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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 ....

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% .......

Representative 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 6th and 7th 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 .......

Acknowledgements

This work was supported by the following grants: CA216248, CA013330, Montefiore'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.

....

Materials

NameCompanyCatalog NumberComments
1% (w/v) solution of enzyme-active detergentAlconox IncN/A concentrated, anionic detergent with protease enzyme for manual and ultrasonic cleaning
2 µm fluorescent microspheresInvitrogenF8827
5 mm coverslipElectron Microscopy Sciences72296-05
5% (w/v) solution of sodium hydroxideSigma-AldrichS8045
5% IsofluraneHenry Schein, Inc29405
5-0 braided silk with RB-1 cutting needleEthicon, Inc.774B
7% (w/v) solution of citric acidSigma-Aldrich251275
8 mm stainless steel window frameN/AN/ACustom made, Supplementary Figure 2
9 cm 2-0 silk tieEthicon, Inc.LA55G
5 mm disposable biopsy punchIntegra 33-35-SH
Blunt micro-dissecting scissorsRobozRS-5980
BuprenorphineHospira0409-2012-32
Cautery penBraintree ScientificGEM 5917
Chlorhexidine gluconate Becton, Dickinson and Company260100ChloraPrep Single swabstick 1.75 mL
Compressed air canisterFalconDPSJB-12
Cyanoacrylate adhesiveHenkel AdhesivesLOC1363589
Fiber-optic illuminatorO.C. White CompanyFL3000
Bead sterilizerCellPoint ScientificGER 5287-120VGerminator 500
Graefe forcepsRobozRS-5135
Infrared heat lampBraintree ScientificHL-1
Insulin syringesBecton Dickinson329424
Isoflurane vaporizerSurgiVetVCT302
Jacobson needle holder with lockKalson SurgicalT1-140
Long cotton tip applicatorsMedline IndustriesMDS202055
NairChurch & Dwight Co., Inc.40002957
Neomycin/polymyxin B/bacitracinJohnson & Johnson501373005Antibiotic ointmen
Ophthalmic ointmentDechra Veterinary Products17033-211-38
Paper tapeFisher ScientificS68702
Murine ventilatorKent ScientificPS-02PhysioSuite
Rectangular Cover GlassCorning2980-225
Rodent intubation standBraintree ScientificRIS 100
Small animal lung inflation bulbHarvard Apparatus72-9083
Stainless steel cutting toolN/AN/ACustom made, Supplementary Figure 1
Sulfamethoxazole and Trimethoprim oral antibioticHi-Tech Pharmacal Co.50383-823-16
SurgiSuite Multi-Functional Surgical Platform for Mice, with WarmingKent ScientificSURGI-M02Heated surgical platform
Tracheal catheterExelint International2674622 G catheter
Vacuum pickup system metal probeTed Pella, Inc.528-112

References

  1. Mehlen, P., Puisieux, A. Metastasis: a question of life or death. Nature Reviews Cancer. 6 (6), 449-458 (2006).
  2. Lee, Y. T. Breast carcinoma: pattern of metastasis at autopsy. Journal of Surgical Oncology. 23 (3), 17....

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