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% ...

<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>
			<td>N/A</td>
			<td>&#160;concentrated, anionic detergent with protease enzyme for manual and ultrasonic cleaning</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Blunt micro-dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5980</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<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>
		</tr>
		<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>
	</tbody>
</table>

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 ...

This protocol allowed to answer an important question in the cancer metastasis field. Including, how tumor cells colonize the lung? How tumor cells remain dormant?And what induce them to exit dormancy and eventually form metastasis. The main advantage of this protocol is that the dynamic metastatic process can be directly studied in vivo, in real time, in a real microenvironment. This method can provide insight into several pulmonary diseases, but it's particularly useful for understanding the behavior individual tumor cells disseminating to the lung, a common site of metastasis.To begin with use a 2-0 silk suture to tie a knot at the base of a 22 gauge catheter, leaving 2 inch long tails. Once the mouse is completely anesthetized, move the mouse to the operation table, intubate the mouse, and then secure the intubation catheter by tying the 2-0 silk suture around the mouse's snout. Connect the ventilator to the intubation catheter.Using paper tape, secure the front cranially and hind limbs caudally to the heated surgical stage. Place a paper tape along the length of the mouse's back to maximize exposure to the surgical field. Lift the skin with the forceps and make an approximately 10 mm circular incision at about 7 mm to the left to the sternum and about 7 mm superior to the subcostal margin.Excise the soft tissue overlying the ribs. Elevate the sixth and seventh rib using forceps. Use a single blade of the blunt micro dissecting scissors with the rounded side towards the lung to carefully pierce the intercostal muscle between the sixth and seventh ribs for entering the intrathoracic space.Delicately discharged the compressed air canister at the defect to collapse the lung and separate the lung from the chest wall. Fire the compressed air in short bursts to prevent iatrogenic lung injury. Place the biopsy punch over the cutting tool and carefully maneuver the cutting tool's base through the intercostal incision.Orient the cutting tool base parallel with the chest wall and punch a 5 mm circular hole through the rib cage. Using a 5-O silk suture, place a purse string stitch, approximately 1 mm from the hole, circumferentially. Next, position the window frame into the chest wall with the edges of the circular defect captured within the windows groove.Tightly tie the 5-O silk suture to securely lock the implanted window. Apply a steady and gentle stream of compressed air for about 10 to 20 seconds to dry the lung. Use the forceps to grip the window frame by its outside edge and gently lift to ensure separation of the lung from the undersurface of the window frame.Dispense a thin layer of cyanoacrylate adhesive along the undersurface of the optical window frame. Gently but firmly press the optical window frame onto the lung tissue for 10 to 20 seconds for attachment. Dispense a 5 mm drop of adhesive on a rectangular cover slip.Use the vacuum pickup tool to pick up a 5 mm cover slip. Dip the undersurface of the cover slip into the adhesive, and then scrape off the excess adhesive three times against the side of the rectangular cover slip to make a very thin layer of adhesive on the cover slip. Carefully position the cover slip at an angle inside the recess at the center of the optical window frame, just above the lung tissue.Briefly clamp the ventilator to generate positive pressure, hyper inflating the lung. Orient the cover slip parallel to the lung tissue, using the rotating motion to create direct deposition between the lung surface and the undersurface of the cover slip. Maintain gentle pressure for approximately 25 seconds to set the cyanoacrylate adhesive.Separate the cover slip from the vacuum pickup tool with forceps. Create a purse string stitch, circumferentially, less than 1 mm from the cut edge of the skin incision using 5-O silk suture. Tuck any excess skin underneath the outer rim of the window frame before tying the window tightly with locking knots.Dispense a small amount of adhesive at the metal glass interface to ensure an airtight seal between the cover slip and window frame. Attach the 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 the syringe to remove any residual air from the thoracic cavity. The multiphoton intravital imaging of a single lung region showed that the microvascular could be relocated over three consecutive days. The definable branch point from a single vessel was identified each consecutive day.The unlabeled erythrocytes created shadows when flowing in larger vessels. The angle of the branch points relative to the vessel can be used to calculate the erythrocyte flow rates. Erythrocyte speed was also quantified by injecting the mouse with fluorescent microspheres and their track lengths were measured divided by the frame acquisition time.The stationary and flowing microspheres were distinguishable. The fate of the disseminated tumor cells was tracked with serial imaging over several days, using the window for high resolution imaging of the lung. The tumor cell arrived at and lodged in the lung vasculature on day one.However, the cell was not detected in the lung vasculature on the second and third days, indicating recirculation or extravasation. The culminating steps of metastatic progression in the lung were visualized starting with the tumor cell arrival, the extravasation of tumor cell into the lung parenchyma and proliferation to form macro metastasis. Take care to avoid trauma to the lung during optical window frame placement.The lung is prone to bleeding, which will later impede cover slip adherence during placement. Other methods that can be performed following this procedure include intravital microscopy, which when employed with a modified approach to microcartography, allows precise relocalization of areas of interest for repeated imaging. This protocol allowed the visualization of the lung in the investigation of a key question about the mechanism of metastasis.This study carry the potential to rebuild a new treatment for cancer patients.

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Cancer Research

3.7K 조회수.  Einstein College of Medicine / Montefiore Medical Center. 이 프로토콜은 암 전이 필드에 있는 중요한 질문에 대답하는 것을 허용했습니다. 포함, 종양 세포는 폐를 식민지? 종양 세포는 어떻게 휴면 상태로 유지합니까?그리고 무엇이 휴면 상태를 종료하고 결국 전이를 형성하도록 유도. 이 프로토콜의 주요 장점은 동적 전이성 프로세스가 실제 미세 환경에서 실시간으로 생체 내에서 직접 연구할 수 있다는 것입니다. 이 방법은...