signature=440cd3973609de77869352807f83ecd7,CD44 + CD24 − prostate cells are early cancer progenitor/...

Cells and media

LNCaP and DU145 cells were obtained from ATCC. LNCaP cells were maintained in RPMI-1640+10% fetal bovine serum (FBS)+2 mM L-glutamine+penicillin and streptomycin, and DU145 cells were maintained in DMEM+10% FBS+2 mM L-glutamine+penicillin and streptomycin. Following cell sorting, cells were maintained in serum-replacement medium consisting of DMEM:F12 plus 10 ng ml−1 bFGF, 20 ng ml−1 EGF, 5 μg ml−1 insulin, and 0.4% BSA. For experiments describing the effects of serum on CD44+CD24− cells, 10% FBS was added to the serum-replacement medium for the indicated time.

Flow cytometric analysis and separation

LNCaP and DU145 cells were detached with trypsin, washed once in FACs buffer (PBS containing 1–2% BSA and 5 mM EDTA), then stained with anti-CD24-FITC (Invitrogen, Carlsbad, CA, USA) and anti-CD44-PE (Invitrogen) using 10 μl of antibody per 106 cells, and incubated at 4°C for 15 min. Following incubation, cells were washed once with FACs buffer. For flow cytometric sorting, cells were resuspended in FACs buffer at 20 × 106 cells ml−1 and separated on either an Aria cell sorter (BD Biosciences, San Jose, CA, USA) or a MoFlo High Performance cell sorter (Dako Cytomation, Carpinteria, CA, USA). Live cells were gated on the basis of forward and side scatter, and single cells were gated on the basis of forward scatter and pulse width (Supplementary Figure 1A). Gates were determined by analysis of unstained cells, isotype-specific stains (Supplementary Figure 1B), and single stains (BD Biosciences). The CD44+CD24− cells were not assessed for purity due to the low numbers of cells obtained.

Real-time polymerase chain reaction

The expression of CD133 was measured on CD44+CD24− and CD44+CD24−-depleted cells using Cells-to-CT kit (Applied Biosystems, Foster City, CA, USA) and a StepOne Real-time PCR machine (Applied Biosystems). A relative CT experiment was performed using the TaqMan Gene Expression Assay reagent (Applied Biosystems) for CD133 (PROM1, Hs00195682_m1) and was normalised using GAPDH (Hs99999905_m1) and the supplied enzyme mix in the Cells-to-CT kit.

Soft agar colony assay

Flow sorted cells were washed once in FACs buffer and 3000 cells were suspended in either serum-replacement medium or DMEM+10% FBS containing 0.4% agarose and overlayed onto a 60-mm dish containing a solidified bottom layer of 0.4% agarose in either serum-replacement or DMEM+10% FBS. Once the top layer solidified, 1 ml of medium was placed on top to keep the plates moist. Plates were incubated for 3 weeks until colonies were visible. The plates were stained with 1 : 75 dilution of 0.33% neutral red solution (Sigma, Saint Louis, MO, USA) at 37°C for 1 h, then counted using an Olympus CK2 microscope (Olympus, Center Valley, PA, USA) and Image Pro Software (MediaCybernetics, Silver Spring, MD, USA).

Mouse xenograft studies

NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal Care was provided in accordance with the procedures outlined in the ‘Guide for Care and Use of Laboratory Animals’ (Institute of Laboratory Animal Resources, 1996). Following separation by flow cytometry, cells were placed in serum-replacement medium overnight at a concentration of 1000 cells ml−1. Cells were trypsinised, washed once with PBS, then resuspended in serum-replacement medium at 1000 cells per 50 μl, mixed with an equal volume of matrigel (BD Biosciences) and injected subcutaneous into male NOD/SCID mice (Jackson Labs, Bar Harbor, ME, USA). Mice were monitored daily for palpable tumour formation for a total of 159 days. Tumour size was measured with calipers. Tumour volume was determined by the following formula: V=W2 × L/2 (Carlsson et al, 1983). Once tumours reached greater than 1.5 mm in one dimension, mice were euthanised and tumours were extracted. For flow cytometric analysis, tumours were dissociated with collagenase IV (Sigma) using 200 units ml−1 in DMEM:F12 and incubated at 37°C for 1.5 h with mechanical disruption every 30 min. Cells were passed through a 70-μm cell strainer and washed twice with FACs buffer.

RNA isolation, amplification, and microarray analysis

Total RNA was isolated with Trizol (Invitrogen). Following isolation, RNA was amplified using Ambion's MessageAmp II kit (Ambion, Austin, TX, USA). Twenty-microgram-amplified RNA was labelled in a reverse transcription (RT) reaction with an oligo-dT primer, and either Cy3-dUTP or Cy5-dUTP. Following the RT reaction, the RNA was hydrolysed by incubation with NaOH. Probes were purified with a Microcon YM-30 column (Millipore, Billerica, MA, USA). Oligonucleotide microarrays printed by the NIH Microarray Core Facility were prehybridised in 5 × SSC, 0.1% SDS, and 1% BSA for 1 h at 42°C, washed in distilled water then 100% ethanol, and air dried. Hybridisation of mixed Cy-3- and Cy-5-labelled probes was done in 50% formamide, 10 × SSC, 0.2% SDS overnight at 42°C. After hybridisation, slides were washed in decreasing concentrations of SSC (2 × SSC+0.1% SDS, 1 × SSC, and 0.2 × SSC). Arrays were scanned in a GenePix 4000B scanner and analysed using GenePix Pro (Molecular Devices, Sunnyvale, CA, USA). Data analysis was performed using the centred hierarchical clustering algorithm in Cluster and visualised using TreeView, offered by Michael B Eisen as freeware (http://rana.lbl.gov/EisenSoftware.htm).