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

ABSTRACT: 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.

SUBMITTER: Smart CE

PROVIDER: S-EPMC3672101 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Smart Chanel E CE Morrison Brian J BJ Saunus Jodi M JM Vargas Ana Cristina AC Keith Patricia P Reid Lynne L Wockner Leesa L Askarian-Amiri Marjan M Sarkar Debina D Simpson Peter T PT Clarke Catherine C Schmidt Chris W CW Reynolds Brent A BA Lakhani Sunil R SR Lopez J Alejandro JA

PloS one 20130604 6

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 immunohistochem ...[more]

PMID: 23750209

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Project description:BackgroundThe breast cancer genome dynamically evolves during malignant progression and recurrence. We investigated the genomic profiles of primary early-stage breast cancers and matched relapses to elucidate the molecular underpinnings of the metastatic process, focusing on potentially actionable alterations in the recurrences.MethodsA mono-institutional cohort of 128 patients with breast cancers (n = 68 luminal B HER2, n = 6 luminal B HER2+, n = 1 HER2+ non-luminal, n = 56 triple negative) and at least one recurrence in a timeframe of 17 years was evaluated. Next-generation sequencing comprehensive genomic profiling was performed on 289 formalin-fixed paraffin-embedded (FFPE) samples, including primary tumors and matched relapses. Correlations of genomic aberrations with clinicopathologic factors and time to breast cancer relapse were analyzed.ResultsGenomic data were available for 188 of 289 FFPE samples that achieved the sequencing quality parameters (failure rate 34.9%), including 106 primary tumors and 82 relapses. All primary and relapse samples harbored at least one genomic alteration, with a median number of six alterations per sample (range 1-16). The most frequent somatic genomic alterations were mutations of TP53 (primary tumors = 49%, relapses = 49%) and PIK3CA (primary tumors = 33%, relapses = 30%). Distinctive genomic alterations of primary tumors were significantly associated with molecular subtypes. TP53, PIK3R1, and NF1 somatic alterations were more frequently detected in triple negative tumors (p value < 0.05); CCND1, FGF3, and FGFR1 copy number gains were recurrently identified in luminal cases (p value < 0.05). Moreover, TP53 mutations and MYC amplification were significantly and independently associated with a shorter time to relapse (p value < 0.05). Molecular subtype changes between primary tumors and relapses were seen in 10 of 128 (7.8%) cases. Most driver genomic alterations (55.8%) were shared between primary tumors and matched recurrences. However, in 39 of 61 cases (63.9%), additional private alterations were detected in the relapse samples only, including 12 patients with potentially actionable aberrations.ConclusionsSpecific genomic aberrations of primary breast cancers were associated with time to relapse. Primary tumors and matched recurrences showed a core of shared driver genomic aberrations but private actionable alterations have been identified in the relapses.

Project description:Tumorigenic breast cancer cells characterized by CD44 expression and low or undetectable CD24 levels (CD44+/CD24-/low) may be resistant to chemotherapy and therefore responsible for cancer relapse. Paired breast cancer core biopsies before and after neoadjuvant chemotherapy or lapatinib were obtained and as single cell suspensions stained using antibodies against CD24, CD44, and lineage markers, and then analyzed by flow cytometry. Mammosphere (MS) formation in culture was compared before and after treatment. Global gene expression differences between cancer cells bearing CD44+/CD24-/low cells and all other sorted cells, and between cancer MS and the primary bulk invasive cancers were analyzed. We report that CD44+/CD24-/low tumorigenic breast cancer cells were intrinsically chemoresistant - chemotherapy led to increased CD44+/CD24-/low cells, increased self-renewal capacity on MS assays, and enhanced tumorigeneicity in immunocompromised SCID/Beige mice. Conversely, in patients with HER2 overexpressing tumors, the EGFR/HER2 tyrosine kinase inhibitor, lapatinib decreased CD44+/CD24-/low cells, with the majority of these patients after conventional therapy achieving pathologic complete response, a validated surrogate marker for long-term survival. Gene transcription pathways that underlie chemoresistant, MS-forming CD44+/CD24-/low cells involve genes belonging to stem cell self-renewal, Wnt signaling, and early development pathways. Experiment Overall Design: Cells from human breast tumors were grown as mammospheres (MS). Experiment Overall Design: Isolated single cell suspensions from primary breast cancers were plated onto non-adherent (polyhema-coated) plastic, counted with a hematocytometer, and 20,000 cells were then seeded into a 6-well ultra-low attachment plate supplemented with 2mL MEGM, with the addition of 2 mL of freshly unfrozen MEGM every 3-4 days. Gene expression profiles were taken of both MS and primary bulk tumors and compared with each other.

Project description:Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies.

Project description:Aromatase inhibitors (AIs), such as letrozole, are considered as first-line treatment for estrogen receptor-positive breast cancer in postmenopausal women. Despite the successful use of letrozole, resistance to therapy, tumor relapse and metastasis remain principal causes of patient mortality. Although there is no therapy currently available for AI-resistant breast cancer, previous reports have demonstrated that AI resistance is associated with hormone independence, increased growth factor signaling, enhanced cellular motility and epithelial to mesenchymal transition (EMT). This suggests a convergence of EMT and cancer stem cells (CSCs) in endocrine resistance. The present study evaluated the contribution of mammospheres in letrozole-resistant breast cancer by characterizing mammospheres and their potential impact on cellular motility. Ovariectomized immunocompromised female mice were inoculated in the mammary fat pad with either letrozole-resistant MCF-7 cells (LTLT-Ca) or letrozole-sensitive MCF-7 cells (AC-1). Subsequently, intratumoral CSC marker expression was assessed by immunohistochemistry. The results indicated that LTLT-Ca tumors were CD44+/CD24+, while AC-1 tumors presented low CD44/CD24 expression. Since mammosphere formation depends on CSCs, both cell lines were cultured either adherently (2D) or as mammospheres (3D) to assess the CD44/CD24 protein expression profile. When 3D culturing both cell lines, higher expression levels of CD44 and CD24 were observed when compared with their adherent counterparts, with the most robust change observed in the LTLT-Ca cell line. To quantitate the breast cancer stem cell activity, mammosphere formation assays were performed, and the LTLT-Ca cells formed mammospheres at a 3.4-fold higher index compared with AC-1 cells. Additionally, targeted gene expression arrays were conducted to compare the LTLT-Ca 3D and 2D cells, revealing that LTLT-Ca 3D cells displayed decreased expression levels of genes involved in cell adhesion and tumor suppression (e. g., E-cadherin, caveolin 1 and β-catenin). To validate this finding, wound healing assays were performed, and LTLT-Ca mammospheres exhibited a 70% wound closure, whereas AC-1 mammospheres exhibited a 39% wound closure. Collectively, the present findings demonstrated a strong association between AI-resistant mammospheres and an increased propensity for migration, which may be indicative of a poor prognosis.

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Dataset Information

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

Publications

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

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