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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
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.
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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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