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Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells
INTRODUCTION: We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. METHODS: Au...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3963897/ https://www.ncbi.nlm.nih.gov/pubmed/24663399 http://dx.doi.org/10.1371/journal.pone.0092511 |
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author | Amann, Arno Zwierzina, Marit Gamerith, Gabriele Bitsche, Mario Huber, Julia M. Vogel, Georg F. Blumer, Michael Koeck, Stefan Pechriggl, Elisabeth J. Kelm, Jens M. Hilbe, Wolfgang Zwierzina, Heinz |
author_facet | Amann, Arno Zwierzina, Marit Gamerith, Gabriele Bitsche, Mario Huber, Julia M. Vogel, Georg F. Blumer, Michael Koeck, Stefan Pechriggl, Elisabeth J. Kelm, Jens M. Hilbe, Wolfgang Zwierzina, Heinz |
author_sort | Amann, Arno |
collection | PubMed |
description | INTRODUCTION: We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. METHODS: Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC. RESULTS: Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. CONCLUSION: We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers. |
format | Online Article Text |
id | pubmed-3963897 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39638972014-03-27 Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells Amann, Arno Zwierzina, Marit Gamerith, Gabriele Bitsche, Mario Huber, Julia M. Vogel, Georg F. Blumer, Michael Koeck, Stefan Pechriggl, Elisabeth J. Kelm, Jens M. Hilbe, Wolfgang Zwierzina, Heinz PLoS One Research Article INTRODUCTION: We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. METHODS: Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and α-smooth muscle actin (α-SMA) was investigated by IHC. RESULTS: Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of α-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. CONCLUSION: We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers. Public Library of Science 2014-03-24 /pmc/articles/PMC3963897/ /pubmed/24663399 http://dx.doi.org/10.1371/journal.pone.0092511 Text en © 2014 Amann et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Amann, Arno Zwierzina, Marit Gamerith, Gabriele Bitsche, Mario Huber, Julia M. Vogel, Georg F. Blumer, Michael Koeck, Stefan Pechriggl, Elisabeth J. Kelm, Jens M. Hilbe, Wolfgang Zwierzina, Heinz Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells |
title | Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells |
title_full | Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells |
title_fullStr | Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells |
title_full_unstemmed | Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells |
title_short | Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells |
title_sort | development of an innovative 3d cell culture system to study tumour - stroma interactions in non-small cell lung cancer cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3963897/ https://www.ncbi.nlm.nih.gov/pubmed/24663399 http://dx.doi.org/10.1371/journal.pone.0092511 |
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