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Imaging Glioma Progression by Intravital Microscopy

  • Fabio Stanchi
  • Ken Matsumoto
  • Holger Gerhardt
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1862)

Abstract

We describe here a method for generating mouse orthotopic gliomas in order to follow their progression over time by multi-photon laser scanning microscopy. After craniotomy of the parietal bone, glioma cells are implanted in the brain cortex and a glass window is cemented atop, allowing chronical imaging of the tumor. The expression of different fluorescent proteins in tumor cells and in specific cell types of a number of currently available transgenic mouse strains allows obtaining multicolor 3D images of the tumor over time. This technique is suitable both to evaluate the effect of pharmacological treatments and to unravel basic mechanisms of tumor-host interactions.

Key words

Intravital imaging Multi-photon Laser scanning microscopy Tumor model Glioma 

Notes

Acknowledgment

The development of the technique here described was supported by the Belgian Cancer Foundation (Stichting Tegen Kanker, grant 2012‐181) and a Hercules type 2 grant (Herculesstichting: AKUL11033). We thank Dr. Till Acker (Institute of Neuropathology, University of Giessen, Germany) and Dr. Thomas N. Seyfried (Biology department, Boston College, USA) for the gift of Glioma261 and CT2A cells, respectively.

References

  1. 1.
    Zipfel WR, Williams RM, Webb WW (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21:1369–1377. https://doi.org/10.1038/nbt899 CrossRefPubMedGoogle Scholar
  2. 2.
    Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940. https://doi.org/10.1038/nmeth818 CrossRefPubMedGoogle Scholar
  3. 3.
    Abe T, Fujimori T (2013) Reporter mouse lines for fluorescence imaging. Dev Growth Differ 55:390–405. https://doi.org/10.1111/dgd.12062 CrossRefPubMedGoogle Scholar
  4. 4.
    Erapaneedi R, Belousov VV, Schäfers M, Kiefer F (2016) A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level. EMBO J 35:102–113. https://doi.org/10.15252/embj.201592775 CrossRefPubMedGoogle Scholar
  5. 5.
    Mathivet T, Bouleti C, Van Woensel M et al (2017) Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth. EMBO Mol Med 9:1629. https://doi.org/10.15252/emmm.201607445 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ricard C, Stanchi F, Rodriguez T et al (2013) Dynamic quantitative intravital imaging of glioblastoma progression reveals a lack of correlation between tumor growth and blood vessel density. PLoS One 8:e72655. https://doi.org/10.1371/journal.pone.0072655 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Ricard C, Stanchi F, Rougon G, Debarbieux F (2014) An orthotopic glioblastoma mouse model maintaining brain parenchymal physical constraints and suitable for intravital two-photon microscopy. J Vis Exp. https://doi.org/10.3791/51108
  8. 8.
    Ausman JI, Shapiro WR, Rall DP (1970) Studies on the chemotherapy of experimental brain tumors: development of an experimental model. Cancer Res 30:2394–2400PubMedGoogle Scholar
  9. 9.
    Seyfried TN, el-Abbadi M, Roy ML (1992) Ganglioside distribution in murine neural tumors. Mol Chem Neuropathol 17:147–167CrossRefGoogle Scholar
  10. 10.
    Mostany R, Portera-Cailliau C (2008) A method for 2-photon imaging of blood flow in the neocortex through a cranial window. J Vis Exp. https://doi.org/10.3791/678
  11. 11.
    Subach OM, Gundorov IS, Yoshimura M et al (2008) Conversion of red fluorescent protein into a bright blue probe. Chem Biol 15:1116–1124. https://doi.org/10.1016/j.chembiol.2008.08.006 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Filonov GS, Piatkevich KD, Ting L-M et al (2011) Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat Biotechnol 29:757–761. https://doi.org/10.1038/nbt.1918 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Muzumdar MD, Tasic B, Miyamichi K, et al (2007) A global double-fluorescent Cre reporter mouse. Genesis 45:593–605. https://doi.org/10.1002/dvg.20335 CrossRefGoogle Scholar
  14. 14.
    Claxton S, Kostourou V, Jadeja S et al (2008) Efficient, inducible Cre-recombinase activation in vascular endothelium. Genesis 46(2):74–80. https://doi.org/10.1002/dvg.20367 CrossRefPubMedGoogle Scholar
  15. 15.
    Bitterman H (2009) Bench-to-bedside review: oxygen as a drug. Crit Care 13:205. https://doi.org/10.1186/cc7151 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Tremoleda JL, Kerton A, Gsell W (2012) Anaesthesia and physiological monitoring during in vivo imaging of laboratory rodents: considerations on experimental outcomes and animal welfare. EJNMMI Res 2:44. https://doi.org/10.1186/2191-219X-2-44 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zoumi A, Yeh A, Tromberg BJ (2002) Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc Natl Acad Sci U S A 99:11014–11019. https://doi.org/10.1073/pnas.172368799 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Fabio Stanchi
    • 1
  • Ken Matsumoto
    • 1
  • Holger Gerhardt
    • 1
    • 2
  1. 1.VIB-KU Leuven Center for Cancer Biology (CCB)LeuvenBelgium
  2. 2.Max Delbrück Center for Molecular MedicineBerlinGermany

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