Abstract
Real-time in vivo imaging has become an integral tool for the investigation and understanding of cellular processes in health and disease at single-cell resolution. This includes the dynamic and complex cellular interactions that occur during cancer progression and the subsequent metastatic dissemination of tumor cells to sites distant from the primary tumor. Herein we outline the methodology for the establishment and intravital imaging of the pulmonary metastatic niche, a preferred site of metastasis for many cancers, and describe the implementation of a lung window to visualize and dissect the intricate behaviour of multiple cell types within this environment. We also address the advantages and limitations of this high-resolution technology.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Secklehner J, Lo Celso C, Carlin LM (2017) Intravital microscopy in historic and contemporary immunology. Immunol Cell Biol 95:506–513
Entenberg D, Rodriguez-Tirado C, Kato Y et al (2015) In vivo subcellular resolution optical imaging in the lung reveals early metastatic proliferation and motility. Intravital 4:1–11
Neupane AS, Kubes P (2022) Imaging reveals novel innate immune responses in lung, liver, and beyond. Immunol Rev 306:244–257
Deniset JF, Kubes P (2016) Intravital imaging of myeloid cells: inflammatory migration and resident patrolling. Microbiol Spectr 4. https://doi.org/10.1128/microbiolspec.MCHD-0042-2016
Jorch SK, Deppermann C (2021) Intravital imaging allows organ-specific insights into immune functions. Front Cell Dev Biol 9:623906
Pittet MJ, Weissleder R (2011) Intravital imaging. Cell 147:983–991
Alizadeh-Tabrizi N, Hall S, Lehmann C (2020) Intravital imaging of pulmonary immune response in inflammation and infection. Front Cell Dev Biol 8:620471
Zomer A, Beerling E, Vlug EJ et al (2011) Real-time intravital imaging of cancer models. Clin Transl Oncol 13:848–854
David K, Meyerholz CJS, Dintzis SM, Frevert CW (2018) Respiratory system. In: Piper M, SMD T, Montine KS (eds) Comparative anatomy and histology, 2nd edn. Academic, pp 147–162
Neupane AS, Willson M, Chojnacki AK et al (2020) Patrolling alveolar macrophages conceal bacteria from the immune system to maintain homeostasis. Cell 183(110-25):e11
Liegeois M, Legrand C, Desmet CJ et al (2018) The interstitial macrophage: a long-neglected piece in the puzzle of lung immunity. Cell Immunol 330:91–96
Granton E, Kim JH, Podstawka J et al (2018) The lung microvasculature is a functional immune niche. Trends Immunol 39:890–899
Budczies J, von Winterfeld M, Klauschen F et al (2015) The landscape of metastatic progression patterns across major human cancers. Oncotarget 6:570–583
Looney MR, Thornton EE, Sen D et al (2011) Stabilized imaging of immune surveillance in the mouse lung. Nat Methods 8:91–96
Yipp BG, Kim JH, Lima R et al (2017) The lung is a host defense niche for immediate neutrophil-mediated vascular protection. Sci Immunol 2:eaam8929
Looney MR, Headley MB (2020) Live imaging of the pulmonary immune environment. Cell Immunol 350:103862
Tarnavski O, McMullen JR, Schinke M et al (2004) Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies. Physiol Genomics 16:349–360
Wu YI, Frey D, Lungu OI et al (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461:104–108
Borriello L, Traub B, Coste A et al (2021) A permanent window for investigating cancer metastasis to the lung. J Vis Exp 173. https://doi.org/10.3791/62761
Jain R, Tikoo S, On K et al (2021) Visualizing murine breast and melanoma tumor microenvironment using intravital multiphoton microscopy. STAR Protoc 2:100722
Wang F, Ma Z, Zhong Y et al (2021) In vivo NIR-II structured-illumination light-sheet microscopy. Proc Natl Acad Sci U S A 118:e2023888118
Wang F, Wan H, Ma Z et al (2019) Light-sheet microscopy in the near-infrared II window. Nat Methods 16:545–552
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
1 Electronic Supplementary Material
Video 1:
Intravital imaging of B16F10 lung metastasis environment. Time series intravital imaging of the tumor environment in the lung 17Â days after mouse melanoma B16F10-mCherry cells (cyan) were intravenously injected. Neutrophils are shown in magenta (anti-Ly6G, BV421), and lung endothelial (yellow, Alexa fluor 647) cells were labeled with anti-CD31 (WMV 6705 kb)
Video 2:
Intravital imaging of E0771 lung metastasis environment. Time lapse intravital imaging of the tumor environment in the lung 14Â days after the injection of mammary E0771-iRFP720 cells (cyan). Neutrophils are shown in magenta (anti-Ly6G, BV421), and lung endothelial (yellow, Alex fluor 594) cells were labeled with anti-CD31 (WMV 9689 kb)
Video 3:
Intravital imaging of E0771 lung metastasis in 4D. Four-dimensional time lapse imaging of lung mammary E0771-iRFP tumor micro-environment. Images over time were acquired at indicated depth (0 μm–70 μm depth; 1.29-μm-thick sections). E0771-iRFP720 cells are shown in cyan, neutrophils labeled with anti-Ly6G are depicted in magenta (BV421), and lung endothelial cells in yellow (anti-CD31, Alexa fluor 594). Total variation denoising tool in the Leica software was used to process all time lapse images. Scale bar 50 μm (WMV 5236 kb)
Video 4:
Intravital imaging of B16F10 lung metastasis in 4D. Four-dimensional time lapse imaging of melanoma B16F10-mCherry lung metastasis at indicted depth (0 μm–32.76 μm; 1.26-μm-thick sections) over time. B16F10-mCherry cells (cyan), neutrophils labeled with anti-Ly6G (magenta, BV421), and lung endothelial (yellow, Alexa fluor 647) cells labeled with anti-CD31. Total variation denoising tool in the Leica software was used to process all time lapse images. Scale bar 50 μm (WMV 8431 kb)
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Babes, L., Yipp, B.G., Senger, D.L. (2023). Intravital Microscopy of the Metastatic Pulmonary Environment. In: Ursini-Siegel, J. (eds) The Tumor Microenvironment. Methods in Molecular Biology, vol 2614. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2914-7_23
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2914-7_23
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2913-0
Online ISBN: 978-1-0716-2914-7
eBook Packages: Springer Protocols