This work was supported by the following grants: CA216248, CA013330, Montefiore&#39;s Ruth L. Kirschstein T32 Training Grant CA200561, METAvivor Early Career Award, the Gruss-Lipper Biophotonics Center and its Integrated Imaging Program, and Jane A. and Myles P. Dempsey. We would like to thank the Analytical Imaging Facility (AIF) at Einstein College of Medicine for imaging support.

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.
1. Passivation of windows
<ol>
	<li>Rinse the optical window frames (Supplemental Figure 2) with a 1% (w/v) solution of enzymatically-active detergent.</li>
	<li>Inside a glass jar, submerge the optical window frames in 5% (w/v) sodium hydroxide solution for 30 min at 70 &#176;C.</li>
	<li>Remove and wash the window frames with deionized water.</li>
	<li>Inside a new glass jar, submerge the optical window frames in 7% (w/v) citric acid solution for 10 min at 55 &#176;C.</li>
	<li>Again, remove and wash the window frames with deionized water.</li>
	<li>Repeat step 1.2; then, remove and wash window frames with deionized water.</li>
</ol>
2. Preparation for surgery
<ol>
	<li>Conduct the surgery in a hood or laminar flow cabinet. To avoid contamination of the operative field, ensure distinct, separated areas for preparation, surgery, and recovery, respectively.</li>
	<li>In advance of the surgery, sterilize all surgical instruments in an autoclave. If subsequent procedures are planned, re-sterilize instruments using a hot bead sterilizer. For this surgical procedure, a&#160;tips-only technique is used.&#160;</li>
	<li>Power on the heated surgical bead and bead sterilizer.</li>
	<li>Anesthetize the mouse with 5% isoflurane in the anesthesia chamber.</li>
	<li>To remove hair, generously apply depilatory cream to the upper-left chest incision site. After no longer than 20 s, firmly wipe away hair and depilatory cream using moistened tissue paper. Repeat as necessary to remove all hair from the surgical site.</li>
	<li>Using 2-0 silk suture, tie a knot at the base of a 22 G catheter, leaving 2-inch long tails (see Figure 1A).</li>
</ol>
3. Lung window surgery
<ol>
	<li>Wash hands using antiseptic soap.</li>
	<li>Prior to each new surgery, don new sterile gloves.</li>
	<li>To prevent corneal drying and damage to the mouse&#39;s eyes, apply ophthalmic ointment to both eyes.</li>
	<li>Dilute 10 &#181;L (0.1 mg/kg) of buprenorphine in 90 &#181;L of sterile PBS, and then inject subcutaneously to ensure preoperative analgesia.</li>
	<li>Intubate the mouse with the silk suture-tied 22 G catheter15. Using an inflation bulb, confirm successful intubation by noting bilateral chest rise upon bulb squeeze.</li>
	<li>Secure the intubation catheter by tying the 2-0 silk suture around the mouse&#39;s snout (see Figure 1B).</li>
	<li>Place the mouse onto the heated surgical stand and position it in the right lateral decubitus to expose the left thorax.</li>
	<li>Connect ventilator to the intubation catheter.</li>
	<li>Ensure controlled, stable ventilation on the ventilator and then lower the isofluorane to 3%. At the procedure&#39;s onset and periodically throughout the duration of the procedure, assess the adequacy of anesthesia by performing a toe pinch test.</li>
	<li>Using paper tape, cranially and caudally secure the front and hind limbs, respectively, to the heated surgical stage. Place another piece of tape along the length of the mouse&#39;s back to maximize exposure to the surgical field (see Figure 1C).</li>
	<li>Open all surgical instruments underneath the hood for the preservation of sterility.</li>
	<li>Sterilize the surgical site by a generous application of antiseptic to the mouse&#39;s skin.</li>
	<li>Using forceps, lift the skin and make an ~10 mm circular incision, ~7 mm to the left of the sternum and ~7 mm superior to the subcostal margin (Figure 1D).</li>
	<li>Carefully identify any major vessels. If the division of vessels is necessary, cauterize at both ends with the electrocautery pen to maintain hemostasis.</li>
	<li>Excise the soft tissue overlying the ribs.</li>
	<li>Elevate the 6th or 7th rib using forceps. Using a single blade of the blunt micro-dissecting scissors, the rounded side towards the lung, carefully pierce the intercostal muscle between the 6th and 7th ribs to enter the intrathoracic space (Figure 1E).</li>
	<li>Delicately discharge compressed air canister at the defect to collapse the lung and separate it from the chest wall. Fire the compressed air in short bursts to prevent iatrogenic lung injury.</li>
	<li>Place the biopsy punch over the cutting tool (Supplementary Figure 1) and carefully maneuver the cutting tool&#39;s base through the intercostal incision (Figure 1F).</li>
	<li>Orient the base of the cutting tool such that it is parallel with the chest wall. Punch a 5 mm circular hole through the rib cage (Figure 1G). 
	NOTE: Ensure that the exposed lung tissue is pink, without signs of damage.</li>
	<li>Using the 5-0 silk suture, create a purse-string stitch ~1 mm from the hole, circumferentially, interlacing with the ribs (Figure 1H).</li>
	<li>Position the window frame such that the edges of the circular defect align within the window&#39;s groove (see Figure 1I).</li>
	<li>Securely lock the implanted window by tightly tying down the 5-0 silk suture.</li>
	<li>Load 100 &#181;L of cyanoacrylate gel adhesive into the 1 mL syringe.</li>
	<li>Dry the lung by applying a steady gentle stream of compressed air for ~10-20 s (Figure 1J).</li>
	<li>Using forceps to grip the window frame by its outside edge, gently lift to ensure separation of the lung from the undersurface of the window frame.</li>
	<li>Dispense a thin layer of cyanoacrylate adhesive along the undersurface of the optical window frame (Figure 1K).</li>
	<li>Increase the positive end-expiratory pressure (PEEP) on the ventilator to inflate the lung.</li>
	<li>Holding for 10-20 s, apply gentle but firm pressure to attach the optical window frame onto the lung tissue (Figure 1L).</li>
	<li>Dispense a 5 mm drop of the remaining cyanoacrylate gel adhesive onto a rectangular coverslip.</li>
	<li>Pick up the 5 mm coverslip using vacuum pickups. Dip the undersurface of the coverslip into the adhesive, and then scrape off excess adhesive three times against the side of the rectangular coverslip, such that only a very thin layer remains (Figure 1M).</li>
	<li>Carefully position the coverslip to fit inside the recess at the center of the optical window frame and is held above the lung tissue at an angle. Briefly clamp the ventilator to generate positive pressure, hyper-inflating the lung. Using a rotating motion, orient the coverslip parallel to the lung tissue to create direct apposition between the lung&#39;s surface and the undersurface of the coverslip. Maintain gentle pressure, allowing the cyanoacrylate adhesive to set (~25 s).</li>
	<li>Use the forceps to separate the coverslip from the vacuum pickups (Figure 1N).</li>
	<li>Using 5-0 silk suture, again create a purse-string stitch, this time &#60;1 mm circumferentially from the cut-edge of the skin incision. Tuck any excess skin underneath the outer rim of the window frame before tying it down tightly with locking knots.</li>
	<li>To ensure an air-tight seal between the coverslip and the window frame, dispense a small amount of liquid cyanoacrylate at the metal-glass interface (see Figure 1O).</li>
	<li>Attach a sterile needle to a 1 mL insulin syringe. Insert the needle below the xiphoid process, advancing toward the left shoulder, entering the thoracic cavity through the diaphragm. Gently draw back on the syringe to remove any residual air from the thoracic cavity (see Figure 1P).</li>
	<li>Remove the tape from the mouse.</li>
	<li>Turn off isoflurane.</li>
	<li>Continue ventilation with 100% oxygen until the mouse appears ready to awaken.</li>
	<li>Carefully cut the 2-0 silk suture around the mouse&#39;s snout and extubate the mouse.</li>
	<li>Transfer the mouse to a clean cage and monitor until fully recovered. Euthanize the mouse if signs of difficulty in breathing are present.</li>
	<li>Provide postoperative analgesia by subcutaneously injecting 10 &#181;L (0.1 mg/kg) of buprenorphine diluted in 90 &#181;L of sterile phosphate buffered solution (PBS).</li>
</ol>

<ol>
	<li>Mehlen, P., Puisieux, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+a+question+of+life+or+death.">Metastasis: a question of life or death.</a> Nature Reviews Cancer. 6 (6), 449-458 (2006).</li><li>Lee, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+carcinoma:+pattern+of+metastasis+at+autopsy.">Breast carcinoma: pattern of metastasis at autopsy.</a> Journal of Surgical Oncology. 23 (3), 175-180 (1983).</li><li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nature Reviews Cancer. 2 (8), 563-572 (2002).</li><li>Coste, A., Oktay, M. H., Condeelis, J. S., 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=Intravital+imaging+techniques+for+biomedical+and+clinical+research.">Intravital imaging techniques for biomedical and clinical research.</a> Cytometry Part A. 95 (5), 448-457 (2019).</li><li>DeClerck, Y. A., Pienta, K. J., Woodhouse, E. C., Singer, D. S., Mohla, 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+tumor+microenvironment+at+a+turning+point+knowledge+gained+over+the+last+decade,+and+challenges+and+opportunities+ahead:+A+white+paper+from+the+NCI+TME+network.">The tumor microenvironment at a turning point knowledge gained over the last decade, and challenges and opportunities ahead: A white paper from the NCI TME network.</a> Cancer Research. 77 (5), 1051-1059 (2017).</li><li>Borriello, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+tumor+microenvironment+in+tumor+cell+intravasation+and+dissemination.">The role of the tumor microenvironment in tumor cell intravasation and dissemination.</a> European Journal of Cell Biology. 99 (6), 151098 (2020).</li><li>Hosseini, 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=Early+dissemination+seeds+metastasis+in+breast+cancer.">Early dissemination seeds metastasis in breast cancer.</a> Nature. 540 (7634), 552-558 (2016).</li><li>Harper, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+early+dissemination+and+metastasis+in+Her2(+)+mammary+cancer.">Mechanism of early dissemination and metastasis in Her2(+) mammary cancer.</a> Nature. 540, 589-612 (2016).</li><li>Risson, E., Nobre, A. R., Maguer-Satta, V., Aguirre-Ghiso, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+paradigm+and+challenges+ahead+for+the+dormancy+of+disseminated+tumor+cells.">The current paradigm and challenges ahead for the dormancy of disseminated tumor cells.</a> Nature Cancer. 1 (7), 672-680 (2020).</li><li>Qian, 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=A+distinct+macrophage+population+mediates+metastatic+breast+cancer+cell+extravasation,+establishment+and+growth.">A distinct macrophage population mediates metastatic breast cancer cell extravasation, establishment and growth.</a> PLoS One. 4 (8), 6562 (2009).</li><li>Qian, B. Z., 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=CCL2+recruits+inflammatory+monocytes+to+facilitate+breast-tumour+metastasis.">CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis.</a> Nature. 475 (7355), 222-225 (2011).</li><li>Miyao, 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=Various+adhesion+molecules+impair+microvascular+leukocyte+kinetics+in+ventilator-induced+lung+injury.">Various adhesion molecules impair microvascular leukocyte kinetics in ventilator-induced lung injury.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 290 (6), 1059-1068 (2006).</li><li>Bernal, P. J., 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=Nitric-oxide-mediated+zinc+release+contributes+to+hypoxic+regulation+of+pulmonary+vascular+tone.">Nitric-oxide-mediated zinc release contributes to hypoxic regulation of pulmonary vascular tone.</a> Circulation Research. 102 (12), 1575-1583 (2008).</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=In+vivo+subcellular+resolution+optical+imaging+in+the+lung+reveals+early+metastatic+proliferation+and+motility.">In vivo subcellular resolution optical imaging in the lung reveals early metastatic proliferation and motility.</a> IntraVital. 4 (3), 1-11 (2015).</li><li>Rodriguez-Tirado, 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=Long-term+High-Resolution+Intravital+Microscopy+in+the+Lung+with+a+Vacuum+Stabilized+Imaging+Window.">Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window.</a> Journal of Visualized Experiments: JoVE. (116), e54603 (2016).</li><li>Looney, M. 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=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">Stabilized imaging of immune surveillance in the mouse lung.</a> Nature Methods. 8 (1), 91-96 (2011).</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=A+permanent+window+for+the+murine+lung+enables+high-resolution+imaging+of+cancer+metastasis.">A permanent window for the murine lung enables high-resolution imaging of cancer metastasis.</a> Nature Methods. 15 (1), 73-80 (2018).</li><li>Dunphy, M. P., Entenberg, D., Toledo-Crow, R., Larson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+microcartography+and+subcellular+imaging+of+tumor+angiogenesis:+a+novel+platform+for+translational+angiogenesis+research.">In vivo microcartography and subcellular imaging of tumor angiogenesis: a novel platform for translational angiogenesis research.</a> Microvascular Research. 78 (1), 51-56 (2009).</li><li>Harney, A. S., Wang, Y., Condeelis, J. S., 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=Extended+time-lapse+intravital+imaging+of+real-time+multicellular+dynamics+in+the+tumor+microenvironment.">Extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment.</a> Journal of Visualized Experiments: JoVE. (112), e54042 (2016).</li><li>Seynhaeve, A. L. B., Ten Hagen, T. L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+tumor-associated+vasculature+using+advanced+dorsal+skinfold+window+chambers+on+transgenic+fluorescent+mice.">Intravital microscopy of tumor-associated vasculature using advanced dorsal skinfold window chambers on transgenic fluorescent mice.</a> Journal of Visualized Experiments: JoVE. (131), e55115 (2018).</li><li>Entenbery, 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=Time-lapsed,+large-volume,+high-resolution+intravital+imaging+for+tissue-wide+analysis+of+single+cell+dynamics.">Time-lapsed, large-volume, high-resolution intravital imaging for tissue-wide analysis of single cell dynamics.</a> Methods. 128, 65-77 (2017).</li><li>Ueki, H., Wang, I. H., Zhao, D., Gunzer, M., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+two-photon+imaging+of+in+vivo+cellular+pathophysiology+upon+influenza+virus+infection+using+the+two-photon+IMPRESS.">Multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon IMPRESS.</a> Nature Protocols. 15 (3), 1041-1065 (2020).</li><li>Ritsma, L., Ponsioen, B., van Rheenen, J. <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+cell+signaling+in+mice.">Intravital imaging of cell signaling in mice.</a> IntraVital. 1 (1), 2-10 (2012).</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>Harney, A. S., 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=Real-time+imaging+reveals+local,+transient+vascular+permeability,+and+tumor+cell+intravasation+stimulated+by+TIE2hi+macrophage-derived+VEGFA.">Real-time imaging reveals local, transient vascular permeability, and tumor cell intravasation stimulated by TIE2hi macrophage-derived VEGFA.</a> Cancer Discovery. 5 (9), 932-943 (2015).</li><li>Karagiannis, G. S., 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=Assessing+tumor+microenvironment+of+metastasis+doorway-mediated+vascular+permeability+associated+with+cancer+cell+dissemination+using+intravital+imaging+and+fixed+tissue+analysis.">Assessing tumor microenvironment of metastasis doorway-mediated vascular permeability associated with cancer cell dissemination using intravital imaging and fixed tissue analysis.</a> Journal of Visualized Experiments: JoVE. (148), e59633 (2019).</li><li>Karagiannis, G. S., 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=Neoadjuvant+chemotherapy+induces+breast+cancer+metastasis+through+a+TMEM-mediated+mechanism.">Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism.</a> Science Translational Medicine. 9 (397), (2017).</li><li>Dreher, M. 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=Tumor+vascular+permeability,+accumulation,+and+penetration+of+macromolecular+drug+carriers.">Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers.</a> Journal of the National Cancer Institute. 98 (5), 335-344 (2006).</li><li>Rizzo, V., Kim, D., Duran, W. N., DeFouw, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+microvascular+permeability+to+macromolecules+in+the+chick+chorioallantoic+membrane+during+normal+angiogenesis.">Ontogeny of microvascular permeability to macromolecules in the chick chorioallantoic membrane during normal angiogenesis.</a> Microvascular Research. 49 (1), 49-63 (1995).</li><li>Hoshino, 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=Tumour+exosome+integrins+determine+organotropic+metastasis.">Tumour exosome integrins determine organotropic metastasis.</a> Nature. 527 (7578), 329-335 (2015).</li><li>Ueki, 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=In+vivo+imaging+of+the+pathophysiological+changes+and+neutrophil+dynamics+in+influenza+virus-infected+mouse+lungs.">In vivo imaging of the pathophysiological changes and neutrophil dynamics in influenza virus-infected mouse lungs.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (28), 6622-6629 (2018).</li><li>Kornfield, T. E., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+retinal+blood+flow+using+fluorescently+labeled+red+blood+cells.">Measurement of retinal blood flow using fluorescently labeled red blood cells.</a> eNeuro. 2 (2), (2015).</li><li>Dasari, S., Weber, P., Makhloufi, C., Lopez, E., Forestier, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+imaging+of+the+liver+following+leishmania+infection:+An+assessment+of+hepatic+hemodynamics.">Intravital microscopy imaging of the liver following leishmania infection: An assessment of hepatic hemodynamics.</a> Journal of Visualized Experiments: JoVE. (101), e52303 (2015).</li><li>Chaigneau, E., Roche, M., Charpak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+analysis+method+for+measurement+of+red+blood+cell+size+and+velocity+with+laser+scanning+microscopy.">Unbiased analysis method for measurement of red blood cell size and velocity with laser scanning microscopy.</a> Frontiers in Neuroscience. 13, 644 (2019).</li><li>Kim, T. 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=Line-scanning+particle+image+velocimetry:+an+optical+approach+for+quantifying+a+wide+range+of+blood+flow+speeds+in+live+animals.">Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals.</a> PLoS One. 7 (6), 38590 (2012).</li><li>Presson, R. 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=Two-photon+imaging+within+the+murine+thorax+without+respiratory+and+cardiac+motion+artifact.">Two-photon imaging within the murine thorax without respiratory and cardiac motion artifact.</a> American Journal of Pathology. 179 (1), 75-82 (2011).</li><li>Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> Journal of Applied Physiology. 104 (2), 338-346 (2008).</li><li>Travis, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+lung+cancer.">Classification of lung cancer.</a> Seminars in Roentgenology. 46 (3), 178-186 (2011).</li><li>Scholten, E. T., Kreel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+lung+metastases+in+the+axial+plane.+A+combined+radiological-pathological+study.">Distribution of lung metastases in the axial plane. A combined radiological-pathological study.</a> Radiologica Clinica (Basel). 46 (4), 248-265 (1977).</li><li>Braman, S. S., Whitcomb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endobronchial+metastasis.">Endobronchial metastasis.</a> Archives of Internal Medicine. 135 (4), 543-547 (1975).</li><li>Herold, C. J., Bankier, A. A., Fleischmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+metastases.">Lung metastases.</a> European Radiology. 6 (5), 596-606 (1996).</li><li>Kimura, 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=Real-time+imaging+of+single+cancer-cell+dynamics+of+lung+metastasis.">Real-time imaging of single cancer-cell dynamics of lung metastasis.</a> Journal of Cellular Biochemistry. 109 (1), 58-64 (2010).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Transactions on Image Processing: A Publication of the IEEE Signal Processing Society. 7 (1), 27-41 (1998).</li><li>Sharma, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+plugin+HyperStackReg+V5.6.">ImageJ plugin HyperStackReg V5.6.</a> Zenodo. , (2018).</li></ol>

The authors disclose no conflicts of interest.

At sites of distant metastasis such as the lung, high-resolution optical imaging provides insight into the elaborate dynamics of tumor cell metastasis. By enabling in vivo visualization of single cancer cells and their interactions with the host tissue, high-resolution intravital imaging has proven instrumental to understanding the mechanisms underlying metastasis.
Described here is an improved surgical protocol for the permanent thoracic implantation of an optical window designed to ...

Responsible for ~90% of deaths, metastas is isthe major cause of cancer-related mortality1.&#160;Among the major sites of clinically observed metastasis (bone, liver, lung, brain)2, the lung has proven particularly challenging for in vivo imaging via intravital microscopy. This is because the lung is a delicate organ in perpetual motion. The lungs&#39; continuous motion, further compounded by intrathoracic cardiac motion, represents a substantial barrier to accurate imaging. Therefore, due to its relative inaccessibility to modalities for high-resolution intravital optical imaging, cancer growth within the lung has often been deemed an occult process3.
In the clinical setting, imaging technologies such as computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI) enable visualization deep within intact vital organs such as the lung4. However, while these modalities provide for excellent views of the gross organ (often even revealing pathology prior to the onset of clinical symptoms), they are of inadequate resolution to detect individual disseminated tumor cells as they advance through the early stages of metastasis. Consequently, by the time the aforementioned modalities provide any indication of metastasis to the lung, metastatic foci are already well established and proliferating. Since the tumor microenvironment plays a pivotal role in cancer progression and metastasis formation5,6, there is great interest in investigating the earliest steps of metastatic seeding in vivo. This interest is further fueled by the increased appreciation that cancer cells disseminate even before the primary tumor is detected7,8 and the increasing evidence that they survive as single cells and in a dormant state for years to decades before outgrowth into macro-metastasis9.
Previously, imaging of the lung at single-cell resolution has necessarily involved ex vivo or explant preparations10,11,12,13, limiting analyses to single time points. While these preparations do provide useful information, they do not provide any insight into the dynamics of tumor cells within the organ connected to an intact circulatory system.
Recent technological advancements in imaging have enabled intravital visualization of the intact lung at single-cell resolution over periods of up to 12 h14,15,16. This was accomplished in a murine model using a protocol that involved mechanical ventilation, resection of the ribcage, and vacuum-assisted lung immobilization. However, despite offering the first single cell-resolution images of the physiologically intact lung, the technique is highly invasive and terminal, thereby precluding further imaging sessions beyond the index procedure. This limitation, therefore, prevents its application to the study of metastatic steps that take longer than 12 h, such as dormancy and re-initiation of growth14,15,16. Further still, patterns of cellular behavior observed using this imaging approach must be cautiously interpreted, given that vacuum-induced pressure differentials are likely to cause diversions in blood flow.
To overcome these limitations, a minimally invasive Window for High-Resolution Imaging of the Lung (WHRIL) was recently developed, facilitating serial imaging over an extended period of days to weeks, without the need for mechanical ventilation17. The technique entails the creation of a &#39;transparent ribcage&#39; with a sealed thoracic cavity for the preservation of normal lung function. The procedure is well-tolerated, permitting the mouse to recover without meaningful alteration to baseline activity and function. To reliably localize exactly the same lung region at each respective imaging session, a technique known as microcartography was applied to this window18. Through this window, it was possible to capture images of cells as they arrive at the vascular bed of the lung, cross the endothelium, undergo cell division, and grow into micro-metastases.
Here, the study presents a detailed description of an improved surgical protocol for implantation of the WHRIL, which simplifies the surgery while simultaneously increasing its reproducibility and quality. While this protocol was designed to enable investigation of the dynamic processes underlying metastasis, the technique may be alternatively applied to investigations of numerous processes of lung biology and pathology.

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			<td>1% (w/v) solution of enzyme-active detergent</td>
			<td>Alconox Inc</td>
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			<td>&#160;concentrated, anionic detergent with protease enzyme for manual and ultrasonic cleaning</td>
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		<tr>
			<td>2 &#181;m fluorescent microspheres</td>
			<td>Invitrogen</td>
			<td>F8827</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mm coverslip</td>
			<td>Electron Microscopy Sciences</td>
			<td>72296-05</td>
			<td></td>
		</tr>
		<tr>
			<td>5% (w/v) solution of sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
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		<tr>
			<td>5% Isoflurane</td>
			<td>Henry Schein, Inc</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 braided silk with RB-1 cutting needle</td>
			<td>Ethicon, Inc.</td>
			<td>774B</td>
			<td></td>
		</tr>
		<tr>
			<td>7% (w/v) solution of citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>8 mm stainless steel window frame</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom made, Supplementary Figure 2</td>
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		<tr>
			<td>9 cm 2-0 silk tie</td>
			<td>Ethicon, Inc.</td>
			<td>LA55G</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mm disposable biopsy punch</td>
			<td>Integra</td>
			<td>&#160;33-35-SH</td>
			<td></td>
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		<tr>
			<td>Blunt micro-dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5980</td>
			<td></td>
		</tr>
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			<td>Buprenorphine</td>
			<td>Hospira</td>
			<td>0409-2012-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery pen</td>
			<td>Braintree Scientific</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorhexidine gluconate</td>
			<td>&#160;Becton, Dickinson and Company</td>
			<td>260100</td>
			<td>ChloraPrep Single swabstick 1.75 mL</td>
		</tr>
		<tr>
			<td>Compressed air canister</td>
			<td>Falcon</td>
			<td>DPSJB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>Henkel Adhesives</td>
			<td>LOC1363589</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-optic illuminator</td>
			<td>O.C. White Company</td>
			<td>FL3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead sterilizer</td>
			<td>CellPoint Scientific</td>
			<td>GER&#160;5287-120V</td>
			<td>Germinator 500</td>
		</tr>
		<tr>
			<td>Graefe forceps</td>
			<td>Roboz</td>
			<td>RS-5135</td>
			<td></td>
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		<tr>
			<td>Infrared heat lamp</td>
			<td>Braintree Scientific</td>
			<td>HL-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>Becton Dickinson</td>
			<td>329424</td>
			<td></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>Long cotton tip applicators</td>
			<td>Medline Industries</td>
			<td>MDS202055</td>
			<td></td>
		</tr>
		<tr>
			<td>Nair</td>
			<td>Church &#38; Dwight Co., Inc.</td>
			<td>40002957</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomycin/polymyxin B/bacitracin</td>
			<td>Johnson &#38; Johnson</td>
			<td>501373005</td>
			<td>Antibiotic ointmen</td>
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		<tr>
			<td>Ophthalmic ointment</td>
			<td>Dechra Veterinary Products</td>
			<td>17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper tape</td>
			<td>Fisher Scientific</td>
			<td>S68702</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine ventilator</td>
			<td>Kent Scientific</td>
			<td>PS-02</td>
			<td>PhysioSuite</td>
		</tr>
		<tr>
			<td>Rectangular Cover Glass</td>
			<td>Corning</td>
			<td>2980-225</td>
			<td></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>Stainless steel cutting tool</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom made, Supplementary Figure 1</td>
		</tr>
		<tr>
			<td>Sulfamethoxazole and Trimethoprim oral antibiotic</td>
			<td>Hi-Tech Pharmacal Co.</td>
			<td>50383-823-16</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>Tracheal catheter</td>
			<td>Exelint International</td>
			<td>26746</td>
			<td>22 G catheter</td>
		</tr>
		<tr>
			<td>Vacuum pickup system metal probe</td>
			<td>Ted Pella, Inc.</td>
			<td>528-112</td>
			<td></td>
		</tr>
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a permanent window for investigating cancer metastasis to the lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

The steps of the surgical procedure described in this protocol are summarized and illustrated&#160;in Figure 1.&#160;Briefly, prior to surgery, mice are anesthetized and the hair over the left thorax is removed. Mice are intubated and mechanically ventilated to enable survival upon breachment of the thoracic cavity. Soft tissue overlying the ribs is excised, and a small circular defect is created, spanning the 6th and 7th ribs. The optical window frame is inserted into ...

Watch this Scientific Journal Video about A Permanent Window for Investigating Cancer Metastasis to the Lung at JoVE.com

A Permanent Window for Investigating Cancer Metastasis to the Lung

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

a-permanent-window-for-investigating-cancer-metastasis-to-the-lung

Research

JoVE Journal

Cancer Research

3.8K Views.  Einstein College of Medicine / Montefiore Medical Center. Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Video: A Permanent Window for Investigating Cancer Metastasis to the Lung

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

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 ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

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

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

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

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

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...