Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Summary

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Abstract

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44⁺/CD24^- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

Introduction

Cancer populations are heterogeneous, with cells presenting different morphologies, proliferation and invasion capacity, due to differential gene expression. Among these cells, a minority population exists named cancer stem cells (CSC)¹, which have the capacity for self-renewal, recapitulating the heterogeneity of the primary tumor niche and producing aberrantly differentiating progenitors that do not respond adequately to homeostatic controls². CSC properties can be directly translated in clinical practice, given the association with events, such as tumorigenicity or resistance to chemotherapy³. ....

Protocol

This protocol was performed complying with the ethical guidelines of the Coimbra Hospital and Universitary Center (CHUC) Tumor Bank, and was approved by CHUC's Ethics Committee for Health and by the Portuguese National Data Protection Commission.

1. Sphere-forming Protocol and Derived Adherent Populations from Continuous Cell Cultures

NOTE: Perform all procedures under strict sterile conditions.

1. Preparation of non-adherent suspension culture flasks o.......

Discussion

This protocol details an approach to obtain tumorspheres from cancer cell lines and primary human samples. Tumorspheres are enriched in a sub-population with stem cell-like properties³⁶. This enrichment in CSC is dependent on viability in an anchorage-free environment while differentiated cells are reliant on adhesion to a substrate³⁷. As primary plating of tumor cells in a low adherence environment that imposes suspension does not ensure enrichment in CSC per se, we provid.......

Materials

Name	Company	Catalog Number	Comments
Absolute ethanol	Merck Millipore	100983
Accutase	Gibco	A1110501	StemPro Accutas Cell Dissociation Reagent
ALDH antibody	Santa Cruz Biotechnology	SC166362
Annexin V FITC	BD Biosciences	556547
Antibiotic antimycotic solution	Sigma	A5955
BCA assay	Thermo Scientific	23225	Pierce BCA Protein Assay Kit
Bovine serum albumin	Sigma	A9418
CD133 antibody	Miteny Biotec	293C3-APC	Allophycocyanin (APC)
CD24 antibody	BD Biosciences	658331	Allophycocyanin-H7 (APC-H7)
CD44 antibody	Biolegend	103020	Pacific Blue (PB)
Cell strainer	BD Falcon	352340	40 µM
Collagenase, type IV	Gibco	17104-019
cOmplete Mini	Roche	118 361 700 0
Dithiothreitol	Sigma	43815
DMEM-F12	Sigma	D8900
DNAse I	Roche	11284932001
ECC-1	ATCC	CRL-2923	Human endometrium adenocarcinoma cell line
Epidermal growth factor	Sigma	E9644
Fibroblast growth factor basic	Sigma	F0291
Haemocytometer	VWR	HERE1080339
HCC1806	ATCC	CRL-2335	Human mammary squamous cell carcinoma cell line
Insulin, transferrin, selenium Solution	Gibco	41400045
MCF7	ATCC	HTB-22	Human mammary adenocarcinoma cell line
Methylcellulose	AlfaAesar	45490
NaCl	JMGS	37040005002212
Poly(2-hydroxyethyl-methacrylate	Sigma	P3932
Putrescine	Sigma	P7505
RL95-2	ATCC	CRL-1671	Human endometrium carcinoma cell line
Sodium deoxycholic acid	JMS	EINECS 206-132-7
Sodium dodecyl sulfate	Sigma	436143
Tris	JMGS	20360000BP152112
Triton-X 100	Merck	108603
Trypan blue	Sigma	T8154
Trypsin-EDTA	Sigma	T4049
��-actin antibody	Sigma	A5316