Background

A growing body of evidence supports the contention that epithelial cancers including the colorectal cancer are diseases driven by a small set of self renewing cells, termed cancer stem cells (CSC) or cancer-initiating cells, that are distinct from the bulk of the cells in the tumor [1]. Initially identified in hematopoietic tumors, CSCs have now been identified and isolated in a variety of solid tumors that include breast, central nervous system, pancreas, skin, head and neck, colon and prostate [2–7]. CSCs share all the fundamental traits of stem cells-self renewal by asymmetric division, reduced proliferation and differentiation and resistance to apoptosis [8]. CSCs are identified by specific surface epitopes, which in the colon include CD44, CD133 and CD166 [9, 10]. To select putative colorectal CSCs, a promising combination of three markers- EpCAM, CD44 and CD166 was described by Dalerba et al. [11]. Although EpCAM previously being considered as pan-epithelial marker in the normal human colon, its frequent expression in CSCs in breast, colon, pancreas and prostate tumors suggests that this surface epitope could be a putative marker for CSCs, particularly in human colon cancer-derived cell lines [12]. More recently Lgr5, Musashi-1 and aldehyde-dehydrogenase 1 (ALDH-1) have been added to the list of stem cell markers for colon cancer [13–15].

One of the recently reported characteristics of tumor derived CSCs is that they can be grown to form spherical colonies in vitro, when plated in limited numbers under anchorage-independent conditions in a serum-free defined media supplemented with growth factors [9]. With the objectives to promoting in vitro expansions of CSCs, methods have been developed to grow and study them in sphere-forming assays as reported for neurospheres [16, 17], mammospheres [18, 19] and colonospheres [13, 20]. Using this approach, we and others have identified and/expanded colon CSCs by generating colonospheres from colon cancer cell lines [10, Formation of colonospheres

The ability of cell lines to form spheres in suspension was evaluated as described by Liu et al. [25], with slight modifications. Briefly, primary colonospheres were generated by incubating the limited number of parental HCT-116 (p53 wild-type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant; K-ras wild-type) cells at a concentration of 100 cells per 200 μL in serum-free stem cell medium (SCM) containing DMEM/F12 (1:1) supplemented with B27 (Life Technologies, Gaithersburg, MD), 20 ng/ml EGF (Sigma, St Louis, MO), 10 ng/ml fibroblast growth factor (Sigma), and antibiotic-anti-mycotic in 24-well ultra low-attachment plates (Corning Inc, Lowell, MA) for 10 days. The colonospheres formed at the end of the incubation period were centrifuged (1000 rpm), dissociated with 0.05% trypsin/EDTA using a 22 gauge needle and then passed through a 40 μM sieve to obtain single cell suspension, as described by Kakarala et al. [19]. The single-cell suspension derived from colonospheres that have undergone 15 or more serial passages were used for all experiments. For spheroids formation, an equal number of cells from adherent cell lines and colonospheres cells were plated at 200 cells/100 μL SCM in each 96 ultra low-attachment well (Corning Inc, Lowell, MA). The colonospheres formed after 5 days were evaluated for their number and size by light microscopy.

Extreme limiting dilution analysis

Extreme limiting dilution analysis (ELDA) was performed as described by Hu and Smyth [45]. Briefly, single cell suspension obtained from adherent or colonospheres-derived cells were plated at concentration of 1000, 100, 10 cells and 1 cell per 100 μl SCM (24 well for each dilution) in 96-well ultra-low attachment and incubated for 5 days. At the end of 5 days, the number of wells showing formation of colonospheres was counted. The frequency of sphere forming cells in a particular cell type was determined using ELDA webtool at http://bioinf.wehi.edu.au/software/elda.

Flow Cytometry Analysis

Single cell suspension from parental monolayer cell cultures and colonospheres were subjected to direct immunofluorescence staining followed by flow cytometry analysis according to our standard protocol [22]. Briefly, the cells were harvested and washed with PBS. Half a million cells were suspended in 90 μl of PBS containing 0.5% BSA. After 10 min at room temperature, 10 μl of fluorophore conjugated anti-human CD44 antibody was added and incubated for 30 min in dark at room temperature. The samples were then washed and analyzed using a FACS DiVa (BD, San Jose, CA). The cells stained with mouse IgG2b (isotype-negative control) served as gating control. The proportion of CD44 positive cells was determined on the basis of fluorescence intensity-spectra of CD44-conjugated PE-Cy7 or PerCP-Cy5.

Immunofluorescence cytochemistry

Single cell suspension, obtained from parental and colonosphere, was washed in PBS and fixed in 2-4% formaldehyde for 10 min at 37 °C. They were washed and re-suspended in 0.5% BSA-PBS (blocking buffer) for 10 min, subsequently incubated in primary antibodies at appropriate dilution for 1 h at room temperature. After rinsing with incubation buffer, the cells were resuspended in fluorophore-conjugated secondary antibodies diluted in incubation buffer according to the manufacturer's recommendations and incubated for 30 minutes at room temperature. The cells were then resuspended in PBS after washing with the incubation buffer. Appropriate aliquot of cells were mounted on glass slides immediately before examining under fluorescence microscope.

Hoechst 33342 dye exclusion assay

Single cell suspension obtained from parental cell lines and colonospheres were washed with PBS (3 times) and stained with Hoechst 33342 or H342 (5 μg/ml, Sigma-Aldrich Inc, St Louis, MO) for 45 minutes at 37°C in HBSS buffer, vortexing gently every 15 min. As a control, a sample was treated with verapamil (Sigma, 50 μM) for ten minutes at room temperature prior to the addition of H342. The stained cells were collected, washed with PBS and resuspended in 3 ml of PBS containing 2 μg/ml of propidium iodide, and subsequently analyzed by flow cytometer-FACS Vantage SE/DiVa SORP (BD Biosciences, San Jose, CA) with all-digital electronics and octagon- and trigon-shaped detector arrays. Excitation of 100 mW at 488 nm was provided by a 177-G argon ion laser (Spectra-Physics, Mountain View, CA) and 200 mW of all-lines UV (351-365 nm) was provided by an Innova 90-5 argon ion laser (Coherent, Palo Alto, CA). Forward and side laser scatter was detected from 488 nm excitation. H342 and propidium iodide fluorescence from UV excitation was split into "blue" and "red" wavelengths by a 505 nm long pass dichroic with a 450/50 bandpass (425-475 nm) filter in front of the "blue" detector and a 630 nm long pass filter in front of the "red" detector. Cell population showing H342 Bright (H342Br) and H342 Low (H342Lo) was determined and the ratio of H342Lo/H3342Br was calculated to evaluate the dye-efflux capacities of the cells. The gating of H342Lo and H342Br cells was based on a verapamil control. Dead cells were gated out based on positive staining with propidium iodide.

Determination of cellular growth

Changes in cellular growth, an assessment of proliferation, of parental and colonosphere-derived cells were assessed by 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay as described previously [22]. Briefly, 4000 cells were plated per well in 96-well plates in 200 μl medium. At each time point (0, 24, 48, 72, 96, 120 h), 20 μl MTT solution was added to each well and the plate was incubated for 1 h at 37°C. Medium was then aspirated from each well, and 100 μl DMSO was added. The intensity of the color developed, which is the reflection of number of live cells, was measured at a wavelength of 570 nm. All values were compared to the corresponding controls. All assays were performed with 6 replicates.

Measurement of Alkaline phosphatase activity

Status of differentiation was determined by measuring the alkaline phosphatase activity using SensoLyte, pNPP alkaline phosphatase assay kit (Anaspec, San Jose, CA) according to the manufacturer's instructions and measuring the absorbance at 405 nm.

β-catenin siRNA transfection

For the transfection of siRNA into the parental and colonosphere derived cells, Oligofectamine reagent (Invitrogen Corp., Carlsbad, CA) and serum-free Opti-MEM (Invitrogen Corp., Carlsbad, CA) medium was used to prepare transfection complexes according to the manufacturer's instructions. Briefly, single cell suspension of colonospheres was plated in 10 cm tissue culture plates with normal growth medium overnight to achieve 25-30% confluence. Next day the medium was removed, washed twice with serum-free Opti-MEM (Invitrogen Corp., Carlsbad, CA) medium prior to adding the complexes containing non-targeted or β-catenin siRNA (Integrated DNA Technologies Inc., Coralville, IA). After 3 days of transfection, the cells were collected and analyzed for protein expression of β-catenin using Western blot and for colonosphere formation assay using SCM.

Activation of relative TCF/LEF-Dual Luciferase assay

The activation of transcription factor TCF/LEF was evaluated by using Cignal TCF/LEF reporter assay kit (SA Biosciences, Frederick, MD). The cells were grown to 25-30% confluence as described above and co-transfected with TCF/LEF reporter constructs and either non-targeted or β-catenin siRNAs (Integrated DNA Technologies Inc., Coralville, IA) using SureFECT transfection reagent (SA Biosciences, Frederick, MD) according to manufacturer's instructions. The TCF/LEF reporter used a mixture of an inducible β-catenin-responsive luciferase construct and a constitutively expressing Renilla element (40:1). At the end of 16-24 h incubation period Opti-MEM medium was changed to DMEM 10% FBS for parental cell lines or SCM for colonosphere derived cells. The cells were allowed to grow for another 3 days, collected and analyzed for TCF/LEF activity using a dual-luciferase assay kit (Promega-Biosciences, San Luis Obispo, CA) following the instructions outlined by the manufacturer.

Overexpression of c-myc gene

Single cell suspension of HCT-116 parental cells was plated in the tissue culture plates to achieve a 90% confluence. Once the 90% confluence is achieved, the adherent cells were transfected using Lipofectamine 2000 and PLUS reagent (Invitrogen Corp., Carlsbad, CA) with plasmid vector-pCMV6-Neo (Origene, Rockville, MD) containing human cDNA clone for c-myc gene or empty plasmid PCMV6-vector in OPTI-MEM medium according to the manufacturer's instruction. After 3 days of transfection, the cells were analyzed for c-myc protein expression by Western blot and re-plated for colonosphere formation assay.

Statistical analysis

Unless otherwise stated, data are expressed as mean ± SD of six observations. Where applicable, the results were analyzed using analysis of variance followed by Fisher protected least significant differences or Scheffé test. p < 0.01 was designated as the level of significance.

Authors' information

Shailender S. Kanwar, Ph.D.: Postdoctoral Research Fellow, Department of Internal Medicine and Veterans Affairs Medical Center, Wayne State University, Detroit, MI 48201, USA. E-mail: sskanwar@gmail.com

Yingjie Yu, M.D., Research Assistant Professor, Department of Internal Medicine and Veterans Affairs Medical Center, Wayne State University, Detroit, MI 48201, USA. E-mail: aa5142@wayne.edu

Jyoti Nautiyal, Ph.D.: Postdoctoral Research Fellow, Department of Internal Medicine and Veterans Affairs Medical Center, Wayne State University, Detroit, MI 48201, USA. E-mail: jyotinautiyal@gmail.com

Bhaumik B. Patel, M.D.: Staff Oncologist and Assistant Professor, Department of Internal Medicine, Veterans Affairs Medical Center and Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA. E-mail: bhaumik.patel@va.gov

Adhip P.N. Majumdar, Ph.D., D.Sc.: Professor and Senior Research Career Scientist, Department of Internal Medicine, Veterans Affairs Medical Center and Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA. E-mail: a.majumdar@wayne.edu