The authors would like to thank Wade Koba for his assistance with micro computed tomography (S10RR029545), Vera DesMarais and Hillary Guzik of the Analytical Imaging Facility for their training and assistance with microscopy, the Einstein Montefiore Cancer Center, the National Cancer Institute (P30CA013330, R01CA21248, R01CA255153), the Gruss Lipper Biophotonics Center, the Integrated Imaging Program for Cancer Research, a Sir Henry Wellcome Postdoctoral Fellowship (221647/Z/20/Z), and a METAvivor Career Development Award.

All procedures described in this protocol have been performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.
Prior to surgery, lung metastases are to be generated in mice using cancer cells that express a photoconvertible/photo-switchable protein; several protocols for lung metastasis generation have been published26,27,28.
1. Preparation for surgery
<ol>
	<li>Prepare distinct work areas for mouse preparation (hair removal and intubation), surgery, and recovery.</li>
	<li>Sterilize all surgical instruments in an autoclave. 
	NOTE: As a tips-only technique is used for this surgery, a hot bead sterilizer may be used to resterilize instruments for subsequent procedures.</li>
	<li>Turn on the heated surgical platform and allow it to reach and stabilize at 37-40 &#176;C.</li>
	<li>Induce anesthesia with 5% isoflurane, and then lower the isoflurane to 3%. Perform toe pinch tests periodically throughout the procedure to assess anesthesia adequacy.</li>
	<li>Place the anesthetized mouse in a right lateral decubitus position. Remove the hair of the upper-left chest/side body (see Figure 1A) by applying depilatory cream to the area. Dampen a piece of gauze or paper towel with water and wipe the hair and depilatory cream away. Repeat as necessary until all hair is removed from the surgical field. 
	NOTE: Do not leave depilatory cream on the mouse for longer than 20 s, as this can damage the skin.</li>
	<li>Tie a double knot ~3 mm above the base of a 22 G catheter with 2-0 silk suture. Leave 2-inch-long tails.</li>
	<li>Intubate the mouse with this catheter as previously described29,30. To confirm successful intubation, attach an inflation bulb to the end of the catheter and gently squeeze. 
	NOTE: Mice that have been intubated successfully will experience bilateral chest rise with bulb squeeze.</li>
	<li>Tightly secure the 2-0 silk suture around the mouse&#39;s snout with a double knot to keep the intubation catheter in place.</li>
</ol>
2. Surgery to expose the lung
NOTE: Perform all steps of the surgery (Figure 1), including photoconversion, in a hood or laminar flow cabinet to avoid contamination of the surgical field.
<ol>
	<li>Wash hands with antiseptic soap and don new sterile gloves. 
	NOTE: As a tips-only technique is used for this surgery, non-sterile gloves may also be used. Sanitizing non-sterile gloves with an alcohol-based disinfectant is recommended.</li>
	<li>Prepare a 0.1 mg/kg dose of buprenorphine (0.03 mg/mL) and inject subcutaneously for analgesia. 
	NOTE: Multimodal analgesia, including local pain blockers and nonsteroidal anti-inflammatory drugs, should be used in conjunction with buprenorphine if they are not expected to interfere with the biology of interest.</li>
	<li>Apply ophthalmic ointment to both mouse eyes to prevent corneal damage.</li>
	<li>Place the mouse in the right lateral decubitus position on the heated surgical platform.</li>
	<li>Connect the ventilator to the intubation catheter. Take care to hold the catheter steady, as even slight movements can disturb the catheter placement and lead to poor ventilation.</li>
	<li>Observe stable, ventilator-controlled, bilateral chest rise and fall.</li>
	<li>Secure the limbs to the surgical platform with labeling tape. Stabilize the surgical field by placing another piece of tape along the mouse&#39;s back and securing the dorsal skin and fur to the surgical platform (Figure 1A).</li>
	<li>Open the sterilized surgical instruments.</li>
	<li>Apply chlorhexidine solution to the mouse&#39;s skin to sterilize the surgical site.</li>
	<li>Identify the area ~7 mm to the left of the sternum and ~7 mm above the subcostal margin. Lift the skin over this area with forceps and make a ~10 mm circular incision using sharp micro-dissecting scissors, taking care to cut only the skin.</li>
	<li>Remove the soft tissue over the ribcage (Figure 1B). Cauterize any major vessels at both ends with a cautery pen. 
	NOTE: In addition to excising all soft tissue underlying the 10 mm circular incision in the skin, removing some additional non-muscular soft tissue from the perimeter facilitates future steps of the procedure.</li>
	<li>With one hand, use forceps to elevate the 6th or 7th rib. With the other hand, use a single blade of the blunt micro-dissecting scissors - angled parallel to the chest wall, rounded edge facing down - and carefully pierce the 6th or 7th intercostal muscle to enter the thoracic cavity (Figure 1C). Gently rotate the scissors as needed and cut to make a ~10 mm incision in the intercostal space, taking care to avoid touching the lung tissue and cutting the ribs.</li>
	<li>Delicately discharge compressed air towards the intercostal incision to increase the space between the lung and the chest wall. Discharge the air first at one&#39;s wrist or hand to find the appropriate flow strength and clear the nozzle of any debris, then in short bursts at the incision to prevent lung injury.</li>
	<li>With one hand, use forceps to elevate the 6th or 7th rib. With the other hand, hold the retractor shut and insert it into the intercostal incision. Orient the blades of the retractor such that they are parallel to the chest wall, taking care to avoid touching the lung itself (Figure 1D).</li>
	<li>Once the retractor is in place, slowly release the handle, allowing the retractor to open and expose the lung (Figure 1E). Figure 2A shows representative images of exposed lung metastases prior to photoconversion, including what they look like to the naked eye, and in the FITC (green, non-photoconverted) and TRITC (red, photoconverted) fluorescent channels while using wide-field illumination.</li>
</ol>
3. Lung metastasis photoconversion
NOTE: Details and variations on the following steps can be found in the Discussion.
<ol>
	<li>Gently apply 2-3 drops of sterile PBS warmed to 37 &#176;C to the exposed lung tissue to prevent drying.</li>
	<li>Place a piece of sterile aluminum foil with a small cutout over the mouse. Position the cutout directly over the exposed lung tissue and size it to expose only the open portion of the thorax and a few millimeters of surrounding soft tissue and skin to ensure photoconversion of only the exposed lung.</li>
	<li>Place a box with a cutout over the mouse and aluminum foil. Ensure that the cutout is directly over the exposed lung.</li>
	<li>Place the photoconversion lamp directly over the cutout on the box (Figure 1F).</li>
	<li>Turn on the photoconversion lamp and allow 6 min of continuous illumination of exposed lung tissue. 
	NOTE: Monitor the mouse&#39;s vital signs using the physiologic monitoring programs on the ventilator.</li>
	<li>After photoconversion, turn off the lamp, and remove the lamp, box, and aluminum foil mask from the mouse.</li>
</ol>
4. Procedure to close chest wall
<ol>
	<li>Repeat step 2.13 to separate the lung from the chest wall.</li>
	<li>Carefully take hold of the retractor handle and squeeze to close the blades.</li>
	<li>Maneuver the closed retractor out of the intercostal space, taking care to avoid touching the lung tissue.</li>
	<li>Using the 5-0 silk suture, close the intercostal incision with a simple continuous suture pattern that incorporates the rib on both sides of the incision (Figure 1G). Pull the suture tight to ensure that the wound edges are appositional and re-establish the integrity of the chest wall (Figure 1H). Take care not to overtighten the suture as this may tear the intercostal muscles.</li>
	<li>Secure the suture by knotting the two ends of the suture together 4x. Cut the suture tails as close to the knot as possible.</li>
	<li>Close the skin with surgical staples.</li>
	<li>Remove excess air from the thoracic cavity with a 1 mL insulin syringe with a 28 G needle attached. Locate the xiphoid process tactilely, and insert the needle just below, advancing toward the left shoulder to enter the thoracic cavity through the diaphragm. Draw back on the syringe to ~1 mL; remove the needle from the mouse and discharge the air from the syringe. Repeat this process 3-4x. 
	NOTE: Take care not to puncture any internal organs. Furthermore, it is important to use a 1 mL syringe and repeat the procedure 3-4x rather than using a larger volume syringe and trying to remove all the air at once. Use of a larger volume syringe may result in too great of a vacuum in the thoracic cavity and lead to lung tissue distension and damage.</li>
	<li>Turn off the isoflurane.</li>
	<li>Continue ventilation with 100% oxygen until the mouse shows signs of awakening.</li>
	<li>Once the mouse shows signs of awakening, disconnect the intubation catheter from the ventilator, cut the suture around the snout, and extubate the mouse.</li>
	<li>Untape the mouse and transfer it to a clean cage placed ~2 feet below a heat lamp. Monitor until fully recovered. If the mouse shows signs of breathing difficulty or lack of mobility, anesthetize it with 5% isoflurane for 5 min, ensure adequate anesthesia by observing a loss of reflexes upon toe pinch, and euthanize via cervical dislocation.</li>
	<li>Once the mouse fully awakens from anesthesia and begins to move, prepare another 0.1 mg/kg dose of buprenorphine and inject it subcutaneously for postoperative analgesia.</li>
	<li>Following the surgery, ensure that the mice are housed individually. Provide antibiotics in the drinking water (enrofloxacin, final concentration 0.4 mg/mL).</li>
	<li>In the 3 days following the surgery, check the mouse twice per day for signs of distress. For pain management, administer 0.1 mg/kg buprenorphine (0.03 mg/mL) subcutaneously every 4-6 hours. Euthanize the mouse if it shows signs of infection, immobility, or breathing difficulty. After the third day, mice can be checked once per day.</li>
</ol>
5. Sample processing and detection of photoconverted cells using tissue clearing
<ol>
	<li>Between 3 and 5 days (or the desired length of time) after photoconversion surgery, anesthetize the mouse with 5% isoflurane, reduce isoflurane from 5% to 2.5% after induction, ensure adequate anesthesia by observing a loss of reflexes upon toe pinch, and perform a terminal transcardial perfusion with 10 mL of room-temperature PBS, followed by 10 mL of 4% paraformaldehyde chilled to 4 &#176;C as previously described31.</li>
	<li>Collect the organs of interest and optically clear them as previously described31.</li>
	<li>Image the cleared tissues with a light sheet microscope as previously described31. Alternatively, place the cleared tissues in a glass bottom dish and image in the FITC and TRITC channels to visualize green (non-photoconverted) and red (photoconverted) cells using a confocal, spinning disk, or multiphoton microscope.</li>
</ol>
6. Sample processing and detection of photoconverted cells using tissue disaggregation
<ol>
	<li>Anesthetize the mouse with 5% isoflurane for 5 min, ensure adequate anesthesia by observing a loss of reflexes upon toe pinch, and sacrifice via cervical dislocation 3-5 days after photoconversion.</li>
	<li>Collect and digest the organs of interest as previously described13.</li>
	<li>Plate the digested tissues and place them in an incubator to adhere overnight as previously described13.</li>
	<li>The following day, image the plated cells in the FITC and TRITC channels to visualize green (non-photoconverted) and red (photoconverted) cells as previously described13.</li>
</ol>

Examining Tumor Cell Dissemination from Lung Metastases

<ol>
	<li>Dillek&#229;s, H., Rogers, M. S., Straume, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+90%+of+deaths+from+cancer+caused+by+metastases.">Are 90% of deaths from cancer caused by metastases.</a> Cancer Medicine. 8 (12), 5574-5576 (2019).</li><li>Gupta, G. P., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+building+a+framework.">Cancer metastasis: building a framework.</a> Cell. 127 (4), 679-695 (2006).</li><li>Nguyen, D. X., Bos, P. D., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nature Reviews. Cancer. 9 (4), 274-284 (2009).</li><li>Paget, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+secondary+growths+in+cancer+of+the+breast.">The distribution of secondary growths in cancer of the breast.</a> Cancer Metastasis Reviews. 8 (2), 98-101 (1989).</li><li>Liu, 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=Self-seeding+circulating+tumor+cells+promote+the+proliferation+and+metastasis+of+human+osteosarcoma+by+upregulating+interleukin-8.">Self-seeding circulating tumor cells promote the proliferation and metastasis of human osteosarcoma by upregulating interleukin-8.</a> Cell Death &amp; Disease. 10 (8), 575 (2019).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+exosomes+promote+tumor+self-seeding+in+hepatocellular+carcinoma+by+transferring+miRNA-25-5p+to+enhance+cell+motility.">Tumor-derived exosomes promote tumor self-seeding in hepatocellular carcinoma by transferring miRNA-25-5p to enhance cell motility.</a> Oncogene. 37 (36), 4964-4978 (2018).</li><li>Kim, M. -. 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=Tumor+self-seeding+by+circulating+cancer+cells.">Tumor self-seeding by circulating cancer cells.</a> Cell. 139 (7), 1315-1326 (2009).</li><li>Zhang, 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=Tumor+self-seeding+by+circulating+tumor+cells+in+nude+mouse+models+of+human+osteosarcoma+and+a+preliminary+study+of+its+mechanisms.">Tumor self-seeding by circulating tumor cells in nude mouse models of human osteosarcoma and a preliminary study of its mechanisms.</a> Journal of Cancer Research and Clinical Oncology. 140 (2), 329-340 (2014).</li><li>Dondossola, E., Crippa, L., Colombo, B., Ferrero, E., Corti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromogranin+A+regulates+tumor+self-seeding+and+dissemination.">Chromogranin A regulates tumor self-seeding and dissemination.</a> Cancer Research. 72 (2), 449-459 (2012).</li><li>Brown, 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=Lymph+node+blood+vessels+provide+exit+routes+for+metastatic+tumor+cell+dissemination+in+mice.">Lymph node blood vessels provide exit routes for metastatic tumor cell dissemination in mice.</a> Science. 359 (6382), 1408-1411 (2018).</li><li>Pereira, E. 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=Lymph+node+metastases+can+invade+local+blood+vessels,+exit+the+node,+and+colonize+distant+organs+in+mice.">Lymph node metastases can invade local blood vessels, exit the node, and colonize distant organs in mice.</a> Science. 359 (6382), 1403-1407 (2018).</li><li>Coste, A., 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=Hematogenous+dissemination+of+breast+cancer+cells+from+lymph+nodes+is+mediated+by+tumor+microenvironment+of+metastasis+doorways.">Hematogenous dissemination of breast cancer cells from lymph nodes is mediated by tumor microenvironment of metastasis doorways.</a> Frontiers in Oncology. 10, 571100 (2020).</li><li>Borriello, L., Condeelis, J., Entenberg, D., Oktay, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cell+re-dissemination+from+lung+metastases-a+mechanism+for+enhancing+metastatic+burden.">Breast cancer cell re-dissemination from lung metastases-a mechanism for enhancing metastatic burden.</a> Journal of Clinical Medicine. 10 (11), 2340 (2021).</li><li>Ullah, 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=Evolutionary+history+of+metastatic+breast+cancer+reveals+minimal+seeding+from+axillary+lymph+nodes.">Evolutionary history of metastatic breast cancer reveals minimal seeding from axillary lymph nodes.</a> The Journal of Clinical Investigation. 128 (4), 1355-1370 (2018).</li><li>Gundem, 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=The+evolutionary+history+of+lethal+metastatic+prostate+cancer.">The evolutionary history of lethal metastatic prostate cancer.</a> Nature. 520 (7547), 353-357 (2015).</li><li>Brown, 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=Phylogenetic+analysis+of+metastatic+progression+in+breast+cancer+using+somatic+mutations+and+copy+number+aberrations.">Phylogenetic analysis of metastatic progression in breast cancer using somatic mutations and copy number aberrations.</a> Nature Communications. 8, 14944 (2017).</li><li>Kalhor, R., Mali, P., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapidly+evolving+homing+CRISPR+barcodes.">Rapidly evolving homing CRISPR barcodes.</a> Nature Methods. 14 (2), 195-200 (2017).</li><li>Kalhor, 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=Developmental+barcoding+of+whole+mouse+via+homing+CRISPR.">Developmental barcoding of whole mouse via homing CRISPR.</a> Science. 361 (6405), eaat9804 (2018).</li><li>McKenna, A., 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=Whole-organism+lineage+tracing+by+combinatorial+and+cumulative+genome+editing.">Whole-organism lineage tracing by combinatorial and cumulative genome editing.</a> Science. 353 (6298), aaf7907 (2016).</li><li>Junker, J. P., 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=Massively+parallel+clonal+analysis+using+CRISPR/Cas9+induced+genetic+scars.">Massively parallel clonal analysis using CRISPR/Cas9 induced genetic scars.</a> bioRxiv. , 056499 (2017).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone+microenvironment+invigorates+metastatic+seeds+for+further+dissemination.">The bone microenvironment invigorates metastatic seeds for further dissemination.</a> Cell. 184 (9), 2471.e20-2486.e20 (2021).</li><li>Kedrin, 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=Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window.">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nature Methods. 5 (12), 1019-1021 (2008).</li><li>Grau, 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=Spatiotemporally+controlled+induction+of+gene+expression+in+vivo+allows+tracking+the+fate+of+tumor+cells+that+traffic+through+the+lymphatics.">Spatiotemporally controlled induction of gene expression in vivo allows tracking the fate of tumor cells that traffic through the lymphatics.</a> International Journal of Cancer. 147 (4), 1190-1198 (2020).</li><li>Riihim&#228;ki, M., Thomsen, H., Sundquist, K., Sundquist, J., Hemminki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+landscape+of+cancer+metastases.">Clinical landscape of cancer metastases.</a> Cancer Medicine. 7 (11), 5534-5542 (2018).</li><li>Gurskaya, N. 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=Engineering+of+a+monomeric+green-to-red+photoactivatable+fluorescent+protein+induced+by+blue+light.">Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.</a> Nature Biotechnology. 24 (4), 461-465 (2006).</li><li>Zhang, G. -. L., Zhang, Y., Cao, K. -. X., Wang, X. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+injection+of+breast+cancer+cells+into+the+mice+mammary+fat+pad.">Orthotopic injection of breast cancer cells into the mice mammary fat pad.</a> Journal of Visualized Experiments. (143), (2019).</li><li>Pavese, J., Ogden, I. M., Bergan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthotopic+murine+model+of+human+prostate+cancer+metastasis.">An orthotopic murine model of human prostate cancer metastasis.</a> Journal of Visualized Experiments. (79), (2013).</li><li>Thies, K. A., Steck, S., Knoblaugh, S. E., Sizemore, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+analysis+of+lung+metastasis+following+lateral+tail-vein+injection+of+tumor+cells.">Pathological analysis of lung metastasis following lateral tail-vein injection of tumor cells.</a> Journal of Visualized Experiments. (159), (2020).</li><li>Das, S., MacDonald, K., Sucie Chang, h. -. y., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+mouse+lung+intubation.">A simple method of mouse lung intubation.</a> Journal of Visualized Experiments. (73), e50318 (2013).</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 Protocols. 4 (7), 1064-1072 (2009).</li><li>Hsu, C. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EZ+Clear+for+simple,+rapid,+and+robust+mouse+whole+organ+clearing.">EZ Clear for simple, rapid, and robust mouse whole organ clearing.</a> eLife. 11, e77419 (2022).</li><li>Entenberg, 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=Setup+and+use+of+a+two-laser+multiphoton+microscope+for+multichannel+intravital+fluorescence+imaging.">Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging.</a> Nature Protocols. 6 (10), 1500-1520 (2011).</li><li>Gambotto, A., 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=Immunogenicity+of+enhanced+green+fluorescent+protein+(EGFP)+in+BALB/c+mice:+identification+of+an+H2-Kd-restricted+CTL+epitope.">Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope.</a> Gene Therapy. 7 (23), 2036-2040 (2000).</li><li>Han, W. G. H., Unger, W. W. J., Wauben, M. H. M. <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+the+immunodominant+CTL+epitope+of+EGFP+in+C57BL/6+mice.">Identification of the immunodominant CTL epitope of EGFP in C57BL/6 mice.</a> Gene Therapy. 15 (9), 700-701 (2008).</li><li>Stripecke, 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=Immune+response+to+green+fluorescent+protein:+implications+for+gene+therapy.">Immune response to green fluorescent protein: implications for gene therapy.</a> Gene Therapy. 6 (7), 1305-1312 (1999).</li><li>Rosenzweig, 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=Induction+of+cytotoxic+T+lymphocyte+and+antibody+responses+to+enhanced+green+fluorescent+protein+following+transplantation+of+transduced+CD34(+)+hematopoietic+cells.">Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34(+) hematopoietic cells.</a> Blood. 97 (7), 1951-1959 (2001).</li><li>Grzelak, C. A., 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=Elimination+of+fluorescent+protein+immunogenicity+permits+modeling+of+metastasis+in+immune-competent+settings.">Elimination of fluorescent protein immunogenicity permits modeling of metastasis in immune-competent settings.</a> Cancer Cell. 40 (1), 1-2 (2022).</li><li>Fluegen, 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=Phenotypic+heterogeneity+of+disseminated+tumour+cells+is+preset+by+primary+tumour+hypoxic+microenvironments.">Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments.</a> Nature Cell Biology. 19 (2), 120-132 (2017).</li><li>Yan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+engrafted+human+cancer+and+therapy+responses+in+immunodeficient+zebrafish.">Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish.</a> Cell. 177 (7), 1903.e14-1914.e14 (2019).</li><li>Borriello, L., Traub, B., Coste, A., Oktay, M. H., Entenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+permanent+window+for+investigating+cancer+metastasis+to+the+lung.">A permanent window for investigating cancer metastasis to the lung.</a> Journal of Visualized Experiments. (173), (2021).</li><li>Tohme, S., Simmons, R. L., Tsung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgery+for+cancer:+a+trigger+for+metastases.">Surgery for cancer: a trigger for metastases.</a> Cancer Research. 77 (7), 1548-1552 (2017).</li><li>Al-Sahaf, O., Wang, J. H., Browne, T. J., Cotter, T. G., Redmond, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+injury+enhances+the+expression+of+genes+that+mediate+breast+cancer+metastasis+to+the+lung.">Surgical injury enhances the expression of genes that mediate breast cancer metastasis to the lung.</a> Annals of Surgery. 252 (6), 1037-1043 (2010).</li><li>Lu, N., Piao, M. -. H., Feng, C. -. S., Yuan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+promotes+epithelial-to-mesenchymal+transition+and+metastasis+of+bladder+cancer+cells+through+HIF-1&#945;-&#946;-catenin/Notch1+pathways.">Isoflurane promotes epithelial-to-mesenchymal transition and metastasis of bladder cancer cells through HIF-1&#945;-&#946;-catenin/Notch1 pathways.</a> Life Sciences. 258, 118154 (2020).</li><li>Jiao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+volatile+anesthetics+and+tumor+progression:+unveiling+the+mystery.">Relationship between volatile anesthetics and tumor progression: unveiling the mystery.</a> Current Medical Science. 38 (6), 962-967 (2018).</li></ol>

The authors have no conflicts of interest to declare.

In this paper, we describe a surgical protocol for the selective photoconversion of tumor cells in the lung. This technique enables researchers to selectively mark tumor cells in the lung and track their fate by reidentifying them throughout the body at a later time point, facilitating the study of metastasis from lung metastases. Using this protocol, it was possible to visualize photoconverted cells in the brain, liver, and non-photoconverted right lung of mice that had undergone the surgery with photoconversion, indicating that these cells redisseminated from the photoconverted lung metastases to these tertiary sites. We did not find any red cells in the organs of mice that did not undergo photoconversion, indicating that natural autofluorescence in cells is not bright enough to be confused with photoconverted tumor cells.
Careful attention to certain aspects of the experiment can increase the success rate of this procedure. First, when generating lung metastases, the protocol for metastasis generation and photoconvertible/photo-switchable protein must be considered. It has been reported that some fluorescent proteins are immunogenic33,34,35,36. Thus, cancer cells expressing such proteins may not spontaneously metastasize in immunocompetent mice. Indeed, we observed that primary tumors form in both syngeneic, immunocompetent (FVB) mice and immunocompromised (Rag2-/-) mice upon orthotopic injection of 6DT1-Dendra2 cells. However, metastases only developed in Rag2-/- mice, indicating that Dendra2 may be somewhat immunogenic. Ways to overcome the potential immunogenicity of fluorescent proteins to generate lung metastases include using an immunocompromised mouse, generating metastases via tail vein injection, using a different photoconvertible/photo-switchable protein, or using a transgenic animal in which the photoconvertible/photo-switchable protein is genetically encoded so that the mice are tolerized to the protein37. The strength and stability of the fluorescent signal in the photoconverted channel must also be considered. For many photoconvertible/photoswitchable proteins, the signal in the photoconverted channel declines over time as the protein gets degraded and diluted with cell division. We and others have previously shown that photoconverted H2B-linked Dendra2 can be reliably detected for 7 days in dividing cells and 16 days in non-dividing cells38,39. Linking the photoconvertible protein to a protein with a low turnover rate can extend the half-life of the photoconverted signal. Methods for troubleshooting the lung metastasis model must depend upon the research aim and cancer cell type being used.
Second, it is important to time the surgery properly based on the disease model of interest. Those who work with disease models with fast-growing lung metastases may have a limited timeframe in which to perform the surgery before the mice become too overwhelmed by tumor burden to recover from surgery and/or survive the desired amount of time after surgery. A thorough understanding of the disease model timeline, as well as the use of non-invasive imaging strategies such as micro computed tomography, may help in timing this procedure.
Third, inadvertent cutting of major vessels during soft tissue removal can cause excessive bleeding, leading to poor visibility of the surgical field and/or exsanguination, as previously described40. Avoiding major vessels and using a cauterizing pen during surgery can prevent this. Fourth, when inserting and removing the retractor, it is crucial to keep the retractor blades parallel to the chest wall at all times. Angling the blades towards the lung increases the risk of lung damage, and angling the blades towards the chest wall increases the risk of breaching the chest wall in other areas. If inserting or removing the retractor in a parallel fashion does not seem possible, the blunt edge micro-dissecting scissors can be used to slightly increase the length of the intercostal incision before trying to insert/remove the retractor again. Finally, care should be taken to ensure that the ribcage is completely resealed at the end of the surgery. When suturing the ribs back together, stitching should be begun ~1 mm before the start of the incision and ended ~1 mm after. This helps to avoid residual openings on the ends of the incision. Additionally, take care to pull and tie the suture tight enough to eliminate space between the ribs, but not so tight that the sutures tear the adjacent intercostal muscles and create additional breaches. If resealing the thoracic cavity cannot be achieved, the mouse should be humanely sacrificed during the surgery, as it will have great difficulty or be unable to breathe on its own when mechanical ventilation is stopped.
The light source and exact methodology for photoconversion may vary between laboratories. Any light source and methodologies that are safe, consistent, and appropriate for the model may be used. For instance, photoconversion may be performed by transferring the mouse to a stereoscope or other microscope capable of shining light of the wavelength and intensity needed for photoconversion on the exposed lung. We use a custom-built 400 nm light-emitting diode array lamp set to its highest intensity to photoconvert Dendra2-expressing lung metastases (Supplementary Figure S1). To support the lamp above the mouse and ensure that the lamp is placed at the same distance above each mouse, we cut a 6.5 x 6.5 cm opening in the bottom of an open-topped cardboard box (18.4 cm (L) x 13.2 cm (W) x 7.3 cm (H)). We place the box upside down over the mouse and rest the 8.5 x 8.5 cm lamp over the 6.5 x 6.5 cm cutout (Figure 1F), so the lamp is supported, and the blue light can reach the exposed lung tissue.
It is important to note that some aspects of this protocol, including surgery and the use of anesthetics, may alter the dissemination kinetics and capacity of tumor cells41,42,43,44. As such, proper controls must be used to ensure that results are not confounded by the procedure.
The main limitation of this technique is that only metastases on the exposed portion of the lung will undergo photoconversion. Therefore, not every tumor cell that redisseminates from the lung will be photoconverted and any quantification of redisseminated cells will be relative. Despite this limitation, this technique can still provide valuable insight into tumor cell redissemination, including determining whether and when it happens, what promotes or prevents it, and the organotropism of redisseminated cells. To our knowledge, this is the first technique that allows selective marking and fate-tracking of tumor cells in the lung. In conclusion, this protocol describes a novel technique that can be used to facilitate and enhance the study of tumor cell redissemination and metastasis-to-metastasis seeding without genomic analysis. Targeting lung metastases makes the procedure useful and relevant to metastasis researchers studying most major cancer types.

Metastasis is the leading cause of cancer-related deaths1.&#160;Metastatic cancer arises when cells from the primary tumor disseminate throughout the body and proliferate into clinically detectable tumors in distant organs2,3.
Although metastasis is commonly thought of as a unidirectional process wherein tumor cells disseminate from the primary tumor and colonize distant organs4, increasing clinical and experimental evidence suggests that a more complex, multi-directional process is at play. It has been shown that circulating tumor cells can reseed the primary tumor (if still in place)5,6,7,8,9, and tumor cells from existing metastatic foci can travel to tertiary sites and give rise to new lesions10,11,12,13. Indeed, evidence from recent genomic analyses suggests that some metastatic lesions arise not from the primary tumor, but from other metastases-a phenomenon known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding&#34;14,15,16. Metastasis-to-metastasis seeding can perpetuate the disease process even after removal of the primary tumor, increasing metastatic burden, and decreasing patient quality of life and survival. Therefore, understanding the processes behind metastasis-to-metastasis seeding is crucial to refining treatment strategies for patients with metastatic disease.
Despite the potentially severe clinical implications, little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Human studies are limited by a paucity of clinical samples. Clinical resection and biopsy of metastatic lesions are uncommon, as is the biopsy of seemingly healthy organs, where single disseminated tumor cells may lurk. This means that human studies are typically only possible using autopsy samples from individuals whose primary tumors are either still in place or were previously resected but are still available to researchers. When such samples are available, lineage analyses of cancer progression must be performed using sequencing methods14. However, bulk sequencing of matched primary tumors and metastases does not have the sensitivity needed for comprehensive lineage tracing. For instance, bulk sequencing of one lesion may reveal a subclone that is undetectable in any of its matched lesions. In this case, one would be unable to determine the origin of this subclone. It may have been present in the primary tumor or another metastasis at a frequency below the limit of detection, or it may have arisen after the initial colonization of the metastatic lesion it was found in. Single-cell sequencing provides increased sensitivity, but its high cost limits the large-scale application of this technique. The retrospective nature of these studies also means that they provide limited insight into transient metastatic events and the disease landscape at different time points.
In animal models, recent technological advances now allow for prospective phylogenetic mapping with high spatial and temporal resolution17,18,19,20. These techniques utilize CRISPR/Cas9 genome editing to engineer cells with an evolving barcode - heritable mutations that accumulate over time. Upon sequencing, the lineage of each cell can be traced based on the mutational profile of its barcode17,18,19,20. Indeed, such technology is already being used to map metastasis-to-metastasis seeding. In a recent paper, Zhang et al. demonstrated that breast and prostate cancer cells in bone metastases redisseminate from the bone to seed secondary metastases in multiple organs21.
While these novel methods have great potential to generate detailed, high-resolution phylogenetic maps of cancer progression, they are highly impractical for those studying the exact timing of metastasis-to-metastasis seeding events and what promotes or prevents them. Filling these knowledge gaps is crucial to refining our understanding and treatment of metastatic cancer, but there is a noticeable lack of technologies to facilitate such studies. To address this need, we recently developed - and present herein - a novel technique that allows us to specifically mark tumor cells via photoconversion in a metastatic site (the lung) and subsequently reidentify them in tertiary organs. Using this technique, we recently showed that breast cancer cells do redisseminate from lung metastases and seed tertiary organs13. This technique may also be used to determine the timing of redissemination events within a narrow window and quantify redisseminated tumor cells, facilitating the study of organotropism of redisseminated cells and what promotes/prevents redissemination.
While photoconversion and locally inducible cre/lox systems that permanently replace one fluorescent protein with another have been previously used to mark and track tumor cells11,22,23, to our knowledge, no approach for spatiotemporal marking of tumor cells has been optimized to target the lung - one of the most common sites of metastasis among men and women diagnosed with any of the 14 most common cancers24. Any cancer cell type and any protocol for lung metastasis generation may be used with our procedure, making it broadly useful for metastasis researchers. All cancer cells used to generate lung metastases should express a photoconvertible or photo-switchable protein, and researchers may choose which protein to use based on their specific needs and resources. In this study, we used 6DT1 breast cancer cells that stably expressed the photoconvertible green-to-red fluorescent protein Dendra2 (6DT1-Dendra2 cells)25 tagged to the histone H2B. We injected 5.0 &#215; 104 6DT1-Dendra2 cells into the fourth mammary fat pad of female Rag2-/- mice. Primary tumors were palpable between 12 and 16 days after injection and were not resected for the duration of the experiment. Spontaneous lung metastases developed between 19 and 26 days after tumor cell injection. Photoconversion surgeries were performed between 26 and 29 days after tumor cell injection. Mice were sacrificed by 72 h post surgery due to lung metastasis burden.

<table><tbody><tr><td>0-30 V, 0-3 A Power Supply</td><td>MPJA</td><td>9616 PS</td><td></td></tr><tr><td>12 VDC, 1.2 A Unregulated Plug Supply</td><td>MPJA</td><td>17563 PD</td><td></td></tr><tr><td>28 G 1 mL BD Insulin Syringe</td><td>BD</td><td>329410</td><td></td></tr><tr><td>400 nm light emitting diode array lamp</td><td>LedEngin Inc.</td><td>897-LZPD0UA00</td><td>Photoconversion lamp, custom-built (individual parts included below)</td></tr><tr><td>5-0 braided silk suture with RB-1 cutting needle</td><td>Ethicon, Inc.</td><td>774B</td><td></td></tr><tr><td>9 cm 2-0 silk tie</td><td>Ethicon, Inc.</td><td>LA55G</td><td></td></tr><tr><td>Baytril 100 (enrofloxacin)</td><td>Bayer (Santa Cruz Biotechnology)</td><td>sc-362890Rx</td><td>Antibiotic used in drinking water</td></tr><tr><td>Buprenorphine</td><td>Hospira</td><td>0409-2012-32</td><td>Analgesic</td></tr><tr><td>Cables (Cable Assemblies) 2.5 JK-ST 72&quot; ZIP CD</td><td>Mouser</td><td>172-0250</td><td></td></tr><tr><td>Chlorhexidine solution</td><td>Durvet</td><td>7-45801-10258-3</td><td>Chlorhexidine Disinfectant Solution</td></tr><tr><td>Compressed air canister</td><td>Falcon</td><td>DPSJB-12</td><td></td></tr><tr><td>Extra Fine Micro Dissecting Scissors 4&quot; Straight Sharp/Sharp 24 mm</td><td>Roboz Surgical</td><td>RS-5912</td><td>Sharp Micro Dissecting Scissors</td></tr><tr><td>Fiber-optic illuminator</td><td>O.C. White Company</td><td>FL3000</td><td>Used during mouse intubation</td></tr><tr><td>Gemini Cautery Kit</td><td>Harvard Apparatus</td><td>726067</td><td>Cautery pen</td></tr><tr><td>Germinator 500</td><td>CellPoint Scientific</td><td>GER&amp;nbsp;5287-120V</td><td>Bead Sterilizer</td></tr><tr><td>Graefe forceps</td><td>Roboz</td><td>RS-5135</td><td></td></tr><tr><td>High power LEDs - single color ultraviolet 90 watts</td><td>Mouser</td><td>LZP-D0UA00</td><td></td></tr><tr><td>Infrared heat lamp</td><td>Braintree Scientific</td><td>HL-1</td><td></td></tr><tr><td>Isoflurane SOL 250 mL PVL</td><td>Covetrus</td><td>29405</td><td>Anesthetic</td></tr><tr><td>Isoflurane vaporizer</td><td>SurgiVet</td><td>VCT302</td><td></td></tr><tr><td>Jacobson needle holder with lock</td><td>Kalson Surgical</td><td>T1-140</td><td></td></tr><tr><td>Labeling tape</td><td>Fisher Scientific</td><td>S68702</td><td></td></tr><tr><td>LED Lighting Reflectors CREE MP-L SNGL LENS REFLECTOR &amp;amp; LOC PIN</td><td>Mouser</td><td>928-C11395TM</td><td></td></tr><tr><td>Long cotton tip applicators</td><td>Medline Industries</td><td>MDS202055</td><td></td></tr><tr><td>Masscool / Soccket 478 / Intel Pentium 4/Celeron up to 3.4GHz / Ball Bearing / Copper Core / CPU Cooling Fan</td><td>CompUSA</td><td>#S457-1023</td><td></td></tr><tr><td>Micro Dissecting Scissors 4&quot; Straight Blunt/Blunt</td><td>Roboz Surgical</td><td>RS-5980</td><td>Blunt Micro Dissecting Scissors</td></tr><tr><td>Murine ventilator</td><td>Kent Scientific&amp;nbsp;</td><td>PS-02</td><td>PhysioSuite</td></tr><tr><td>Nair Hair Removal Lotion</td><td>Amazon</td><td>B001RVMR7K</td><td>Depilatory cream</td></tr><tr><td>Personnet mini retractor</td><td>Roboz</td><td>RS-6504</td><td>Retractor</td></tr><tr><td>Phosphate Buffered Saline 1x</td><td>Fisher Scientific</td><td>14190144</td><td>PBS</td></tr><tr><td>pLenti.CAG.H2B-Dendra2.W</td><td>Addgene</td><td>51005</td><td>Dendra2 lentivirus</td></tr><tr><td>Puralube</td><td>Henry Schein Animal Health</td><td>008897</td><td>Eye Lubricant</td></tr><tr><td>Rodent intubation stand</td><td>Braintree Scientific</td><td>RIS 100</td><td></td></tr><tr><td>Small animal lung inflation bulb</td><td>Harvard Apparatus</td><td>72-9083</td><td></td></tr><tr><td>SurgiSuite Multi-Functional Surgical Platform for Mice, with Warming</td><td>Kent Scientific</td><td>SURGI-M02</td><td>Heated surgical platform</td></tr><tr><td>Test Leads 48&quot; TEST LEAD BANANA - Black</td><td>Mouser</td><td>565-1440-48-0</td><td></td></tr><tr><td>Test Leads 48&quot; TEST LEAD BANANA - Red</td><td>Mouser</td><td>565-1440-48-2</td><td></td></tr><tr><td>Tracheal catheter&amp;nbsp;</td><td>Exelint International</td><td>26746</td><td>22 G catheter</td></tr><tr><td>Wound closing system veterinary kit</td><td>Clay Adams</td><td>IN015</td><td>Veterinary surgical stapling kit</td></tr></tbody></table>

tracking tumor cell dissemination from lung metastases using photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

The steps of the surgery described in this protocol are illustrated in Figure 1. In brief, the mouse is anesthetized, and hair is removed from the left thorax. The mouse is then intubated and ventilated, which allows the mouse to receive oxygen while the thoracic cavity is open. Soft tissue is removed to expose the ribcage, and an incision is made in the 6th or 7th intercostal muscle. A retractor is inserted in the intercostal breach and released to spread the adjacent ribs and expose the left lung and any photoconvertible metastases thereupon. The lung and metastases are then exposed to blue light to photoconvert the exposed lesions. Following photoconversion, the retractor is removed, the ribs are sutured back together, and the overlying skin is closed. Finally, excess air is removed from the thoracic cavity to alleviate pressure on the lungs and allow the mouse to resume independent, spontaneous respiration.
The protocol described here can be adapted to a number of different photoconvertible/photo-switchable proteins. Ensuring that photoconverted cells reach their peak fluorescence intensity in the photoconverted channel can facilitate their identification in other organs. The conditions needed to achieve peak fluorescence intensity after photoconversion should be determined empirically, as they may vary with other aspects of the experiment such as the photoconvertible protein being used and the intensity of the light used to photoconvert. Figure 2B,C demonstrate how the fluorescence intensity changed in both the FITC (non-photoconverted) and TRITC (photoconverted) channels as a function of the duration of exposure to blue light in an ex vivo sample. We determined that 6 min of exposure to blue light produced the brightest signal in the photoconverted channel and that additional exposure led to photobleaching. We then measured the fluorescence intensity in both channels before and after 6 min of blue light exposure, in vivo, and obtained similar results (Figure 2D).
Next, the brain, liver, and non-photoconverted right lung were harvested from mice that underwent this surgery with (experimental group) and without (control group) the photoconversion lamp turned on. The tissues were optically cleared as previously described31, placed in a glass bottom dish, and imaged on a custom-built multiphoton microscope32 using a 25x 1.0 NA objective lens and an excitation wavelength of 880 nm. Fluorescence was captured for the green and red channels using the bandpass filters 525/35 nm and 580/60 nm, respectively. Photoconverted and non-photoconverted cells could be clearly identified in all three tissue types from mice that underwent the surgery with blue light exposure (Figure 3). As expected, only non-photoconverted tumor cells were found in the tissues of mice that underwent the surgery without blue light exposure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65732/65732fig01.jpg" /> 
Figure 1: Overview of the surgical protocol for the lung photoconversion surgery. (A) Depilated mouse placed on the surgical stage and taped in position. (B) A 10 mm circular clearing of skin and soft tissue over the ribcage. (C) Initial breach of intercostal muscle with blunt-edge micro-dissecting scissors. (D) Closed retractor inserted in 10 mm intercostal incision. (E) Open retractor exposing lung tissue. (F) Photoconversion lamp in place directly above open thorax of the mouse. (G) Simple continuous suture that incorporates the rib on both sides of the intercostal incision. (H) Suture tightened and secured to reestablish chest wall integrity. (I) Removal of air from thoracic cavity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65732/65732fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65732/65732fig02.jpg" /> 
Figure 2: Determining the conditions needed to achieve peak fluorescence intensity in photoconverted channel. (A) Representative images of exposed Dendra2-expressing lung metastases prior to blue light exposure in room lighting without any filters and at low magnification (1) and high magnification (2), and high magnification in the FITC (3) and TRITC channels with widefield illumination (4). (B) Fluorescence images of a Dendra2-expressing lung metastasis before blue light exposure, and after 2 min, 4 min, 6 min, and 8 min of blue light exposure, ex vivo. (C) Normalized mean fluorescence intensity of a Dendra2-expressing lung metastasis as a function of the duration of exposure to blue light, ex vivo. (D) Normalized mean fluorescence intensity of a Dendra2-expresssing lung metastasis before and after 6 min of exposure to blue light, in vivo. Scale bars = 2 mm (A), 400 &#181;m (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65732/65732fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65732/65732fig03.jpg" /> 
Figure 3: Representative images of photoconverted (top row) and non-photoconverted (bottom row) tumor cells found in the contralateral lung, brain, and liver of mice that underwent photoconversion surgery. Scale bars = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65732/65732fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: Custom-built 400 nm LED Array lamp.(A) Off and (B) on. CF = Cooling fan, R = Reflector, LED = High-power 400 nm LED. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65732/65732_supplementary figure 1_v1 MF.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread at JoVE.com

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

tracking-tumor-cell-dissemination-from-lung-metastases-using

Nanyang Technological University

Research

JoVE Journal

Cancer Research

976 Views.  Albert Einstein College of Medicine/Montefiore Medical Center. Recent bewijs suggereert dat bestaande metastatische laesies kunnen fungeren als een bron van verspreiding van tumorcellen die aanleiding geven tot nieuwe laesies. Dit proces wordt metastase naar metastase seeding genoemd en kan de tumorlast verhogen en de kwaliteit van leven en overleving van kankerpatiënten verminderen. Onze methode is ontwikkeld om metastase naar metastase zaaien en de factoren die dit beïnvloeden te bestuderen.Deze techniek heeft ons laboratorium geholpen vast te...

Video: Auteur in de schijnwerpers: het decoderen van metastase-naar-metastase-zaaien met behulp van een nieuwe in-vivotechniek voor het volgen van de verspreiding van borstkanker 

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

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 ...

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Medicine

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

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 ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video