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Scientific ReportsVolume 6, 3 June 2016, Article number 27072

A 3D in vitro model to explore the inter-conversion between epithelial and mesenchymal states during EMT and its reversion(Article)(Open Access)

  • aI3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, Porto, 4200-135, Portugal
  • bINEB - Instituto de Engenharia Biomédica, Rua Alfredo Allen, 208, Porto, 4200-135, Portugal
  • cExpression Regulation in Cancer Group, Institute of Molecular Pathology and Immunology, University of Porto, Rua Alfredo Allen, 208, Porto, 4200-135, Portugal
  • dDepartment of Pathology and Oncology, Faculty of Medicine, University of Porto, Al. Prof. Hernâni Monteiro, Porto, 4200-319, Portugal
  • eInstituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, Porto, 4050-313, Portugal

Abstract

Epithelial-to-mesenchymal transitions (EMT) are strongly implicated in cancer dissemination. Intermediate states, arising from inter-conversion between epithelial (E) and mesenchymal (M) states, are characterized by phenotypic heterogeneity combining E and M features and increased plasticity. Hybrid EMT states are highly relevant in metastatic contexts, but have been largely neglected, partially due to the lack of physiologically-relevant 3D platforms to study them. Here we propose a new in vitro model, combining mammary E cells with a bioengineered 3D matrix, to explore phenotypic and functional properties of cells in transition between E and M states. Optimized alginate-based 3D matrices provided adequate 3D microenvironments, where normal epithelial morphogenesis was recapitulated, with formation of acini-like structures, similar to those found in native mammary tissue. TGFβ1-driven EMT in 3D could be successfully promoted, generating M-like cells. TGFβ1 removal resulted in phenotypic switching to an intermediate state (RE cells), a hybrid cell population expressing both E and M markers at gene/protein levels. RE cells exhibited increased proliferative/clonogenic activity, as compared to M cells, being able to form large colonies containing cells with front-back polarity, suggesting a more aggressive phenotype. Our 3D model provides a powerful tool to investigate the role of the microenvironment on metastable EMT stages.

Indexed keywords

EMTREE drug terms:actinalginic acidbiological markercadherinE-cadherin protein, mouseglucuronic acidhexuronic acidKi 67 antigenMki67 protein, mouseoccludinOcln protein, mousetransforming growth factor beta1
EMTREE medical terms:animalcell culture techniquecell linechemistrycytologydrug effectsepithelial mesenchymal transitionepithelium cellfemalegene expressiongeneticsmetabolismmousemulticellular spheroidphenotypeudder
MeSH:ActinsAlginatesAnimalsBiomarkersCadherinsCell Culture TechniquesCell LineEpithelial CellsEpithelial-Mesenchymal TransitionFemaleGene ExpressionGlucuronic AcidHexuronic AcidsKi-67 AntigenMammary Glands, AnimalMiceOccludinPhenotypeSpheroids, CellularTransforming Growth Factor beta1

Chemicals and CAS Registry Numbers:

alginic acid, 28961-37-7, 29894-36-8, 9005-32-7, 9005-38-3; glucuronic acid, 36116-79-7, 576-37-4, 6556-12-3; occludin, 176304-61-3;

Actins; Alginates; alginic acid; Biomarkers; Cadherins; E-cadherin protein, mouse; Glucuronic Acid; Hexuronic Acids; Ki-67 Antigen; Mki67 protein, mouse; Occludin; Ocln protein, mouse; Transforming Growth Factor beta1

Funding details

Funding sponsor Funding number Acronym
Office of Electricity Delivery and Energy ReliabilityPTDC/BBB-ECT/2518/2014,i3S/IPATIMUPOE
007274,UID/BIM/04293,PEst-C/SAU/LA0003/2013,NORTE-07-0162-FEDER-000067
Fundação para a Ciência e a Tecnologia
See opportunities
POCI-01-0145-FEDER-016627
SFRH/BPD/80571/2011,SFRH/BPD/89764/2012,PD/BI/113971/2015

  • 1

    IPATIMUP and INEB integrate the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work was funded by: 1) FCT/MEC through National Funds and, when applicable, co-financed by the FEDER via the PT2020 Partnership Agreement under the 4293 Unit I&D.; 2) FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through the FCT and under the projects "PEst-C/SAU/LA0003/2013" and FCT/MEC through National Funds and co-financed by the FEDER through the PT2020 Partnership Agreement under the project no. 007274 (UID/BIM/04293); 3) NORTE-07-0162-FEDER-000118-Contributos para o refor?o da capacidade do IPATIMUP enquanto actor do sistema regional de inova??o" and NORTE-07-0162-FEDER-000067 - Refor?o e consolida??o da capacidade infraestrutural do IPATIMUP para o sistema regional de inova??o", both supported by Programa Operacional Regional do Norte (ON.2 - O Novo Norte), through FEDER funds under the Quadro de Refer?ncia Estrat?gico Nacional (QREN); 4) FCT Fellowships SFRH/BPD/80571/2011 to SJB and SFRH/BPD/89764/2012 to PO and PD/BI/113971/2015 to SR; 5) Research position (IF, Investigator FCT) funded by FCT and POPH/ESF (EC) to CCB; 6) Projected funded by POCI via FEDER (POCI-01-0145-FEDER-016627) and by FCT via OE (PTDC/BBB-ECT/2518/2014). The authors are grateful to Dra Joana Paredes (i3S/IPATIMUP) for providing the H&E stained normal breast tissue samples.

  • ISSN: 20452322
  • Source Type: Journal
  • Original language: English
  • DOI: 10.1038/srep27072
  • PubMed ID: 27255191
  • Document Type: Article
  • Publisher: Nature Publishing Group

  Barrias, C.C.; I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, Porto, Portugal;
© Copyright 2018 Elsevier B.V., All rights reserved.

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