In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to...

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Autores principales:	Chanel E Smart, Brian J Morrison, Jodi M Saunus, Ana Cristina Vargas, Patricia Keith, Lynne Reid, Leesa Wockner, Marjan Askarian-Amiri, Debina Sarkar, Peter T Simpson, Catherine Clarke, Chris W Schmidt, Brent A Reynolds, Sunil R Lakhani, J Alejandro Lopez
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/eeecb8106f5545398056fc442c0bcdb0
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spelling	oai:doaj.org-article:eeecb8106f5545398056fc442c0bcdb02021-11-18T07:43:08ZIn vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.1932-620310.1371/journal.pone.0064388https://doaj.org/article/eeecb8106f5545398056fc442c0bcdb02013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23750209/?tool=EBIhttps://doaj.org/toc/1932-6203Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.Chanel E SmartBrian J MorrisonJodi M SaunusAna Cristina VargasPatricia KeithLynne ReidLeesa WocknerMarjan Askarian-AmiriDebina SarkarPeter T SimpsonCatherine ClarkeChris W SchmidtBrent A ReynoldsSunil R LakhaniJ Alejandro LopezPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 6, p e64388 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Chanel E Smart Brian J Morrison Jodi M Saunus Ana Cristina Vargas Patricia Keith Lynne Reid Leesa Wockner Marjan Askarian-Amiri Debina Sarkar Peter T Simpson Catherine Clarke Chris W Schmidt Brent A Reynolds Sunil R Lakhani J Alejandro Lopez In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.
description	Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1(+)) and basal/myoepithelial (CD10(+)) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally 'enriching' for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity.
format	article
author	Chanel E Smart Brian J Morrison Jodi M Saunus Ana Cristina Vargas Patricia Keith Lynne Reid Leesa Wockner Marjan Askarian-Amiri Debina Sarkar Peter T Simpson Catherine Clarke Chris W Schmidt Brent A Reynolds Sunil R Lakhani J Alejandro Lopez
author_facet	Chanel E Smart Brian J Morrison Jodi M Saunus Ana Cristina Vargas Patricia Keith Lynne Reid Leesa Wockner Marjan Askarian-Amiri Debina Sarkar Peter T Simpson Catherine Clarke Chris W Schmidt Brent A Reynolds Sunil R Lakhani J Alejandro Lopez
author_sort	Chanel E Smart
title	In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.
title_short	In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.
title_full	In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.
title_fullStr	In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.
title_full_unstemmed	In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.
title_sort	in vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/eeecb8106f5545398056fc442c0bcdb0
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In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity.

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