Biomedical Microdevices

, Volume 15, Issue 4, pp 645–655 | Cite as

Enrichment of tumor-initiating breast cancer cells within a mammosphere-culture microdevice

  • Katayoon Saadin
  • Jeffrey M. Burke
  • Neerav P. Patel
  • Rebecca E. Zubajlo
  • Ian M. WhiteEmail author


We report for the first time a microdevice that enables the selective enrichment, culture, and identification of tumor-initiating cells on native polydimethylsiloxane (PDMS). For nearly a decade, researchers have identified tumor-initiating breast cancer cells within heterogeneous populations of breast cancer cells by utilizing low-attachment serum-free culture conditions, which lead to the formation of spheroidal colonies (mammospheres) that are enriched for tumor-initiating cells. However, the utility of this assay has been limited by difficulties in combining this culture-plate-based technique with other cellular and molecular analyses. Integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions, such as cell sorting, drug screens, and molecular assays. In this work, we demonstrate mammosphere culture within a PDMS microdevice. We first prove that a native hydrophobic PDMS surface is as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then experimentally assess the PDMS microdevice. Time-lapse images of mammosphere formation within the microdevice show that mammospheres form from single cells or small clusters of cells. Following formation of the mammospheres, it is desirable to evaluate the cells within the spheroids for enrichment of tumor initiating cells. To perform assays such as this (which require the loading and rinsing of reagents) without flushing the cells (which are in suspension) from the device, the culture chamber is separated from a reagent reservoir by a commercially available microporous membrane, and thus reagents are exchanged between the reservoir and the culture chamber by diffusion only. Using this capability, we verify that the mammospheres are enriched for tumor initiating cells by staining aldehyde dehydrogenase activity, a cancer stem cell marker. To the best of our knowledge, this is the first assay that enables the direct observation of tumor-initiating cells within a suspended mammosphere.


Mammosphere Metastasis PDMS Cancer stem cells 



Funding for this work was provided by the University of Maryland Research Board (SEED Grant). The authors also acknowledge the University of Maryland Nanocenter Fab Lab facility for microfabrication support. The authors thank Dr. Stuart Martin and Dr. Amy Fulton of the Greenebaum Cancer Center at the University of Maryland for fruitful discussions, and Nathalie Dagenais for technical assistance.

Supplementary material

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  1. M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez et al., Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci 100, 3983–3988 (2003). doi: 10.1073/pnas.0530291100 CrossRefGoogle Scholar
  2. Y. Arima, H. Iwata, Effect of wettability and surface functional groups on protein adsorption and cell adhesion using well-defined mixed self-assembled monolayers. Biomaterials 28, 3074–3082 (2007). doi: 10.1016/j.biomaterials.2007.03.013 CrossRefGoogle Scholar
  3. A. Bandyopadhyay, L. Wang, J. Agyin et al., Doxorubicin in combination with a small TGFb inhibitor: a potential novel therapy for metastatic breast cancer in mouse models. PLoS One 5, e10365 (2010). doi: 10.1371/journal.pone.0010365 CrossRefGoogle Scholar
  4. G.I. Botchkina, E.S. Zuniga, M. Das et al., New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells. Mol Cancer 9, 192 (2010)CrossRefGoogle Scholar
  5. G. Dontu, W.M. Abdallah, J.M. Foley et al., In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17, 1253–1270 (2003). doi: 10.1101/gad.1061803 CrossRefGoogle Scholar
  6. K. Engelmann, H. Shen, O.J. Finn, MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1. Cancer Res 68, 2419–2426 (2008)CrossRefGoogle Scholar
  7. D. Fang, T.K. Nguyen, K. Leishear et al., A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65, 9328–9337 (2005). doi: 10.1158/0008-5472.CAN-05-1343 CrossRefGoogle Scholar
  8. H. Fukazawa, S. Mizuno, Y. Uehara, A microplate assay for quantitation of anchorage-independent growth of transformed cells. Anal Biochem 228, 83–90 (1995). doi: 10.1006/abio.1995.1318 CrossRefGoogle Scholar
  9. C. Ginestier, M.H. Hur, E. Charafe-Jauffret et al., ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567 (2007). doi: 10.1016/j.stem.2007.08.014 CrossRefGoogle Scholar
  10. M.J. Grimshaw, L. Cooper, K. Papazisis et al., Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res 10, R52 (2008). doi: 10.1186/bcr2106 CrossRefGoogle Scholar
  11. P.B. Gupta, T.T. Onder, G. Jiang et al., Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138, 645–659 (2009). doi: 10.1016/j.cell.2009.06.034 CrossRefGoogle Scholar
  12. H.D. Hemmati, I. Nakano, J. Lazareff et al., Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci 100, 15178–15183 (2003). doi: 10.1073/pnas.2036535100 CrossRefGoogle Scholar
  13. A.Y. Hsiao, Y. Torisawa, Y.-C. Tung et al., Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids. Biomaterials 30, 3020–3027 (2009). doi: 10.1016/j.biomaterials.2009.02.047 CrossRefGoogle Scholar
  14. P.J. Hung, P.J. Lee, P. Sabounchi et al., Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays. Biotechnol Bioeng 89, 1–8 (2005). doi: 10.1002/bit.20289 CrossRefGoogle Scholar
  15. E. Kang, Y.Y. Choi, Y. Jun et al., Development of a multi-layer microfluidic array chip to culture and replate uniform-sized embryoid bodies without manual cell retrieval. Lab on a Chip 10, 2651–2654 (2010). doi: 10.1039/c0lc00005a CrossRefGoogle Scholar
  16. C.F.B. Kim, E.L. Jackson, A.E. Woolfenden et al., Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121, 823–835 (2005). doi: 10.1016/j.cell.2005.03.032 CrossRefGoogle Scholar
  17. C. Kim, K.S. Lee, J.H. Bang et al., 3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body. Lab on a Chip 11, 874–882 (2011). doi: 10.1039/c0lc00516a CrossRefGoogle Scholar
  18. A.H. Klopp, L. Lacerda, A. Gupta et al., Mesenchymal stem cells promote mammosphere formation and decrease E-cadherin in normal and malignant breast cells. PLoS One 5, e12180 (2010). doi: 10.1371/journal.pone.0012180 CrossRefGoogle Scholar
  19. M. Kok, R.H. Koornstra, T.C. Margarido et al., Mammosphere-derived gene set predicts outcome in patients with ER-positive breast cancer. J Pathol 218, 316–326 (2009)CrossRefGoogle Scholar
  20. C.-T. Kuo, C.-L. Chiang, R. Yun-Ju Huang et al., Configurable 2D and 3D spheroid tissue cultures on bioengineered surfaces with acquisition of epithelial–mesenchymal transition characteristics. NPG Asia Mater 4, e27 (2012). doi: 10.1038/am.2012.50 CrossRefGoogle Scholar
  21. D.A. Lawson, L. Xin, R.U. Lukacs et al., Isolation and functional characterization of murine prostate stem cells. Proc Natl Acad Sci 104, 181–186 (2007). doi: 10.1073/pnas.0609684104 CrossRefGoogle Scholar
  22. J. Lee, G. Khang, J. Lee, H. Lee, Interaction of different types of cells on polymer surfaces with wettability gradient. J Colloid Interface Sci 205, 323–330 (1998). doi: 10.1006/jcis.1998.5688 CrossRefGoogle Scholar
  23. J.H. Lee, C. Jung, P. Javadian-Elyaderani et al., Pathways of proliferation and antiapoptosis driven in breast cancer stem cells by stem cell protein Piwil2. Cancer Res 70, 4569–4579 (2010)CrossRefGoogle Scholar
  24. K. Lee, C. Kim, J.Y. Yang et al., Gravity-oriented microfluidic device for uniform and massive cell spheroid formation. Biomicrofluidics 6, 014114 (2012). doi: 10.1063/1.3687409 CrossRefGoogle Scholar
  25. C. Li, D.G. Heidt, P. Dalerba et al., Identification of pancreatic cancer stem cells. Cancer Res 67, 1030–1037 (2007a). doi: 10.1158/0008-5472.CAN-06-2030 CrossRefGoogle Scholar
  26. F. Li, B. Tiede, J. Massagué, Y. Kang, Beyond tumorigenesis: cancer stem cells in metastasis. Cell Res 17, 3–14 (2007b). doi: 10.1038/ CrossRefGoogle Scholar
  27. T.A. Mahmood, S. Miot, O. Frank et al., Modulation of chondrocyte phenotype for tissue engineering by designing the biologic-polymer carrier interface. Biomacromolecules 7, 3012–3018 (2006). doi: 10.1021/bm060489+ CrossRefGoogle Scholar
  28. S. Mani, W. Guo, M.-J. Liao et al., The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704–715 (2008). doi: 10.1016/j.cell.2008.03.027 CrossRefGoogle Scholar
  29. R. Pardal, M.F. Clarke, S.J. Morrison, Applying the principles of stem-cell biology to cancer. Nat Rev Cancer 3, 895–902 (2003). doi: 10.1038/nrc1232 CrossRefGoogle Scholar
  30. D. Ponti, A. Costa, N. Zaffaroni et al., Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65, 5506–5511 (2005). doi: 10.1158/0008-5472.CAN-05-0626 CrossRefGoogle Scholar
  31. G.J. Prud’homme, Y. Glinka, A. Toulina et al., Breast cancer stem-like cells are inhibited by a non-toxic aryl hydrocarbon receptor agonist. PLoS One 5, e13831 (2010)CrossRefGoogle Scholar
  32. G. Rappa, J. Mercapide, F. Anzanello et al., Growth of cancer cell lines under stem cell-like conditions has the potential to unveil therapeutic targets. Exp Cell Res 314, 2110–2122 (2008). doi: 10.1016/j.yexcr.2008.03.008 CrossRefGoogle Scholar
  33. T. Reya, S.J. Morrison, M.F. Clarke, I.L. Weissman, Stem cells, cancer, and cancer stem cells. Nature 414, 105–111 (2001). doi: 10.1038/35102167 CrossRefGoogle Scholar
  34. B. Reynolds, S. Weiss, Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 175, 1–13 (1996). doi: 10.1006/dbio.1996.0090 CrossRefGoogle Scholar
  35. P. Sansone, G. Storci, S. Tavolari et al., IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland. J Clin Investig 117, 3988–4002 (2007)CrossRefGoogle Scholar
  36. M. Shackleton, E. Quintana, E.R. Fearon, S.J. Morrison, Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell 138, 822–829 (2009). doi: 10.1016/j.cell.2009.08.017 CrossRefGoogle Scholar
  37. S.K. Singh, I.D. Clarke, M. Terasaki et al., Identification of a cancer stem cell in human brain tumors. Cancer Res 63, 5821–5828 (2003)Google Scholar
  38. M. Smalley, A. Ashworth, Stem cells and breast cancer: a field in transit. Nat Rev Cancer 3, 832–844 (2003). doi: 10.1038/nrc1212 CrossRefGoogle Scholar
  39. J. Stingl, C. Caldas, Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer 7, 791–799 (2007). doi: 10.1038/nrc2212 CrossRefGoogle Scholar
  40. A. Vescovi, B. Reynolds, D. Fraser, S. Weiss, bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells. Neuron 11, 951 (1993)CrossRefGoogle Scholar
  41. H.-Y. Wang, N. Bao, C. Lu, A microfluidic cell array with individually addressable culture chambers. Biosens Bioelectron 24, 613–617 (2008). doi: 10.1016/j.bios.2008.06.005 CrossRefGoogle Scholar
  42. Y.-Y. Wang, L.-X. Lü, J.-C. Shi et al., Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility. Biomacromolecules 12, 551–559 (2011). doi: 10.1021/bm100886w CrossRefGoogle Scholar
  43. K. Webb, V. Hlady, P. Tresco, Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization. J Biomed Mater Res 41, 422–430 (1998)CrossRefGoogle Scholar
  44. M.-H. Wu, S.-B. Huang, G.-B. Lee, Microfluidic cell culture systems for drug research. Lab on a Chip 10, 939–956 (2010). doi: 10.1039/b921695b CrossRefGoogle Scholar
  45. L. Xin, D.A. Lawson, O.N. Witte, The Sca-1 cell surface marker enriches for a prostate-regenerating cell subpopulation that can initiate prostate tumorigenesis. Proc. Natl. Acad. Sci. 6942–6947 (2005) doi: 10.1073/pnas.0502320102

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Katayoon Saadin
    • 1
  • Jeffrey M. Burke
    • 2
  • Neerav P. Patel
    • 2
  • Rebecca E. Zubajlo
    • 2
  • Ian M. White
    • 1
    • 2
    Email author
  1. 1.Chemical Physics ProgramUniversity of MarylandCollege ParkUSA
  2. 2.Fischell Department of BioengineeringUniversity of MarylandCollege ParkUSA

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