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Endometrial Cancer Spheres Show Cancer Stem Cells Phenotype and Preference for Oxidative Metabolism

  • Maria João Carvalho
  • Mafalda Laranjo
  • Ana Margarida Abrantes
  • João Casalta-Lopes
  • Daniela Sarmento-Santos
  • Tânia Costa
  • Beatriz Serambeque
  • Nuno Almeida
  • Telmo Gonçalves
  • Catarina Mamede
  • João Encarnação
  • Rui Oliveira
  • Artur Paiva
  • Rui de Carvalho
  • Filomena Botelho
  • Carlos Oliveira
Original Article

Abstract

This study aimed to characterize endometrial cancer regarding cancer stem cells (CSC) markers, regulatory and differentiation pathways, tumorigenicity and glucose metabolism. Endometrial cancer cell line ECC1 was submitted to sphere forming protocols. The first spheres generation (ES1) was cultured in adherent conditions (G1). This procedure was repeated and was obtained generations of spheres (ES1, ES2 and ES3) and spheres-derived cells in adherent conditions (G1, G2 and G3). Populations were characterized regarding CD133, CD24, CD44, aldehyde dehydrogenase (ALDH), hormonal receptors, HER2, P53 and β-catenin, fluorine-18 fluorodeoxyglucose ([18F]FDG) uptake and metabolism by NMR spectroscopy. An heterotopic model evaluated differential tumor growth. The spheres self-renewal was higher in ES3. The putative CSC markers CD133, CD44 and ALDH expression were higher in spheres. The expression of estrogen receptor (ER)α and P53 decreased in spheres, ERβ and progesterone receptor had no significant changes and β-catenin showed a tendency to increase. There was a higher 18F-FDG uptake in spheres, which also showed a lower lactate production and an oxidative cytosol status. The tumorigenesis in vivo showed an earlier growth of tumours derived from ES3. Endometrial spheres presented self-renewal and differentiation capacity, expressed CSC markers and an undifferentiated phenotype, showing preference for oxidative metabolism.

Keywords

Endometrial neoplasms Neoplastic stem cells Glucose metabolism 

Abbreviations

ALDH

aldehyde dehydrogenase

ATCC

American Type Culture Collection

bFGF

basic fibroblast growth factor

BSA

bovine serum albumin solution

CPM

counts per minute

CSC

cancer stem cells

ECC-1

human endometrioid carcinoma type I cell line

EGF

epidermal growth factor

EMT

epithelial to mesenchymal transition

ER

oestrogen receptors

ES1

first sphere generation

ES2

second sphere generation

ES3

third sphere generation

18F-FDG

fluorine-18 fluorodeoxyglucose

G1

first generation of adherent cells derived from the spheres

G2

second generation of adherent cells derived from the spheres

G3

third generation of adherent cells derived from the spheres

H&E

hematoxylin and eosin

MFI

mean fluorescence intensity

PR

progesterone receptors

RPMI

Rooswell Park Memorial Institute 1640 Medium

TBS-T

Tris-buffered saline Tween-20

[U-13C]

uniformly enriched 13C isotopomer glucose

Notes

Acknowledgements

This study was funded by the Foundation for Science and Technology, Portugal, through individual support to Carvalho MJ (SFRH/SINTD/60068/2009), 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 NMR spectrometer is part of the National NMR Network and was purchased as part of the Portuguese National Programme for Scientific Re-equipment (REDE/1517/RMN/2005), with funds from POCI 2010 (European Fund for Regional Development) and from the Foundation for Science and Technology, Portugal. The authors thank to the Pathology Service of the University Hospital Centre of Coimbra for technical support and David Anthony Tucker for the manuscript review.

Compliance with Ethical Standards

Conflits of Interest

Nothing to declare.

Ethics Approval

The experimental protocol was approved by the Ethics Committee of the Medicine Faculty of Coimbra University (Ref: Of IBB/48/09). All experiments were performed in accordance with guidelines and regulations of the European Union.

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Copyright information

© Arányi Lajos Foundation 2018

Authors and Affiliations

  • Maria João Carvalho
    • 1
    • 2
    • 3
    • 4
    • 5
  • Mafalda Laranjo
    • 2
    • 3
    • 4
  • Ana Margarida Abrantes
    • 2
    • 3
    • 4
  • João Casalta-Lopes
    • 2
    • 3
    • 4
    • 6
  • Daniela Sarmento-Santos
    • 2
  • Tânia Costa
    • 2
  • Beatriz Serambeque
    • 2
  • Nuno Almeida
    • 2
  • Telmo Gonçalves
    • 2
  • Catarina Mamede
    • 2
    • 3
  • João Encarnação
    • 2
    • 3
  • Rui Oliveira
    • 7
  • Artur Paiva
    • 8
  • Rui de Carvalho
    • 9
  • Filomena Botelho
    • 2
    • 3
    • 4
  • Carlos Oliveira
    • 3
  1. 1.Gynecology ServiceCoimbra Hospital and Universitary CentreCoimbraPortugal
  2. 2.Biophysics Institute, Faculty of MedicineUniversity of CoimbraCoimbraPortugal
  3. 3.Institute for Clinical and Biomedical Research (iCBR), area of Environment Genetics and Oncobiology (CIMAGO), Faculty of MedicineUniversity of CoimbraCoimbraPortugal
  4. 4.CNC.IBILIUniversity of CoimbraCoimbraPortugal
  5. 5.Universitary Clinic of GynecologyUniversity of CoimbraCoimbraPortugal
  6. 6.Radiotherapy ServiceCoimbra Hospital and Universitary CentreCoimbraPortugal
  7. 7.Pathology ServiceCoimbra Hospital and Universitary CentreCoimbraPortugal
  8. 8.Flow Cytometry UnitCoimbra Hospital and Universitary CentreCoimbraPortugal
  9. 9.Centre for Functional Ecology, Department of Life Sciences, Faculty of Science and TechnologyUniversity of CoimbraCoimbraPortugal

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