We thank the University of California Los Angeles' Crump Preclinical Imaging Technology Center for their assistance with the PET/CT imaging of the mice, the Translational Pathology Core Laboratory and Statistics Core at the University of California Los Angeles' David Geffen School of Medicine for their assistance with the tumor sample preparation and analysis. For funding, David B. Shackelford was supported by the CTSI and KL2 Translational Science Award grant numbers KL2TR000122 and UL1TR000124 at the David Geffen School of Medicine at UCLA and by the Department of Defense Lung Cancer Research Program Translational Research Partnership W81XWH-13-1-0459 and ACS RSG-16-234-01-TBG. Sean T. Bailey was supported by an NIH T32 training grant HL072752 through the David Geffen School of Medicine at UCLA. Anthony Jones is supported by the UCLA Tumor Cell Biology Training Program (USHHS Ruth L. Kirschstein Institutional National Research Service Award # T32 CA009056). Gihad Abdelhady is supported by an NIH/NCI Diversity Supplement R01CA208642.

All procedures described in the protocol have been approved by the institutional animal care and use committee (IACUC) at the University of California, Los Angeles.
1. 18F-FDG PET and CT Imaging in Mice
CAUTION: Use protective equipment when handling radioactivity. Follow all applicable regulatory procedures when handling radioactivity.
<ol>
	<li>Place the cage with the mice to be imaged on a warm bed at 37 &#176;C 1 h prior to the 18F-FDG injection to reduce the brown fat consumption of 18F-FDG. 
	NOTE: Fasting the mice for 4 - 16 h can help reduce the myocardial consumption of 18F-FDG.</li>
	<li>Weigh the mouse and record its weight.</li>
	<li>Anesthetize the mouse using 2 - 3% isoflurane in oxygen at 0.5 - 2 L/min for 2 - 3 min using an anesthesia chamber kept at 37 &#176;C. Ensure that mouse has been anesthetized by pinching the toe; no reaction will be observed if the mouse has been anesthetized. Apply ophthalmic ointment to the eyes to prevent any dryness during the anesthesia.</li>
	<li>Dilute 18F-FDG (109 min radioactive half-life) in sterile saline at an adjusted decay-corrected injection concentration of 70 - 75 &#181;Ci/100 &#181;L. 
	NOTE: Follow the PET scanner manufacturer&#39;s recommended 18F dose for optimal scanner imaging.</li>
	<li>Draw 70 - 75 &#181;Ci with an insulin syringe with a 28 G needle, measure the radioactivity dose using a dose calibrator, and record the measurement and time. Place the syringe in a lead syringe holder. 
	NOTE: The amount of 18F-FDG radioactivity in each dose is measured with a dose calibrator, which is calibrated against a standard reference material, such as cesium-137, according to the manufacturer&#39;s protocols. The time of the reading is also recorded to determine the decay correction.</li>
	<li>Prick the distal end of the mouse tail and measure the mouse&#39;s blood glucose with a glucometer.</li>
	<li>Warm the tail for 1 - 2 min with a gauze soaked in warm water. Wipe the tail with 70% isopropanol to dilate the tail vein just prior to the injection. Administer 100 &#181;L of 18F-FDG (the entire volume in the syringe) with a bolus injection via the lateral tail vein and record the time of injection. Measure the remaining dose in the syringe using the dose calibrator and record the measurement and time. 
	NOTE: There will be some amount of probe left in the syringe. The use of insulin syringes is preferred over syringes connected to needles via Luer locks due to the decreased amount of the dose trapped in the syringe/needle after the injection has been administered.</li>
	<li>Place the injected mouse in the anesthesia chamber kept under 1.5 - 2% isoflurane at 37 &#176;C to allow the probe to be distributed via the mouse&#39;s systemic circulation for 1 h prior to the PET scan. 
	NOTE: It might be beneficial to void the bladder prior to the scanning to allow for an easier 18F-FDG PET visualization of the tumors implanted in the lower flanks of the mouse.</li>
	<li>After 1 h, place the mouse in an imaging chamber under nose-cone isoflurane anesthesia and at 37 &#176;C, and secure its limbs in place with medical tape in a supine position.</li>
	<li>Place the imaging chamber in the PET/CT scanner.</li>
	<li>Acquire the PET and CT scans as described in the PET/CT scanner manual28. 
	NOTE: The PET images are acquired for 600 s with an energy window of 150 - 650 keV, reconstructed using maximum-likelihood expectation maximization with corrections for photon attenuation, detector normalization, and radioisotope decay (a scatter correction was not applied). The CT images are acquired in a continuous mode for 50 s using a 50 kVp, 200 &#181;A X-ray source and a flat-panel detector, and they are reconstructed using the Feldkamp algorithm.</li>
	<li>After the PET/CT is completed, remove the mouse from the imaging chamber and allow it to recover in its cage. Monitor the mouse until it has fully regained consciousness and can maintain sternal recumbency.</li>
	<li>Import the reconstructed PET/CT images into AMIDE software by clicking File, then Open, and selecting the appropriate file.</li>
	<li>Convert the PET data to the unit of percent-injected dose per gram (%ID/g) by entering the dose at the time of injection after accounting for any residual dose left in the syringe, or to the unit of standardized uptake value (SUV) by additionally entering the subject&#39;s weight. To do this, right-click on the PET data set and locate the %ID/g field on the Basic Info tab. Enter the %ID/g recorded earlier.</li>
	<li>Draw the regions-of-interest (ROIs) on the tumors and normal tissues (liver, muscle, lung, heart, brain, and subcutaneous fat). To do this, click on Edit, select Add ROI, select ROI shape and give the ROI a name. Draw ROIs over tumors and tissues and adjust their dimensions to cover tissue of interest in all 3 axes. 
	NOTE: To account for differences in PET probe biodistribution between animals, tumor ROI values can be further normalized to ROI values of the liver, a well-perfused organ with minimal glycolytic activity representing 18F-FDG in circulation. ROI analyses of tumor and normal tissues are performed on the same mouse. Lung tumor lesions are generally identified by 18F-FDG PET since the 18F-FDG retention in a normal lung is relatively low. CT is also used to identify lesions, particularly lesions that are 18F-FDG non-avid. An ex vivo analysis of isolated lungs also helps to localize tumor lesions.</li>
</ol>
2. 18F-FDG Autoradiography
<ol>
	<li>Prepare the mouse for imaging with 18F-FDG by following steps 1.1 - 1.12, except now, dilute 18F-FDG in sterile saline at an adjusted decay-corrected injection concentration of 1,000 &#181;Ci/200 &#181;L. 
	NOTE: Higher doses of 18F-FDG are used for autoradiography to account for additional sample processing time and an optimal detection by phosphor plates.</li>
	<li>Euthanize the mouse via a lethal inhalation of isoflurane at 5% or by CO2 (an IACUC-approved procedure). 
	NOTE: Cervical dislocation should not be used as this can damage the lung tissue.</li>
	<li>Pin down the mouse with the ventral surface exposed and spray it with 70% ethanol to mat down its hair prior to the incision.</li>
	<li>Open the thoracic cavity by applying a midline incision, cutting away the diaphragm and removing the chest walls. Expose the trachea by carefully removing the salivary gland. Place the bulldog clamp on the trachea as close to the jaw as possible, ensuring a tight fit on the trachea. Place a 23 G needle attached to a 3 mL syringe inside the trachea below the bulldog clamp and inject ~2 mL of an OCT:PBS (1:1) (Optimal Cutting Temperature:Phosphate Buffered Saline) solution. 
	NOTE: OCT is very viscous and is mixed with PBS to allow for an easier injection into the lungs.</li>
	<li>Remove the needle from the trachea and use forceps to clamp on the injection point to prevent any leaking of the OCT:PBS solution.</li>
	<li>Carefully remove the lungs from the thoracic cavity and separate the left lobe from the rest of the lungs. Place the left lobe in labeled cryomold filled with a few drops of OCT. Once the lung lobe is inside the mold, fill the cryomold to the top with OCT.</li>
	<li>Repeat the same procedure with the right half of the lungs. 
	NOTE: If the 18F-FDG signal in the heart is expected to be high or if the lung tumors are located close to the heart, it might be beneficial to remove the heart in order to prevent any tracer leakage. Alternatively, entire lungs can be embedded into a single cryomold. It is important to avoid air bubbles when working with OCT.</li>
	<li>Using long forceps, place the prepared cryomold in a closed-cell extruded polystyrene foam container containing a mixture of dry ice and isopentane. 
	NOTE: This mixture should be at approximately -70 &#176;C before placing the cryomold in it for freezing. After hardening, the OCT compound will become white. If multiple samples are processed at the same time, the frozen samples in OCT cryomolds can be temporarily stored on dry ice.</li>
	<li>Remove the frozen block from the cryomold and mount it onto a cryostat for sectioning. Section the block at a 4 &#181;m thickness using microtome blades (34&#176;/80 mm, high profile). Transfer the tissue sections to a glass slide that has been stored at room temperature.</li>
	<li>Place the specimen slides onto a phosphor imaging plate. Place the plate in the cassette and gently close it to prevent slides from shifting. Store the cassette in a -20 &#176;C freezer for the plate exposure, generally overnight. 
	NOTE: The plates and cassettes need to be pre-chilled to -20 &#176;C before use. Placing the samples at -80 &#176;C is acceptable as well.</li>
	<li>After the exposure, remove the slides from the plate and read the plate on the image reader.</li>
	<li>The slides can be wrapped in plastic wrap and stored at -80 &#176;C, or they can be prepared for hematoxylin &#38; eosin staining or immunohistochemistry.</li>
</ol>
3. Harvesting Lung Tissue for Histology
<ol>
	<li>Follow steps 2.2 - 2.4, except now, instead of using the OCT:PBS solution, inject 2 - 3 mL of 10% normal buffered formalin to fix the lungs.</li>
	<li>Remove the needle from the trachea and use forceps to clamp on the injection point to prevent any leaking of the formalin. Carefully remove the lungs from the thoracic cavity and place them into a 50 mL conical tube containing ~20 mL of 10% normal buffered formalin for 16 - 24 h to ensure a complete fixation. 
	NOTE: Fixing the lungs allows for the preservation of the optimal anatomical features.</li>
	<li>The next day, transfer the fixed lungs from the formalin to 70% ethanol and prepare the lungs to be placed in a tissue cassette.</li>
	<li>Prepare the lungs for histology by carefully dissecting lobes using scissors to cut at branch points between 5 lobes, numbered 1 - 5 as shown in Figure 1D, and place them in a non-overlapping orientation in the tissue cassette. Place a foam pad gently on the lung tissue to keep the orientation intact.</li>
	<li>Store the dissected lung lobes in 70% ethanol until the paraffin embedding.</li>
	<li>Paraffin-embed the tissue in cassettes and cut 4 &#181;m thick sections for the staining, using standard procedures.</li>
</ol>
4. Tissue Segmentation and Quantification Using Commercial Software
<ol>
	<li>Image hematoxylin and eosin (H&#38;E) stained lung sections at a 1.25X magnification using a commercial multispectral imaging system.</li>
	<li>Convert the images to digital image cubes and load (click on File, then on Load Spectral Library) pre-made spectral libraries for H&#38;E. 
	NOTE: The spectral libraries were developed in advance by acquiring spectral images from singly stained lung sections, one section stained only with eosin and the other only stained with hematoxylin, which were saved in an open-source proprietary spectral library file (.csl). The imaging system has operating software that acquires an image at each wavelength that is needed (as defined by the acquisition protocol). Those images (the &#34;image cube&#34;) are stored in an open-source proprietary multispectral file format (.im3). Spectra are extracted from the image cube using the morphometric software and stored in separate spectral library files.</li>
	<li>Spectrally unmix the pseudo-colored H&#38;E images of the whole lungs by clicking on the Unmix button. 
	NOTE: Unmixing takes less than 1 s. The number of pixels in each tissue type were quantified using morphometric image analysis software.</li>
	<li>Visualize each image cube by converting the multispectral data into the equivalent 3-color (red, green, blue) image by using the eye&#39;s wavelength-dependent color response convolved with the intensity of each image. 
	NOTE: The resulting 3 images are displayed as a standard 24-bit color image.</li>
	<li>Pseudo-color each unmixed image by scaling that image in a user-chosen color (e.g., red, green, purple, etc.) and add that together with the other pseudo-colored unmixed images into one standard 24-bit color image.</li>
	<li>Use the default settings for all analyses. 
	NOTE: In general, since this kind of segmentation relies on a visual assessment of the results (i.e., an assessment of how well the segmentation on the images outside of the training set works), it is important to properly divide the images up into training, test, and validation sets.</li>
	<li>Start with 2 - 3 images as a training set (10 - 15 for human biopsy samples), train on those until the results look good, and then apply that algorithm to another 2 - 3 images. Then, apply the resulting algorithm to the full validation set. 
	NOTE: Most likely, some retraining will be needed.</li>
	<li>Analyze the tumor area in whole lung sections by calculating the total pixel count for the red pseudo-colored tumors in lobes 1 - 5 of each mouse. 
	NOTE: The normal tissue was pseudo-colored green and blood/blood vessels were pseudo-colored pink as shown in Figure 3. The mean tumor burden for each treatment group was calculated by measuring the total pixel count for each mouse in the treatment group.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+cancer+with+positron+emission+tomography.">Molecular imaging of cancer with positron emission tomography.</a> Nature Reviews Cancer. 2 (9), 683-693 (2002).</li><li>Keng, P. 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Kevin P. Francis is an employee of Perkin Elmer. James Mansfield is a public stockholder of PerkinElmer, Inc (PKI) shares on NASDAQ. The authors have nothing else to disclose.

This article described an imaging-based experimental approach that utilized 18F-FDG PET/CT imaging with qIHC in order to measure both the metabolic and molecular responses in lung tumors following the delivery of the mTOR inhibitor MLN0128. MLN0128 effectively reduced the 18F-FDG consumption, indicating a significant metabolic response in the tumors. By linking PET/CT imaging to immunohistochemistry, we were able to spatially register sectioned tumors to the 3D PET/CT images and perform a detailed examination of whole tumors at a cellular and molecular level. This made it possible to confirm that MLN0128 inhibited the mTOR signaling, thus confirming an on-target molecular response to the drug in the tumors. Lastly, by taking advantage of quantitative histology, we were able to map and separate distinct tumor pathologies, such as overall tumor mass from tumor necrosis, define adenocarcinoma from squamous cell carcinomas, and complement microPET imaging.
MicroPET is currently limited by a spatial resolution of approximately 1 mm. In addition, 18F-FDG retention in certain tissues can be affected by various factors, including plasma glucose levels, the type and duration of anesthetic exposure, the environmental temperature, and the general health of the animal, which may impact 18F-FDG pharmacokinetics30. These parameters have been optimized for this protocol but should be optimized for each animal model. Reproducibility studies of 18F-FDG imaging of subcutaneous tumors in mice demonstrate a coefficient of variation for the mean %ID/g of approximately 15%, suggesting that the tumor therapeutic response of an individual mouse assessed by 18F-FDG PET should be larger than this threshold to be considered reliable and significant31.
The cellular and even subcellular distribution of PET tracers can be assessed by tissue autoradiography with the sections subsequently stained and co-registered with qIHC. Co-registering PET with CT allows a PET image to be put in an anatomical context; this is extremely valuable, even with low soft tissue contrast. The lack of soft tissue contrast by CT can be overcome with magnetic resonance imaging (MRI). In addition, biomarkers for fluorescence imaging can be used to assess glycolysis in vivo, but photon absorption and scatter in the lung cavity may affect the accurate quantitation or detection sensitivity32. In summary, utilizing whole animal PET/CT imaging with quantitative histology provides an accurate and real-time map of tumor biology following therapeutic intervention.
Multispectral imaging (MSI) is applicable in any situation where a color image might be used. At the very least, MSI provides the same information as a color image, and for some applications, MSI can provide more detailed information about the spectral properties of a sample than a simple broad-band three-color (RGB) image. In general, the limitations of MSI are those of color imaging, except that MSI is slower and takes more time to acquire images. The morphometric software was used to obtain reproducible, accurate segmentation results for the images and is described in the Table of Materials. There are additional commercially available products that can be used for tissue segmentation and the quantification of histology.
The complexity of cancer metabolism extends beyond the Warburg Effect and glucose metabolism33,34. It is highly likely that tumors will readily adapt to single agent treatments that inhibit glycolysis. The reliance on amino acid metabolism has been well documented in cancer, and it is expected that tumors rely on a host of amino acids such as glutamine, glycine, and serine, as well as other metabolites such as free fatty acids35,36,37. In addition to 18F-FDG, probes such as 18F- and 11C-labeled glutamine, choline, acetate, 1-(2&#39;-Deoxy-2&#39;-fluoroarabinofuranosyl)cytosine (FAC), and fluorothymidine (FLT) have been successfully used to image amino acid, nucleotide, and lipid metabolism in animal models of cancer38,39,40,41. Automation and microscale tracer radiochemistry technologies coupled with higher resolution, higher sensitivity PET scanners will improve the accessibility of PET for measuring various biological procedures42,43. As the understanding of metabolism increases, it is logical that the repertoire of PET radiotracers will increase as well, enabling researchers and physicians to noninvasively profile tumor metabolism.
The utilization of PET/CT imaging and quantitative histology addresses a clinical need, which is to rapidly translate bench discoveries into clinical use. To accomplish this, researchers must be able to accurately measure the therapeutic response as well as the acquired resistance to drugs, which PET/CT imaging enables. Moreover, the PET/CT and immunohistochemical analysis of lung tumors are used as standard of care for patients, and thus, are directly translatable into clinical practice. Importantly, PET/CT imaging readily identifies therapy-resistant tumors, which researchers can isolate and interrogate at a molecular level in order to better understand the mechanisms of disease. This is an iterative process that has made it possible to better understand the mechanisms of resistance and design more effective therapeutic strategies for the clinical translation.

Our research has focused on investigating and targeting cancers with mutations in the Liver kinase B1 (LKB1, also referred to as STK11) mutant cancers1. LKB1 is a master tumor suppressor that represses mTOR complex 1 (mTORC1) through the activation of the AMP kinase (AMPK) leading to the regulation of growth and metabolism. Therefore, the loss of LKB1 leads to an unrestrained mTORC1 activation, the activation of the HIF1-alpha resultant in a glycolytic metabolic phenotype commonly referred to as the Warburg Effect2,3,4. LKB1 inactivating mutations directly lead to the development of a rare familial cancer pre-disposition syndrome known as Peutz-Jeghers Syndrome (PJS) that is characterized by the development of benign gastrointestinal polyps known as hamartomas5,6,7. Moreover, LKB1 frequently co-mutates with oncogenic KRAS resulting in hypermetabolic and aggressive human lung tumors8,9.
Lkb1-related diseases are readily modeled in mice. The heterozygous inactivation of Lkb1 in mice leads to the development of hamartomas accurately modeling PJS10,11,12,13. Additionally, Lkb1 mutations that are readily modeled in mice accurately recapitulate cancer phenotypes of the lung, skin, pancreas, and breast14. The co-mutation of Kras/Lkb1 in the lung tissue of transgenic mice, using a Cre recombinase-mediated activation of the oncogenic KrasG12D allele and biallelic deletion of Lkb1, results in the formation of aggressive and metastatic lung tumors15,16. The characterization of KrasG12D;Lkb1-/- (KL) lung tumors isolated from mice shows these tumors have a high mTORC1 activation and are highly glycolytic, using both direct metabolite measurements of glucose and lactate or measuring the consumption of [18F]-2-fluoro-2-deoxy-D-glucose (18F-FDG) by positron emission tomography (PET) with computed tomography (CT) 17. The mTORC1 hyper-activation in LKB1 mutant tumors provides a clear rationale for testing both allosteric and catalytic kinase inhibitors of mTOR to treat these cancers.
In a previous study, we demonstrated that the allosteric mTORC1 inhibitor rapamycin (RAPA) successfully inhibited the growth of and glycolysis in gastrointestinal (GI) tumors using an Lkb1+/- transgenic mouse model of PJS3. RAPA is currently approved as a single agent therapy for the treatment of renal cell carcinoma but showed limited efficacy in NSCLC18,19,20. RAPA is an allosteric mTORC1 inhibitor and may be improved upon by the development of next-generation mTOR catalytic kinase inhibitors that deliver a more nearly complete inhibition of the mTOR complexes 1 and 2 (mTORC1 and mTORC2, respectively)21. Drugs such as MLN0128 are now being evaluated in preclinical studies and early phase clinical trials22,23. A recent study from our laboratory demonstrated that MLN0128 is a potent mTOR inhibitor in human lung tumor cell lines and in vivo in KL GEMMs of lung cancer15,16. MLN0128 suppressed the lung tumor growth and glucose metabolism in these mice24.
In this study, we take advantage of the well-characterized adenoviral Cre-induced mouse models of lung cancer initiated by a conditionally activated Lox-Stop-Lox-KRASG12D oncogene15,25. These KrasG12D mice were crossed with mice having floxed alleles of Lkb1 (Lkb1L/L) to generate KrasG12D; Lkb1L/L (KL) mice16. Following the intranasal delivery of adeno- or lentivirus expressing Cre recombinase, the KL mice develop early lesions by 4 weeks post-tumor induction. By 6 weeks, the tumors in the KL mice change from adenomatous tumors to a more malignant, aggressive tumor phenotype typical of lung carcinomas, and by 8 - 10 weeks, the mice develop frank carcinomas with a 100% penetrance16,26.
Both PET/CT imaging and quantitative immunohistochemistry may be utilized to determine the molecular and metabolic responses as well as the therapeutic responses in tumors following the delivery of targeted therapies such as MLN012817,26,27. Described here is an experimental protocol that utilizes 18F-FDG PET imaging to measure the metabolic response to an MLN0128-targeted therapy. Coupling PET imaging with quantitative histology enables the measurement of the molecular response to mTOR inhibition as well as the quantification of the tumor burden and tumor histology.

<table><tbody><tr><td>G8 PET/CT</td><td>Perkin Elmer</td><td>CLS139564</td><td>Used for 18F-FDG PET and CT imaging of mice</td></tr><tr><td>Axio Imager.M2</td><td>Zeiss</td><td>490020-0003-000</td><td>Acquiring images of FFPE lung tumor sections</td></tr><tr><td>Inform software</td><td>Perkin Elmer</td><td>CLS135781</td><td>Morphometric used for image analysis of tumor pathologies</td></tr><tr><td>Glut1 antibody</td><td>Alpha Diagnostics</td><td>GT12-A</td><td>IHC staining of FFPE lung tumor sections</td></tr><tr><td>Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP&amp;trade; Rabbit mAb</td><td>Cell Signaling Technologies</td><td>4858</td><td>IHC staining of FFPE lung tumor sections</td></tr><tr><td>MX35 Premier microtome blades&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>3051835</td><td>Microtome blades for sectioning tissue for autoradiography</td></tr></tbody></table>

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.

18F-FDG PET imaging was performed on KL mice and demonstrated that the tumors in these mice were highly glycolytic as shown by an elevated 18F-FDG consumption (Figure 1A), agreeing with previously published studies26,29. A resection of whole lungs revealed the presence of several tumors (Figure 1B). Mouse lungs can be divided up into 5 separate lobes represented in Figures 1C and 1D. Lobes 1 - 5 were labeled on sectioned lungs that were stained with H&#38;E or glucose transporter 1 (Glut1) (Figure 1D). Glut1 is a primary transporter of both glucose and 18F-FDG and its expression and localization to the plasma membrane of tumor cells directly correlate with 18F-FDG SUV29. A higher resolution analysis of Glut1 staining (40X) in 18F-FDG-avid lung tumors shows an elevated expression and localization of the transporter at the plasma membrane (Figure 1D).
Due to the limited resolution of PET imaging, both PET/CT and tissue autoradiography were performed. The higher resolution of autoradiography might identify smaller tumors and/or heterogeneity of the tumor 18F-FDG distribution. After the tumor induction, 18F-FDG PET/CT imaging was performed on KL mice (Figure 2A) followed by autoradiography on lungs isolated from these mice (Figures 2B and 2C). As seen in Figures 2B and 2C, the autoradiography identified two additional smaller tumors that were positive for 18F-FDG yet were not readily visible by PET. Following autoradiography, the slides with tissue could also be used for the immunohistochemical (IHC) staining of biomarker(s). H&#38;E staining for the tumors confirmed the presence of tumors in the left lobe (Figure 2D).
Next, 18F-FDG PET imaging was performed on MLN0128-treated KrasG12D;Lkb1-/- mice in order to utilize 18F-FDG as a functional biomarker of glucose metabolism in lung tumors (Figure 3). We identified that a treatment with MLN0128 robustly inhibited the mTORC1 signaling and glycolysis as shown by a reduced 18F-FDG consumption (Figures 3A and 3B). These results agree with preclinical studies assessing MLN0128 in KL mice as previously published by our laboratory17,27. Lastly, IHC staining was performed on the tumors (Figure 3C). Tumors were stained either for H&#38;E or with antibodies against phospho-S6, which is a conserved substrate of mTORC1 and is used to indicate an mTORC1 activation (P-S6) vs. inactivation (S6). Figure 3C shows a robust inhibition of P-S6 by MLN0128 in KL tumors compared to those treated with a vehicle, which agrees with previously published work17. In addition to KRAS, oncogenic drivers such as the epidermal growth factor receptor (EGFR) support the glycolytic metabolism in lung tumors as well. Therefore, we tested whether the inhibition of constitutively active mutant EGFR with erlotinib suppressed the 18F-FDG metabolism in mouse xenografts. Figures 3D and 3E show that the human lung tumor line HCC827, which harbors an EGFR del19 mutation, showed a significantly reduced 18F-FDG consumption following five days of erlotinib treatment.
Lastly, morphometric tissue analysis was performed on sectioned lungs and lung tumors to quantify the total tumor burden as well as to differentiate tumor pathologies that included tissue subtype, necrosis, blood vessels from normal lung tissue, and air space. The KL GEMMs developed a complex and pathologically heterogeneous disease that presented itself with lung tumors of varying histopathologies. These include adenocarcinomas (ADC) and squamous cell carcinomas (SCC)-this heterogeneity makes the treatment of this cancer a formidable challenge. Figure 4A shows a single H&#38;E stained lung lobe with two large tumors present. The higher magnification images shown in Figure 4B identify a normal lung, vessels, and airspace, and tumor necrosis as well as adenocarcinoma, characterized by a well-defined papillary structure and a squamous cell carcinoma. Figure 4C represents the pseudo-coloring of the lung lobe and tumor using Inform morphometric software. Figure 4D shows the percentages of normal lung, vessels, and individual pathologies such as tumor necrosis and tumor subtypes that segmented well-differentiated adenocarcinoma from squamous cell carcinoma.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57167/57167fig1.jpg" /> 
Figure 1: Metabolically active KrasG12D; Lkb1-/- (KL) mutant lung tumors are 18F-FDG positive and express high levels of glucose transporter 1 (Glut1). Panels A and B show a maximal intensity projection [also referred to as a 3-dimensional (3D) image] of the 18F-FDG-PET and CT analysis on some FDG-avid KL mice harboring squamous lung tumors. Shown are (A) a 3D reconstruction and (B) transverse, sagittal and coronal views of lung tumor(s) as (T). (C) This panel shows a whole lung histology of the KL mouse imaged in panels A and B, either stained for H&#38;E (top panel) or with an antibody specific for Glut1 (bottom panel). The lung lobes are numbered. The scale bar = 2 mm. (D) This diagram represents the orientation and numbers of lobes in the mice (top panel) and higher resolution 40X images of the H&#38;E- or Glut1-stained tumors from the slides shown in panel C for H&#38;E (middle panel) or stained with an antibody specific for Glut1 (bottom panel). The scale bar = 25 &#181;m. <a href="https://www.jove.com/files/ftp_upload/57167/57167fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57167/57167fig2.jpg" /> 
Figure 2:&#160;18F-FDG autoradiography can identify small tumors that are metabolically active. (A) This 18F-FDG PET/CT image shows 18F-FDG-avid tumors in a KL mouse shown as a maximum-intensity projection image. T1 and T2 = tumors, H = the heart, B = the bladder, K = the kidneys. (B) This panel shows the ex vivo autoradiography on serial sections of the right and left lung lobes of the mouse. The lungs in the left and right panels are identical. The lungs in the left panels are pseudo colored orange. The lungs in the right panels are colored in black and white. The tumors (T1, T2, and T3) are indicated with arrows. (C) This is a magnified view of the autoradiography pseudocolored orange (top panel) and black and white (bottom panel). (D) This panel shows the H&#38;E staining of the top slice of the left lobe shown in panel B. The scale bar = 200 &#181;m <a href="https://www.jove.com/files/ftp_upload/57167/57167fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57167/57167fig3.jpg" /> 
Figure 3: The mTOR inhibitor MLN0128 suppresses glucose consumption in lung tumors of KL mice as detected by 18F-FDG PET. (A) This panel shows representative 18F-FDG PET/CT images of KL mice treated with a vehicle (18F-FDG avid, left) or MLN0128 (18F-FDG non-avid, right). The transverse (top panel), coronal (middle panel), and sagittal (lower panel) views are shown. The tumors are outlined with red lines; H = the heart, L = the liver. (B) This panel shows a quantification of SUVmax (%ID/g) between the vehicle- and MLN0128-treated tumors. (C) This panel shows the H&#38;E and P-S6 staining of whole lung sections from KL mice treated with vehicle or MLN0128. The scale bar = 25 &#181;m. (D) This panel shows representative 18F-FDG-PET and CT images of HCC827 EGFR (del19) xenografts pre- and post-erlotinib treatment. The tumor (T) is indicated with an arrow, K = the kidney, B = the brain. (E) This panel shows a quantification of the SUVmax (%ID/g) for HCC827 xenografts before and after the erlotinib treatment. n = 10 tumors/group. <a href="https://www.jove.com/files/ftp_upload/57167/57167fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57167/57167fig4.jpg" /> 
Figure 4: Tumor burden and tumor histology are quantified using morphometric software. 
(A) This panel shows the H&#38;E staining of a single mouse lung lobe with a tumor collected from a KL mouse. (B) These higher resolution images show the squamous cell carcinoma (top left), the normal lung, the vessels, and the air space (top right), and the well-differentiated papillary adenocarcinoma (bottom left) and necrosis (bottom right). (C) This panel shows the pseudocoloring of the H&#38;E-stained lung lobe using morphometric software. (D) This panel shows the percentages for the individual lung lobe and tumor pathologies measured by Inform. <a href="https://www.jove.com/files/ftp_upload/57167/57167fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer at JoVE.com

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.

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 ...

utilizing-18f-fdg-petct-imaging-quantitative-histology-to-measure

Research

JoVE Journal

Cancer Research

17.8K Views.  University of California Los Angeles David Geffen School of Medicine. In dit protocol beschrijven we het gebruik van [18F]-2-fluoro-2-deoxy-D-glucose positron emissie tomografie en computertomografie (18F-FDG PET/CT) denkbaar voor het meten van de tumor metabole respons op de gerichte therapie MLN0128 in een Kras/Lkb1 mutant muis model van longkanker en gekoppelde imaging met hoge resolutie ex vivo autoradiografie en kwantitatieve histologie.

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

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

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 ...

Intrapulmonary Inoculation of Multicellular Spheroids to Model Orthotopic NSCLC

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.
Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease&#39;s progression and facilitate the development of potential anticancer drugs.

Author Spotlight: Establishing a Murine Non-Small Cell Lung Cancer Model for Developing Nanoformulations of Anticancer Drugs

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Immunology and Infection

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Medicine

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Biology

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.

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 ...

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. ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods are essential to provide critical insights into these thermogenic adipose depots. Here, the protocol presents a PET/MR imaging-based method to evaluate the activity of brown and beige adipocytes in mouse interscapular brown adipose tissue (iBAT) and inguinal subcutaneous white adipose tissue (iWAT). Visualization and quantification of the thermogenic adipose depots were achieved using [18F]FDG, the non-metabolizable glucose analog, as the radiotracer, when combined with the precise anatomical information provided by MR imaging. The PET/MR imaging was conducted 7 days after cold acclimation and quantitation of [18F]FDG signal in different adipose depots was conducted to assess the relative mobilization of thermogenic adipose tissues. Removal of iBAT substantially increased cold-evoked [18F]FDG uptake in iWAT of the mice.

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

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

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