Three-Dimensional Cell Culture Model Utilization in Renal Carcinoma Cancer Stem Cell Research

  • Kamila Maliszewska-Olejniczak
  • Klaudia K. Brodaczewska
  • Zofia F. Bielecka
  • Anna M. Czarnecka
Part of the Methods in Molecular Biology book series (MIMB, volume 1817)


Specific 3D conditions of cancer cell lines have been optimized over last years, with growing significance of serum-free and xeno-free culture variants. The choice of proper culture media enables cancer stem cells proliferation in primary and stable cell lines. To obtain renal cell cancer stem-like phenotype, we employed media dedicated for mesenchymal cells and adult stem cells. Developed RCC cell line 3D culture system enables effective drug testing, including tyrosine kinase inhibitor anti-cancer cell toxicity. To induce formation of 3D spheroids by RCC cell lines, StemXvivo and NutriStem media must be used. Usage of laminin- or poly-d-lysine coated plates enhances also the formation of spheroids in 3D-promoting media. Seeding is optimal with Caki-1 or ACHN cell lines as well as 786-O or HKCSC cells. Our bio-mimic 3D RCC cell culture model promotes cell viability and stem-related gene expression including E-cadherin, N-cadherin, HIF1, HIF2, VEGF, Sox2, Pax2, and Nestin. 3D spheroid formation ability and spheroid volume increase are disturbed upon drug treatment. Untreated 3D structures reach ~100 μm in diameter at the end of 14-day long experiment. Sorter-based cell cycle analysis and Ki-67 staining should be conducted to verify specific toxicity. We suggest that due to the more complex architecture 3D RCC culture is more relevant to investigate the in vivo-like tumor drug response.

Key words

Renal carcinoma cancer stem cells 3D cell culture Tyrosine kinase inhibitor 



This research was supported by the National Centre for Research and Development (NCBR, Poland) LIDER grant no. Lider/031/625/L-4/NCBR/2013. KMO was supported by the Foundation for Polish Science (FNP) START Program during preparation of this manuscript.


  1. 1.
    Ferlay J, Autier P, Boniol M et al (2007) Estimates of the cancer incidence and mortality in Europe in 2006. Ann Oncol 18:581–592CrossRefPubMedGoogle Scholar
  2. 2.
    Bussolati B, Brossa A, Camussi G (2011) Resident stem cells and renal carcinoma. Int J Nephrol 2011:286985CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Escudier B, Eisen T, Stadler WM et al (2007) Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med 356:125–134CrossRefPubMedGoogle Scholar
  4. 4.
    Motzer RJ, Hutson TE, Tomczak P et al (2007) Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med 356:115–124CrossRefPubMedGoogle Scholar
  5. 5.
    Bellmunt J, Eisen T, Szczylik C et al (2011) A new patient-focused approach to the treatment of metastatic renal cell carcinoma: establishing customized treatment options. BJU Int 107:1190–1199CrossRefPubMedGoogle Scholar
  6. 6.
    Bussolati B, Bruno S, Grange C et al (2008) Identification of a tumor-initiating stem cell population in human renal carcinomas. FASEB J 22:3696–3705CrossRefPubMedGoogle Scholar
  7. 7.
    Oates JE, Grey BR, Addla SK et al (2009) Hoechst 33342 side population identification is a conserved and unified mechanism in urological cancers. Stem Cells Dev 18:1515–1522CrossRefPubMedGoogle Scholar
  8. 8.
    Bruno S, Bussolati B, Grange C et al (2006) CD133+ renal progenitor cells contribute to tumor angiogenesis. Am J Pathol 169:2223–2235CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Debeb BG, Zhang X, Krishnamurthy S et al (2010) Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells. Mol Cancer 9:180CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ueda K, Ogasawara S, Akiba J et al (2013) Aldehyde dehydrogenase 1 identifies cells with cancer stem cell-like properties in a human renal cell carcinoma cell line. PLoS One 8:e75463CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Gassenmaier M, Chen D, Buchner A et al (2013) CXC chemokine receptor 4 is essential for maintenance of renal cell carcinoma-initiating cells and predicts metastasis. Stem Cells 31:1467–1476CrossRefPubMedGoogle Scholar
  12. 12.
    Pode-Shakked N, Shukrun R, Mark-Danieli M et al (2013) The isolation and characterization of renal cancer initiating cells from human Wilms’ tumour xenografts unveils new therapeutic targets. EMBO Mol Med 5:18–37CrossRefPubMedGoogle Scholar
  13. 13.
    Nishizawa S, Hirohashi Y, Torigoe T et al (2012) HSP DNAJB8 controls tumor-initiating ability in renal cancer stem-like cells. Cancer Res 72:2844–2854CrossRefPubMedGoogle Scholar
  14. 14.
    Huang B, Huang YJ, Yao ZJ et al (2013) Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P. PLoS One 8:e68293CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Lu J, Cui Y, Zhu J et al (2013) Biological characteristics of Rh123(high) stem-like cells in a side population of 786-O renal carcinoma cells. Oncol Lett 5:1903–1908CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chen L, Xiao Z, Meng Y et al (2012) The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs. Biomaterials 33:1437–1444CrossRefPubMedGoogle Scholar
  17. 17.
    Khan MI, Czarnecka AM, Lewicki S et al (2016) Comparative gene expression profiling of primary and metastatic renal cell carcinoma stem cell-like cancer cells. PLoS One 11:e0165718CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Matak D, Brodaczewska KK, Szczylik C et al (2017) Functional significance of CD105-positive cells in papillary renal cell carcinoma. BMC Cancer 17:21CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Matak D, Brodaczewska KK, Lipiec M et al (2017) Colony, hanging drop, and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines. Cytotechnology 69:565–578CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Lee GY, Kenny PA, Lee EH, Bissell MJ (2007) Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4:359–365CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kano J, Ishiyama T, Nakamura N et al (2003) Establishment of hepatic stem-like cell lines from normal adult porcine liver in a poly-D-lysine-coated dish with NAIR-1 medium. Vitro Cell Dev Biol Anim 39:440–448CrossRefGoogle Scholar
  22. 22.
    Lepiller Q, Abbas W, Kumar A et al (2013) HCMV activates the IL-6-JAK-STAT3 axis in HepG2 cells and primary human hepatocytes. PLoS One 8:e59591CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Rajasingh S, Thangavel J, Czirok A et al (2015) Generation of functional cardiomyocytes from efficiently generated human iPSCs and a novel method of measuring contractility. PLoS One 10:e0134093CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Williams RD, Elliott AY, Stein N, Fraley EE (1976) In vitro cultivation of human renal cell cancer. I. Establishment of cells in culture. In Vitro 12:623–627CrossRefPubMedGoogle Scholar
  25. 25.
    Glube N, Giessl A, Wolfrum U, Langguth P (2007) Caki-1 cells represent an in vitro model system for studying the human proximal tubule epithelium. Nephron Exp Nephrol 107:47–56CrossRefGoogle Scholar
  26. 26.
    Kochevar J (1990) Blockage of autonomous growth of ACHN cells by anti-renal cell carcinoma monoclonal antibody 5F4. Cancer Res 50:2968–2972PubMedGoogle Scholar
  27. 27.
    Windmüller C, Bronger H, Davoodi M et al (2015) The role of CXCR3/ligand axis in cancer. Int Trends Immun 3:19–25Google Scholar
  28. 28.
    Brodaczewska KK, Szczylik C, Fiedorowicz M et al (2016) Choosing the right cell line for renal cell cancer research. Mol Cancer 15:83CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Roskoski R (2007) Sunitinib: a VEGF and PDGF receptor protein kinase and angiogenesis inhibitor. Biochem Biophys Res Commun 356:323–328CrossRefPubMedGoogle Scholar
  30. 30.
    Karaman MW, Herrgard S, Treiber DK et al (2008) A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol 26:127–132CrossRefPubMedGoogle Scholar
  31. 31.
    Zhang C, Liu Z, Bunker E et al (2017) Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-parkin pathway and modulate cellular drug response. J Biol Chem 292:15105–15120CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Kelly RJ, Rixe O (2010) Axitinib (AG-013736). Recent Results Cancer Res 184:33–44CrossRefPubMedGoogle Scholar
  33. 33.
    Ho TH, Jonasch E (2011) Axitinib in the treatment of metastatic renal cell carcinoma. Fut Oncol 7:1247–1253CrossRefGoogle Scholar
  34. 34.
    Ho AL, Dunn L, Sherman EJ et al (2016) A phase II study of axitinib (AG-013736) in patients with incurable adenoid cystic carcinoma. Ann Oncol 27:1902–1908CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Oudard S, Medioni J, Aylllon J et al (2009) Everolimus (RAD001): an mTOR inhibitor for the treatment of metastatic renal cell carcinoma. Expert Rev Anticancer Ther 9:705–717CrossRefPubMedGoogle Scholar
  36. 36.
    Czarnecka AM, Kornakiewicz A, Lian F, Szczylik C (2015) Future perspectives for mTOR inhibitors in renal cell cancer treatment. Fut Oncol 11:801–817CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Kamila Maliszewska-Olejniczak
    • 1
    • 2
  • Klaudia K. Brodaczewska
    • 1
  • Zofia F. Bielecka
    • 1
    • 3
  • Anna M. Czarnecka
    • 1
  1. 1.Department of Oncology with Laboratory of Molecular OncologyMilitary Institute of MedicineWarsawPoland
  2. 2.National Centre for Nuclear ResearchSołtanaPoland
  3. 3.School of Molecular MedicineWarsaw Medical UniversityWarsawPoland

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