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Evaluation of Tumor Vasculature Using a Syngeneic Tumor Model in Wild-Type and Genetically Modified Mice

  • Francisco Javier Rodríguez-Baena
  • Silvia Redondo-García
  • María del Carmen Plaza-Calonge
  • Rubén Fernández-Rodríguez
  • Juan Carlos Rodríguez-ManzanequeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1731)

Abstract

The relevance of tumor vasculature has been extensively recognized, and it is still the focus of numerous lines of research for basic, translational, and clinical scientists. Indeed, the knowledge of some of its regulatory mechanisms has provoked the generation of ongoing cancer therapies. Within the context of the tumor microenvironment, the information that the analysis of the vasculature provides is very valuable, and it might reveal not just its quality and the response against a specific therapy but also its close relationship with neighboring stromal and tumor players.

Studies during last decades already supported the contribution of extracellular proteases in neovascularization events, including ADAMTS. However, deeper analyses are still required to better understand the modulation of their proteolytic activity in the tumor microenvironment. Future studies will clearly benefit from existing and ongoing genetically modified mouse models.

Here we emphasize the use of syngeneic models to study the vasculature during tumor progression, supported by their intact immunocompetent capacities and also by the range of possibilities to play with engineered mice and with modified tumor cells. Although various high-tech and sophisticated approaches have already been reported to evaluate tumor neovascularization, here we describe a simple and easily reproduced methodology based in the immunofluorescence detection of vascular-specific molecules. A final in silico analysis guarantees an unbiased quantification of tumor vasculature under different conditions.

Key words

Extracellular microenvironment In silico analysis Metalloproteinase Tumor stroma Vasculature 

Notes

Acknowledgment

Work in the author’s laboratory has been supported by grants from the Ministerio de Economía y Competitividad and Instituto de Salud Carlos III from Spain and cofinanced by FEDER (PI13/00168 and PI16/00345 to JCRM). SRG is supported by a contract from Garantía Juvenil (PEJ-2014-A-38416-MINECO-FSE).

For data shown, all mice were kept in the Centro de Investigaciones Biomédicas-UGR Animal Facility under pathogen-free conditions and according to institutional guidelines.

References

  1. 1.
    Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature 473(7347):298–307. https://doi.org/10.1038/nature10144 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    De Sousa E, Melo F, Vermeulen L et al (2013) Cancer heterogeneity – a multifaceted view. EMBO Rep 14(8):686–695. https://doi.org/10.1038/embor.2013.92 CrossRefGoogle Scholar
  3. 3.
    Jain RK (2014) Antiangiogenesis strategies revisited: from starving tumors to alleviating hypoxia. Cancer Cell 26:605–622. https://doi.org/10.1016/j.ccell.2014.10.006 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Turk B (2006) Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 5(9):785–799. https://doi.org/10.1038/nrd2092 CrossRefPubMedGoogle Scholar
  5. 5.
    Handsley MM, Edwards DR (2005) Metalloproteinases and their inhibitors in tumor angiogenesis. Int J Cancer 115(6):849–860. https://doi.org/10.1002/ijc.20945 CrossRefPubMedGoogle Scholar
  6. 6.
    Lu P, Takai K, Weaver VM et al (2011) Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb Perspect Biol 3:a005058. https://doi.org/10.1101/cshperspect.a005058 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bix G, Iozzo RV (2005) Matrix revolutions: “tails” of basement-membrane components with angiostatic functions. Trends Cell Biol 15:52–60. https://doi.org/10.1016/j.tcb.2004.11.008 CrossRefPubMedGoogle Scholar
  8. 8.
    Rodríguez-Manzaneque JC, Fernández-Rodríguez R, Rodríguez-Baena FJ et al (2015) ADAMTS proteases in vascular biology. Matrix Biol 44–46:38–45. https://doi.org/10.1016/j.matbio.2015.02.004 CrossRefPubMedGoogle Scholar
  9. 9.
    Reynolds LE, Watson AR, Baker M et al (2010) Tumour angiogenesis is reduced in the Tc1 mouse model of Down’s syndrome. Nature 465(7299):813–817. https://doi.org/10.1038/nature09106 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Martino-Echarri E, Fernández-Rodríguez R, Rodríguez-Baena FJ et al (2013) Contribution of ADAMTS1 as a tumor suppressor gene in human breast carcinoma. Linking its tumor inhibitory properties to its proteolytic activity on nidogen-1 and nidogen-2. Int J Cancer 133(10):2315–2324. https://doi.org/10.1002/ijc.28271 CrossRefPubMedGoogle Scholar
  11. 11.
    Casal C, Torres-Collado AX, Plaza-Calonge MCDC et al (2010) ADAMTS1 contributes to the acquisition of an endothelial-like phenotype in plastic tumor cells. Cancer Res 70(11):4676–4686. https://doi.org/10.1158/0008-5472.CAN-09-4197 CrossRefPubMedGoogle Scholar
  12. 12.
    Lu X, Wang Q, Hu G et al (2009) ADAMTS1 and MMP1 proteolytically engage EGF-like ligands in an osteolytic signaling cascade for bone metastasis. Genes Dev 23(16):1882–1894. https://doi.org/10.1101/gad.1824809 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ricciardelli C, Frewin KM, Tan IDA et al (2011) The ADAMTS1 protease gene is required for mammary tumor growth and metastasis. Am J Pathol 179(6):3075–3085. https://doi.org/10.1016/j.ajpath.2011.08.021 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Rocks N, Paulissen G, Quesada-Calvo F et al (2008) ADAMTS-1 metalloproteinase promotes tumor development through the induction of a stromal reaction in vivo. Cancer Res 68(22):9541–9550. https://doi.org/10.1158/0008-5472.CAN-08-0548 CrossRefPubMedGoogle Scholar
  15. 15.
    Fernández-Rodríguez R, Rodríguez-Baena FJ, Martino-Echarri E et al (2016) Stroma-derived but not tumor ADAMTS1 is a main driver of tumor growth and metastasis. Oncotarget 7(23):34507–34519. 10.18632/oncotarget.8922 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Wieczorek E, Jablonska E, Wasowicz W et al (2015) Matrix metalloproteinases and genetic mouse models in cancer research: a mini-review. Tumor Biol 36(1):163–175. https://doi.org/10.1007/s13277-014-2747-6 CrossRefGoogle Scholar
  17. 17.
    Poste G, Doll J, Hart IR et al (1980) In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties. Cancer Res 40(5):1636–1644PubMedGoogle Scholar
  18. 18.
    Bertram JSJ, Janik PP (1980) Establishment of a cloned line of Lewis lung carcinoma cells adapted to cell culture. Cancer Lett 11(1):63–73. https://doi.org/10.1016/0304-3835(80)90130-5 CrossRefPubMedGoogle Scholar
  19. 19.
    Simons M, Alitalo K, Annex BH et al (2015) State-of-the-art methods for evaluation of angiogenesis and tissue vascularization: a scientific statement from the American Heart Association. Circ Res 116(11):e99–e132. https://doi.org/10.1161/RES.0000000000000054 CrossRefPubMedGoogle Scholar
  20. 20.
    Vakoc BJ, Lanning RM, Tyrrell JA et al (2009) Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nat Med 15(10):1219–1223. https://doi.org/10.1038/nm.1971 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Missbach-Guentner J, Hunia J, Alves F (2011) Tumor blood vessel visualization. Int J Dev Biol 55:535–546. https://doi.org/10.1387/ijdb.103229jm CrossRefPubMedGoogle Scholar
  22. 22.
    Robertson RT, Levine ST, Haynes SM et al (2015) Use of labeled tomato lectin for imaging vasculature structures. Histochem Cell Biol 143(2):225–234. https://doi.org/10.1007/s00418-014-1301-3 CrossRefPubMedGoogle Scholar
  23. 23.
    Rodriguez-Manzaneque JC, Lane TF, Ortega MA et al (2001) Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor. Proc Natl Acad Sci U S A 98(22):12485–12490. https://doi.org/10.1073/pnas.171460498 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Francisco Javier Rodríguez-Baena
    • 1
  • Silvia Redondo-García
    • 1
  • María del Carmen Plaza-Calonge
    • 1
  • Rubén Fernández-Rodríguez
    • 1
  • Juan Carlos Rodríguez-Manzaneque
    • 1
    Email author
  1. 1.GENYO, Centre for Genomics and Oncological ResearchPfizer/Universidad de Granada/Junta de AndalucíaGranadaSpain

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