Culture and Characterization of Mammary Cancer Stem Cells in Mammospheres

Abstract

Mammospheres (MMs) are a model for culturing and maintaining mammary gland stem cells (SCs) or cancer stem cells (CSCs) ex situ. As MMs recapitulate the micro-niche of the mammary gland or a tumor, MMs are a model for studying the properties of SCs or CSCs, and for mapping, isolating, and characterizing the SC/CSC generated lineages. Cancer stem cells share with normal SCs the properties of self-renewal and the capacity to generate all cell types and organ structures of the mammary gland. Analysis of human tumor samples suggests that CSCs are heterogeneous in terms of proliferation and differentiation potential. Mammospheres from CSCs likewise display heterogeneity. This heterogeneity makes analysis of CSC generated MMs challenging. To identify the unique and diverse properties of MM derived CSCs, comparative analysis with MMs obtained from normal SCs is required. Here we present protocols for identifying and enriching cells with SC features from a cancer cell line using the LA7CSCs as a model. A comprehensive and comparative approach for identifying, isolating, and characterizing MMs from SCs and CSCs from human breast is also introduced. In addition, we describe detailed procedures for identifying, isolating, and characterizing mammary gland specific cell types, generated during MM formation.

FGF2 and EGF Are Required for Self-Renewal and Organoid Formation of Canine Normal and Tumor Breast Stem Cells

“…We and others have shown that normal mammary SCs are characterized by a relatively low proliferation rate during MM formation, and therefore when stained with fluorescent dye retain the highest levels of membrane labeling with respect to all cells generated within MMs [Pece et al, ; Tosoni et al, ; Piscitelli et al, ]. In addition, since luminal differentiated and certain progenitor cells have high ALDH activity, the ALDEFLUOR assay was also used to characterize different population of cells generated within MMs during MM formation [Piscitelli et al, ; Honeth et al, ]. The ALDEFLUOR assay also allowed to distinguish MMs from cysts (in vitro generated organoid‐like structures representative of mammary alveolar luminal structures, Booth et al, ) since in suspension cultures both are often indistinguishable in their early phase of formation [Zucchi et al, ].…”

“…The ALDEFLUOR assay also allowed to distinguish MMs from cysts (in vitro generated organoid‐like structures representative of mammary alveolar luminal structures, Booth et al, ) since in suspension cultures both are often indistinguishable in their early phase of formation [Zucchi et al, ]. Experiments performed by combining Maroon dye labeling and the ALDEFLUOR assay suggest that cyst cells have a more homogeneous proliferation rate and lower membrane dye dilution during their formation/expansion in suspension cultures and higher levels of ALDH activity, compared to MM cells [Piscitelli et al, ].…”

“…Since we previously determined that sera (such as bovine serum) contain undefined factors that by inducing differentiation of SCs and cell adherence to the tissue plate surface may be incompatible with SC self‐renewal and MM regeneration [Piscitelli et al, ] for all the conditions tested here therefore serum replacement (SR) which is composed of defined factors that normally do not induce SC differentiation was used. As summarized in Table , condition C1 represents medium in which EGF and FGF2 are not included.…”

“…C3 is the medium in which FGF2 is introduced. C3 is a medium that from our previous work was identified as optimal for maintaining human mammary SC/CSC self‐renewal and MM regeneration [Piscitelli et al, ]. C4 medium has an increased concentration of FGF2 without change of EGF, to determine the effects of increased level of FGF2.…”

“…Since SCs and CSCs have the unique in vitro capacities to survive and proliferate in an anchorage‐independent manner and generate mammospheres (MMs) in suspension culture conditions [Dontu et al, ; Medina et al, ], we and others have utilized the property of MM formation to culture SCs/CSCs ex situ. Additionally, as SCs are characterized by a low rate of cell proliferation ex situ, SCs can be enriched during MM formation by their capacity to retain higher levels of a membrane‐labeling dye compared to the progeny cells generated from the SCs [Dontu et al, ; Zucchi et al, , ; Pece et al, ; Tosoni et al, ; Piscitelli et al, ; Koshkin et al, ].…”