We thank Dr. I. Verma for the lentiviral Ca2Cre-shp53 vector. The work was supported by funds from the Canadian Institutes of Health Research (CIHR MOP 137113) to AEK.

Animal studies were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of McGill University and procedures were approved by the Animal Welfare Committee of McGill University (animal use protocol # 2009-5754).
1. Generation of CA2Cre-shp53 Lentiviral Titre
NOTE: The following protocol is the same as that described in Xia et al.6, with minor modifications.
<ol>
	<li>Preparation of lentivirus (for 15 cm x 10 cm dishes)
	<ol>
		<li>On day 1, plate healthy HEK293T cells (7.5 x 106 cells per 10 cm dish) with 10 mL of Dulbecco&#39;s modified Eagle medium) (DMEM), 10% fetal bovine serum (FBS), and 1% pen/strep. Culture in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>Prepare the mix for calcium-phosphate transfection (mixture for 15 plates). Prepare tube A containing 225 &#181;g (15 &#181;g/plate) of the lentiviral vector (Ca2Cre-shp53), 75 &#181;g (5 &#181;g/plate) of PsPAX2 plasmid (containing HIV-1 gag and HIV-1 pol genes), 75 &#181;g (5 &#181;g/plate) of pMD2.G plasmid (containing VSV-G gene) for packaging, a final concentration of 0.15 M of CaCl2 and fill the tube with distilled H2O up to 3.75 mL. Prepare tube B containing 3.75 mL of 2x HEPES-buffered saline (HBS; 50 mM HEPES, pH 7.05, 280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4&#183;2H2O, and 12 mM D-dextrose).</li>
		<li>Vortex tube A and add it dropwise to tube B under continuous vortexing. 
		NOTE: The total volume will be 7.5 mL.</li>
		<li>Incubate at room temperature in the dark for 20&#8722;30 min.</li>
		<li>Approximately 9 h after plating the cells, add 500 &#181;L of the transfection mixture dropwise into the cell medium (7.5 mL/15 dishes: 0.5 mL per dish).</li>
		<li>Swirl gently each dish to mix, and incubate all dishes at 37 &#176;C, 5% CO2.</li>
		<li>On day 2, 12&#8722;18 h after transfection, replace the media with 10 mL of antibiotic-free reduced serum media (Table of Materials) per 10 cm dish. Place dishes back in the 37 &#176;C, 5% CO2 incubator.</li>
		<li>On day 3, collect the media containing lentiviruses expressing Ca2Cre-shp53 and filter through 0.45 &#956;m filters. Replenish the media with fresh antibiotic-free reduced serum media. 
		NOTE: The collected media can be kept at 4 &#176;C for no more than 3 days.</li>
		<li>On day 4, collect, for a second time, the media containing the lentivirus and filter through a 0.45 &#181;m filter. Keep at 4 &#176;C (for no more than 3 days).</li>
		<li>Combine the virus supernatants from steps 1.1.8 and 1.1.9. Concentrate them through centrifugal filter units (Table of Materials) by centrifuging at 1,372 x g for 30 min at 4 &#176;C. Repeat the process until all the collected media has passed through the columns. 
		NOTE: After each centrifugation, 100&#8722;200 &#956;L of concentrated filtrate can be harvested.</li>
		<li>Collect and mix the concentrated filtrates in a 15 mL ice-cold tube. Mix the concentrated lentiviruses well and then aliquot (e.g., 100 &#956;L/ tube). Store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Lentiviral titration 
	NOTE: Immortalized mouse embryonic fibroblasts (MEFs) expressing a loxP-flanked allele of green fluorescent protein (GFP) are used in this protocol for the quantification of viral titer. However, any cell line with a loxP-GFP allele should be suitable for this step.
	<ol>
		<li>Culture cells expressing a loxP-flanked allele of GFP with DMEM, 10% FBS and 1% pen/strep at 37 &#176;C, 5% CO2.</li>
		<li>Plate 2 x 105 cells in two 50 mm wells of a 6-well plate. 
		NOTE: The cells of one well will be used for lentiviral infection while that of the other will be used as a negative control.</li>
		<li>The next day, replenish the cells with 2 mL of DMEM, 10% FBS, and 1% pen/strep 2 h before lentiviral infection.</li>
		<li>Add 20 &#181;L of the CA2Cre-shp53 lentivirus (the volume may vary when needed) in the lentiviral infection well.</li>
		<li>After 3 days in culture, determine the frequency of the Cre-induced GFP-positive cells by flow cytometry, as shown in Figure 1. Wash cells with phosphate-buffered saline (PBS), detach by trypsinization and collect cells by centrifugation at 112 x g.</li>
		<li>Wash the cells twice with PBS. Finally, suspend the cells in 100 &#181;L of PBS. Determine the percentage of GFP positive cells by a cell analyzer (Table of Materials).</li>
		<li>Calculate the lentivirus infectious/functional titer using the following formula: 
		<img alt="Equation 1" src="/files/ftp_upload/60565/60565equ01.jpg" /> 
		where F is the frequency of GFP-positive cells and calculated by subtracting the frequency of GFP -positive cells after infection (Fi) from the frequency of GFP-positive cells (background) prior to infection (Fc) as shown in Figure 1 (F = Fi-Fc), Cn is the number of plated cells (2 x 105), V is the volume of the inoculum (mL), and DF is the virus dilution factor. 
		NOTE: The infectious/functional concentration = ~2 x 106 TU/mL.</li>
	</ol>
	</li>
</ol>
2. Intratracheal Intubation of Lentiviruses in LSL-KRASG12D Mice
NOTE: The method of intratracheal intubation was used as described in the published protocol by Vandivort et al.9. In this protocol, mice LSL-KRASG12D mice in C57BL/6 background are used at age 6&#8722;8 weeks. A home-made working procedure board is used as described in Vandivort et al.9. The board is positioned in front of the experimenter in a convenient workspace (approximately 1 m2).
<ol>
	<li>Prepare a spirometer by removing the plunger of a 1 mL syringe and loading 60 &#181;L of PBS into the syringe.</li>
	<li>Attach a 22 G catheter tip into the syringe and set aside.</li>
	<li>Anaesthetize the mice by intraperitoneal injection of a 1 &#181;L/g of mouse body weight of ketamine (50 mg/mL)/xylazine (5 mg/mL)/acepromazine (1 mg/mL) cocktail. Ensure proper sedation of the mouse by a low respiratory rate (1 breath every 2 s).</li>
	<li>Aspirate 20 &#181;L of CA2Cre-shp53 lentivirus into a pipettor and set aside.</li>
	<li>Position the sedated mouse on the working procedure board by hooking its upper incisors into the thread of the board. 
	NOTE: The mouse&#39;s dorsum should be flat against the platform.</li>
	<li>Tape the caudal portion of the thoracic cavity to the platform to ensure alignment of the mouse during the procedure.</li>
	<li>Adjust a gooseneck illuminator between 80&#8722;100% intensity and place the light 1&#8722;2 cm from the skin surface.</li>
	<li>From behind the platform, draw the tongue out of the oral cavity of the mouse using sterile forceps.</li>
	<li>While securing the tongue, insert a depressor into the oral cavity of the mouse then release the tongue.</li>
	<li>Position the gooseneck on the main stem bronchi to illuminate trachea. 
	NOTE: The trachea may be visible through the action of respiration, causing fluctuation of the light.</li>
	<li>When the trachea is clearly viewed, insert the spirometer prepared (syringe with PBS and catheter) into the tracheal path.</li>
	<li>Remove the depressor and observe the rise and fall of PBS in the syringe with each breath. 
	NOTE: This is an indicator that the catheter is properly positioned into the trachea.</li>
	<li>Remove the syringe containing PBS while maintaining the catheter inside the trachea as the previous position.</li>
	<li>Deposit the 20 &#181;L CA2Cre-shp53 lentivirus into the center of the catheter.</li>
	<li>While keeping the catheter in place, inject 300 &#181;L of air into the catheter using an empty syringe to ensure proper distribution of lentivirus in the lungs.</li>
	<li>Keep the catheter in place and reinsert the spirometer into the catheter. 
	NOTE: The rise and fall of the PBS bubble will ensure that the procedure is successful.</li>
	<li>Remove the catheter and tape. Place the animal in a warm and dry place until it is revived.</li>
</ol>
3. Ultrasound Imaging of Lung Tumors in Mice
NOTE: Ultrasound imaging was performed after 7 and 18 weeks of lentiviral intubation using the system listed in Table of Materials; however, any model can be used for the analysis.
<ol>
	<li>One day prior to imaging, remove fur from the chest area of the intubated mouse. 
	NOTE: Mouse should be sedated during this step by placing them in an induction chamber of 3% isoflurane and 2L/minute O2.</li>
	<li>On the day of imaging, set up the workspace as shown in Figure 2. Turn on the heating pump for ultrasound gel and the temperature monitor.</li>
	<li>Set up a warm 33 &#176;C incubator to place the mice in post-imaging.</li>
	<li>Place the three-dimensional (3D) motor (Figure 2F) on the integrated rail system.</li>
	<li>Make sure that the 3D motor and the transducer mounting system are securely in place.</li>
	<li>Connect a preferred transducer (frequency: 40 MHz; Figure 2E and Table of Materials) for tumor measurements perpendicular to the 3D motor.</li>
	<li>Start a new study on the ultrasound software.
	<ol>
		<li>Select Study Browser, then select New at the bottom of the screen.</li>
		<li>Select New Study, a new window will appear that enables the addition of a Study Name in addition to further information about the study, i.e., date of study, name of researcher, etc.</li>
		<li>Fill in the information in Series Name, i.e., Animal ID, Strain, Weight, Date of Birth, etc.</li>
		<li>Select Done, the program will change for B-mode.</li>
	</ol>
	</li>
	<li>Put a heating lamp in a convenient position above the animal platform.</li>
	<li>Place the mouse in the induction chamber (3.5% isoflurane). 
	NOTE: Proper anesthetization is confirmed by unconsciousness of the mouse, and slower respiratory rate of around 1 breath per 2 seconds.</li>
	<li>When the mouse is sedated, change the connection of the anesthetic machine to be directed towards the animal platform, reduce isoflurane to 2.5%.</li>
	<li>Place the mouse on the animal platform in decubitus ventral, with its oral cavity directed towards the anesthetics tube.</li>
	<li>Apply lubricant on the eyes of the mouse.</li>
	<li>Place the mouse in decubitus dorsal and tape its hands and feet firmly onto the animal platform.</li>
	<li>Apply a small layer of ultrasound gel on the mouse chest.</li>
	<li>Lower the acquisition probe using the height-control knob to touch the surface of the mouse chest. Position the probe such that the heart of the mouse is approximately centered.</li>
	<li>Use the micro-knobs to acquire images of the whole chest, from both extremities, in the transverse orientation ideally gathering 500 frames per mouse (number of frames may vary depending on personal choice).</li>
	<li>Once imaging is done, remove the gel from the chest of the mouse and place the mouse in the warm incubator.</li>
</ol>
4. 2D Analysis of Ultrasound Images
<ol>
	<li>After opening acquired frames on the ultrasound software, scan the frames for tumors.</li>
	<li>For small initiating tumors, count the number of B-lines periodically every 10 frames for the full length of the 500 frames acquired. 
	Therefore, B-lines are counted in a total of 50 images, each image is separated by 10 frames. B-lines are characterized by longitudinal white straight lines fully traversing the screen.</li>
	<li>For 2D measurements of large tumors, select the linear tool and measure the width and length of the tumor present.</li>
	<li>To calculate the volume of the tumors, use the following formula: 
	<img src="/files/ftp_upload/60565/60565equ02.jpg" /> 
	where L and W are the length and width of the tumor, respectively.</li>
</ol>

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The authors have nothing to disclose.

We demonstrate a method that can assess lung tumor growth in the Cre-inducible LSL-KRAS G12D mouse model by ultrasound. This method can be used for evaluating the effect of pharmacological inhibitors on lung tumor growth. It can also be used to compare lung tumor growth between mice of different genetic backgrounds. Using this technique does not require specialized computational skills, however, it is important to be systematic in the number of frames used for analysis to allow for proper comparison if the method is used for comparing different groups of mice.
The intratracheal intubation of ~2 x 106 TU/mL Ca2Cre-shp53 in LSL-KRAS G12D mice led to the formation of precursor lesions after 7 weeks of infection. These appear as longitudinal white lines on sonographic images and are called B-lines (Figure 3B)12. The average number of B-lines per mouse was 141.6 &#177; 41.52 (Figure 3E). We chose to visualize initiating tumors at 7 weeks post-infection since this timepoint would include various types of precursor lesions; atypical adenomatous hyperplasia, epithelial hyperplasia and adenoma3. The formation of precursor lesions on the lung was verified by H&#38;E staining after 8 weeks of lentiviral infection (Figure 4A). After 18 weeks of viral intubation, the tumors are large enough to allow 2D volume measurements in the ultrasound software (Figure 3C). Analysis of ultrasound images was done at the 18-week timepoint in order to include the quantification of well-developed adenoma and adenocarcinoma3. The formation of these types of tumors was confirmed at 20 weeks post-infection by H&#38;E staining analysis (Figure 4B). 3D volume measurements are also possible by the ultrasound software but require high level of expertise. The titre described in this protocol is optimal for the formation of lung tumors that can be properly visualized and quantified in 2D.
Other imaging techniques can also be used to monitor lung tumor growth in mice, such as CT imaging and BLI13. However, CT imaging requires delivering a radiation dose to the tumor, which might affect tumor growth upon repeated assays13. Moreover, it requires the use of contrast agents for accurate tumor measurements13. BLI is an important imaging technique that is often used for quantifying tumor proliferation; however, its main limitation is its dependence on metabolic activities and the presence of ATP and O213. The advantage of ultrasound imaging lies in it being a non-invasive, non-irradiating, speedy and inexpensive technique for acquiring images of the lung parenchyma13. Ultrasound has been previously used to monitor growth of orthotopic human tumors xenografted in lung and pancreas of mice13,14. Raes et al. have been successful in longitudinally monitoring the growth of a single human lung tumor implanted near the posterior diaphragmic surface in mice via ultrasound13. The difference in the LSL-KRAS G12D mouse model is having spontaneous development of lung tumors that can be monitored from initiation to progression and this development can be compared between mice groups. Also, there are numerous tumors being developed on the lung of the LSL-KRAS G12D mouse model, that eventually grow, and might even merge together to form large tumors (Figure 4B). This urged the need to use a method that can monitor lung tumor initiation and progression in this mouse model. We optimized the 2D brightness (B) mode analysis to allow quantitative assessment of lung tumors developed in this mouse model.
A possible limitation with ultrasound imaging is the presence of false positives15. These are seen when a highly reflective surface is present (diaphragm or liver) that interrupts the trajectory of the beam and might create a virtual object mimicking a true object15. Such images could be false tumors but can be bypassed upon proper examination; a large tumor is usually dynamic whereas a virtual object would be static.
Ultrasound imaging for the LSL-KRAS mouse model is ideal due to the non-invasive nature of this technique. The method of analysis used in our lab to monitor lung tumor progression in the LSL-KRAS mouse model is fast and user-friendly.

As the leading cause of cancer-related deaths worldwide, lung cancer remains refractory to treatments, mainly due to lack of relevant pre-clinical models that can recapitulate the disease in the lab1. Around 25% of lung cancer cases are due to mutations in the KRAS oncogene2. KRAS-driven lung cancer is often associated with poor prognosis and low response to therapy, highlighting the importance of further studies in this disease2.
We optimized a method that allows the relative evaluation of lung tumor growth in real time in KRAS lung cancer-induced immune-competent mice. We use Lox-Stop-Lox KRAS G12D (LSL-KRAS G12D) mice in which the KRAS G12D oncogene can be expressed by Cre lentiviral vectors3,4. These vectors are driven by carbonic anhydrase 2, allowing the viral infection to take place specifically in alveolar epithelial cells5. In addition, to accelerate the initiation and progression of lung tumors, the lentiviral construct also expresses P53 shRNA from an U6/H1 promoter (the lentiviral construct herein will be referred to as Ca2Cre-shp53)6. The biological relevance of this method lies in the natural course of lung tumor development in mice as opposed to xenografts of non-orthotopic tumors in mice. An obstacle using the orthotopic method is monitoring lung tumor growth without sacrificing the mouse. To overcome this limitation, we optimized ultrasound imaging to permit the analysis of lung tumor progression in two-dimensional (2D) mode in this mouse model. Initiating tumors at 7 weeks post-infection are reflected as B-lines in ultrasound images, which can be counted, but will not reflect the exact number of tumors present on the lung. B-lines are characterized by laser-like vertical white lines arising from the pleural line in the lung parenchyma7,8. Large tumors can be visualized after 18 weeks of infection. The relative volume of these tumors is quantified by 2D measurements done on ultrasound.
This method is optimal for researchers investigating the effect of pharmacological drugs on lung tumor growth in the LSL-KRAS G12D mouse model. In addition, lung tumor progression can be compared between mice with different genetic lineages, to examine the importance of the presence or absence of certain genes/proteins on the development of lung tumor volume.

<table><tbody><tr><td>0.45 &amp;mu;m Acrodisc Syringe Filters</td><td>Pall Corporation</td><td>PN 4614</td><td></td></tr><tr><td>100-mm Cell Cultre Plate</td><td>CELLSTAR</td><td>664 160</td><td></td></tr><tr><td>6-well Cell Culture Plate</td><td>CELLSTAR</td><td>657 160</td><td></td></tr><tr><td>Amicon Ultra - 15 Centrifugal Filter Units</td><td>Merck Millipore Ltd.</td><td>UFC910024</td><td></td></tr><tr><td>BD LSR-Fortessa</td><td>BD Biosciences</td><td>649225B 3024</td><td></td></tr><tr><td>CA2Cre-shp53 lentiviral vector</td><td></td><td></td><td>From Dr. I Verma Laboratory</td></tr><tr><td>DMEM</td><td>Multicell</td><td>319-005-CL</td><td></td></tr><tr><td>FBS</td><td>Multicell</td><td>80450</td><td></td></tr><tr><td>LSL-KRASG12D mouse</td><td>JAX Mice</td><td>8179</td><td></td></tr><tr><td>MX550S; Centre Transmit: 40 MHz</td><td>FUJIFILM VisualSonics</td><td>51070</td><td></td></tr><tr><td>OptiMEM</td><td>gibco</td><td>11058-021</td><td></td></tr><tr><td>Pen/strep</td><td>Multicell</td><td>450-201-EL</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>PsPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>VEVO-3100</td><td>FUJIFILM VisualSonics</td><td>51072-50</td><td></td></tr></tbody></table>

detection of lung tumor progression in mice by ultrasound imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

After obtaining a lentiviral infectious titer of ~2 x 106 TU/mL (Figure 1), the Ca2Cre-shp53 lentivirus was intratracheally injected when LSL-KRAS G12D mice reached an appropriate age (6&#8722;8 weeks)9. Ultrasound imaging was performed after 7 weeks of infection upon initiation of tumors (Figure 3B). Imaging was done at 7 weeks in order to include the various types of precursor lesions that occur in the LSL-KRASG12D mouse model, ranging from hyperplasia to adenoma3 as shown in Figure 4A after 8 weeks of infection. Imaging earlier than 6 weeks will not ensure the inclusion of adenoma-forming tumors in the analysis3. These precursor lesions are visualized as B-lines in ultrasound and can be counted by eye since they can be identified as narrow beams of white light vertically traversing the screen and are the result of a strong reflection of ultrasound wave (Figure 3B)10. B-lines represent small tumors that are on the surface of the lungs; tumors being initiated in deeper structures of the lung such as close to the trachea will not be reflected as B-lines. The number of tumors being initiated on the pleural surface can be quantified by counting the B-lines, since they are too small for volume measurements. We acquired 500 frames spanning the full lung area in 5 mice. B-lines were counted every 10 frames; thus, they were counted in a total of 50 images per mouse that span the full 500 frames acquired. The scatter plot in Figure 3D shows the sum of B-lines counted in every 10 images (100 frames) for 5 mice. The number of B-lines per mouse is then summed up to include B-lines present in the full lung area. The mean quantity of B-lines representing tumors in the five mice was 141.6 &#177; 41.52 (Figure 3E). Figure 3A shows an ultrasound image acquired of a non-infected mouse lung, for comparison.
According to Jackson et al.3, 16 weeks post-intubation includes adenoma as well as adenocarcinoma, which are tumor types of interest in KRAS lung cancer. To ensure inclusion of these types of tumors when analyzing volume measurements via ultrasound in our model, we imaged the lungs of mice at 18 weeks post-infection. In order to quantify the volume of the large tumors via ultrasound, we used 2D analysis on the ultrasound software. Large tumors appear as deep clefts interrupting the pleural surface as shown in the image of Figure 3C. When a tumor is spotted, we measure the length (L) and width (W) of the tumor, using the measurement tool of the ultrasound software. Once obtained, L and W are applied in the conventional formula used for calculating tumor volume (protocol step 4.4). We analyzed the tumor volume of 10 mice, as shown in Figure 3F, where the number of tumors formed in each mouse ranged from 5 to 26 tumors. The volumes of all tumors formed per mouse are then summed up to represent the tumor volume per mouse. For this analysis, we assume that the tumors formed in the lungs of mice are epileptic in shape, thus the width of the tumor is considered as perpendicular to the imaging plane. Relative volume quantification can be done when linear size of tumors reaches 0.3 mm (either length or width). After analyzing the lung parenchyma of 10 mice, the average tumor volume obtained was 31.8 &#177; 6.61 mm3 (Figure 3G). Hematoxylin and Eosin (H&#38;E) staining analysis on lung sections confirmed the formation of large tumors at 20 weeks post-infection (Figure 4B) and confirmed formation of adenoma and adenocarcinoma. It should be noted that as tumor growth progresses, different tumors might merge together to form large ones, as shown in Figure 4B. Although the ultrasound software permits 3D analysis of tumors, this type of analysis proved difficult in our model, mainly due to the high dynamic movement of the primary tumors formed that is consistent with lung sliding (movement of the pleural line with respiration) as well as cardiac beats11. We reasoned that 2D analysis would provide consistency in relative tumor volume measurements, especially when comparing lung tumor progression between different groups of mice.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60565/60565fig01.jpg" /> 
Figure 1: Flow cytometry analysis of MEFs with a GFP LoxP-allele. Representative flow cytometry images showing frequency of GFP-positive MEFs that are non-infected (Fc) (A) and infected (Fi) with the Ca2Cre-shp53 lentivirus (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60565/60565fig01large.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/60565/60565fig02.jpg" /> 
Figure 2: Workspace of integrated rail system of ultrasound imaging. (A) X-axis micro-knob. (B) Y-axis micro-knob. (C) Animal platform. (D) Height control knob of scan head-probe. (E) Transducer. (F) 3D motor. (G) Heating lamp. (H) Anesthetics tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60565/60565fig02large.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/60565/60565fig03.jpg" /> 
Figure 3: Representative ultrasound images of mouse lungs before and after lung tumor induction. (A) Image of a non-infected mouse lung section. Red arrows point to ribs (R) and the pleural surface (PS) of the lung. (B) Image of a mouse lung 7 weeks after tumor induction by intratracheal intubation of lentivirus. Arrows indicate B-lines reflecting small tumors on the lung surface. (C) Image of a mouse lung section 18 weeks after tumor induction. Blue lines indicate measurements of the length (L: mm) and width (W: mm) of the tumor shown. (D) Scatter plot showing B-line quantification in 10 images spanning 500 frames (each image is separated by 10 frames). (E) Quantification of relative tumor number in lungs at 7 weeks post-induction. (F) Scatter plot showing the relative volume of the different tumors formed in the lungs of 10 mice at 18 weeks post-infection. (G) Quantification of lung tumor volume in mice (n = 10). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60565/60565fig03large.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/60565/60565fig04.jpg" /> 
Figure 4: Representative H&#38;E staining of LSL-KRAS G12D lung sections. (A) Lung section at 8 weeks post-infection at 500 &#181;m magnification. Arrows point at precursor lesions formed at this timepoint, which are reflected as B-lines in ultrasound. Black arrows point at magnified lesions at 60 &#181;m. (B) Lung section at 20 weeks post-infection at 2 mm magnification. Arrowheads of the same color show different tumors that have merged together. Arrows show magnified tumors at 60 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60565/60565fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Detection of Lung Tumor Progression in Mice by Ultrasound Imaging at JoVE.com

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

detection-of-lung-tumor-progression-in-mice-by-ultrasound-imaging

Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.6K Views.  Sir Mortimer B. Davis-Jewish General Hospital. De betekenis van dit protocol ligt in de real-time detecteerbaarheid van de longtumoren die zich spontaan ontwikkelden in dit muismodel, van tumorinitiatie tot tumorprogressie. Het voordeel van deze techniek is dat niet-invasief is, alleen basisvaardigheid vereist en ons in staat stelt het effect van het medicijn en de behandeling van belang direct te controleren. De progressie van de longtumor kan ook worden geëvalueerd in de aanwezigheid of afwezigheid van een gen van belang.Met beh...

Video: Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

Medicine

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

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 ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

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