Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

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 ALDH^highCD44⁺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 21^st century, where a small population of CD44⁺CD24^-/low breast cancer cells were found to be capable of generating heterogenous tumors in mice^1,2. Subsequently, it was observed that human breast cancer cells with high enzymatic activity of aldehyde dehydrogenase (ALDH^high) 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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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 metastasis^{26,43,44,45,46} and therapy resistance^21,47,48. Therefore.......

Materials

Name	Company	Catalog Number	Comments
7-Aminoactinomycin D (7AAD)	BD	51-68981E	suggested: 0.25 µg/1x106 cells
Acetone	Fisher	A18-1
Aldehyde dehydrogenase (ALDH) substrate	Stemcell Technologies	1700	Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer's instructions for ALDH substrate preparation
Basement membrane extract (BME)	Corning	354234	Sold under the commercial name Matrigel
Cell culture plates: 6 well	Corning	877218
Cell culture plates: 60mm	Corning	353002
Cell culture plates: 96-well ultra low attachment	Corning	3474
Cell strainer: 40 micron	BD	352340
Collagen	Stemcell Technologies	7001	Prepare 1:30 dilution of 3 mg/mL collagen in PBS
Collagenase	Sigma	11088807001	1x
Conical tubes: 50 mL	Fisher scientific	05-539-7
Crystal violet	Sigma	C6158	Use 0.05% crystal violet solution in water for staining
Dispase	Stemcell Technologies	7913	5U/mL
DMEM:F12	Gibco	11330-032	1x, With L-glutamine and 15 mM HEPES
DNAse	Sigma	D5052	0.1 mg/mL final concentration
FBS	Avantor Seradigm Lifescience	97068-085
Flow tubes: 5ml	BD	352063	Polypropylene round-bottom tubes
Methanol	Fisher	84124
mouse anti-Human CD24 antibody	BD	561646	R-phycoerythrin and Cyanine dye conjugated Clone: ML5
mouse anti-Human CD44 antibody	BD	555479	R-phycoerythrin conjugated, Clone: G44-26
N,N-diethylaminobenzaldehyde (DEAB)	Stemcell Technologies	1700	Sold commerically as part of the ALDEFLOUR Assay kit; follow manufacturer's instructions DEAB preparation
PBS	Wisent Inc	311-425-CL	1x, Without calcium and magnesium
Trypsin-EDTA	Gibco	25200-056
Mammosphere Media Composition
B27	Gibco	17504-44	1x
bFGF	Sigma	F2006	10 ng/mL
BSA	Bioshop	ALB003	04%
DMEM:F12	Gibco	11330-032	1x, With L-glutamine and 15 mM HEPES
EGF	Sigma	E9644	20 ng/mL
Insulin	Sigma	16634	5 µg/mL
3D Organoid Media Composition
A8301	Tocris	2939	500 nM
B27	Gibco	17504-44	1x
DMEM:F12	Gibco	11330-032	1x, With L-glutamine and 15 mM HEPES
EGF	Sigma	E9644	5 ng/mL
FGF10	Peprotech	100-26	20 ng/mL
FGF7	Peprotech	100-19	5 ng/mL
GlutaMax	Invitrogen	35050-061	1x
HEPES	Gibco	15630-080	10 mM
N-acetylcysteine	Sigma	A9165	1.25 mM
Neuregulin β1	Peprotech	100-03	5 nM
Nicotinamide	Sigma	N0636	5 mM
Noggin	Peprotech	120-10C	100 ng/mL
R-spondin3	R&D	3500	250 ng/mL
SB202190	Sigma	S7067	500 nM
Y-27632	Tocris	1254	5 µM