Skip to main content

Advertisement

SpringerLink
Log in

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

  • Original Article
  • Published:
Pathology & Oncology Research

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

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

References

  1. Allegra A, Alonci A, Penna G, Innao V, Gerace D, Rotondo F, Musolino C (2014) The cancer stem cell hypothesis: a guide to potential molecular targets. Cancer Investig 32:470–495. https://doi.org/10.3109/07357907.2014.958231

    Article  Google Scholar 

  2. Chan RWS, Schwab KE, Gargett CE (2004) Clonogenicity of human endometrial epithelial and stromal cells. Biol Reprod 70:1738–1750. https://doi.org/10.1095/biolreprod.103.024109

    Article  CAS  PubMed  Google Scholar 

  3. Kato K, Yoshimoto M, Kato K, Adachi S, Yamayoshi A, Arima T, Asanoma K, Kyo S, Nakahata T, Wake N (2007) Characterization of side-population cells in human normal endometrium. Hum Reprod 22:1214–1223. https://doi.org/10.1093/humrep/del514

    Article  CAS  PubMed  Google Scholar 

  4. Chan RWS, Gargett CE (2006) Identification of label-retaining cells in mouse endometrium. Stem Cells (Dayton, Ohio) 24:1529–1538. https://doi.org/10.1634/stemcells.2005-0411

    Article  CAS  Google Scholar 

  5. Carvalho MJ, Laranjo M, Abrantes AM, Torgal I, Botelho MF, Oliveira CF (2015) Clinical translation for endometrial cancer stem cells hypothesis. Cancer Metastasis Rev 34:401–416. https://doi.org/10.1007/s10555-015-9574-0

    Article  CAS  PubMed  Google Scholar 

  6. Hubbard SA, Friel AM, Kumar B et al (2009) Evidence for cancer stem cells in human endometrial carcinoma. Cancer Res 69:8241–8248. https://doi.org/10.1158/0008-5472.CAN-08-4808

    Article  CAS  PubMed  Google Scholar 

  7. Kato K, Takao T, Kuboyama A, Tanaka Y, Ohgami T, Yamaguchi S, Adachi S, Yoneda T, Ueoka Y, Kato K, Hayashi S, Asanoma K, Wake N (2010) Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage. Am J Pathol 176:381–392. https://doi.org/10.2353/ajpath.2010.090056

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kusunoki S, Kato K, Tabu K, Inagaki T, Okabe H, Kaneda H, Suga S, Terao Y, Taga T, Takeda S (2013) The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells. Gynecol Oncol 129:598–605. https://doi.org/10.1016/j.ygyno.2013.03.005

    Article  CAS  PubMed  Google Scholar 

  9. Götte M, Greve B, Kelsch R et al (2011) The adult stem cell marker Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via Notch-1 and p21 WAF1/CIP1. Int J Cancer 129:2042–2049. https://doi.org/10.1002/ijc.25856

    Article  CAS  PubMed  Google Scholar 

  10. Zhou X, Zhou Y-P, Huang G-R et al (2011) Expression of the stem cell marker, Nanog, in human endometrial adenocarcinoma. Int J Gynecol Pathol 33:262–270. https://doi.org/10.1097/PGP.0b013e3182055a1f

    Article  Google Scholar 

  11. Rutella S, Bonanno G, Procoli A et al (2009) Cells with characteristics of cancer stem/progenitor cells express the CD133 antigen in human endometrial tumors. Clin Cancer Res 15:4299–4311. https://doi.org/10.1158/1078-0432.CCR-08-1883

    Article  CAS  PubMed  Google Scholar 

  12. Friel AM, Zhang L, Curley MD et al (2010) Epigenetic regulation of CD133 and tumorigenicity of CD133 positive and negative endometrial cancer cells. Reprod Biol Endocrinol 8:147. https://doi.org/10.1186/1477-7827-8-147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rahadiani N, Ikeda J, Mamat S, Matsuzaki S, Ueda Y, Umehara R, Tian T, Wang Y, Enomoto T, Kimura T, Aozasa K, Morii E (2011) Expression of aldehyde dehydrogenase 1 (ALDH1) in endometrioid adenocarcinoma and its clinical implications. Cancer Sci 102:903–908. https://doi.org/10.1111/j.1349-7006.2011.01864.x

    Article  CAS  PubMed  Google Scholar 

  14. Tang DG (2012) Understanding cancer stem cell heterogeneity and plasticity. Cell Res 22:457–472. https://doi.org/10.1038/cr.2012.13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17:1253–1270. https://doi.org/10.1101/gad.1061803

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511. https://doi.org/10.1158/0008-5472.CAN-05-0626

    Article  CAS  PubMed  Google Scholar 

  17. Wilson H, Huelsmeyer M, Chun R, Young KM, Friedrichs K, Argyle DJ (2008) Isolation and characterisation of cancer stem cells from canine osteosarcoma. Vet J 175:69–75. https://doi.org/10.1016/j.tvjl.2007.07.025

    Article  CAS  PubMed  Google Scholar 

  18. Franken N a P, Rodermond HM, Stap J et al (2006) Clonogenic assay of cells in vitro. Nat Protoc 1:2315–2319. https://doi.org/10.1038/nprot.2006.339

    Article  CAS  PubMed  Google Scholar 

  19. Santos K, Laranjo M, Abrantes AM, Brito AF, Gonçalves C, Sarmento Ribeiro AB, Botelho MF, Soares MIL, Oliveira ASR, Pinho e Melo TMVD (2014) Targeting triple-negative breast cancer cells with 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles. Eur J Med Chem 79:273–281. https://doi.org/10.1016/j.ejmech.2014.04.008

    Article  CAS  PubMed  Google Scholar 

  20. Abrantes AM, Serra MES, Gonçalves AC, Rio J, Oliveiros B, Laranjo M, Rocha-Gonsalves AM, Sarmento-Ribeiro AB, Botelho MF (2010) Hypoxia-induced redox alterations and their correlation with 99mTc-MIBI and 99mTc-HL-91 uptake in colon cancer cells. Nucl Med Biol 37:125–132. https://doi.org/10.1016/j.nucmedbio.2009.11.001

    Article  CAS  PubMed  Google Scholar 

  21. Carvalho RA, Rodrigues TB, Zhao P et al (2004) A13C isotopomer kinetic analysis of cardiac metabolism: influence of altered cytosolic redox and [Ca2+]o. Am J Phys Heart Circ Phys 287:H889–H895. https://doi.org/10.1152/ajpheart.00976.2003

    Article  CAS  Google Scholar 

  22. Sherry AD, Jeffrey FMH, Malloy CR (2004) Analytical solutions for 13C isotopomer analysis of complex metabolic conditions: substrate oxidation, multiple pyruvate cycles, and gluconeogenesis. Metab Eng 6:12–24. https://doi.org/10.1016/j.ymben.2003.10.007

    Article  CAS  PubMed  Google Scholar 

  23. Visvader JE, Lindeman GJ (2008) Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8:755–768. https://doi.org/10.1038/nrc2499

    Article  CAS  PubMed  Google Scholar 

  24. Nieto-Estévez V, Pignatelli J, Araúzo-Bravo MJ, Hurtado-Chong A, Vicario-Abejón C (2013) A global transcriptome analysis reveals molecular hallmarks of neural stem cell death, survival, and differentiation in response to partial FGF-2 and EGF deprivation. PLoS One 8:e53594. https://doi.org/10.1371/journal.pone.0053594

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Weiswald L-B, Bellet D, Dangles-Marie V (2015) Spherical Cancer models in tumor biology. Neoplasia 17:1–15. https://doi.org/10.1016/j.neo.2014.12.004

    Article  PubMed  PubMed Central  Google Scholar 

  26. Prasetyanti PR, Zimberlin C, De Sousa E, Melo F, Medema JP (2013) Isolation and propagation of colon cancer stem cells. Methods Mol Biol (Clifton, NJ) 1035:247–259. https://doi.org/10.1007/978-1-62703-508-8_21

    Article  CAS  Google Scholar 

  27. Bortolomai I, Canevari S, Facetti I, de Cecco L, Castellano G, Zacchetti A, Alison MR, Miotti S (2010) Tumor initiating cells: development and critical characterization of a model derived from the A431 carcinoma cell line forming spheres in suspension. Cell Cycle 9:1194–1206. https://doi.org/10.4161/cc.9.6.11108

    Article  CAS  PubMed  Google Scholar 

  28. Liu Y, Nenutil R, Appleyard MV, Murray K, Boylan M, Thompson AM, Coates PJ (2014) Lack of correlation of stem cell markers in breast cancer stem cells. Br J Cancer 110:2063–2071. https://doi.org/10.1038/bjc.2014.105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chung L, Tang S, Wu Y et al (2015) Galectin-3 augments tumor initiating property and tumorigenicity of lung cancer through interaction with β-catenin. Oncotarget 6:4936–4952. https://doi.org/10.18632/oncotarget.3210

    Article  Google Scholar 

  30. Kryczek I, Liu S, Roh M, Vatan L, Szeliga W, Wei S, Banerjee M, Mao Y, Kotarski J, Wicha MS, Liu R, Zou W (2012) Expression of aldehyde dehydrogenase and CD133 defines ovarian cancer stem cells. Int J Cancer 130:29–39. https://doi.org/10.1002/ijc.25967

    Article  CAS  PubMed  Google Scholar 

  31. Tirino V, Desiderio V, Paino F, de Rosa A, Papaccio F, la Noce M, Laino L, de Francesco F, Papaccio G (2013) Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J 27:13–24. https://doi.org/10.1096/fj.12-218222

    Article  CAS  PubMed  Google Scholar 

  32. Hartomo T, Van Huyen Pham T, Yamamoto N et al (2014) Involvement of aldehyde dehydrogenase 1A2 in the regulation of cancer stem cell properties in neuroblastoma. Int J Oncol 46:1089–1098. https://doi.org/10.3892/ijo.2014.2801

    Article  CAS  PubMed  Google Scholar 

  33. Rivlin N, Koifman G, Rotter V (2014) P53 orchestrates between Normal differentiation and Cancer. Semin Cancer Biol 32:10–17. https://doi.org/10.1016/j.semcancer.2013.12.006

    Article  CAS  PubMed  Google Scholar 

  34. Cui J, Li P, Liu X et al (2015) Abnormal expression of the notch and Wnt/β-catenin signaling pathways in stem-like ALDHhiCD44+ cells correlates highly with Ki-67 expression in breast cancer. Oncol Lett 9:1600–1606. https://doi.org/10.3892/ol.2015.2942

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Wang Y, van der Zee M, Fodde R, Blok LJ (2010) Wnt/Β-catenin and sex hormone signaling in endometrial homeostasis and cancer. Oncotarget 1:674–684. https://doi.org/10.18632/oncotarget.101007

    Article  PubMed  PubMed Central  Google Scholar 

  36. Gargett CE, Chan RWS, Schwab KE (2008) Hormone and growth factor signaling in endometrial renewal: role of stem/progenitor cells. Mol Cell Endocrinol 288:22–29. https://doi.org/10.1016/j.mce.2008.02.026

    Article  CAS  PubMed  Google Scholar 

  37. Hapangama DK, Kamal a M, Bulmer JN (2014) Estrogen receptor : the guardian of the endometrium. Hum Reprod Update 21:174–193. https://doi.org/10.1093/humupd/dmu053

    Article  CAS  PubMed  Google Scholar 

  38. R a G, Gillies RJ (2004) Why do cancers have high aerobic glycolysis? Nat Rev Cancer 4:891–899. https://doi.org/10.1038/nrc1478

    Article  CAS  Google Scholar 

  39. Morfouace M, Lalier L, Bahut M, Bonnamain V, Naveilhan P, Guette C, Oliver L, Gueguen N, Reynier P, Vallette FM (2012) Comparison of spheroids formed by rat glioma stem cells and neural stem cells reveals differences in glucose metabolism and promising therapeutic applications. J Biol Chem 287:33664–33674. https://doi.org/10.1074/jbc.M111.320028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Palorini R, Votta G, Balestrieri C, Monestiroli A, Olivieri S, Vento R, Chiaradonna F (2014) Energy metabolism characterization of a novel cancer stem cell-like line 3AB-OS. J Cell Biochem 115:368–379. https://doi.org/10.1002/jcb.24671

    Article  CAS  PubMed  Google Scholar 

  41. Jang H, Yang J, Lee E, Cheong J-H (2015) Metabolism in embryonic and cancer stemness. Arch Pharm Res 38:381–388. https://doi.org/10.1007/s12272-015-0558-y

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Gynecology Service, Coimbra Hospital and Universitary Centre, Coimbra, Portugal

    Maria João Carvalho

  2. Biophysics Institute, Faculty of Medicine, University of Coimbra, Coimbra, Portugal

    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 & Filomena Botelho

  3. Institute for Clinical and Biomedical Research (iCBR), area of Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

    Maria João Carvalho, Mafalda Laranjo, Ana Margarida Abrantes, João Casalta-Lopes, Catarina Mamede, João Encarnação, Filomena Botelho & Carlos Oliveira

  4. CNC.IBILI, University of Coimbra, Coimbra, Portugal

    Maria João Carvalho, Mafalda Laranjo, Ana Margarida Abrantes, João Casalta-Lopes & Filomena Botelho

  5. Universitary Clinic of Gynecology, University of Coimbra, Coimbra, Portugal

    Maria João Carvalho

  6. Radiotherapy Service, Coimbra Hospital and Universitary Centre, Coimbra, Portugal

    João Casalta-Lopes

  7. Pathology Service, Coimbra Hospital and Universitary Centre, Coimbra, Portugal

    Rui Oliveira

  8. Flow Cytometry Unit, Coimbra Hospital and Universitary Centre, Coimbra, Portugal

    Artur Paiva

  9. Centre for Functional Ecology, Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal

    Rui de Carvalho

Authors
  1. Maria João Carvalho
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mafalda Laranjo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ana Margarida Abrantes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. João Casalta-Lopes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daniela Sarmento-Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tânia Costa
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Beatriz Serambeque
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Nuno Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Telmo Gonçalves
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Catarina Mamede
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. João Encarnação
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Rui Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Artur Paiva
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Rui de Carvalho
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Filomena Botelho
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Carlos Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria João Carvalho.

Ethics declarations

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Carvalho, M.J., Laranjo, M., Abrantes, A.M. et al. Endometrial Cancer Spheres Show Cancer Stem Cells Phenotype and Preference for Oxidative Metabolism. Pathol. Oncol. Res. 25, 1163–1174 (2019). https://doi.org/10.1007/s12253-018-0535-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12253-018-0535-0

Keywords

Search

Navigation