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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Abstract

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

Introduction

Understanding the cellular and molecular mechanisms of human breast cancer stem cells (BCSCs) is crucial for addressing the challenges encountered in breast cancer treatment. The emergence of the BCSC concept dates back to the early 21st century, where a small population of CD44+CD24-/low breast cancer cells were found to be capable of generating heterogenous tumors in mice1,2. Subsequently, it was observed that human breast cancer cells with high enzymatic activity of aldehyde dehydrogenase (ALDHhigh) also displayed similar stem cell-like properties

Protocol

Collection of patient-derived surgical or biopsy samples directly from consenting breast cancer patients were carried out under approved human ethics protocol approved by the institutional ethic board. All mice used to generate patient-derived xenograft models were maintained and housed in an institution approved animal facility. The tumor tissue from patient-derived xenograft models using mice were generated as per approved ethics protocol approved by the institutional animal care committee.

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Representative Results

The described protocol allows isolation of human BCSCs from a heterogenous population of breast cancer cells, either from cell lines or from dissociated tumor tissue. For any given cell line or tissue sample, it is crucial to generate a uniform single cell suspension to isolate BCSCs at maximum purity as contaminating non-BCSC populations could result in variable cellular responses, especially if the study aim is to evaluate the efficacy of therapeutic agents targeting BCSCs. Application of a stringent sorting strategy w.......

Discussion

Breast cancer metastasis and resistance to therapy have become major cause of mortality in women worldwide. The existence of a sub-population of breast cancer stem cells (BCSCs) contributes to enhanced metastasis26,43,44,45,46 and therapy resistance21,47,48. Therefore.......

Acknowledgements

We thank members of our laboratory for their helpful discussions and support. Our research on breast cancer stem cells and the tumor microenvironment is funded by grants from the Canadian Cancer Research Society Research Institute and the U.S. Army Department of Defense Breast Cancer Program (Grant # BC160912). V.B. is supported by a Western Postdoctoral Fellowship (Western University), and both A.L.A. and V.B. are supported by the Breast Cancer Society of Canada. C.L. is supported by a Vanier Canada Graduate Scholarship from the Government of Canada.

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Materials

NameCompanyCatalog NumberComments
7-Aminoactinomycin D (7AAD)BD51-68981Esuggested: 0.25 µg/1x106 cells
AcetoneFisherA18-1
Aldehyde dehydrogenase (ALDH) substrateStemcell Technologies1700Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer's instructions for ALDH substrate preparation
Basement membrane extract (BME)Corning354234Sold under the commercial name Matrigel
Cell culture plates: 6 wellCorning877218
Cell culture plates: 60mmCorning353002
Cell culture plates: 96-well ultra low attachmentCorning3474
Cell strainer: 40 micronBD352340
CollagenStemcell Technologies7001Prepare 1:30 dilution of 3 mg/mL collagen in PBS
CollagenaseSigma110888070011x
Conical tubes: 50 mLFisher scientific05-539-7
Crystal violetSigmaC6158Use 0.05% crystal violet solution in water for staining
DispaseStemcell Technologies79135U/mL
DMEM:F12Gibco11330-0321x, With L-glutamine and 15 mM HEPES
DNAseSigmaD50520.1 mg/mL final concentration
FBSAvantor Seradigm Lifescience97068-085 
Flow tubes: 5mlBD352063Polypropylene round-bottom tubes
MethanolFisher84124
mouse anti-Human CD24 antibodyBD561646R-phycoerythrin and Cyanine dye conjugated Clone: ML5
mouse anti-Human CD44 antibodyBD555479R-phycoerythrin conjugated, Clone: G44-26
N,N-diethylaminobenzaldehyde (DEAB)Stemcell Technologies1700Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer's instructions DEAB preparation
PBSWisent Inc311-425-CL1x, Without calcium and magnesium
Trypsin-EDTAGibco25200-056
Mammosphere Media Composition
B27Gibco17504-441x
bFGFSigmaF200610 ng/mL
BSABioshopALB00304%
DMEM:F12Gibco11330-0321x, With L-glutamine and 15 mM HEPES
EGFSigmaE964420 ng/mL
InsulinSigma166345 µg/mL
3D Organoid Media Composition
A8301Tocris2939500 nM
B27Gibco17504-441x
DMEM:F12Gibco11330-0321x, With L-glutamine and 15 mM HEPES
EGFSigmaE96445 ng/mL
FGF10Peprotech100-2620 ng/mL
FGF7Peprotech100-195 ng/mL
GlutaMaxInvitrogen35050-0611x
HEPESGibco15630-08010 mM
N-acetylcysteineSigmaA91651.25 mM
Neuregulin β1Peprotech100-035 nM
NicotinamideSigmaN06365 mM
NogginPeprotech120-10C100 ng/mL
R-spondin3R&D3500250 ng/mL
SB202190SigmaS7067500 nM
Y-27632Tocris12545 µM

References

  1. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., Clarke, M. F. Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America. 100 (7), 3983-3988 (2003).
  2. Shipitsin, M., et al.

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Breast Cancer Stem CellsCell IsolationTissue DissociationSingle Cell SuspensionALDH AssayColony Forming AssayFunctional CharacterizationTumorigenicityMetastasis

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