Skip to main content

Advertisement

SpringerLink
Log in

Molecular characterization of CD44+/CD24−/Ck+/CD45− cells in benign and malignant breast lesions

  • Original Article
  • Published:
Virchows Archiv Aims and scope Submit manuscript

Abstract

Breast cancer epithelial cells with the CD44+/CD24−/low phenotype possess tumor-initiating cells and epithelial-mesenchymal transition (EMT) capacity. Massive parallel sequencing can be an interesting approach to deepen the molecular characterization of these cells. We characterized CD44+/CD24−/cytokeratin(Ck)+/CD45− cells isolated through flow cytometry from 43 biopsy and 6 mastectomy samples harboring different benign and malignant breast lesions. The Ion Torrent Ampliseq Cancer Hotspot panel v2 (CHPv2) was used for the identification of somatic mutations in the DNA extracted from isolated CD44+/CD24−/Ck+/CD45− cells. E-Cadherin and vimentin immunohistochemistry was performed on sections from the corresponding formalin-fixed, paraffin-embedded (FFPE) blocks. The percentage of CD44+/CD24−/Ck+/CD45− cells increased significantly from non-malignant to malignant lesions and in association with a significant increase in the expression of vimentin. Non-malignant lesions harbored only a single-nucleotide polymorphism (SNP). Mutations in the tumor suppressor p53 (TP53), NOTCH homolog 1 (NOTCH1), phosphatase and tensin homolog (PTEN), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) genes were found in isolated CD44+/CD24−/Ck+/CD45− cells from ductal carcinomas in situ (DCIS). Additional mutations in the colony-stimulating factor 1 receptor (CSF1R), ret proto-oncogene (RET), and TP53 genes were also identified in invasive ductal carcinomas (IDCs). The use of massive parallel sequencing technology for this type of application revealed to be extremely effective even when using small amounts of DNA extracted from a low number of cells. Additional studies are now required using larger cohorts to design an appropriate mutational profile for this phenotype.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Abraham BK, Fritz P, McClellan M, Hauptvogel P, Athelogou M, Brauch H (2005) Prevalence of CD44+/CD24−/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis. Clin Cancer Res 11:1154–1159

    CAS  PubMed  Google Scholar 

  2. Ginestier C, Min MH, Charafe-Jauffret E et al (2007) 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Symmans WF, Liu J, Knowles DM, Inghirami G (1995) Breast cancer heterogeneity: evaluation of clonality in primary and metastatic lesions. Hum Pathol 26:210–216

    Article  CAS  PubMed  Google Scholar 

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

    Article  Google Scholar 

  5. Neumeister v, Agarwal S, Bordeaux J, Camp RL, Rimm DL (2010) In situ identification of putative cancer stem cells by multiplexing ALDH1, CD44, and cytokeratin identifies breast cancer patients with poor prognosis. Am J Pathol 176:2131–2138

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Xiao Y, Ye Y, Yearsley K, Jones S, Barsky SH (2008) The lymphovascular embolus of inflammatory breast cancer expresses a stem cell-like phenotype. Am J Pathol 173:561–574

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Croker AK, Goodale D, Chu J, Postenka C, Hedley BD, Hess DA, Allan AL (2009) High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability. J Cell Mol Med 13:2236–2252

    Article  PubMed  Google Scholar 

  8. Da Cruz PA, Marques O, Rosa AM, Faria MDF, Rema A, Lopes C (2014) Co-expression of stem cell markers ALDH1 and CD44 in non-malignant and neoplastic lesions of the breast. Anticancer Res 34:1427–1434

    Google Scholar 

  9. Da Cruz PA, Marques O et al (2016) Characterization of CD44+ ALDH1+ Ki-67− cells in non-malignant and neoplastic lesions of the breast. Anticancer Res 36:4629–4638

    Article  Google Scholar 

  10. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100:3983–3988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ponti D, Costa A et al (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511

    Article  CAS  PubMed  Google Scholar 

  12. Fillmore CM, Kuperwasser C (2008) Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res 10:R25

    Article  PubMed  PubMed Central  Google Scholar 

  13. Sheridan C, Kishimoto H et al (2006) CD44+/CD24-breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res 8:R59

    Article  PubMed  PubMed Central  Google Scholar 

  14. Balic M et al (2006) Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res 12:5615–5621

    Article  CAS  PubMed  Google Scholar 

  15. Theodoropoulos PA, Polioudaki H et al (2010) Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer. Cancer Lett 288:99–106

    Article  CAS  PubMed  Google Scholar 

  16. Wang N, Shi L et al (2012) Detection of circulating tumor cells and tumor stem cells in patients with breast cancer by using flow cytometry. Tumor Biol 33:561–569

    Article  CAS  Google Scholar 

  17. Hernandez L, Wilkerson PM, Lambros MB et al (2012) Genomic and mutational profiling of ductal carcinomas in situ and matched adjacent invasive breast cancers reveals intra-tumour genetic heterogeneity and clonal selection. J Pathol 227:42–52

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Navin N, Kendall J, Troge J et al (2011) Tumour evolution inferred by single-cell sequencing. Nature 472:90–94

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jorge RF (2009) Next-generation sequencing. Breast Cancer Res Treat 11:S12

    Google Scholar 

  20. Liu X, Mody K, de Abreu FB et al (2014) Molecular profiling of appendiceal epithelial tumors using massively parallel sequencing to identify somatic mutations. Clin Chem 60:1004–1011

    Article  CAS  PubMed  Google Scholar 

  21. Amato E, dal Molin M, Mafficini A et al (2014) Targeted next-generation sequencing of cancer genes dissects the molecular profiles of intraductal papillary neoplasms of the pancreas. J Pathol 233:217–227

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cheang MCU, Chia SK, Voduc D et al (2009) Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. J Natl Cancer Inst 101:736–750

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Riethdorf S, Fritsche H 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 13:920–928

    Article  CAS  PubMed  Google Scholar 

  24. Cristofanilli M, Budd GT et al (2004) Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351:781–791

    Article  CAS  PubMed  Google Scholar 

  25. Hayes DF, Cristofanilli M et al (2006) Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res 15:4218–4224

    Article  Google Scholar 

  26. Cohen SJ, Punt CJ et al (2008) Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol 26:3213–3221

    Article  PubMed  Google Scholar 

  27. De Bono JS, Scher et al (2008) Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res 14:6302–6309

    Article  CAS  PubMed  Google Scholar 

  28. Hardt O, Wild S et al (2012) Highly sensitive profiling of CD44+/CD24− breast cancer stem cells by combining global mRNA amplification and next generation sequencing: evidence for a hyperactive PI3K pathway. Cancer Lett 325:165–174

    Article  CAS  PubMed  Google Scholar 

  29. Geens M, Van de Velde H et al (2007) The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients. Hum Reprod 22:733–742

    Article  CAS  PubMed  Google Scholar 

  30. Bane A, Viloria-Petit A, Pinnaduwage D, Mulligan AM, O’Malley FP, Andrulis IL (2013) Clinical–pathologic significance of cancer stem cell marker expression in familial breast cancers. Breast Cancer Res Treat 140:195–205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Makki J, Myint O, Wynn AA, Samsudin AT, John DV (2014) Expression distribution of cancer stem cells, epithelial to mesenchymal transition, and telomerase activity in breast cancer and their association with clinicopathologic characteristics. Clin Med Insights Pathol 8:1–16

    Google Scholar 

  32. Vuoriluoto K, Haugen H, Kiviluoto S et al (2011) Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene 30:1436–1448

    Article  CAS  PubMed  Google Scholar 

  33. Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz T, Rosen JM (2007) WNT/β-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci U S A 104:618–623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Choi Y, Lee HJ, Jang MH et al (2013) Epithelial-mesenchymal transition increases during the progression of in situ to invasive basal-like breast cancer. Hum Pathol 44:2581–2589

    Article  CAS  PubMed  Google Scholar 

  35. Diehn M, Cho RW, Lobo NA et al (2009) Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature 458:780–783

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. de Leng W, Gadellaa-van Hooijdonk CG, Barendregt-Smouter FA, Koudijs MJ et al (2016) Targeted next generation sequencing as a reliable diagnostic assay for the detection of somatic mutations in tumours using minimal DNA amounts from formalin fixed paraffin embedded material. PLoS One 11:e0149405

    Article  PubMed  PubMed Central  Google Scholar 

  37. Chang CJ, Chao CH, Xia W et al (2011) p53 regulates epithelial-mesenchymal transition and stem cell properties through modulating miRNAs. Nat Cell Biol 13:317–323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Farnie G, Clarke RB, Spence K et al (2007) Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. J Natl Cancer Inst 99:616–627

    Article  CAS  PubMed  Google Scholar 

  39. Koh M, Woo Y, Valiathan RR et al (2015) Discoidin domain receptor 1 is a novel transcriptional target of ZEB1 in breast epithelial cells undergoing H-Ras-induced epithelial to mesenchymal transition. Intl J Cancer 6:E508–E520

    Article  Google Scholar 

  40. Vuoriluoto K, Haugen H, Kiviluoto S et al (2011) Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene 12:1436–1448

    Article  Google Scholar 

  41. Zhou J, Wulfkuhle J, Zhang H et al (2007) Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance. Proc Natl Acad Sci 104:16158–16163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zhu QS, Rosenblatt K, Huang KL et al (2011) Vimentin is a novel AKT1 target mediating motility and invasion. Oncogene 30:457–470

    Article  CAS  PubMed  Google Scholar 

  43. Tsongalis GJ, Peterson JD et al (2014) Routine use of the Ion Torrent AmpliSeq™ Cancer Hotspot Panel for identification of clinically actionable somatic mutations. Clinical Chem Lab Med 52:707–714

    Article  CAS  Google Scholar 

  44. Kacinski BM, Scata KA, Carters D et al (1991) FMS (CSF-l receptor) and CSF-l transcripts and protein are expiessed. Oncogene 6:941–952

    CAS  PubMed  Google Scholar 

  45. Sapi E, Flick MB, Rodov S et al (1996) Independent regulation of invasion and anchorage-independent growth by different autophosphorylation sites of the macrophage colony-stimulating factor 1 receptor. Cancer Res 56:5704–5712

    CAS  PubMed  Google Scholar 

  46. Gao J, Aksoy BA, Dresdner G et al (2013) Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal. Sci Signal 269:pl1

    Google Scholar 

  47. Gattelli A, Nalvarte I, Boulay A et al (2013) Ret inhibition decreases growth and metastatic potential of estrogen receptor positive breast cancer cells. EMBO Mol Med 5:1335–1350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kan Z, Jaiswal BS, Stinson J et al (2010) Diverse somatic mutation patterns and pathway alterations in human cancers. Nature 466:869–873

    Article  CAS  PubMed  Google Scholar 

  49. Mimori K, Inoue H, Shiraishi T et al (2002) A single-nucleotide polymorphism of SMARCB1 in human breast cancers. Genomics 80:254–258

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was funded by the Foundation for Science and Technology (Portugal). Scholarships references: SFRH/BD/74307/2010 (ACP) and SFRH/BD/2011/78184 (OM). The authors would like to thank the invaluable help of Manuela Certo and Maria José Oliveira during breast tissue collection.

Author information

Authors and Affiliations

  1. Pathology and Molecular Immunology Department, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Jorge Viterbo Ferreira Street, n° 288, 4050-313, Porto, Portugal

    Arnaud Da Cruz Paula, Oriana Marques, Alexandra Rêma, Maria de Fátima Faria & Carlos Lopes

  2. Department of Pathology, Portuguese Oncology Institute (IPO), Porto, Portugal

    Arnaud Da Cruz Paula & Carlos Lopes

  3. Institute of Research and Health Innovation, University of Porto, Porto, Portugal

    Catarina Leitão, Oriana Marques, Ana Margarida Rosa & José Luís Costa

  4. IBMC – Institute of Molecular and Cell Biology, Porto, Portugal

    Catarina Leitão, Ana Helena Santos & Margarida Lima

  5. Unit for Multidisciplinary Biomedical Research (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal

    Oriana Marques

  6. Basic and Clinical Research on Iron Biology, Institute of Molecular and Cell Biology (IBMC)/i3s, Porto, Portugal

    Oriana Marques & Ana Margarida Rosa

  7. Laboratory of Cytometry, Department of Hematology, Santo António Hospital (HSA), Porto Hospital Centre (CHP), Porto, Portugal

    Ana Helena Santos & Margarida Lima

  8. IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal

    Ana Rocha & José Luís Costa

  9. Medical Faculty of the University of Porto, Porto, Portugal

    Ana Rocha & José Luís Costa

Authors
  1. Arnaud Da Cruz Paula
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Catarina Leitão
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Oriana Marques
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ana Margarida Rosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ana Helena Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alexandra Rêma
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maria de Fátima Faria
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ana Rocha
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. José Luís Costa
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Margarida Lima
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Carlos Lopes
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Arnaud Da Cruz Paula or Carlos Lopes.

Ethics declarations

This project was approved by the following ethical boards: Porto Hospital Centre Research Ethics Health Committee (reference 203-CES) and by Porto Hospital Centre Department of Education, Development and Research (reference 135-DEFI).

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Margarida Lima and Carlos Lopes contributed equally to the supervision of this work.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Da Cruz Paula, A., Leitão, C., Marques, O. et al. Molecular characterization of CD44+/CD24−/Ck+/CD45− cells in benign and malignant breast lesions. Virchows Arch 470, 311–322 (2017). https://doi.org/10.1007/s00428-017-2068-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00428-017-2068-4

Keywords

Search

Navigation