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.

The procedures below have been ethically approved by Imperial College, London.
1. Generation of Primary Mammospheres from Human Breast Cancer Cell Lines
NOTE: Perform the following steps under a sterile culture hood.
<ol>
	<li>Prepare Mammosphere Media containing DMEM/F12 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin. Prepare complete media immediately before use by adding 20 ng/ml recombinant human epidermal growth factor (EGF; Sigma), 10 ng/ml recombinant human basic fibroblast growth factor (bFGF; R&#38;D Systems) and 1x B27 supplement.</li>
	<li>Aspirate media from flask containing adherent MCF-7 or MDA-MB-231 cells (or a breast cancer cell line of your choice; 70-80% confluence), wash twice in 1x PBS, and trypsinize cells.</li>
	<li>Centrifuge cells at 200 x g at room temperature for 5 min. Decant supernatant and resuspend cells in 1-5 ml of mammosphere media. Pipette up and down and if necessary use a 40 &#181;m cell strainer cap filter to obtain single-cell suspension. Use a hemocytometer to ensure a single cell suspension has formed (if not, use a 25 G needle to syringe the cell suspension up to 3 times).</li>
	<li>If necessary isolate CD44+CD24- cellular subset via FACS using anti-CD24-phycoerythrin (PE) and anti-CD44-fluorescein isothiocyanate (FITC) monoclonal antibodies9. Alternatively, isolate such population using a magnetic-activated cell sorting (MACS) system with anti-CD44 and anti-CD24-biotin combined microbeads as per manufacturer&#8217;s instructions. Perform positive selection using LS columns, and negative selection using LD columns and confirm the phenotypes of all isolated cells by flow cytometry17.</li>
	<li>Calculate the number of viable cells per ml using trypan blue. 
	NOTE: Cell viability is calculated as the number of viable cells divided by the total number of cells within the grids on the hemocytometer. Cells that take up trypan blue are considered non-viable. The following procedure is used to accurately determine proportion of viable cells.
	<ol>
		<li>Prepare a 0.4% solution of trypan blue in PBS. Add 0.1 ml of trypan blue stock solution to 1 ml of cells. Load a hemocytometer and examine immediately under a microscope at low magnification. Count the number of total cells and the number of blue staining cells. 
		Viable cells = [1.00 &#8211; (Number of blue cells &#247; Number of total cells)] &#215; 100.</li>
		<li>To calculate the number of viable cells per ml of culture, use the formula below. Remember to correct for the dilution factor. 
		Number of viable cells &#215; 104 &#215; 1.1 = cells/ml culture</li>
	</ol>
	</li>
	<li>Resuspend a pre-determined amount of cells in 2 ml of complete mammosphere media in each well of a 6-well ultra-low adherent plate. The seeding density is normally 500-4,000 cells/cm2 cell per well. Optionally use low attachment 24-well plates while seeding cells at the same density in 0.5 ml of media. 
	NOTE: We recommend optimization of seeding density and time of culture for each cell lines of interest.</li>
	<li>Incubate ultra low-attachment 6-well plates at 37 &#176;C and 5% CO2 for 5-10 days (depending on the cell line and the size of mammospheres) without disturbing the plates, particularly during the growth phase of the first 5 days.</li>
</ol>
2. Generation of Primary Mammospheres from Human Breast Cancer Clinical Samples
NOTE: Human breast cancer tissue can be obtained from patients undergoing surgery for the removal of breast tumors. Store tissue on ice for up to 24 hr in 50 ml sterile tubes in culture media containing 100 U/ml penicillin and 100 U/ml streptomycin. Perform the following steps under a sterile culture hood.
<ol>
	<li>Transfer the sample into a 100 mm tissue Petri dish with a small volume of media. Remove the adipose tissue using sterile scissors, scapel and tweezers.</li>
	<li>Add 2-3 ml of DMEM/F12 and mince the sample into 1 mm3 pieces with a sterile scapel or razor blade until no large pieces remain.</li>
	<li>Resuspend the tissue pieces in pre-warmed 10 ml DMEM containing proteolytic enzymes (3,000 U/ml collagenase and 1,000 U/ml hyaluronidase) and incubate at 37 &#176;C in a rotary shaker until all the tissue fragments are digested. Usually complete digestion takes from 1-3 hr. Adjust the digestion time according to pathological characters of tumors. (For example breast adenocarcinoma is usually harder to digest compared to mucinous carcinoma which is more fragile and needs shorter digestion time). Assess the degree of digestion every &#189; hour by observing 20 &#181;l suspension under hemocytometer.</li>
	<li>Allow the fragments to sediment for 5 min, then transfer the supernatant in a 15 ml conical polypropylene tube and centrifuge at 200 x g for 10 min at room temperature. Carefully decant off the supernatant and resuspend cells in 1 - 5 ml of mammosphere media.</li>
	<li>Follow the steps 1.1-1.4 described above for cell lines to obtain and plate single cell suspensions. Pass cells through the 25 G syringe a maximum of 2 times if necessary to limit damaging cells.</li>
</ol>
3. Mammosphere Forming Efficiency (%) Calculation
<ol>
	<li>After the culture period, count the mammospheres (greater than 40 &#181;m) diameter under a microscope at 40X magnification. Digitally image 5 random fields using a digital camera on a light microscope and determine the mammospheres size using the acquisition software. Use any Image Analysis Software.</li>
	<li>Calculate Mammosphere Forming Efficiency (MFE%) using the following equation: 
	MFE (%) = (# of mammospheres per well) / (# of cells seeded per well) x 100</li>
</ol>
4. Serial Passaging of Mammospheres for Assessment of Self-renewal
<ol>
	<li>Pipette the media containing the mammospheres from each well into a 15 ml tube. Wash the wells with PBS and add to the collected media. Centrifuge at 115 x g for 10 min at room temperature.</li>
	<li>Discard supernatant and resuspend the pellet in 500 &#181;l of pre-warmed 0.5% trypsin/0.2% EDTA. Incubate for 2-3 min.</li>
	<li>Add 500 &#181;l of FCS to neutralize trypsin. Centrifuge at 500 x g for 5 min. Discard supernatant and resuspend pellet in 100 &#181;l of mammospheres media, pipetting up and down to disaggregate spheres.</li>
	<li>Count cells with hemocytometer and determine if cells are in single cell suspension. If not pass through a 25 G syringe up to 3 times to obtain single cells. Seed the cells into a new ultra low-attachment 6-well plate at the same density used in the primary generation.</li>
	<li>After 5-10 days, count spheres &#62;40 &#181;m and calculate sphere-forming efficiency using the formula reported before.</li>
</ol>

<ol>
	<li>Iliopoulos, D., Hirsch, H. a., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epigenetic+switch+involving+NF-kappaB,+Lin28,+Let-7+MicroRNA,+and+IL6.">An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6.</a> Cell. 139 (4), 693-706 (2009).</li><li>Iliopoulos, D., Hirsch, H. a., Wang, G., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+formation+of+breast+cancer+stem+cells+and+their+dynamic+equilibrium+with+non-stem+cancer+cells+via+IL6+secretion.">Inducible formation of breast cancer stem cells and their dynamic equilibrium with non-stem cancer cells via IL6 secretion.</a> Proc Natl Acad Sci U S A. 108 (4), 1397-1402 (2011).</li><li>Visvader, J. E., Lindeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+current+status+and+evolving+complexities.">Cancer stem cells: current status and evolving complexities.</a> Cell stem cell. 10 (6), 717-728 (2012).</li><li>Rosen, J. M., Jordan, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+complexity+of+the+cancer+stem+cell+paradigm.">The increasing complexity of the cancer stem cell paradigm.</a> Science. 324 (5935), 1670-1673 (2009).</li><li>Rokavec, M., Wu, W., Luo, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL6-mediated+suppression+of+miR-200c+directs+constitutive+activation+of+inflammatory+signaling+circuit+driving+transformation+and+tumorigenesis.">IL6-mediated suppression of miR-200c directs constitutive activation of inflammatory signaling circuit driving transformation and tumorigenesis.</a> Mol Cell. 45 (6), 777-789 (2012).</li><li>Chen, J., Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+restricted+cell+population+propagates+glioblastoma+growth+after+chemotherapy.">A restricted cell population propagates glioblastoma growth after chemotherapy.</a> Nature. 488 (7412), 522-526 (2012).</li><li>Driessens, G., Beck, B., Caauwe, A., Simons, B. D., Blanpain, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+mode+of+tumour+growth+by+clonal+analysis.">Defining the mode of tumour growth by clonal analysis.</a> Nature. 488 (7412), 527-530 (2012).</li><li>Schepers, A. G., Snippert, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+tracing+reveals+Lgr5++stem+cell+activity+in+mouse+intestinal+adenomas.">Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas.</a> Science. 337 (6095), 730-735 (2012).</li><li>Sheridan, C., Kishimoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+/CD24-+breast+cancer+cells+exhibit+enhanced+invasive+properties:+an+early+step+necessary+for+metastasis.">CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis.</a> Breast cancer research: BCR. 8 (5), R59 (2006).</li><li>Ginestier, C., Hur, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALDH1+is+a+marker+of+normal+and+malignant+human+mammary+stem+cells+and+a+predictor+of+poor+clinical+outcome.">ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.</a> Cell stem cell. 1 (5), 555-567 (2007).</li><li>Liu, S., Cong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+Stem+Cells+Transition+between+Epithelial+and+Mesenchymal+States+Reflective+of+their+Normal+Counterparts.">Breast Cancer Stem Cells Transition between Epithelial and Mesenchymal States Reflective of their Normal Counterparts.</a> Stem cell reports. 2 (1), 78-91 (2014).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255 (5052), 1707-1710 (1992).</li><li>Pastrana, E., Silva-Vargas, V., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyes+wide+open:+a+critical+review+of+sphere-formation+as+an+assay+for+stem+cells.">Eyes wide open: a critical review of sphere-formation as an assay for stem cells.</a> Cell stem cell. 8 (5), 486-498 (2011).</li><li>Dontu, G., Abdallah, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem+/+progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem / progenitor cells.</a> Genes Dev. , 1253-1270 (2003).</li><li>Ponti, D., Costa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+in+vitro+propagation+of+tumorigenic+breast+cancer+cells+with+stem/progenitor+cell+properties.">Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties.</a> Cancer Res. 65 (13), 5506-5011 (2005).</li><li>Grimshaw, M. J., Cooper, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammosphere+culture+of+metastatic+breast+cancer+cells+enriches+for+tumorigenic+breast+cancer+cells.">Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells.</a> Breast Cancer Res. 10 (3), R52 (2008).</li><li>Pham, P. V., Phan, N. L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+breast+cancer+stem+cells+by+knockdown+of+CD44:+promising+differentiation+therapy.">Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy.</a> J Transl Med. 9 (1), 209 (2011).</li><li>Manuel Iglesias, J., Beloqui, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammosphere+formation+in+breast+carcinoma+cell+lines+depends+upon+expression+of+E-cadherin.">Mammosphere formation in breast carcinoma cell lines depends upon expression of E-cadherin.</a> PloS one. 8 (10), e77281 (2013).</li><li>Coles-Takabe, B. L. K., Brain, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don&#8217;t+look:+growing+clonal+versus+nonclonal+neural+stem+cell+colonies.">Don&#8217;t look: growing clonal versus nonclonal neural stem cell colonies.</a> Stem Cells. 26 (11), 2938-2944 (2008).</li><li>Stingl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+mammary+gland+stem+cells.">Detection and analysis of mammary gland stem cells.</a> J Pathol. 217 (2), 229-241 (2009).</li><li>Kreso, A., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+cancer+stem+cell+model.">Evolution of the cancer stem cell model.</a> Cell stem cell. 14 (3), 275-291 (2014).</li><li>Al-Hajj, M., Clarke, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+solid+tumor+stem+cells.">Self-renewal and solid tumor stem cells.</a> Oncogene. 23 (43), 7274-7282 (2004).</li><li>Yu, F., Yao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=let-7+regulates+self+renewal+and+tumorigenicity+of+breast+cancer+cells.">let-7 regulates self renewal and tumorigenicity of breast cancer cells.</a> Cell. 131 (6), 1109-1123 (2007).</li></ol>

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

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...

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&#8211;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&#8211;8, sphere-forming assays remain a popular and convenient approach to estimate breast cancer cells&#8217; 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).

<table border="0" cellpadding="0" cellspacing="0" width="1049">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Name of Material/ Equipment</td>
			<td style="width:139px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td style="width:139px;">Lonza</td>
			<td>CC-3151</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:625px;">2mM L-Glutamine</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>G8540</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:625px;">100U/ml Penicillin &#38; Streptomycin</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>P4083</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:625px;">20ng/ml recombinant human epidermal growth factor (EGF)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>E9644</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:625px;">20ng/ml recombinant human basic fibroblast growth factor (bFGF)</td>
			<td style="width:139px;">R&#38;D systems</td>
			<td>233-FB-025</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:625px;">1x B27 supplement&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Phosphate buffered saline (PBS);</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>12399902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;0.5% trypsin-0.2%EDTA;</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>59418C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum</td>
			<td style="width:139px;">First Link UK</td>
			<td>02-00-850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Trypan Blue</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>93595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Low attachment 6 well plates</td>
			<td style="width:139px;">Corning</td>
			<td>CLS3814</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type 1A</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>C9891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyaluronidase</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile razor blades</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>12443170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Sterile scalpel</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">11758353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Sterile micro-dissecting scissors</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>S3146</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammosphere formation assay from human breast cancer tissues and cell lines

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.

Different samples or those subjected to different treatments may vary in the number of mammospheres &#62;40 &#181;m that form after normalizing for initial cells seeded. Calculate mammosphere forming efficiency (MFE) for each treatment grown in triplicate. This enables experiments with different seeding densities to be compared. Data is best displayed on a bar graph, ideally with positive and negative controls, and displaying the standard deviation across the triplicate wells. Consistently transfer adherent cells into no...

Watch this Scientific Journal Video about Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines at JoVE.com

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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.

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 ...

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33.1K Views.  Imperial College London. 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.

Video: Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...