual energy CT (DECT) and positron emission tomography/computed tomography (PET/CT) platforms for the detection and monitoring of gastric tumor metastasis in nude mice models. We have shown that weekly continuous monitoring with DECT and PET/CT can identify dynamic changes in peritoneal metastasis. The sFRP1-overexpression in gastric cancer mice models showed positive radiological performance, a higher FDG uptake and increasing enhancement, and the SUVmax (standardized uptake value) of nodules demonstrated an obvious alteration trend in response to targeted therapy of TGF-&#946;1 inhibitor. In this article, we described the detailed non-invasive imaging procedures to conduct more complex research on gastric cancer peritoneal metastasis using animal models and provided representative imaging results. The use of non-invasive imaging techniques should enable us to better understand the mechanisms of tumorigenesis, monitor tumor growth, and evaluate the effect of therapeutic interventions for gastric cancer.

DECT and PET/CT scanning were performed on nude mice after two weeks of cell line injections. GSI images yielded excellent results for displaying subcutaneous metastasis beyond the contour of the abdomen for the sFRP1 overexpression group, and metastasis with peripheral enhancement was confirmed by color-scale image (Figure 1a-c). PET/CT images depicted focally abnormal FDG uptake of metastasis, including in the peritoneal and subcutaneous me...

Watch this Scientific Journal Video about Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT at JoVE.com

Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

This protocol describes the value of dual energy CT and PET/CT imaging methods in tumor imaging and efficacy evaluation. This article demonstrates the research methods and results acquired by dual energy CT and PET/CT to evaluate the gene regulation and targeted treatment of gastric cancer peritoneal metastasis.

Gastric cancer remains fourth in cancer incidence worldwide with a five-year survival of only 20%-30%. Peritoneal metastasis is the most frequent type of ...

gene-regulation-targeted-therapy-gastric-cancer-peritoneal-metastasis

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Medicine

Unit 1

Cancer

SCIENTISTS IN ACTION

10.9K Views.  Shanghai Jiao Tong University School of Medicine. This protocol describes the value of dual energy CT and PET/CT imaging methods in tumor imaging and efficacy evaluation. This article demonstrates the research methods and results acquired by dual energy CT and PET/CT to evaluate the gene regulation and targeted treatment of gastric cancer peritoneal metastasis.

Video: Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Cancer Research

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 ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

PET Imaging and Biodistribution Analysis in Mice Using BCAMs and SaaS Tools

Preclinical Positron Emission Tomography with Body Conforming Animal Molds for Cloud-Based Automated Image Analysis in Mice

Positron Emission Tomography (PET) is a molecular imaging modality that can be used to investigate a multitude of pharmacological questions, such as biomarker modulation, receptor occupancy, and biodistribution of compounds of interest. In biodistribution studies, experimental subjects are often longitudinally imaged after receiving the test article. The images are then analyzed to derive the compound&#39;s distribution profile in various organs at different timepoints. This constitutes a crucial step in drug development to understand the distribution and potentially binding profile of an investigative compound. Standard/manual methods of PET imaging-based biodistribution analyses, however, are labor-intensive and time-consuming and are often associated with high inter-operator variability. Further, it is challenging to keep the animals&#39; positions consistent across different timepoints. To address these shortcomings, a series of mouse Body Conforming Animal Molds (BCAMs) were used to enable rigid and consistent positioning of animals during PET/CT imaging acquisition. Further, a Software-as-a-Service (SaaS) platform consisting of a cloud-based Organ Probability Map (OPM) and an artificial intelligence-powered segmentation tool were employed to enable reliable and automated quantitation of in vivo PET imaging data. The workflow presented here includes (1) prepping mice for imaging with the BCAMs, including the proper implantation of subcutaneous tumors to be compatible with the molds, (2) acquiring PET/CT images with BCAMs using the G8 scanner, and 3) performing automated organ segmentation and biodistribution analysis using the cloud-based SaaS. [18F]FDG was used as an exemplar tracer here, but other biomarkers and/or radio-labeled compounds can be readily adapted into the workflow. This procedure can be executed accurately and effectively with minimal training, and the automated PET data analysis yielded satisfactory results consistent with the manual method.

Author Spotlight: Standardizing Mouse In Vivo PET Imaging with Body Conforming Molds and Automated Analysis

This protocol describes the procedure for performing in vivo PET imaging on mice using Body Conforming Animal Molds (BCAMs) in the G8 PET/CT scanner. Technical details on mouse preparation, including proper tumor implantation, optimal positioning, BCAM-assisted PET/CT image acquisition, and data analysis, are provided.

Positron Emission Tomography (PET) is a molecular imaging modality that can be used to investigate a multitude of pharmacological questions, such as ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Immunology and Infection

SNP-Guided 5-FU Therapy in Gastric Cancer Using Ion Torrent Sequencing

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although 5-fluorouracil (5-FU) is widely used for treatment, its therapeutic sensitivity and drug tolerance still need to be determined, which emphasizes the importance of individualized administration. Pharmacogenetics can guide the clinical implementation of individualized treatment. Single nucleotide polymorphisms (SNPs), as a genetic marker, contribute to the selection of appropriate chemotherapy regimens and dosages. Some SNPs are associated with folate metabolism, the therapeutic target of 5-FU. Methylenetetrahydrofolate reductase (MTHFR) rs1801131 and rs1801133, dihydrofolate reductase (DHFR) rs1650697 and rs442767, methionine synthase (MTR) rs1805087, gamma-glutamyl hydrolase (GGH) rs11545078 and solute carrier family 19 member 1 (SLC19A1) rs1051298 have been investigated in different kinds of cancers and antifolate antitumor drugs, which have potential forecasting and guiding significance for application of 5-FU. The ion torrent next-generation semiconductor sequencing technology can rapidly detect gastric cancer-related SNPs. Each time a base is extended in a DNA chain, an H+ will be released, causing local pH changes. The ionic sensor detects pH changes and converts chemical signals into digital signals, achieving sequencing by synthesis. This technique has low sample requirement, simple operation, low cost, and fast sequencing speed, which is beneficial for guiding individualized chemotherapy by SNPs.

Author Spotlight: Genetic Profiling for Fluorouracil Response in Gastric Cancer

This protocol proposes holistic laboratory procedures required to detect single nucleotide polymorphisms in gastric cancer samples based on an ion semiconductor sequencing platform. The target sequences, ligating adapters, library amplification and purification, and quality control criteria are also described in detail.

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although ...

Gene Regulation and Targeted Therapy in Gastric Cancer Peritoneal Metastasis: Radiological Findings from Dual Energy CT and PET/CT

Summary

Explore More Videos