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In This Article

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

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.......

Representative Results

The sphere-forming protocol allows spherical colonies to be obtained in suspension from several endometrial and breast cancer cell lines (Figure 2A) or after gentle enzymatic digestion of tissue from human tumor samples (Figure 2E). In both cases, a few days after plating, monoclonal spherical colonies in suspension are obtained. Both endometrial and breast cancer spheres give rise to a cell monolayer with similar morphology to .......

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.......

Acknowledgements

This study was funded by the Portuguese Society of Gynecology through the 2016 Research Prize and by CIMAGO. CNC.IBILI is supported through the Foundation for Science and Technology, Portugal (UID/NEU/04539/2013), and co-funded by FEDER-COMPETE (POCI-01-0145-FEDER-007440). The Coimbra Hospital and Universitary Center (CHUC) Tumor Bank, approved by CHUC's Ethics Committee for Health and by the Portuguese National Data Protection Commission, was the source of endometrial samples of patients followed at the institution's Gynecology Service. Figure 1 was produced using Servier Medical Art, available from www.servier.com

Materials

NameCompanyCatalog NumberComments
Absolute ethanolMerck Millipore100983
AccutaseGibcoA1110501StemPro Accutas Cell Dissociation Reagent
ALDH antibodySanta Cruz BiotechnologySC166362
Annexin V FITCBD Biosciences556547
Antibiotic antimycotic solutionSigmaA5955
BCA assayThermo Scientific23225Pierce BCA Protein Assay Kit
Bovine serum albuminSigmaA9418
CD133 antibodyMiteny Biotec293C3-APCAllophycocyanin (APC)
CD24 antibodyBD Biosciences658331Allophycocyanin-H7 (APC-H7)
CD44 antibodyBiolegend103020Pacific Blue (PB)
Cell strainerBD Falcon35234040 µM
Collagenase, type IVGibco17104-019
cOmplete MiniRoche118 361 700 0
DithiothreitolSigma43815
DMEM-F12SigmaD8900
DNAse IRoche11284932001
ECC-1ATCCCRL-2923Human endometrium adenocarcinoma cell line
Epidermal growth factorSigmaE9644
Fibroblast growth factor basicSigmaF0291
HaemocytometerVWRHERE1080339
HCC1806ATCCCRL-2335Human mammary squamous cell carcinoma cell line
Insulin, transferrin, selenium SolutionGibco41400045
MCF7ATCCHTB-22Human mammary adenocarcinoma cell line
MethylcelluloseAlfaAesar45490
NaClJMGS37040005002212
Poly(2-hydroxyethyl-methacrylateSigmaP3932
PutrescineSigmaP7505
RL95-2ATCCCRL-1671Human endometrium carcinoma cell line
Sodium deoxycholic acidJMSEINECS 206-132-7
Sodium dodecyl sulfateSigma436143
TrisJMGS20360000BP152112
Triton-X 100Merck108603
Trypan blueSigmaT8154
Trypsin-EDTASigmaT4049
��-actin antibodySigmaA5316

References

  1. Hardin, H., Zhang, R., Helein, H., Buehler, D., Guo, Z., Lloyd, R. V. The evolving concept of cancer stem-like cells in thyroid cancer and other solid tumors. Laboratory Investigation. 97 (10), 1142 (2017).
  2. Plaks, V., Kong, N., Werb, Z.

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