This study was funded by grants from the National Cancer Institute (R01CA166376 and R01CA191489 to D.X. Nguyen) and the Department of Defense (W81XWH-16-1-0227 to D.X. Nguyen).

All experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Yale University.
1. Set Up / Preparation of the Reagents.
<ol>
	<li>Bleomycin 
	Caution: Based on the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals, bleomycin is classified as a GHS08 health hazard.
	<ol>
		<li>Prepare bleomycin in a chemical hood. Resuspend 15 U into 5 mL of sterile phosphate buffered saline (PBS).</li>
		<li>Aliquot 100-200 &#181;L of the solution into glass vials and freeze them at -20 &#176;C for future use. Properly label the tubes with the date of resuspension. Use within 6 months from this date. 
		NOTE: Each mouse strain has a different sensitivity to bleomycin26. Testing different doses of bleomycin for each mouse strain is recommended. The mouse strains and the corresponding doses of bleomycin used in representative experiments are listed in Table 1.</li>
	</ol>
	</li>
	<li>Mice
	<ol>
		<li>Purchase mice needed for the experiment and allow mice 7 days to acclimate before injection. Perform infusions in a biosafety hood. Transfer animals housed in a non-BSL2 compliant room to a BSL2 room using standard institutional procedures prior to bleomycin infusion. Inject mice at 6-8 weeks of age. 
		Caution: Inject mice following institutional guidelines for hazardous agents, biosafety level 2 (BSL2) and IACUC approval. 
		NOTE: Mice purchased for the representative experiments are listed in the Table of Materials. Only male mice have been used.</li>
	</ol>
	</li>
	<li>Lung Cancer Cell Lines
	<ol>
		<li>Culture desired cell lines in their respective media. Before injections, perform short tandem repeat DNA profiling or genetic testing to ensure proper cell line identification. To facilitate in vivo and ex vivo imaging, infect cell lines with a lentivirus expressing the thymidine kinase, green fluorescent protein (GFP) and luciferase fusion reporter27, and sort reporter positive cells by fluorescence activated cell sorting (FACS) prior to engraftment. Test lines for mycoplasma every 6 months.
		<ol>
			<li>Culture H2030 human cancer cell line as recommended by the manufacturer using Roswell Park Memorial Institute medium (RPMI) with 10% Fetal Bovine Serum (FBS), penicillin-streptomycin, and amphotericin B.</li>
			<li>Culture 368T1 murine cell line (derived from a KrasG12D;p53-/- mouse) using Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) with 10% FBS, penicillin-streptomycin, and amphotericin B.</li>
			<li>Culture PC9 human cancer cell line using RPMI with 10% FBS, penicillin-streptomycin, and amphotericin B.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Bleomycin Treatment
<ol>
	<li>Plan to inject mice intratracheally with bleomycin 14 days prior to tumor cell engraftment.</li>
	<li>Thaw bleomycin stock on ice 2 h prior to injection.</li>
	<li>Once thawed, dilute bleomycin to the desired working concentration (0.02 U/50 &#181;L or 0.005 U/50 &#181;L), and keep it on ice. 
	NOTE: Concentration of bleomycin depends on the mouse strain used for the experiments (Table 1). Titration of bleomycin in mice should be performed to determine optimal dose.</li>
	<li>Prepare anesthesia by diluting ketamine and xylazine to a final concentration of 10 mg/mL and 1 mg/mL, respectively, Using a 1 mL syringe and 27 G needle, anesthetize each mouse by injecting intraperitoneally ketamine/xylazine solution at 100/10 mg/kg respectively.</li>
	<li>Monitor the breathing of mice and employ a toe pinch. Confirm proper anesthetization when mouse does not respond to toe pinch. Apply vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Place 1 mouse at a time on an intubation platform by hanging its front teeth with its back against the platform.</li>
	<li>Illuminate the upper chest using a fiber-optic illuminator to help with visualization of the trachea. Open the mouth of the mouse and pull the tongue out gently with sterile flat forceps.</li>
	<li>Use an intravenous catheter without the needle to avoid blocking breathing. Position the catheter over the white light emitted from the opening of the trachea.</li>
	<li>Insert the catheter into the trachea until the top of the catheter reaches the front teeth. Confirm proper placement of the catheter in the trachea by visualizing the white light shining through the opening of the catheter in the mouth.</li>
	<li>Using a pipette, dispense 50 &#181;L of bleomycin or vehicle (PBS) directly into the catheter to ensure that the entire volume is inhaled. Perform this step under sterile conditions.</li>
	<li>If the catheter is correctly inserted, the mouse will immediately inhale the contents of the catheter. Wait a few seconds until the entire volume travels down the catheter. Then remove the catheter from the trachea and dispose in 10% bleach solution. 
	NOTE: The average time per mouse of steps 2.7-2.11 is around 5 min.</li>
	<li>If the mouse is not inhaling the liquid, carefully monitor breathing and adjust the catheter position. If the mouse stops breathing, remove the catheter immediately and allow the mouse to resume breathing normally before re-inserting the catheter.</li>
	<li>Place the injected mouse on an IACUC approved heating pad on their back on a flat surface for recovery. The whole procedure will take 10 min per mouse. Do not return an animal that has undergone injection to the company of other animals until fully recovered.</li>
	<li>Place a &#34;Hazardous Chemical in Use&#34; Card on the cage for 24 h.</li>
</ol>
3. Monitoring Mice Post-Intubation
<ol>
	<li>Monitor the mice for respiratory distress immediately after intubation and then every 15 min until mice wake up from anesthesia. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Inject ketoprofen (5 mg/kg) intraperitoneally to alleviate pain.</li>
	<li>Examine the mice 24 h post intubation and 3 times a week for any signs of morbidity.</li>
	<li>Keep a record of the date of procedure, animal identification, type of procedure, type of anesthetic, and post-operative monitoring observations with times.</li>
	<li>Monitor the mice for weight loss, respiratory distress, behavioral abnormalities, and a body condition score &#60;2 (segmentation of vertebral column evident/dorsal pelvic bones are palpable) bi-weekly following injection of bleomycin.</li>
	<li>Euthanize mice under respiratory distress or mice that have experienced 15% loss of body mass by CO2 or by ketamine/xylazine followed by cervical dislocation. Confirm euthanasia by conducting palpation for respiratory activity.</li>
</ol>
4. Engraftment of Lung Adenocarcinoma Cell Lines.
NOTE: Perform engraftment of cells 14 days after the injection of bleomycin (step 2.1).
<ol>
	<li>Allow cells to grow in 75 cm2 flasks undisturbed for 3 days prior to the day of injection with corresponding media as stated in step 1.3. 
	NOTE: They should be 80% confluent at the day of injection (which corresponds to 2-3 x 106 in each flask depending on the cell line).</li>
	<li>Wash the cells growing on 75 cm2 flasks with 5 mL of PBS at room temperature.</li>
	<li>Aspirate PBS and add 1.5 mL of trypsin to the cells and incubate the cells at 37 &#176;C until the cells detach (typically 2-5 min). 
	NOTE: We recommend checking every 2 min for cell detachment from the plastic by simple visualization of the bottom of the flask or under a light microscope. Do not exceed 5 min of incubation with trypsin.</li>
	<li>Neutralize trypsin by adding 4 mL of media containing 10% FBS (same media as step 4.1). Collect the cells into a 15 mL tube and centrifuge it at 200 x g for 3 min at room temperature.</li>
	<li>Aspirate the media and resuspend the cell pellet in 5 mL of PBS to wash cells. Centrifuge the cells at 200 x g for 3 min at room temperature to pellet cells. Repeat this 1 time.</li>
	<li>Aspirate the PBS and resuspend the pellet in 1.5 mL of PBS per flask.</li>
	<li>Mix 50 &#181;L of cell mixture with 50 &#181;L of trypan blue and count the cells using a Cell Counter/Hemocytometer chamber28.</li>
	<li>Prepare cell suspension by diluting the cells in PBS to inject 1 x 105 cells (or other amount indicated) in 50 &#181;L per mouse. Keep the cells on ice prior to injection. 
	NOTE: Prepare at least two times more cells for injection to avoid running out of cell suspension.</li>
	<li>Prepare anesthesia by diluting ketamine and xylazine to a final concentration of 10 mg/mL and 1 mg/mL, respectively, Using a 1 mL syringe and 27 G needle, anesthetize each mouse by injecting intraperitoneally ketamine/xylazine solution at 100/10 mg/kg respectively.</li>
	<li>Monitor the breathing of mice and employ a toe pinch. Confirm proper anesthetization when mouse does not respond to toe pinch. Apply vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Place 1 mouse at a time on an intubation platform by hanging its front teeth with its back against the platform.</li>
	<li>Illuminate the upper chest using a fiber-optic illuminator to help with visualization of the trachea.</li>
	<li>Resuspend the cells gently using a p200 pipette to make sure they do not form clumps. Open the mouth of the mouse and pull the tongue out gently with sterile flat forceps.</li>
	<li>Use an intravenous catheter with the needle removed to avoid blocking breathing. Position the catheter over the white light emitted from the opening of the trachea.</li>
	<li>Insert the catheter into the trachea until the top of the catheter reaches the front teeth. Confirm proper placement of the catheter in the trachea by visualizing the white light shining through the opening of the catheter in the mouth.</li>
	<li>Using a pipette, dispense 50 &#181;L of cells directly into the catheter to ensure that the entire volume is inhaled. Perform this step under sterile conditions. 
	NOTE: If the catheter is correctly inserted, the mouse will immediately inhale the contents of the catheter.</li>
	<li>Wait a few seconds until the entire volume travels down the catheter, and then remove the catheter from the trachea and dispose in 10% bleach solution.</li>
	<li>If the mouse is not inhaling the liquid, carefully monitor breathing and adjust the catheter position. If the mouse stops breathing, remove the catheter immediately and allow the mouse to resume breathing normally before re-inserting the catheter. 
	NOTE: The average time per mouse of steps 4.11-4.18 is around 5 min.</li>
	<li>Place the injected mouse on an IACUC approved heating pad on their back on a flat surface for recovery. 
	NOTE: The whole procedure will take 10 min per mouse. Do not return an animal that has undergone injection to the company of other animals until fully recovered.</li>
	<li>Shave the entire rib area both ventrally and dorsally of B6129SF1/J and other mouse strains with dark hair using an electric shaver prior to imaging.</li>
	<li>2-3 min after cell injection, inject 100 &#181;L of luciferin retro-orbitally (15 mg/mL) using an insulin needle. Alternate the eye used for injection every imaging session.</li>
	<li>After at least 2 min, place up to 5 mice in an animal bioluminescence imager in the dorsal recumbency position and acquire a ventral picture using luminescence settings29.</li>
	<li>Under the control panel adjust the resolution and sensitivity settings to measure luminescence of a given cell line. For most applications, start with 3 min (or &#34;Auto&#34; if saturated), binning=Medium (4), F/Stop=1, Field of View=D, Subject Height=1.50. 
	NOTE: If the injection is successful, luminescence signal is detected in the upper chest area, in one lung or both lungs.</li>
	<li>Flip mice onto sternal recumbency position and acquire a dorsal picture as above. 
	NOTE: If cell injection is not performed properly, cells may cluster at the entrance of the trachea or in the neck.</li>
	<li>Inject ketoprofen 5 mg/kg intraperitoneally to alleviate pain.</li>
	<li>Move mice back to their cage and place a &#34;BSL-2 Agent in Use&#34; Card on the cage.</li>
	<li>Monitor mice for respiratory distress immediately after intubation, and then every 15 min until mice wake up from anesthesia. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Examine the mice 24 h post intubation and three times a week for any signs of morbidity.</li>
	<li>Keep record in the logbook of the date of procedure, animal identification, type of procedure, type of anesthetic and post-operative monitoring observations and times.</li>
	<li>Monitor mice for weight loss, respiratory distress, behavioral abnormalities, and a body condition score &#60;2 (segmentation of vertebral column evident/dorsal pelvic bones are palpable) bi-weekly following inoculation of bleomycin. 
	NOTE: Mice with lung tumors may exhibit respiratory irritation, weight loss, cachexia, and/or death.</li>
	<li>Euthanize mice under respiratory distress or mice that have experienced 15% loss of body mass by CO2 or by ketamine/xylazine followed by cervical dislocation if collecting tissues. Confirm euthanasia by conducting palpation for respiratory activity.</li>
</ol>
5. Monitoring of Tumor Growth by Bioluminescence Imaging
<ol>
	<li>Repeat imaging of the mice at day 3 post-engraftment and weekly thereafter.</li>
	<li>Anesthetize mice by injecting ketamine/xylazine (as described in steps 2.4-2.5) or by inhaled isoflurane (2%). Monitor breathing of mice and employ a toe pinch to confirm proper anesthetization. Apply vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Shave the entire rib area both ventrally and dorsally of B6129SF1/J and other mouse strains with dark hair using an electric shaver prior to imaging.</li>
	<li>Once the mice are under anesthesia, inject 100 &#181;L of luciferin retro-orbitally (15 mg/mL) using an insulin needle. Wait 2 min. Alternate the eye used for injection every imaging session.</li>
	<li>Place mice in the imager and acquire dorsal and ventral images as described in steps 4.22-4.24.</li>
	<li>Place mice back into the cage after imaging, on their back on a flat surface for recovery. 
	NOTE: The whole procedure will take 5 min per group of 5 mice. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Analyze the images using the imager/bioluminescence analysis software.
	<ol>
		<li>Select the appropriate images and load them as a group in the bioluminescence analysis software.</li>
		<li>Click ROI tool | square to add a square region of interest (ROI). Place the ROI over the upper chest area, covering the entire lung image. Repeat this for each mouse image.</li>
		<li>Right click and select Copy all ROI function to apply the same ROI to all images in the group and position them in the upper chest of the mice, both ventral and dorsal views.</li>
		<li>In the upper left corner of the image window, select the Photons function. Click the Measure control panel function to measure the ROIs. Copy and paste the output table containing total flux (photons/s) into a software of choice to analyze the data further.</li>
		<li>Normalize the daily ROI value of each mouse to its ROI value measured on day 0.</li>
	</ol>
	</li>
</ol>
6. Tissue Isolation and Processing.
<ol>
	<li>Anesthetize mice by injecting ketamine/xylazine (as described in steps 2.4-2.5 or by inhaled isoflurane (2%). Monitor breathing of mice and employ a toe pinch. Confirm proper anesthetization when mouse does not respond to toe pinch. Apply vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Inject 100 &#181;L of luciferin retro-orbitally (15 mg/mL) using an insulin needle. Wait 2 min.</li>
	<li>Image mice following steps 4.22-4.24 from this protocol.</li>
	<li>Inject into the other eye from step 6.2 another 100 &#181;L of luciferin retro-orbitally using an insulin needle prior to euthanasia.</li>
	<li>Euthanize the mice by intraperitoneal injection of ketamine/xylazine (see step 2.4 for details) followed by cervical dislocation if mice are under deep anesthesia in accordance with guidelines provided by IACUC. Confirm euthanasia by conducting palpation for respiratory activity.</li>
	<li>Using sterile surgical scissors make a skin incision below the sternum. Open the muscle layer along the diaphragm using forceps and surgical scissors and expose the thoracic cavity by cutting through the rib cage on either of the lateral sides avoiding the internal thoracic arteries.</li>
	<li>Perfuse the mouse by making a small incision in the right atrium of the heart and inject 5 mL of PBS through the left ventricle. The color of lung tissue will go from red to pale pink-white if the blood perfusion is done properly.</li>
	<li>Excise the lungs from the thoracic cavity carefully by grabbing the esophagus or heart with forceps. Gently pull the tissue and use surgical scissors to carefully cut connecting tissue avoiding puncture of the lung tissue. Preserve the trachea as much as possible for inflation of the lung airways at a later step.</li>
	<li>Wash the lung by submerging and swirling the tissue 3 times in a 6 well plate filled with 3 mL of PBS to remove excess blood.</li>
	<li>Place the lung onto the lid of a 6 well plate, and place the lid containing the tissue in the bioluminescent imager and acquire a luminescent image30. 
	NOTE: We recommend selecting the following settings from the &#34;Acquisition control panel&#34; &#34;Field of view=A&#34; and &#34;Subject Height=0.75 cm&#34; and then &#34;Auto exposure&#34; to avoid getting saturated images.</li>
	<li>Excise and image other organs, as done in step 6.9-6.10, where metastasis have been identified by luminescence, such as brain, liver, adrenal, bone, lymph nodes, kidney or spleen31.</li>
	<li>Perfuse the lung with 1 mL of 4% paraformaldehyde by inserting the needle into the trachea of the mouse. The lungs will inflate if well-perfused. 
	Caution: 4% paraformaldehyde is a toxic, a health hazard GHS07 and GHS08.</li>
	<li>Transfer the lungs into a 15 mL conical with 5 mL of 4% paraformaldehyde.</li>
	<li>Fix the lungs or other tissues by incubating the tissue with 4% paraformaldehyde overnight at 4 &#176;C in a shaking device at 30-50 rpm.</li>
	<li>Embed lungs (or other tissues) in paraffin or optimum cutting temperature compound depending on the application32. 
	NOTE: Paraffin is the preferred method to preserve the lung structure and for Masson Trichrome and Hematoxylin-Eosin staining.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+the+nude+(nu/nu)+mutation+on+bleomycin-induced+pulmonary+fibrosis.+A+biochemical+evaluation.">The effects of the nude (nu/nu) mutation on bleomycin-induced pulmonary fibrosis. A biochemical evaluation.</a> Am Rev Respir Dis. 127, 614-617 (1983).</li><li>Ponomarev, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+triple-modality+reporter+gene+for+whole-body+fluorescent,+bioluminescent,+and+nuclear+noninvasive+imaging.">A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging.</a> Eur J Nucl Med Mol I. 31, 740-751 (2004).</li><li>Morten, B. C., Scott, R. J., Avery-Kiejda, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Three+Different+Methods+for+Determining+Cell+Proliferation+in+Breast+Cancer+Cell.">Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell.</a> J. Vis. Exp. , (2016).</li><li>Tseng, J. C., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+bioluminescence+imaging+of+mouse+tumor+models.">Quantitative bioluminescence imaging of mouse tumor models.</a> Cold Spring Harbor protocols. , (2015).</li><li>Byrne, F. L., McCarroll, J. A., Kavallaris, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+Tumor+Burden+In+Vivo+and+Metastasis+Ex+Vivo+Using+Luciferase-Expressing+Cancer+Cells+in+an+Orthotopic+Mouse+Model+of+Neuroblastoma.">Analyses of Tumor Burden In Vivo and Metastasis Ex Vivo Using Luciferase-Expressing Cancer Cells in an Orthotopic Mouse Model of Neuroblastoma.</a> Methods Mol Biol. 1372, 61-77 (2016).</li><li>Parkinson, C. M., 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=Diagnostic+necropsy+and+selected+tissue+and+sample+collection+in+rats+and+mice.">Diagnostic necropsy and selected tissue and sample collection in rats and mice.</a> J. Vis. Exp. , (2011).</li><li>Tammela, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Wnt-producing+niche+drives+proliferative+potential+and+progression+in+lung+adenocarcinoma.">A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma.</a> Nature. 545, 355-359 (2017).</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> Nat Protoc. 4, 1064-1072 (2009).</li><li>Byrne, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+open+issues+in+cancer+precision+medicine+with+patient-derived+xenografts.">Interrogating open issues in cancer precision medicine with patient-derived xenografts.</a> Nat Rev Cancer. 17, 254-268 (2017).</li><li>Nguyen, D. X., 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=WNT/TCF+signaling+through+LEF1+and+HOXB9+mediates+lung+adenocarcinoma+metastasis.">WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis.</a> Cell. 138, 51-62 (2009).</li><li>Raghu, G., Nyberg, F., Morgan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+interstitial+lung+disease+and+its+association+with+lung+cancer.">The epidemiology of interstitial lung disease and its association with lung cancer.</a> Brit J Cancer. 91, S3-S10 (2004).</li><li>Hubbard, R., Venn, A., Lewis, S., Britton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer+and+cryptogenic+fibrosing+alveolitis.+A+population-based+cohort+study.">Lung cancer and cryptogenic fibrosing alveolitis. A population-based cohort study.</a> Am J Resp Crit Care. 161, 5-8 (2000).</li><li>Nagai, A., Chiyotani, A., Nakadate, T., Konno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer+in+patients+with+idiopathic+pulmonary+fibrosis.">Lung cancer in patients with idiopathic pulmonary fibrosis.</a> Tohoku J Exp Med. 167, 231-237 (1992).</li><li>Rock, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+stromal+populations+contribute+to+pulmonary+fibrosis+without+evidence+for+epithelial+to+mesenchymal+transition.">Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition.</a> Proc Natl Acad Sci USA. 108, E1475-E1483 (2011).</li><li>Saito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+after+surgery+for+pathologic+stage+IA+non-small+cell+lung+cancer+associated+with+idiopathic+pulmonary+fibrosis.">Survival after surgery for pathologic stage IA non-small cell lung cancer associated with idiopathic pulmonary fibrosis.</a> Ann Thorac Surg. 92, 1812-1817 (2011).</li><li>Stevens, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+Receptor+Expression+in+Subtypes+of+Lung+Adenocarcinoma+Potentiates+Outgrowth+of+Micrometastases.">Extracellular Matrix Receptor Expression in Subtypes of Lung Adenocarcinoma Potentiates Outgrowth of Micrometastases.</a> Cancer Res. 77, 1905-1917 (2017).</li><li>Harrison, J. H., Lazo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+dose+continuous+infusion+of+bleomycin+in+mice:+a+new+model+for+drug-induced+pulmonary+fibrosis.">High dose continuous infusion of bleomycin in mice: a new model for drug-induced pulmonary fibrosis.</a> J Pharmacol Exp Ther. 243, 1185-1194 (1987).</li><li>Shcherbo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+far-red+fluorescent+protein+for+whole-body+imaging.">Bright far-red fluorescent protein for whole-body imaging.</a> Nature Methods. 4, 741-746 (2007).</li><li>Aso, Y., Yoneda, K., Kikkawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+biochemical+study+of+pulmonary+changes+induced+by+bleomycin+in+mice.">Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice.</a> Lab Invest. 35, 558-568 (1976).</li><li>Kim, C. 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=Identification+of+bronchioalveolar+stem+cells+in+normal+lung+and+lung.">Identification of bronchioalveolar stem cells in normal lung and lung.</a> Cell. 121, 823-835 (2005).</li><li>Fichtner, I., 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=Establishment+of+patient-derived+non-small+cell+lung+cancer+xenografts+as+models+for+the+identification+of+predictive+biomarkers.">Establishment of patient-derived non-small cell lung cancer xenografts as models for the identification of predictive biomarkers.</a> Clin Cancer Res. 14, 6456-6468 (2008).</li><li>Zhang, X. 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The authors declare no competing financial interests.

Striking clinical parallels have been documented between lung cancer and other chronic diseases of the lung36. In particular, patients with idiopathic pulmonary fibrosis (IPF) have an increased predilection for developing lung cancer, and this association is independent of smoking history37,38. IPF is characterized by progressive destruction of lung architecture and impaired respiratory function through deposition of ECM3...

Lung cancer is the leading cause of cancer related deaths worldwide1. Patients with lung cancer eventually succumb from metastasis to distant organs, notably to the central nervous system, liver, adrenal glands, and bones2,3,4. Thoracic malignancies have been traditionally classified as small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC)5. NSCLC is the most frequently diagnosed malignancy and can be subdivided into different histological subtypes, including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC)6. Genomic analysis of resected human primary lung cancers has revealed that tumors within a given histotype can also express diverse molecular perturbations, further contributing to their divergent clinical progression and confounding patient prognosis. The remarkable heterogeneity of lung cancers represents a significant challenge to the rational design, pre-clinical testing, and implementation of effective therapeutic strategies. Consequently, there is a need to expand the repertoire of tractable experimental lung cancer models to study the diverse cellular origins, molecular subtypes, and stages of this disease.
Various approaches using animal models have been employed to study lung cancer in vivo, each with their own advantages and disadvantages depending on the biological question(s) of interest. Genetically engineered mouse models (GEMMs) can target specific genetic alterations in a given progenitor cell type, resulting in tumors that progress within an immunocompetent host7. While extremely powerful and clinically relevant, the latency, variability, and/or lung tumor morbidity associated with GEMMs can be prohibitive to certain quantitative measurements and the detection of late stage metastasis in distant organs8. A complementary approach is the use of allograft models, whereby lung cancer cells, obtained either directly from a mouse tumor or derived first as established cell lines in culture, are re-introduced into syngeneic hosts. Analogously, lung cancer xenografts are established from human cell lines or patient derived tumor samples. Human cell line xenografts or patient derived xenografts (PDXs) are generally maintained in immunocompromised mice and therefore preclude complete immune-surveillance9. Despite this drawback, they provide an avenue to propagate limiting amounts of human biospecimens and study fundamental in vivo properties of human cancer cells, which encode for more complex genomic aberrations than GEMM tumors.
One useful property of allografts and xenografts is that they are amenable to traditional limiting cell dilution assays, employed to quantify the frequency of tumor initiating cells (TICs) within a malignant cell population10. In these experiments, a defined number of cells are injected subcutaneously into the flank of animals and the frequency of TICs can be estimated based on tumor take rate. Subcutaneous tumors however can be more hypoxic11 and may not model key physiological constraints of the lung tumor microenvironment. Intratracheal delivery of epithelial stem or progenitor cells into the lungs of mice is a method to study pulmonary regeneration and airway stem cell biology12. However, the engraftment rate from this technique can be relatively low, unless the lungs are first subjected to physiological forms of injury, such as viral infection13,14. Support from inflammatory stromal cells and/or the disruption of the lung basement membrane may improve retention of transplanted cells into relevant stem cell niches in the distal airways15. Fibrosis inducing agents can also pre-condition the lungs to enhance engraftment of induced pluripotent cells16 and mesenchymal stem cells17. Whether similar forms of airway injury can affect the engraftment rate, tumor initiating capacity, and outgrowth of lung cancer cells has yet to be systematically assessed.
In this study, we describe a method to increase the efficiency of orthotopic lung cancer cell engraftment, by pre-conditioning the lungs of mice with injury. LUAD arises in the distal airways with a significant subset of these cancers developing a fibrotic stroma18 that often correlates with poor prognosis19. Bleomycin, a natural nonribosomal hybrid peptide-polyketide, has been extensively utilized to induce pulmonary fibrosis in mice20. Airway instillation of bleomycin first promotes epithelial attrition in the alveoli and recruitment of inflammatory cells, including macrophages, neutrophils and monocytes21. This is followed by tissue remodeling in the distal airways, basement membrane reorganization22,23 and extracellular matrix (ECM) deposition24. The effects of a single bleomycin injection are transient, with fibrosis resolving after 30 days in most studies25. Using both allograft and xenograft models, we tested if pre-conditioning the airways of mice with bleomycin could significantly increase the take rate of LUAD cells in the lungs.

<table>
	<tbody>
		<tr>
			<td>Bleomycin</td>
			<td>Sigma</td>
			<td>B5507-15UN</td>
			<td>CAUTION Health hazard GHS08</td>
		</tr>
		<tr>
			<td>Exel Catheter 24G</td>
			<td>Fisher</td>
			<td>1484121</td>
			<td>Remove needle. For intratracheal injection</td>
		</tr>
		<tr>
			<td>Ketamine (Ketaset inl 100 mg/mL C3N 10 mL)</td>
			<td>Butler Schein</td>
			<td>56344</td>
			<td>To anesthetize mice</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Butler Schein</td>
			<td>33198</td>
			<td>To anesthetize mice</td>
		</tr>
		<tr>
			<td>Ketoprofen, 5,000 mg</td>
			<td>Cayman Chemical</td>
			<td>10006661</td>
			<td>Analgesic</td>
		</tr>
		<tr>
			<td>Puralube Veterinary Ophthalmic Ointment</td>
			<td>BUTLER ANIMAL HEALTH COMPANY LLC</td>
			<td>8897</td>
			<td>To prevent eye dryness while under anesthesia</td>
		</tr>
		<tr>
			<td>D-Luciferin powder</td>
			<td>Perkin Elmer Health Sciences Inc</td>
			<td>122799</td>
			<td>For luminescent imaging. Reconstitute powder with PBS for a working concentration of 15mg/mL. Protect from Light</td>
		</tr>
		<tr>
			<td>Rodent Intubation stand</td>
			<td>Braintree Scientific</td>
			<td>RIS-100</td>
			<td>Recommended stand for intratracheal injection</td>
		</tr>
		<tr>
			<td>MI-150 ILLUMINATOR 150W MI-150</td>
			<td>DOLAN-JENNER INDUSTRIES</td>
			<td>MI-150 / EEG2823M</td>
			<td>To illuminate and visualize trachea</td>
		</tr>
		<tr>
			<td>Graefe Forceps, 2.75 (7 cm) long serrat</td>
			<td>Roboz</td>
			<td>RS-5111</td>
			<td>For intratracheal injection</td>
		</tr>
		<tr>
			<td>Syringe Luer-Lok Sterile 5ml</td>
			<td>BD / Fisher</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Satiny Smooth by Conair Dual Foil Wet/Dry Rechargeable Shaver</td>
			<td>Conair</td>
			<td>-</td>
			<td>To shave mice</td>
		</tr>
		<tr>
			<td>Bonn Scissors, 3.5&#34; straight 15 mm sharp/sharp sure cut blades</td>
			<td>Roboz</td>
			<td>RS-5840SC</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>BD / Fisher</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>USA SCIENTIFIC INC</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vial Scintillation 7 mL Borosilicate Glass GPI</td>
			<td>Fisher</td>
			<td>701350</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter pipette tips (200 &#956;L)</td>
			<td>USA SCIENTIFIC INC</td>
			<td>1120-8710</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum USDA</td>
			<td>Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A2942-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain 0.4%</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Flask T/C 75cm sq canted neck, blue cap</td>
			<td>Fisher / Corning</td>
			<td>353135</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum Xenogen Bioluminiscence</td>
			<td>Perkin Elmer Health Sciences Inc</td>
			<td>124262</td>
			<td>For in vivo bioluminescence imaging</td>
		</tr>
		<tr>
			<td>Living image software</td>
			<td>Perkin Elmer Health Sciences Inc</td>
			<td>128113</td>
			<td>For in vivo bioluminescence analysis</td>
		</tr>
		<tr>
			<td>XGI-8 Gas Anesthesia System</td>
			<td>Perkin Elmer Health Sciences Inc</td>
			<td>118918</td>
			<td>For Isoflurane anesthesia</td>
		</tr>
		<tr>
			<td>BD Ultra-Fine II Short Needle Insulin Syringe 1 cc. 31 G x 8 mm (5/16 in)</td>
			<td>BD / Fisher</td>
			<td>BD328418</td>
			<td>For retro-orbital luciferin injection</td>
		</tr>
		<tr>
			<td>Syringe 1ml</td>
			<td>BD / Fisher</td>
			<td>14-823-434</td>
			<td>For intraperitoneal injections</td>
		</tr>
		<tr>
			<td>26 G x 1/2 in. needle</td>
			<td>BD / Fisher</td>
			<td>305111</td>
			<td>For intraperitoneal injections</td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>VWR</td>
			<td>43368-9M</td>
			<td>CAUTION Health hazard GHS07, GHS08. For fixing tissue</td>
		</tr>
		<tr>
			<td>Pipet-Lite Pipette, Unv. SL-200XLS+</td>
			<td>METTLER-TOLEDO INTERNATIONAL</td>
			<td>17014411</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#39;s Hematoxylin</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>517-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y stain 0.25% (w/v) in 57%</td>
			<td>Fisher</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson Trichrome Stain Kit</td>
			<td>IMEB Inc</td>
			<td>K7228</td>
			<td>For masson trichrome stain to visualize collagen</td>
		</tr>
		<tr>
			<td>Superfrost plus glass slides</td>
			<td>Fisher</td>
			<td>1255015</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Corning</td>
			<td>C3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Mycoplasma Detection Kit</td>
			<td>ATCC</td>
			<td>30-1012K</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Embedding compound</td>
			<td>ELECTRON MICROSCOPY SCIENCES</td>
			<td>62550-12</td>
			<td>For embedding tissue for frozen sections</td>
		</tr>
		<tr>
			<td>Leica CM3050 S Research Cryostat</td>
			<td>Leica</td>
			<td>CM3050 S</td>
			<td>To section tissue for staining analysis</td>
		</tr>
		<tr>
			<td>Keyence All-in One Fluorescence Microscope</td>
			<td>Keyence</td>
			<td>BZ-X700</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>US National Institutes of Health</td>
			<td>IJ1.46</td>
			<td>http://rsbweb.nih.gov/ij/ download.html</td>
		</tr>
		<tr>
			<td>Prism 7.0 for Mac OS X</td>
			<td>GraphPad Software, Inc.</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Athymic (Crl:NU(NCr)-Foxn1nu) mice</td>
			<td>Charles River</td>
			<td>NIH-553</td>
			<td></td>
		</tr>
		<tr>
			<td>NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice</td>
			<td>Jackson Laboratories</td>
			<td>5557</td>
			<td></td>
		</tr>
		<tr>
			<td>B6129SF1/J mice</td>
			<td>Jackson Laboratories</td>
			<td>101043</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH-H2030 cells</td>
			<td>ATCC</td>
			<td>CRL-5914</td>
			<td></td>
		</tr>
		<tr>
			<td>368T1</td>
			<td>generously provided by Monte Winslow (Standford University)</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>PC9 cells</td>
			<td>Nguyen DX et al. Cell. 2009;138:51&#8211;62</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>H2030 BrM3 cells</td>
			<td>Nguyen DX et al. Cell. 2009;138:51&#8211;62</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

pre-conditioning the airways of mice with bleomycin increases the efficiency of orthotopic lung cancer cell engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

To increase the efficiency of LUAD cancer cell engraftment into the lungs of mice, we developed a protocol that first pre-conditions the airways using bleomycin followed by orthotopic tumor cell injection (Figure 1). We confirmed that even when administered into immunocompromised athymic mice, bleomycin induced transient fibrosis by day 14 as evidenced by loss of airway architecture and increased collagen deposition (Figure 2). G...

Watch this Scientific Journal Video about Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment at JoVE.com

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

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13.5K Views.  Yale University School of Medicine. We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts. 

Video: Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...