Advertisement

Preparation and Testing of Cells Expressing Fluorescent Proteins for Intravital Imaging of Tumor Microenvironment

  • S. S. VodopyanovEmail author
  • M. A. Kunin
  • A. S. Garanina
  • N. F. Grinenko
  • K. Yu. Vlasova
  • P. A. Mel’nikov
  • V. P. Chekhonin
  • K. K. Sukhinich
  • A. V. Makarov
  • V. A. Naumenko
  • M. A. Abakumov
  • A. G. Majouga
Translated from Kletochnye Tekhnologii v Biologii i Meditsine (Cell Technologies in Biology and Medicine)

Intravital microscopy is widely used for in vivo studies of the mechanisms of carcinogenesis and response to antitumor therapy. For visualization of tumor cells in vivo, cell lines expressing fluorescent proteins are needed. Expression of exogenous proteins can affect cell growth rate and their tumorigenic potential. Therefore, comprehensive analysis of the morphofunctional properties of transduced cells is required for creating appropriate models of tumor microenvironment. In the present study, six lines of mouse tumor cells expressing green and red fluorescent proteins were derived. Analysis of cells morphology, growth kinetics, and response to chemotherapy in vitro revealed no significant differences between wild-type and transduced cell lines. Introduction of fluorescent proteins into the genome of 4T1 (murine breast cancer) and B16-F10 (murine melanoma) cells did not affect tumor growth rate after subcutaneous implantation to mice, while both CT26-GFP and CT26-RFP cells (murine colon cancer) were rejected starting from day 8 after implantation. Elucidation of the mechanisms underlying CT26-GFP/RFP rejection is required to modify transduction technique for creating the models of tumor microenvironment accessible for in vivo visualization. Transduced 4T1 and B16-F10 cell lines can be used for intravital microscopic imaging of tumor cells, neoplastic vasculature, and leukocyte subpopulations.

Key Words

fluorescent cell lines transduction tumor uptake intravital microscopy 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Ansari AM, Ahmed AK, Matsangos AE, Lay F, Born LJ, Marti G, Harmon JW, Sun Z. Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments. Stem Cell Rev. 2016;12(5):553-559.CrossRefGoogle Scholar
  2. 2.
    Beagles KE, Peterson L, Zhang X, Morris J, Kiem HP. Cyclosporine inhibits the development of green fluorescent protein (GFP)-specific immune responses after implantation of GFPexpressing hematopoietic repopulating cells in dogs. Hum. Gene Ther. 2005;16(6):725-733.CrossRefGoogle Scholar
  3. 3.
    Castano AP, Liu Q, Hamblin MR. A green fluorescent proteinexpressing murine tumour but not its wild-type counterpart is cured by photodynamic therapy. Br. J. Cancer 2006;94(3):391-397.CrossRefGoogle Scholar
  4. 4.
    Caysa H, Hoffmann S, Luetzkendorf J, Mueller LP, Unverzagt S, Mäder K, Mueller T. Monitoring of xenograft tumor growth and response to chemotherapy by non-invasive in vivo multispectral fluorescence imaging. PLoS One. 2012;7(10):e47927. doi:  https://doi.org/10.1371/journal.pone.0047927.CrossRefGoogle Scholar
  5. 5.
    Chishima T, Miyagi Y, Wang X, Yamaoka H, Shimada H, Moossa AR, Hoffman RM. Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression. Cancer Res. 1997;57(10):2042-2047.Google Scholar
  6. 6.
    Gambotto A, Dworacki G, Cicinnati V, Kenniston T, Steitz J, Tüting T, Robbins PD, DeLeo AB. Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene Ther. 2000;7(23):2036-2040.CrossRefGoogle Scholar
  7. 7.
    Ganini D, Leinisch F, Kumar A, Jiang J, Tokar EJ, Malone CC, Petrovich RM, Mason RP. Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells. Redox Biol. 2017;12:462-468.CrossRefGoogle Scholar
  8. 8.
    Goto H, Yang B, Petersen D, Pepper KA, Alfaro PA, Kohn DB, Reynolds CP. Transduction of green fluorescent protein increased oxidative stress and enhanced sensitivity to cytotoxic drugs in neuroblastoma cell lines. Mol. Cancer Ther. 2003;2(9):911-917.Google Scholar
  9. 9.
    Hoffman RM. The multiple uses of fluorescent proteins to visualize cancer in vivo. Nat. Rev. Cancer 2004;5(10):796-806.CrossRefGoogle Scholar
  10. 10.
    Huang WY, Aramburu J, Douglas PS, Izumo S. Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy. Nat. Med. 2000;6(5):482-483.CrossRefGoogle Scholar
  11. 11.
    Immunobiology: The Immune System in Health and Disease. Janeway СA, Travers P, Walport M, Shlomchik M, eds. New York, 2001. Russian.Google Scholar
  12. 12.
    Inoue H, Ohsawa I, Murakami T, Kimura A, Hakamata Y, Sato Y, Kaneko T, Takahashi M, Okada T, Ozawa K, Francis J, Leone P, Kobayashi E. Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem. Biophys. Res. Commun. 2005;329(1):288-295.CrossRefGoogle Scholar
  13. 13.
    Lechner MG, Karimi SS, Barry-Holson K, Angell TE, Murphy KA, Church CH, Ohlfest JR, Hu P, Epstein AL. Immunogenicity of murine solid tumor models as a defining feature of in vivo behavior and response to immunotherapy. J. Immunother. 2013 Vol. 36(9):477-489.CrossRefGoogle Scholar
  14. 14.
    Liu HS, Jan MS, Chou CK, Chen PH, Ke NJ. Is green fluorescent protein toxic to the living cells? Biochem. Biophys. Res. Commun. 1999;260(3):712-717.CrossRefGoogle Scholar
  15. 15.
    Miller MA, Weissleder R. Imaging the pharmacology of nanomaterials by intravital microscopy: toward understanding their biological behavior. Adv. Drug Deliv. Rev. 2017;113:61-86.CrossRefGoogle Scholar
  16. 16.
    Skelton D, Satake N, Kohn DB. The enhanced green fluorescent protein (eGFP) is minimally immunogenic in C57BL/6mice. Gene Ther. 2001;8(23):1813-1814.CrossRefGoogle Scholar
  17. 17.
    Stripecke R, Carmen Villacres M, Skelton D, Satake N, Halene S, Kohn D. Immune response to green fluorescent protein: implications for gene therapy. Gene Ther. 1999;6(7):1305-1312.CrossRefGoogle Scholar
  18. 18.
    Taghizadeh RR, Sherley JL. CFP and YFP, but not GFP, provide stable fluorescent marking of rat hepatic adult stem cells. J. Biomed. Biotechnol. 2008;2008. ID 453590. doi:  https://doi.org/10.1155/2008/453590.
  19. 19.
    Yang M, Baranov E, Jiang P, Sun FX, Li XM, Li L, Hasegawa S, Bouvet M, Al-Tuwaijri M, Chishima T, Shimada H, Moossa AR, Penman S, Hoffman RM. Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc. Natl Acad. Sci. USA. 2000;97(3):1206-1211.CrossRefGoogle Scholar
  20. 20.
    Yuzhakova DV, Shirmanova MV, Serebrovskaya EO, Lukyanov KA, Druzhkova IN, Shakhov BE, Lukyanov SA, Zagaynova EV. CT26 murine colon carcinoma expressing the red fluorescent protein KillerRed as a highly immunogenic tumor model. J. Biomed. Opt. 2015;20(8):ID 88002. doi:  https://doi.org/10.1117/1.JBO.20.8.088002.

Copyright information

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

Authors and Affiliations

  • S. S. Vodopyanov
    • 1
    Email author
  • M. A. Kunin
    • 2
  • A. S. Garanina
    • 1
  • N. F. Grinenko
    • 3
  • K. Yu. Vlasova
    • 2
  • P. A. Mel’nikov
    • 3
  • V. P. Chekhonin
    • 3
    • 4
  • K. K. Sukhinich
    • 5
  • A. V. Makarov
    • 3
  • V. A. Naumenko
    • 1
  • M. A. Abakumov
    • 1
    • 4
  • A. G. Majouga
    • 1
    • 2
    • 6
  1. 1.Laboratory of Biomedical Nanomaterials, National University of Science and Technology (MISIS)MoscowRussia
  2. 2.M. V. Lomonosov Moscow State UniversityMoscowRussia
  3. 3.V. P. Serbsky Federal Medical Research Center for Psychiatry and Narcology, Ministry of Health of the Russian FederationMoscowRussia
  4. 4.N. I. Pirogov Russian National Research Medical University, Ministry of Health of the Russian FederationMoscowRussia
  5. 5.N. K. Kol’tsov Institute of Developmental Biology, Russian Academy of SciencesMoscowRussia
  6. 6.D. I. Mendeleev University of Chemical TechnologyMoscowRussia

Personalised recommendations