A Permanent Window for Investigating Cancer Metastasis to the Lung

Summary

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.

Abstract

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.

Introduction

Responsible for ~90% of deaths, metastas is isthe major cause of cancer-related mortality¹. 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' 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 formation^5,6, 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 detected^7,8 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 preparations^10,11,12,13, 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 h^14,15,16. 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 growth^14,15,16. 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 'transparent ribcage' 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.

Protocol

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

1. Rinse the optical window frames (Supplemental Figure 2) with a 1% (w/v) solution of enzymatically-active detergent.
2. Inside a glass jar, submerge the optical window frames in 5% (w/v) sodium hydroxide solution for 30 min at 70 °C.
3. Remove and wash the window frames with deionized water.
4. Inside a new glass jar, submerge the optical window frames in 7% (w/v) citric acid solution for 10 min at 55 °C.
5. Again, remove and wash the window frames with deionized water.
6. Repeat step 1.2; then, remove and wash window frames with deionized water.

2. Preparation for surgery

1. 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.
2. 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 tips-only technique is used. 
3. Power on the heated surgical bead and bead sterilizer.
4. Anesthetize the mouse with 5% isoflurane in the anesthesia chamber.
5. 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.
6. Using 2-0 silk suture, tie a knot at the base of a 22 G catheter, leaving 2-inch long tails (see Figure 1A).

3. Lung window surgery

1. Wash hands using antiseptic soap.
2. Prior to each new surgery, don new sterile gloves.
3. To prevent corneal drying and damage to the mouse's eyes, apply ophthalmic ointment to both eyes.
4. Dilute 10 µL (0.1 mg/kg) of buprenorphine in 90 µL of sterile PBS, and then inject subcutaneously to ensure preoperative analgesia.
5. 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.
6. Secure the intubation catheter by tying the 2-0 silk suture around the mouse's snout (see Figure 1B).
7. Place the mouse onto the heated surgical stand and position it in the right lateral decubitus to expose the left thorax.
8. Connect ventilator to the intubation catheter.
9. Ensure controlled, stable ventilation on the ventilator and then lower the isofluorane to 3%. At the procedure's onset and periodically throughout the duration of the procedure, assess the adequacy of anesthesia by performing a toe pinch test.
10. 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's back to maximize exposure to the surgical field (see Figure 1C).
11. Open all surgical instruments underneath the hood for the preservation of sterility.
12. Sterilize the surgical site by a generous application of antiseptic to the mouse's skin.
13. 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).
14. Carefully identify any major vessels. If the division of vessels is necessary, cauterize at both ends with the electrocautery pen to maintain hemostasis.
15. Excise the soft tissue overlying the ribs.
16. Elevate the 6^th or 7^th 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 6^th and 7^th ribs to enter the intrathoracic space (Figure 1E).
17. 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.
18. Place the biopsy punch over the cutting tool (Supplementary Figure 1) and carefully maneuver the cutting tool's base through the intercostal incision (Figure 1F).
19. 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.
20. Using the 5-0 silk suture, create a purse-string stitch ~1 mm from the hole, circumferentially, interlacing with the ribs (Figure 1H).
21. Position the window frame such that the edges of the circular defect align within the window's groove (see Figure 1I).
22. Securely lock the implanted window by tightly tying down the 5-0 silk suture.
23. Load 100 µL of cyanoacrylate gel adhesive into the 1 mL syringe.
24. Dry the lung by applying a steady gentle stream of compressed air for ~10-20 s (Figure 1J).
25. 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.
26. Dispense a thin layer of cyanoacrylate adhesive along the undersurface of the optical window frame (Figure 1K).
27. Increase the positive end-expiratory pressure (PEEP) on the ventilator to inflate the lung.
28. Holding for 10-20 s, apply gentle but firm pressure to attach the optical window frame onto the lung tissue (Figure 1L).
29. Dispense a 5 mm drop of the remaining cyanoacrylate gel adhesive onto a rectangular coverslip.
30. 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).
31. 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's surface and the undersurface of the coverslip. Maintain gentle pressure, allowing the cyanoacrylate adhesive to set (~25 s).
32. Use the forceps to separate the coverslip from the vacuum pickups (Figure 1N).
33. Using 5-0 silk suture, again create a purse-string stitch, this time <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.
34. 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).
35. 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).
36. Remove the tape from the mouse.
37. Turn off isoflurane.
38. Continue ventilation with 100% oxygen until the mouse appears ready to awaken.
39. Carefully cut the 2-0 silk suture around the mouse's snout and extubate the mouse.
40. Transfer the mouse to a clean cage and monitor until fully recovered. Euthanize the mouse if signs of difficulty in breathing are present.
41. Provide postoperative analgesia by subcutaneously injecting 10 µL (0.1 mg/kg) of buprenorphine diluted in 90 µL of sterile phosphate buffered solution (PBS).

Results

The steps of the surgical procedure described in this protocol are summarized and illustrated in Figure 1. 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 6^th and 7^th ribs. The optical window frame is inserted into ...

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
1% (w/v) solution of enzyme-active detergent	Alconox Inc	N/A	concentrated, anionic detergent with protease enzyme for manual and ultrasonic cleaning
2 µm fluorescent microspheres	Invitrogen	F8827
5 mm coverslip	Electron Microscopy Sciences	72296-05
5% (w/v) solution of sodium hydroxide	Sigma-Aldrich	S8045
5% Isoflurane	Henry Schein, Inc	29405
5-0 braided silk with RB-1 cutting needle	Ethicon, Inc.	774B
7% (w/v) solution of citric acid	Sigma-Aldrich	251275
8 mm stainless steel window frame	N/A	N/A	Custom made, Supplementary Figure 2
9 cm 2-0 silk tie	Ethicon, Inc.	LA55G
5 mm disposable biopsy punch	Integra	33-35-SH
Blunt micro-dissecting scissors	Roboz	RS-5980
Buprenorphine	Hospira	0409-2012-32
Cautery pen	Braintree Scientific	GEM 5917
Chlorhexidine gluconate	Becton, Dickinson and Company	260100	ChloraPrep Single swabstick 1.75 mL
Compressed air canister	Falcon	DPSJB-12
Cyanoacrylate adhesive	Henkel Adhesives	LOC1363589
Fiber-optic illuminator	O.C. White Company	FL3000
Bead sterilizer	CellPoint Scientific	GER 5287-120V	Germinator 500
Graefe forceps	Roboz	RS-5135
Infrared heat lamp	Braintree Scientific	HL-1
Insulin syringes	Becton Dickinson	329424
Isoflurane vaporizer	SurgiVet	VCT302
Jacobson needle holder with lock	Kalson Surgical	T1-140
Long cotton tip applicators	Medline Industries	MDS202055
Nair	Church & Dwight Co., Inc.	40002957
Neomycin/polymyxin B/bacitracin	Johnson & Johnson	501373005	Antibiotic ointmen
Ophthalmic ointment	Dechra Veterinary Products	17033-211-38
Paper tape	Fisher Scientific	S68702
Murine ventilator	Kent Scientific	PS-02	PhysioSuite
Rectangular Cover Glass	Corning	2980-225
Rodent intubation stand	Braintree Scientific	RIS 100
Small animal lung inflation bulb	Harvard Apparatus	72-9083
Stainless steel cutting tool	N/A	N/A	Custom made, Supplementary Figure 1
Sulfamethoxazole and Trimethoprim oral antibiotic	Hi-Tech Pharmacal Co.	50383-823-16
SurgiSuite Multi-Functional Surgical Platform for Mice, with Warming	Kent Scientific	SURGI-M02	Heated surgical platform
Tracheal catheter	Exelint International	26746	22 G catheter
Vacuum pickup system metal probe	Ted Pella, Inc.	528-112