Phenotypic and microRNA characterization of the neglected CD24+ cell population in MCF-7 breast cancer 3-dimensional spheroid culture.

BACKGROUND: In vitro 3-dimensional (3D) spheroid culture has been widely used as model to enrich CD44CD24 cancer stem cells (CSC) with high aldehyde dehydrogenase 1 (ALDH1) activity. Although CD24 subpopulation was known to be present in 3D spheroids and may influence cancer drug therapies, its characteristics and CSC properties were not well defined. METHODS: In this study, CD24 population from the Michigan Cancer Foundation-7 (MCF-7) spheroid was sorted and subjected to spheroid formation test, stem cell markers immunofluorescence, invasion and migration test, as well as microRNA expression profiling. RESULTS: Sorted MCF-7 CD24 cells from primary spheroids were able to reform its 3D spheroid shape after 7 days in nonadherent culture conditions. In contrast to the primary spheroids, the expression of SOX-2, CD44, CD49f, and Nanog was dim in MCF-7 CD24 cells. Remarkably, MCF-7 CD24 cells were found to show high expression of ALDH1 protein which may have resulted in these cells exhibiting higher resistance against doxorubicin and cisplatin when compared with that of the parental cells. Moreover, microRNA profiling has shown that the absence of CSC properties was consistent with the downregulation of major CSCs-related pathways including Hedgehog, wingless-related integration site (Wnt), and microtubule associated protein kinase (MAPK) signaling pathways. However, the upregulated pathways such as adherens junctions, focal adhesion, and tight junction suggest that CD24 cells were probably at an epithelial-like state of cell transition. CONCLUSION: In conclusion, neglected CD24 cells in MCF-7 spheroid did not exhibit typical breast CSCs properties. The presence of miRNAs and their analyzed pathways suggested that these cells could be a distinct intermediate cell state in breast CSCs.

Phenotypic and microRNA transcriptomic profiling of the MDA-MB-231 spheroid-enriched CSCs with comparison of MCF-7 microRNA profiling dataset.

Breast cancer spheroids have been widely used as in vitro models of cancer stem cells (CSCs), yet little is known about their phenotypic characteristics and microRNAs (miRNAs) expression profiles. The objectives of this research were to evaluate the phenotypic characteristics of MDA-MB-231 spheroid-enriched cells for their CSCs properties and also to determine their miRNAs expression profile. Similar to our previously published MCF-7 spheroid, MDA-MB-231 spheroid also showed typical CSCs characteristics namely self-renewability, expression of putative CSCs-related surface markers and enhancement of drug resistance. From the miRNA profile, miR-15b, miR-34a, miR-148a, miR-628 and miR-196b were shown to be involved in CSCs-associated signalling pathways in both models of spheroids, which highlights the involvement of these miRNAs in maintaining the CSCs features. In addition, unique clusters of miRNAs namely miR-205, miR-181a and miR-204 were found in basal-like spheroid whereas miR-125, miR-760, miR-30c and miR-136 were identified in luminal-like spheroid. Our results highlight the roles of miRNAs as well as novel perspectives of the relevant pathways underlying spheroid-enriched CSCs in breast cancer.

MiRNA Transcriptome Profiling of Spheroid-Enriched Cells with Cancer Stem Cell Properties in Human Breast MCF-7 Cell Line.

Breast cancer is the second leading cause of cancer-related mortality worldwide as most patients often suffer cancer relapse. The reason is often attributed to the presence of cancer stem cells (CSCs). Recent studies revealed that dysregulation of microRNA (miRNA) are closely linked to breast cancer recurrence and metastasis. However, no specific study has comprehensively characterised the CSC characteristic and miRNA transcriptome in spheroid-enriched breast cells. This study described the generation of spheroid MCF-7 cell in serum-free condition and the comprehensive characterisation for their CSC properties. Subsequently, miRNA expression differences between the spheroid-enriched CSC cells and their parental cells were evaluated using next generation sequencing (NGS). Our results showed that the MCF-7 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, spheroid-enriched CSCs possessed greater cell proliferation, migration, invasion, and wound healing ability. A total of 134 significantly (p<0.05) differentially expressed miRNAs were identified between spheroids and parental cells using miRNA-NGS. MiRNA-NGS analysis revealed 25 up-regulated and 109 down-regulated miRNAs which includes some miRNAs previously reported in the regulation of breast CSCs. A number of miRNAs (miR-4492, miR-4532, miR-381, miR-4508, miR-4448, miR-1296, and miR-365a) which have not been previously reported in breast cancer were found to show potential association with breast cancer chemoresistance and self-renewal capability. The gene ontology (GO) analysis showed that the predicted genes were enriched in the regulation of metabolic processes, gene expression, DNA binding, and hormone receptor binding. The corresponding pathway analyses inferred from the GO results were closely related to the function of signalling pathway, self-renewability, chemoresistance, tumorigenesis, cytoskeletal proteins, and metastasis in breast cancer. Based on these results, we proposed that certain miRNAs identified in this study could be used as new potential biomarkers for breast cancer stem cell diagnosis and targeted therapy.

Probable impact of age and hypoxia on proliferation and microRNA expression profile of bone marrow-derived human mesenchymal stem cells.

Decline in the therapeutic potential of bone marrow-derived mesenchymal stem cells (MSC) is often seen with older donors as compared to young. Although hypoxia is known as an approach to improve the therapeutic potential of MSC in term of cell proliferation and differentiation capacity, its effects on MSC from aged donors have not been well studied. To evaluate the influence of hypoxia on different age groups, MSC from young (<30 years) and aged (>60 years) donors were expanded under hypoxic (5% O2) and normal (20% O2) culture conditions. MSC from old donors exhibited a reduction in proliferation rate and differentiation potential together with the accumulation of senescence features compared to that of young donors. However, MSC cultured under hypoxic condition showed enhanced self-renewing and proliferation capacity in both age groups as compared to normal condition. Bioinformatic analysis of the gene ontology (GO) and KEGG pathway under hypoxic culture condition identified hypoxia-inducible miRNAs that were found to target transcriptional activity leading to enhanced cell proliferation, migration as well as decrease in growth arrest and apoptosis through the activation of multiple signaling pathways. Overall, differentially expressed miRNA provided additional information to describe the biological changes of young and aged MSCs expansion under hypoxic culture condition at the molecular level. Based on our findings, the therapeutic potential hierarchy of MSC according to donor's age group and culture conditions can be categorized in the following order: young (hypoxia) > young (normoxia) > old aged (hypoxia) > old aged (normoxia).