Name: isolation of stem-like cells from 3-dimensional spheroid cultures
Uploaded: 2019-12-13T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures at JoVE.com

This study was supported by grants from the National Cancer Institute R01-CA172220 (GSP, WYH), R01-ES02207 (GSP, WYH). We thank the Flow Cytometry Core at the University of Illinois at Chicago for assistance on cell sorting.

All cell handling and media preparations should be performed with aseptic technique in a Class II biological safety cabinet (BSC).
1. Culture and maintenance of HPrEC Cells in 2D
<ol>
	<li>Coat 100 mm culture dishes with 2 mL of 2.5 &#181;g/cm2 fibronectin solution overnight at room temperature (RT). Aspirate the solution and let the culture dishes air dry in the BSC for 45 min. Fibronectin-coated culture dishes can be stored 2&#8211;4 weeks at 4 &#730;C.</li>
	<li>Add 9 mL of the...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+identification+of+tumorigenic+prostate+cancer+stem+cells.">Prospective identification of tumorigenic prostate cancer stem cells.</a> Cancer Research. 65, 10946-10951 (2005).</li><li>Vander Griend, D. 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=The+role+of+CD133+in+normal+human+prostate+stem+cells+and+malignant+cancer-initiating+cells.">The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.</a> Cancer Research. 68, 9703-9711 (2008).</li><li>Wang, 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=Symmetrical+and+asymmetrical+division+analysis+provides+evidence+for+a+hierarchy+of+prostate+epithelial+cell+lineages.">Symmetrical and asymmetrical division analysis provides evidence for a hierarchy of prostate epithelial cell lineages.</a> Nature Communications. 5, 4758 (2014).</li><li>Neumuller, R. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+cellular+asymmetry:+asymmetric+cell+division+and+its+implications+for+stem+cells+and+cancer.">Dividing cellular asymmetry: asymmetric cell division and its implications for stem cells and cancer.</a> Genes and Development. 23, 2675-2699 (2009).</li><li>Cicalese, 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=The+tumor+suppressor+p53+regulates+polarity+of+self-renewing+divisions+in+mammary+stem+cells.">The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells.</a> Cell. 138, 1083-1095 (2009).</li><li>Klein, A. M., Simons, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+patterns+of+stem+cell+fate+in+cycling+adult+tissues.">Universal patterns of stem cell fate in cycling adult tissues.</a> Development. 138, 3103-3111 (2011).</li><li>Cairns, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+selection+and+the+natural+history+of+cancer.">Mutation selection and the natural history of cancer.</a> Nature. 255, 197-200 (1975).</li><li>Karthaus, W. R., 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=Identification+of+multipotent+luminal+progenitor+cells+in+human+prostate+organoid+cultures.">Identification of multipotent luminal progenitor cells in human prostate organoid cultures.</a> Cell. 159, 163-175 (2014).</li><li>Hu, W. 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=Isolation+and+functional+interrogation+of+adult+human+prostate+epithelial+cells+at+single+cell+resolution.">Isolation and functional interrogation of adult human prostate epithelial cells at single cell resolution.</a> Stem Cell Research. 23, 1-12 (2017).</li><li>Bryant, D. M., Stow, 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=The+ins+and+outs+of+E-cadherin+trafficking.">The ins and outs of E-cadherin trafficking.</a> Trends in Cell Biology. 14, 427-434 (2004).</li><li>Clevers, H., Loh, K. M., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+signaling.+An+integral+program+for+tissue+renewal+and+regeneration:+Wnt+signaling+and+stem+cell+control.">Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control.</a> Science. 346, 1248012 (2014).</li><li>Madueke, 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=The+role+of+WNT10B+in+normal+prostate+gland+development+and+prostate+cancer.">The role of WNT10B in normal prostate gland development and prostate cancer.</a> The Prostate. 79 (14), 1692-1704 (2019).</li><li>Guan, J. 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=Autophagy+in+stem+cells.">Autophagy in stem cells.</a> Autophagy. 9, 830-849 (2013).</li></ol>

Flow cytometry using multiple stem cell surface markers is a commonly used approach for stem cell research despite lacking both specificity and selectivity1,5,6. While spheroid formation in a 3D culture system is another useful method in enriching the rare stem cell population from primary epithelial cells, including HPrEC, the resulting spheroids are still a heterogeneous mixture of stem and progenitor cells2<...

The human prostate gland contains luminal epithelium with secretory function and basal cells underlying it along with an unusual neuroendocrine cell component. The epithelial cells, in this case, are generated from a rare population of prostate stem cells that are relatively quiescent in vivo and act as a repair system to maintain glandular homeostasis throughout life1. Despite many advances, the identification and functional isolation of prostate stem cells remains a major challenge in the field. Stem cell biomarkers, including cell surface marker-based methodologies combined with flow cytometry are commonly used for stem cell research2,3,4. However, results for enrichment and isolation vary widely as a function of marker combinations and antibody specificity5,6, raising questions about the identity of the isolated cells. Another widely used approach for stem-like cell enrichment is three-dimensional (3D) spheroid culture2,3,4. While resident stem cells are relatively quiescent in vivo with niche restraints, they undergo cell division in 3D matrix culture (both symmetric and asymmetric), generating both stem and progenitor cells that rapidly reproduce toward the lineage commitment7,8. The formed spheroids are a heterogeneous mixture containing both stem cells and progenitor cells at various stages of lineage commitment, including early and late stage progenitor cells. Thus, assays using the whole spheroids are not stem cell exclusive, making the identification of unique stem cell properties inconclusive. Therefore, it is critical to create assays to identify and separate prostate stem cells from their daughter progenitors. Towards this end, the goal of the current protocol is to establish an assay system that allows for the efficient identification and isolation of stem cells from human prostate tissues followed by robust downstream analysis of their biological functions.
Long-term 5-bromo-2'-deoxyuridine (BrdU) label-retention is widely used for in vivo and in vitro lineage tracing of stem cells based on their prolonged doubling time9,10. The current approach for prostate stem cell identification and isolation described herein is based on their relative quiescent characteristic and label-retention properties within a mixed epithelial population. Furthermore, based on the immortal strand DNA hypothesis, only stem cells can undergo asymmetric cell division. The stem cell represents the daughter cell that contains the older parental DNA while the progenitor cell, which is a committed daughter cell, receives the newly synthesized DNA. The unique stem cell property described above is exploited to perform BrdU labeling in parental stem cells in primary cultures and then track their label following BrdU-washout upon transfer to 3D anchor-free spheroid culture. While the majority of primary prostate epithelial cells retain a basal and transit amplifying phenotype in 2D culture, there is also a rare population of multipotent stem cells replenishing and maintaining the epithelial homeostasis as evidenced by formation of spheroids or fully differentiated organoids with corresponding culture media upon transfer to 3D systems3,12. In our current protocol, by using HPrEC prostate spheroids or prostasphere-based BrdU, CFSE, or Far Red retention assays followed by fluorescence activated cell sorting (FACS), we identify label-retaining stem cells in spheroids at a single cell level13.
Importantly, we further confirmed the stem cell characteristics of label-retaining cells within early-stage spheroids compared to the progenitor cells with lineage commitment. These include stem cell asymmetric division, in vitro spheroid formation ability and in vivo tissue regeneration capacity, elevated autophagy activity, augmented ribosome biogenesis and decreased metabolic activity. Subsequently, RNAseq analysis was performed. Differentially expressed genes in label-retaining spheroid cells were observed that may serve as novel biomarkers for human prostate stem cells. This spheroid-based label-retaining approach can apply to cancer specimens to similarly identify a small number of cancer stem-like cells, thus providing translational opportunities to manage the therapeutic resistant populations13. Presented below is the prostasphere-based label-retention assay using human primary prostate epithelial cells (HPrEC) as an example.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL tuberculin syringes</td>
			<td>Bectin Dickinson</td>
			<td>BD 309625</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes, sterile</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dishes</td>
			<td>Corning/Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>Corning/Falcon</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Corning/Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm coverslips, sq</td>
			<td>Corning</td>
			<td>284522</td>
			<td>For MatTek 35 mm culture dish</td>
		</tr>
		<tr>
			<td>24 x 50 mm coverslips</td>
			<td>Corning</td>
			<td>2975245</td>
			<td></td>
		</tr>
		<tr>
			<td>26G x 1.5 inch hypodermic needle</td>
			<td>Monoject</td>
			<td>1188826112</td>
			<td></td>
		</tr>
		<tr>
			<td>2N HCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm culture dish with cover glass bottom</td>
			<td>MatTek Corp</td>
			<td>P35G-0-10-C</td>
			<td>Glass bottom No. 0, uncoated, <img src="files/ftp_upload/60357/60357img1.jpg"/> irradiated</td>
		</tr>
		<tr>
			<td>40 &#181;m pore nylon cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2 culture incubator, 37 &#176;C</td>
			<td>Forma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Corning/Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Polystyrene Round-Bottom Tube with strainer snap cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>35 &#181;m nylon mesh</td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slides</td>
			<td>Millipore Sigma</td>
			<td>PEZGS0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting medium containing DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>A nuclear fluorescent dye</td>
		</tr>
		<tr>
			<td>Biological safety cabinet, Level 2 certified</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BrdU (5-bromo-2'-deoxyuridine)</td>
			<td>Sigma-Aldrich</td>
			<td>B5002</td>
			<td>1 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Centrifuge for 1.5 mL microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 15 mL tubes</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Allegra 6</td>
		</tr>
		<tr>
			<td>CFSE (carboxyfluorescein succinimidyl ester)</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34554</td>
			<td>5 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>cytochalasin D</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHZ1063</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase 1U/mL</td>
			<td>StemCell Technologies</td>
			<td>07923</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS CellSorter MoFlo XDP</td>
			<td>Beckman Coulter</td>
			<td>s</td>
			<td></td>
		</tr>
		<tr>
			<td>Far-Red pro-dye</td>
			<td>Thermo Fisher</td>
			<td>C34564</td>
			<td>5 mM stock solution in DMSO</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F0895</td>
			<td>For coating 100 mm culture dishes</td>
		</tr>
		<tr>
			<td>Fluorescent microscope with color digital camera</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Axioskop 20 fluorescent microscope; color digital Axiocamera</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>HPrEC (Primary normal human prostate epithelial cells)</td>
			<td>Lifeline Cell Technology</td>
			<td>FC-0038</td>
			<td>Pooled from 3 young (19-21yr od) disease-free organ donors; 1 x 105 cells/mL; stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>ice bucket and ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microsope with digital camera</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, low growth factor, phenol-red free</td>
			<td>Corning</td>
			<td>356239</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Corning</td>
			<td>A452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-BrdU antibody</td>
			<td>Cell Signaling</td>
			<td>5292S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG antibody (negative control)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS), pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors and tips, various sizes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PrEGM (ProstaLife Epithelial Cell Growth Medium)</td>
			<td>Lifeline Cell Technology</td>
			<td>LL-0041</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide (PI)</td>
			<td>R &#38; D Systems</td>
			<td>5135/10</td>
			<td>10 &#956;g/mL PI in PBS stored at 4 &#176;C in the dark</td>
		</tr>
		<tr>
			<td>Serological pipets, various sizes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for sphere counting and size measurements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software: 3D images using Imaris an imageing software with freeform drawing capabilities</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath, 37 &#176;C</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>z-stack images using a transmitted light inverted fluorescent confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of stem-like cells from 3-dimensional spheroid cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Primary normal human prostate epithelial cells are placed into fibronectin-coated culture dishes and cell growth is maintained in 2D culture (Figure 1a). Upon transfer into 3D culture with a basement membrane matrix, differentiated epithelial cells slowly die out. Only prostate stem cells can survive in an anchor-free culture and form spheroids in 5 days (Figure 1b).
Dual labeling of prostate epitheli...

Watch this Scientific Journal Video about Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures at JoVE.com

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Stem cell isolation remains a major challenge due to co-isolation of progenitor cells using current methods. We developed a novel biomarker-free assay to isolate quiescent stem cells from mixed progenitors. The main advantage of this spheroid-based, label-retention assay, it can expand and isolate live stem cells from their daughter progenitors by FACS using CFSE or Far Red labeling.While conventional therapies eradicate most prostate cancer cells, cancer stem cells remain due to therapeutic resistance, thus driving disease progression. Stem-like cell isolation will permit identification of novel genes that can be therapeutically co-targeted for effective disease remission. Our label-retention assay's applicable for normal stem cell isolation from various tissues and cells, and for cancer stem cell research.Importantly, this assay's applicable to other epithelial cell cancers, such as breast and colorectal cancers. Begin by coating 100-millimeter culture dishes with two milliliters of 2.5 microgram fibronectin solution and incubate them overnight at room temperature. Then aspirate the solution and let the culture dishes air dry in the biosafety cabinet for 45 minutes.Add nine milliliters of the prostate epithelial cell growth medium to a fibronectin-coated dish and keep it warm in carbon dioxide incubator at 37 degrees Celsius. Thaw one vial of frozen human prostate epithelial cells in a 37 degrees Celsius water bath, and resuspend the cells in 10 milliliters of warm medium. Centrifuge the cell suspension at 500 times g for five minutes.Then aspirate and discard the supernatant. Resuspend the cells in one milliliter of warm culture medium and transfer one milliliter of the suspension directly to the fibronectin-coated dish with the pre-warmed medium. Then incubate the dish in the carbon dioxide incubator at 37 degrees Celsius.If co-labeling the cells, prepare BrdU-labeled cells in 10-days 2D culture as described in the text manuscript. Then add five micromolar CFSE or Far Red and incubate the cells for 30 minutes. After the incubation, carefully aspirate the medium with the dye and wash the cells twice with five milliliters of warm PBS.Next, perform enzymatic digestion with 05%trypsin EDTA according to the manuscript directions. Centrifuge the cells at 500 times g for five minutes and discard the supernatant. Cells were resuspended in ice-cold, one-to-one basement membrane matrix and culture medium mix for 3D prostasphere formation.To prepare prostasphere culture in a 3D basement matrix system, thaw the basement membrane matrix at four degrees Celsius overnight and keep it on ice prior to use. Then gently add one milliliter of ice-cold basement membrane matrix into an equal volume of ice-cold culture medium. Pipette up and down to mix, taking care to not introduce air bubbles.Resuspend five times 10 to 1/4 human prostate epithelial cells in ice-cold one-to-one basement membrane matrix and culture media mix for total volume of 500 microliters. Pipette the solution to the bottom rim of each well and swirl the plate to evenly distribute the mixture. Place the plate in a 37 degrees Celsius carbon dioxide incubator for 30 minutes to allow the matrix to solidify.Then cover it with one milliliter of warm culture medium per well, making sure to not disturb the ring. To harvest the CFSE-labeled prostaspheres replace the medium with two milliliters dispase and pipette up and down to mix several times. Incubate the plate at 37 degrees Celsius for 30 minutes to digest the matrix, then collect the sphere mixture into a 15-milliliter centrifuge tube.Centrifuge the spheres at 500 times g for five minutes and discard the supernatant. Resuspend the pellet in 500 microliters of warm 05%trypsin EDTA, and transfer them into a 1.5-milliliter centrifuge tube. Incubate the spheres at 37 degrees Celsius for five minutes, then add 500 microliters of warm PBS with 10%FBS.Pass them through a one-milliliter syringe with a 26-gauge needle to completely dissociate the spheres. Centrifuge the cells at 500 times g for five minutes. Then aspirate and discard the supernatant.Resuspend the cells in one milliliter of warm culture medium with one microgram per milliliter propidium iodide to stain the dead cells and incubate them for one minute at room temperature. Repeat the centrifugation, and discard the supernatant. Then wash the cells with one milliliter of warm medium.Repeat the centrifugation, and discard the supernatant. Resuspend the cells in one milliliter of warm culture medium and collect the cells in five-milliliter polystyrene round bottom tubes by filtering them through 35-micron pore size cell strainer snap caps. Perform FACS analysis of trypsin-dispersed CFSE-labeled prostasphere cells.Using non-labeled cells as a negative control and CFSE-labeled cells as a positive control to set the gates, run the sample to sort and collect the subpopulations of fractionated CFSE-high and CFSE-low cells. Primary normal human prostate epithelial cells were placed into fibronectin-coated culture dishes and cell growth was maintained in 2D culture. Upon transfer into a 3D culture with a basement membrane matrix, differentiated epithelial cells slowly died out and only prostate stem cells remained.Dual-labeling of prostate epithelial cells in 2D culture followed by spheroid formation in 3D culture showed colocalization of BrdU, CFSE, and Far Red in the same label-retaining cells. Dual immunostaining showed that label-retaining cells exhibit lower levels of cytokeratin protein keratin 14, decreased cell junction protein E-cadherin, increased stem cell early marker proteins Wnt10b and ALDH1A1, increased autophagy protein LC3, and increased myosin IIB. Furthermore, the spheroid-based, label-retention assay successfully detects cancer stem-like cells in prostate cancer specimens.CFSE-labeled retaining stem-like cancer cells and spheroids derived from human prostate cancer specimens exhibited reduced E-cadherin protein relative to the non-labeled progenitor cells. When attempting this protocol, it is important to maintain matrix in liquid form for culture medium preparation. Store it overnight at four degrees, and chill pipette tips in ice-cold PBS prior to dispensing matrix.Following isolation of stem cells using the spheroid-based label-retention assay, single-cell RNA sequencing and proteomic analysis can be performed to identify specific genes and novel biomarkers in stem cells. Isolation of live cancer stem cells by the spheroid-based label-retention assay makes it possible for researchers to grow cancer stem cell-derived tumoroids in vitro and to screen novel anti-cancer drugs.

Research

JoVE Journal

Cancer Research

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...