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Spheroid Cultures of Primary Urothelial Cancer Cells: Cancer Tissue-Originated Spheroid (CTOS) Method

  • Takahiro Yoshida
  • Hiroaki Okuyama
  • Hiroko Endo
  • Masahiro InoueEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1655)

Abstract

Increasingly, it has been recognized that studying cancer samples from individual patients is important for the development of effective therapeutic strategies and in endeavors to overcome therapy resistance. Primary cultures of cancer cells acutely dissected from individual patients can provide a platform that enables the study and characterization of individual tumors. To that end, we have developed a method for preparing cancer cells in the form of multi-cellular spheroids. The cells can be derived from patient tumors (primary cells), from patient-derived xenografts, or from genetically- or chemically induced animal tumors. This method of culturing spheroids composed of cells derived from cancer tissues can be applied to various types of cancer, including urothelial cancer. The method is based on the principle of retaining cell-cell contact throughout cancer cell preparation and culturing. The first step is a partial digestion of the tumor specimen into small fragments; these fragments spontaneously form spheroidal shapes within several hours. The spheroid is referred to as a cancer tissue-originated spheroid (CTOS). The advantage of the CTOS method is that it allows one to prepare pure cancer cells at high yield. CTOSs can be stably cultured in serum-free conditions. The CTOS method can be applied to drug sensitivity assays, drug screening, and analyses of intracellular signaling. Moreover, the CTOS method provides a platform for studying the nature of cancer cell clusters.

Key words

Urothelial cancer Bladder cancer Primary cell culture Spheroid Organoid CTOS 

References

  1. 1.
    Sharma SV, Haber DA, Settleman J (2010) Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents. Nat Rev Cancer 10(4):241–253CrossRefPubMedGoogle Scholar
  2. 2.
    Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S et al (2012) The cancer cell line encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483(7391):603–607CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Yu M, Bardia A, Wittner BS, Stott SL, Smas ME, Ting DT et al (2013) Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 339(6119):580–584CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA et al (2014) Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158(5):1110–1122CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Yoshida T, Okuyama H, Nakayama M, Endo H, Tomita Y, Nonomura N et al (2015) Dynamic change in p63 protein expression during implantation of urothelial cancer clusters. Neoplasia 17(7):574–585CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Bedard PL, Hansen AR, Ratain MJ, Siu LL (2013) Tumour heterogeneity in the clinic. Nature 501(7467):355–364CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Cancer Genome Atlas Research N, Weinstein JN, Collisson EA, Mills GB, Shaw KR, Ozenberger BA et al (2013) The cancer genome atlas pan-cancer analysis project. Nat Genet 45(10):1113–1120CrossRefGoogle Scholar
  8. 8.
    Mitra A, Mishra L, Li S (2013) Technologies for deriving primary tumor cells for use in personalized cancer therapy. Trends Biotechnol 31(6):347–354CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kondo J, Endo H, Okuyama H, Ishikawa O, Iishi H, Tsujii M et al (2011) Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer. Proc Natl Acad Sci U S A 108(15):6235–6240CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Takeda T, Okuyama H, Nishizawa Y, Tomita S, Inoue M (2012) Hypoxia inducible factor-1alpha is necessary for invasive phenotype in Vegf-deleted islet cell tumors. Sci Rep 2:494CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Okuyama H, Yoshida T, Endo H, Nakayama M, Nonomura N, Nishimura K et al (2013) Involvement of heregulin/HER3 in the primary culture of human urothelial cancer. J Urol 190(1):302–310CrossRefPubMedGoogle Scholar
  12. 12.
    Yoshida T, Okuyama H, Nakayama M, Endo H, Nonomura N, Nishimura K et al (2015) High-dose chemotherapeutics of intravesical chemotherapy rapidly induce mitochondrial dysfunction in bladder cancer-derived spheroids. Cancer Sci 106(1):69–77CrossRefPubMedGoogle Scholar
  13. 13.
    Kiyohara Y, Yoshino K, Kubota S, Okuyama H, Endo H, Ueda Y et al (2016) Drug screening and grouping by sensitivity with a panel of primary cultured cancer spheroids derived from endometrial cancer. Cancer Sci 107(4):452–460CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Endo H, Okami J, Okuyama H, Kumagai T, Uchida J, Kondo J et al (2013) Spheroid culture of primary lung cancer cells with neuregulin 1/HER3 pathway activation. J Thorac Oncol 8(2):131–139CrossRefPubMedGoogle Scholar
  15. 15.
    Nakajima A, Endo H, Okuyama H, Kiyohara Y, Kimura T, Kamiura S et al (2015) Radiation sensitivity assay with a panel of patient-derived spheroids of small cell carcinoma of the cervix. Int J Cancer 136(12):2949–2960CrossRefPubMedGoogle Scholar
  16. 16.
    Knowles MA, Hurst CD (2015) Molecular biology of bladder cancer: new insights into pathogenesis and clinical diversity. Nat Rev Cancer 15(1):25–41CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Takahiro Yoshida
    • 1
  • Hiroaki Okuyama
    • 2
  • Hiroko Endo
    • 2
  • Masahiro Inoue
    • 2
    • 3
    Email author
  1. 1.Department of Urology, The James Buchanan Brady Urological InstituteThe Johns Hopkins University School of MedicineBaltimoreUSA
  2. 2.Department of BiochemistryOsaka International Cancer InstituteOsakaJapan
  3. 3.Department of Clinical and Experimental PathophysiologyOsaka University Graduate School of Pharmaceutical SciencesOsakaJapan

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