Enrichment of a Population of Mammary Gland Cells that Form Mammospheres and Have In vivo Repopulating Activity

Massachusetts Institute of Technology, Cambridge, Massachusetts, United States
Cancer Research (Impact Factor: 9.33). 10/2007; 67(17):8131-8. DOI: 10.1158/0008-5472.CAN-06-4493
Source: PubMed


The identification of mammary gland stem cells (MGSC) or progenitors is important for the study of normal breast development and tumorigenesis. Based on their immunophenotype, we have isolated a population of mouse mammary gland cells that are capable of forming "mammospheres" in vitro. Importantly, mammospheres are enriched for cells that regenerate an entire mammary gland on implantation into a mammary fat pad. We also undertook cytogenetic analyses of mammosphere-forming cells after prolonged culture, which provided preliminary insight into the genomic stability of these cells. Our identification of new cell surface markers for enriching mammosphere-initiating cells, including endoglin and prion protein, will facilitate the elucidation of the cell biology of MGSC.

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    • "From a mechanistic perspective , the ability of ILK to stimulate Notch signaling through γ-secretase activation might provide a molecular basis to account for the unique ability of IL-6 to facilitate the conversion of non–stem cancer cells to CSCs [45]. To verify this hypothesis, we assessed the effect of ILK inhibition on mammosphere formation in anchorageindependent , serum-free culture conditions, which represents a surrogate measure of CSC expansion [21] [46]. Stable clones of ILK shRNA-expressing MDA-MB-231 and SUM-159 cells exhibited a diminished ability to form mammospheres relative to the respective Figure 4. ILK regulates the localization of the γ-secretase complex at caveolae/lipid rafts. "
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    ABSTRACT: Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6-driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6-driven Notch1 activation by ILK in IL-6-producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7(IL-6) cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase-mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6-induced breast CSCs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Neoplasia (New York, N.Y.) 06/2015; 105(6). DOI:10.1016/j.neo.2015.06.001 · 4.25 Impact Factor
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    • "Dissociated epithelial cells were obtained from wild-type mice and from MMTV-Wnt1 transgenic animals. Such mammosphere cultures provide an assay system for stem cell-initiated sphere growth, independent of previously identified stem cell enrichment markers [12], [13]. Single cell suspensions being assayed for mammosphere formation were labeled with the lipid-soluble vital dye Di-I in order to track cell division. "
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    ABSTRACT: The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.
    PLoS ONE 07/2014; 9(7):e101800. DOI:10.1371/journal.pone.0101800 · 3.23 Impact Factor
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    • "To demonstrate repopulation capacity of cancer stem/progenitor cells, the progenitor cell candidates are usually injected in an immunocompromised mouse in the general vicinity of the target organ in which prior elimination of the target population has taken place (as the progenitor cells are supposed to replace them) [29]. If the progenitor cell candidates (e.g., mammary gland stem cell) possess repopulating potential, then they will be found in the stead of the original target cell population (e.g., mammary gland). "
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    ABSTRACT: Introduction: JEG3 is a choriocarcinoma--and HTR8/SVneo a transformed extravillous trophoblast--cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of "stemness-" associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.
    11/2013; 2013(9):243649. DOI:10.1155/2013/243649
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