Name: a permanent window for investigating cancer metastasis to the lung
Uploaded: 2021-07-01T15:00:00
Description: Watch this Scientific Journal Video about A Permanent Window for Investigating Cancer Metastasis to the Lung at JoVE.com

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>
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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 ...

Research

JoVE Journal

Cancer Research

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 ...

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. ...

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 ...

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 ...

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 ...

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 ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

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. ...

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 ...

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 ...