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>

<ol>
	<li>Eisenstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Personalized+medicine:+Special+treatment.">Personalized medicine: Special treatment.</a> Nature. 513, 8 (2014).</li><li>Karachaliou, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KRAS+mutations+in+lung+cancer.">KRAS mutations in lung cancer.</a> Clinical Lung Cancer. 14 (3), 205-214 (2013).</li><li>Jackson, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+lung+tumor+initiation+and+progression+using+conditional+expression+of+oncogenic+K-ras.">Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.</a> Genes &amp; Development. 15 (24), 3243-3248 (2001).</li><li>DuPage, M., Dooley, A. L., Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+mouse+lung+cancer+models+using+adenoviral+or+lentiviral+delivery+of+Cre+recombinase.">Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase.</a> Nature Protocol. 4 (7), 1064-1072 (2009).</li><li>Chen, J., Lecuona, E., Briva, A., Welch, L. C., Sznajder, J. 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(108), e53771 (2016).</li><li>Saraogi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+ultrasound:+Present+and+future.">Lung ultrasound: Present and future.</a> Lung India. 32 (3), 250-257 (2015).</li><li>Gargani, L., Volpicelli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+I+do+it:+lung+ultrasound.">How I do it: lung ultrasound.</a> Cardiovascular Ultrasound. 12, 25 (2014).</li><li>Soldati, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Physical+Basis+of+Pulmonary+Sonographic+Interstitial+Syndrome.">On the Physical Basis of Pulmonary Sonographic Interstitial Syndrome.</a> Journal of Ultrasound in Medicine. 35 (10), 2975 (2016).</li><li>Raes, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Ultrasound+and+Photoacoustic+Imaging+of+Orthotopic+Lung+Cancer+in+Mice:+New+Perspectives+for+Onco-Pharmacology.">High-Resolution Ultrasound and Photoacoustic Imaging of Orthotopic Lung Cancer in Mice: New Perspectives for Onco-Pharmacology.</a> PLoS One. 11 (4), 15 (2016).</li><li>Lakshman, M., Needles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+quantification+of+the+tumor+microenvironment+with+micro-ultrasound+and+photoacoustic+imaging.">Screening and quantification of the tumor microenvironment with micro-ultrasound and photoacoustic imaging.</a> Nature Methods. 12 (4), 372 (2015).</li><li>Chichra, A., Makaryus, M., Chaudhri, P., Narasimhan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+for+the+Pulmonary+Consultant.">Ultrasound for the Pulmonary Consultant.</a> Clinical Medicine Insights: Circulatory Respiratory and Pulmonary Medicine. 10, 9 (2016).</li></ol>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;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-KR...

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

Research

JoVE Journal

Cancer Research

6.6K Views.  Sir Mortimer B. Davis-Jewish General Hospital. 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.

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

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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

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

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

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

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled.
These authors&#39; laboratories&#39; contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

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 ...