Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines
Summary
Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.
Abstract
Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.
Introduction
The existence of tumor cell lineages headed by stem-like cancer stem cells has greatly added to our understanding of tumor heterogeneity. While some phenotypic diversity in tumors does arise from the clonal outgrowth of genetically distinct clones, a substantial component appears to result from epigenetic differences: cancer cells can transition (sometimes reversibly) between stem, progenitor, and differentiated states via activation or repression of specific gene expression programs1–3. This may reflect cell intrinsic or extrinsic factors, reflecting the gene expression program currently being expressed in a cell with its resultant autocrine signalling in conjunction with paracrine signalling from neighboring cancer, stromal or immune cells delivering modulatory factors, and microenviromental conditions such as the degree of hypoxia2,4,5.
Although innovative lineage tracing approaches are advancing our ability to study putative cancer stem cells in their in vivo niche6–8, sphere-forming assays remain a popular and convenient approach to estimate breast cancer cells’ potential to behave like stem cells, at least under the assay conditions used. It is often used alongside retrospective methods for purifying cancer stem cells, by their expression of membrane markers CD44 and CD249, and activity levels of the enzyme ALDH (aldehyde dehydrogenase)10, markers that have been proposed to correspond to more mesenchymal- and epithelial-like cancer stem cells respectively11. The sphere formation approach was first developed as the neurosphere assay, enabling the growth of putative stem cells from single clones in non-adherent, serum free conditions with the addition of epithelial growth factor (EGF)12, later being usefully applied to normal and cancerous breast tissues.
The identity of the sphere-forming founder cell, and the mixed cell types making up the sphere mass, are relevant for the inferences that can be made from mammo-, or other-sphere forming assays. Long-term quiescent bone fide stem cells, thought to rest in G0 phase, will not experience the precise combination of factors that would favor activation in vivo. The mammosphere assay instead enables the growth of cells either poised for mitotic division or already dividing13. These progenitors, although not a truly quiescent cell, may be the cell stage that proliferates with the EGF and basic fibroblast growth factor (bFGF) mitogens used in the assay. Nevertheless, they contain a range of activated stem cell-associated signalling pathways14. In addition, the rate of their formation relates to the tumorigenicity of the tissue they were taken from, when measured by their potency in limited dilution assays in mouse xenografts2,15,16.
Here we provide detailed protocols to isolate single cells and generate primary mammospheres from both human breast cancer cell lines and clinical samples of breast tumors. We also describe how to perform serial passages of primary mammospheres to assess self-renewal, and how to calculate sphere forming efficiency that allows comparison across different seeding densities (see scheme in Figure 1).
Protocol
Representative Results
Discussion
Successful assessment of primary and secondary mammospheres relies on cells being plated at sufficiently low densities that mammospheres form from single clones, with minimal sphere aggregation. However at densities that are too low, too few mammospheres may form to distinguish the effects of treatments statistically. Seeding density should be optimised for each cell line used since they can vary considerably in their sphere forming efficiencies (those expressing low E-cadherin may form less stable and shorter term mammo…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work is supported by the Imperial BRC, the National Institute for Health Research, and Action Against Cancer with special mention to Hilary Craft and Sir Douglas Myers.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| DMEM/F12 | Lonza | CC-3151 | |
| 2mM L-Glutamine | Sigma Aldrich | G8540 | |
| 100U/ml Penicillin & Streptomycin | Sigma Aldrich | P4083 | |
| 20ng/ml recombinant human epidermal growth factor (EGF) | Sigma Aldrich | E9644 | |
| 20ng/ml recombinant human basic fibroblast growth factor (bFGF) | R&D systems | 233-FB-025 | |
| 1x B27 supplement | Invitrogen | 17504-044 | |
| Phosphate buffered saline (PBS); | Thermo Scientific | 12399902 | |
| 0.5% trypsin-0.2%EDTA; | Sigma Aldrich | 59418C | |
| Fetal Calf Serum | First Link UK | 02-00-850 | |
| Trypan Blue | Sigma Aldrich | 93595 | |
| Low attachment 6 well plates | Corning | CLS3814 | |
| Collagenase type 1A | Sigma Aldrich | C9891 | |
| Hyaluronidase | Sigma Aldrich | H3506 | |
| Sterile razor blades | Fisher Scientific | 12443170 | |
| Sterile scalpel | Fisher Scientific | 11758353 | |
| Sterile micro-dissecting scissors | Sigma Aldrich | S3146 |
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