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>

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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.

<table><tbody><tr><td>1% (w/v) solution of enzyme-active detergent</td><td>Alconox Inc</td><td>N/A</td><td>&amp;nbsp;concentrated, anionic detergent with protease enzyme for manual and ultrasonic cleaning</td></tr><tr><td>2 &amp;micro;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, &lt;strong&gt;Supplementary Figure 2&lt;/strong&gt;</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>&amp;nbsp;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>Brass window tool holder</td><td>N/A</td><td>N/A</td><td>Custom-made, Supplemental Figure 3</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>&amp;nbsp;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&amp;nbsp;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 &amp;amp; Dwight Co., Inc.</td><td>40002957</td><td></td></tr><tr><td>Neomycin/polymyxin B/bacitracin</td><td>Johnson &amp;amp; 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, &lt;strong&gt;Supplementary Figure 1&lt;/strong&gt;</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 ...

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Cancer Research

3.9K 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

Lung Tumor Cell Recruitment Assay

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

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

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Medicine

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 ...

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

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

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

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

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

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

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

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

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 ...

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

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

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

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

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

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

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

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

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

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

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

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

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

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

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

A Permanent Window for Investigating Cancer Metastasis to the Lung

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Lung Tumor Cell Recruitment Assay

Experimental Metastasis Assay

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

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

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Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion