Skip to main content

Advertisement

Log in

Development and verification of a three-dimensional (3D) breast cancer tumor model composed of circulating tumor cell (CTC) subsets

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Breast cancer is one of the most common cancer types among women in which early tumor invasion leads to metastases and death. EpCAM (epithelial cellular adhesion molecule) and HER2 (human epidermal growth factor receptor 2) are two main circulating tumor cell (CTC) subsets in HER2+ breast cancer patients. In this regard, the main aim of this study is to develop and characterize a three-dimensional (3D) breast cancer tumor model composed of CTC subsets to evaluate new therapeutic strategies and drugs. For this reason, EpCAM(+) and HER2(+) sub-populations were isolated from different cell lines to establish 3D tumor model that mimics in situ (in vivo) more closely than two-dimensional (2D) models. EpCAM(+)/HER2(+) cells had a high proliferation rate and low tendency to attach to the surface in comparison with parental MDA-MB-453 cells as CTC subsets. Aggressive breast cancer subpopulations cultured in 3D porous chitosan scaffold had enhanced cell–cell and cell–matrix interactions compared to 2D cultured cells and these 3D models showed more aggressive morphology and behavior, expressed higher levels of pluripotency marker genes, Nanog, Sox2 and Oct4. For the verification of the 3D model, the effects of doxorubicin which is a chemotherapeutic agent used in breast cancer treatment were examined and increased drug resistance was determined in 3D cultures. The 3D tumor model comprising EpCAM(+)/HER2(+) CTC subsets developed in this study has a promising potential to be used for investigation of an aggressive CTC microenvironment in vitro that mimics in vivo characteristics to test new drug candidates against CTCs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Maltoni R et al (2015) Circulating tumor cells in early breast cancer: a connection with vascular invasion. Cancer Lett 367:43–48. https://doi.org/10.1016/j.canlet.2015.06.020

    Article  CAS  PubMed  Google Scholar 

  2. Wang HY et al (2015) Detection of circulating tumor cell-specific markers in breast cancer patients using the quantitative RT-PCR assay. Int J Clin Oncol 20:878–890. https://doi.org/10.1007/s10147-015-0798-3

    Article  CAS  PubMed  Google Scholar 

  3. Fauci AS, Braunwald E, Kasper DL (2008) Breast cancer Harrison’s principles of internal Medicine, 17th edn. McGraw-Hill Companies, Inc, New York

    Google Scholar 

  4. Joosse SA, Gorges TM, Pantel K (2015) Biology, detection, and clinical implications of circulating tumor cells. EMBO Mol Med 7:1–11. https://doi.org/10.15252/emmm.201303698

    Article  CAS  PubMed  Google Scholar 

  5. Zeune L et al (2017) Quantifying HER-2 expression on circulating tumor cells by ACCEPT. PLoS ONE 12:e0186562. https://doi.org/10.1371/journal.pone.0186562

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Andree KC, van Dalum G, Terstappen LW (2016) Challenges in circulating tumor cell detection by the cell search system. Mol Oncol 10:395–407. https://doi.org/10.1016/j.molonc.2015.12.002

    Article  CAS  PubMed  Google Scholar 

  7. Ashworth TRA (1869) A case of cancer in which cells similar to those in the tumours were seen in the blood after death. Med J Aust 14:146–147

    Google Scholar 

  8. Esmaeilsabzali H, Beischlag TV, Cox ME, Parameswaran AM, Park EJ (2013) Detection and isolation of circulating tumor cells: principles and methods. Biotechnol Adv 31:1063–1084. https://doi.org/10.1016/j.biotechadv.2013.08.016

    Article  CAS  PubMed  Google Scholar 

  9. Gadalla SE, Ojemalm K, Vasquez PL, Nilsson I, Ericsson C, Zhao J, Nister M (2013) EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells. Biochem Biophys Res Commun 439:203–208. https://doi.org/10.1016/j.bbrc.2013.08.059

    Article  CAS  PubMed  Google Scholar 

  10. Zieglschmid V, Hollmann C, Gutierrez B, Albert W, Strothoff D, Gross E, Bocher O (2005) Combination of immunomagnetic enrichment with multiplex RT-PCR analysis for the detection of disseminated tumor cells. Anticancer Res 25:1803–1810

    CAS  PubMed  Google Scholar 

  11. Yeo SK, Guan JL (2017) Breast cancer: multiple subtypes within a tumor? Trends Cancer 3:753–760. https://doi.org/10.1016/j.trecan.2017.09.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Riethdorf S et al (2007) Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the cell search system. Clin Cancer Res 13:920

    Article  CAS  Google Scholar 

  13. Pestrin M et al (2009) Correlation of HER2 status between primary tumors and corresponding circulating tumor cells in advanced breast cancer patients. Breast Cancer Res Treat 118:523–530. https://doi.org/10.1007/s10549-009-0461-7

    Article  CAS  PubMed  Google Scholar 

  14. Iqbal N, Iqbal N (2014) Human epidermal growth factor receptor 2 (HER2) in cancers: overexpression and therapeutic ımplications. Mol Biol Int 2014:852748. https://doi.org/10.1155/2014/852748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JM (2003) Expression of the HER1-4 family of receptor tyrosine kinases in breast cancer. J Pathol 200:290–297. https://doi.org/10.1002/path.1370

    Article  CAS  PubMed  Google Scholar 

  16. Fehm T, Maimonis P, Katalinic A, Jäger WH (1998) The prognostic significance of c-erbB-2 serum protein in metastatic. Breast Cancer Oncol 55:33–38. https://doi.org/10.1159/000011832

    Article  CAS  Google Scholar 

  17. Pantel K, Alix-Panabieres C (2010) Circulating tumour cells in cancer patients: challenges and perspectives. Trends Mol Med 16:398–406. https://doi.org/10.1016/j.molmed.2010.07.001

    Article  PubMed  Google Scholar 

  18. Man Y, Wang Q, Kemmner W (2011) Currently used markers for CTC isolation—advantages, limitations and ımpact on cancer prognosis. J Clin Exp Pathol. https://doi.org/10.4172/2161-0681.1000102

    Article  Google Scholar 

  19. Caballero D, Kaushik S, Correlo VM, Oliveira JM, Reis RL, Kundu SC (2017) Organ-on-chip models of cancer metastasis for future personalized medicine: from chip to the patient. Biomaterials 149:98–115. https://doi.org/10.1016/j.biomaterials.2017.10.005

    Article  CAS  PubMed  Google Scholar 

  20. Balalaeva IV, Sokolova EA, Puzhikhina AD, Brilkina AA, Deyev SM (2017) Spheroids of HER2-positive breast adenocarcinoma for studying anticancer ımmunotoxins ın vitro. Acta Naturae 9:38–43

    Article  CAS  Google Scholar 

  21. Mahmoudzadeh A, Mohammadpour H (2016) Tumor cell culture on collagen-chitosan scaffolds as three-dimensional tumor model: a suitable model for tumor studies. J Food Drug Anal 24:620–626. https://doi.org/10.1016/j.jfda.2016.02.008

    Article  CAS  PubMed  Google Scholar 

  22. Ozcelikkale A et al (2017) Differential response to doxorubicin in breast cancer subtypes simulated by a microfluidic tumor model. J Control Release 266:129–139. https://doi.org/10.1016/j.jconrel.2017.09.024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sant S, Johnston PA (2017) The production of 3D tumor spheroids for cancer drug discovery. Drug Discov Today Technol 23:27–36. https://doi.org/10.1016/j.ddtec.2017.03.002

    Article  PubMed  PubMed Central  Google Scholar 

  24. Martelotto LG, Ng CK, Piscuoglio S, Weigelt B, Reis-Filho JS (2014) Breast cancer intra-tumor heterogeneity. Breast Cancer Res 16:210

    Article  Google Scholar 

  25. Marusyk A, Almendro V, Polyak K (2012) Intra-tumour heterogeneity: a looking glass for cancer? Nat Rev Cancer 12:323

    Article  CAS  Google Scholar 

  26. Brooks MD, Burness ML, Wicha MS (2015) Therapeutic implications of cellular heterogeneity and plasticity in breast cancer. Cell Stem Cell 17:260–271

    Article  CAS  Google Scholar 

  27. McGranahan N, Swanton C (2015) Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell 27:15–26

    Article  CAS  Google Scholar 

  28. Arpino G, Milano M, De Placido S (2015) Features of aggressive breast cancer. Breast 24:594–600

    Article  Google Scholar 

  29. Parekh A, Das D, Das S, Dhara S, Biswas K, Mandal M, Das S (2018) Bioimpedimetric analysis in conjunction with growth dynamics to differentiate aggressiveness of cancer cells. Sci Rep 8:783

    Article  Google Scholar 

  30. Spizzo G et al (2011) EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis. J Clin Pathol 64:415–420

    Article  Google Scholar 

  31. Gastl G, Spizzo G, Obrist P, Dünser M, Mikuz G (2000) Ep-CAM overexpression in breast cancer as a predictor of survival. Lancet 356:1981–1982

    Article  CAS  Google Scholar 

  32. Yarden Y (2001) Biology of HER2 and its importance in breast cancer. Oncology 61:1–13

    Article  CAS  Google Scholar 

  33. Rijal G, Li W (2016) 3D scaffolds in breast cancer research. Biomaterials 81:135–156

    Article  CAS  Google Scholar 

  34. Cheung RCF, Ng TB, Wong JH, Chan WY (2015) Chitosan: an update on potential biomedical and pharmaceutical applications. Mar Drugs 13:5156–5186

    Article  CAS  Google Scholar 

  35. Chang P-H, Sekine K, Chao H-M, Hsu S-h, Chern E (2017) Chitosan promotes cancer progression and stem cell properties in association with Wnt signaling in colon and hepatocellular carcinoma cells. Sci Rep 7:45751

    Article  Google Scholar 

  36. Kievit FM et al (2014) Proliferation and enrichment of CD133 + glioblastoma cancer stem cells on 3D chitosan-alginate scaffolds. Biomaterials 35:9137–9143

    Article  CAS  Google Scholar 

  37. Sims-Mourtada J, Niamat RA, Samuel S, Eskridge C, Kmiec EB (2014) Enrichment of breast cancer stem-like cells by growth on electrospun polycaprolactone-chitosan nanofiber scaffolds. Int J Nanomed 9:995

    Article  Google Scholar 

  38. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, Weinberg RA (2008) An embryonic stem cell–like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 40:499

    Article  CAS  Google Scholar 

  39. Savci-Heijink CD, Halfwerk H, Hooijer GK, Horlings HM, Wesseling J, van de Vijver MJ (2015) Retrospective analysis of metastatic behaviour of breast cancer subtypes. Breast Cancer Res Treat 150:547–557

    Article  CAS  Google Scholar 

  40. Gabriel MT, Calleja LR, Chalopin A, Ory B, Heymann D (2016) Circulating tumor cells: a review of non–EpCAM-based approaches for cell enrichment and isolation. Clin Chem 62:571–581

    Article  CAS  Google Scholar 

  41. Elliott NT, Yuan F (2011) A review of three-dimensional in vitro tissue models for drug discovery and transport studies. J Pharm Sci 100:59–74. https://doi.org/10.1002/jps.22257

    Article  CAS  PubMed  Google Scholar 

  42. Luo YT et al (2018) The viable circulating tumor cells with cancer stem cells feature, where is the way out? J Exp Clin Cancer Res 37:38. https://doi.org/10.1186/s13046-018-0685-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Madihally SV, Matthew HW (1999) Porous chitosan scaffolds for tissue engineering. Biomaterials 20:1133–1142

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This project was supported by the research fund as Ege University Scientific Research Project (Project Number: 15-MUH-038) and by TUBITAK-1919B011503631 under the supervision of Sultan GULCE-IZ. The authors would like to thank Dr Aylin SENDEMIR-URKMEZ for providing the chitosan scaffolds.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sultan Gulce-Iz.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Anil-Inevi, M., Sağlam-Metiner, P., Kabak, E.C. et al. Development and verification of a three-dimensional (3D) breast cancer tumor model composed of circulating tumor cell (CTC) subsets. Mol Biol Rep 47, 97–109 (2020). https://doi.org/10.1007/s11033-019-05111-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-019-05111-z

Keywords

Navigation