CD133+CD44+ Population Efficiently Enriches Colon Cancer Initiating Cells
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- Haraguchi, N., Ohkuma, M., Sakashita, H. et al. Ann Surg Oncol (2008) 15: 2927. doi:10.1245/s10434-008-0074-0
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Previous reports have demonstrated that CD133+ cells or CD44+ cells might be cancer initiating cells (CIC) of colon cancer. However, the association between the two cell types is unclear. In this study, we evaluated the tumorigenicity of each population of human colon cancer divided by CD133 and CD44 using non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice.
Using the colon cancer cell lines HT29 and Caco2 we evaluated the change of expression status of CD133 or CD44 by a treatment with sodium butyrate (NaBT) that can induce cellular differentiation. Next, we prepared ten clinical samples of colon cancer and analyzed the expression and tumorigenicity of CD133 and CD44.
With NaBT treatment, CD44 expression was greatly downregulated in both HT29 and Caco2 (HT29: nontreatment versus treatment; 77.8% versus 0.6%, Caco2: 14.0% versus 0.4%, respectively), more than CD133 expression (HT29: nontreatment versus treatment; 90.1% versus 67.7%, Caco2: 98.9% versus 76.3%, respectively). In clinical samples, the percentages of CD133+ cells and CD44+ cells varied from 0.3% to 82.0% (mean 35.5%), and from 11.5% to 58.4% (mean 30.0%), respectively. Subcutaneous injection of CD133+ or CD44+ cells made a tumor in all mice (3/3 and 4/4, respectively). The combined analysis of CD133 and CD44 revealed that only the CD133+CD44+ population had the ability to produce a tumor (3/3).
The findings demonstrate that, at present, the CD133+CD44+ population may be the best to identify tumor initiating cells of human colon cancer.
KeywordsCD133CD44Colon cancerColon cancer initiating cellsSodium butyrate
Recently the cancer stem cell theory has been introduced not only in leukemia1 but also in several kinds of solid cancers.2–6 According to this theory, only cancer stem cells that exist as a minor population in a tumor can produce a tumor and maintain it.7 CD133+ cells or CD44+ cells were reported to be cancer stem cells or tumor initiating cells that produce a tumor of human colon cancer.8–11 However, there have been few studies demonstrating the correlation between the two populations identified by CD133 or CD44, and evaluating the ability of tumor formation using both markers. In this study we therefore aimed to clarify the correlation between these two populations and whether it is possible to better identify tumor initiating cells using both CD markers. The results indicate that both CD133+ and CD44+ cells might be real tumor initiating cells of human colon cancer.
MATERIALS AND METHODS
Tumor Cell Preparation and Cell Culture
Human colorectal cancer cell lines, HT29 and Caco2, were used in the following analysis. HT29 was cultured in McCoy’s medium 5A (Invitrogen Corporation, CA, USA)/10% fetal bovine serum (FBS; Equitech-Bio, TX, USA) with 2 mM L-glutamine (Invitrogen Corporation, CA, USA), 100 μg/ml penicillin G, and 100 unit/ml streptomycin (Invitrogen Corporation, CA, USA). Caco2 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen Corporation, CA, USA)/10% FBS with 100 μg/ml penicillin G and 100 unit/ml streptomycin. The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. HT29 was obtained from American Type Culture Collection (ATCC), and Colo201 was obtained from Japanese Collection of Research Bioresources Cell Bank (JCRB).
Ten samples from cases of colorectal cancer were obtained from Kyushu University at Beppu and Oita Red Cross Hospital after obtaining patients’ informed consent and approval by the Research Ethics Board at Kyushu University in Japan. Surgical specimens were subsequently subjected to mechanical and enzymatic digestion as described below. Digested tumor cells were prepared for flow cytometric analysis and cell sorting, or were injected into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice to obtain further large numbers of tumor cells.
Sodium Butyrate Treatment and Alkaline Phosphatase Assay
Cells of HT29 and Caco2 colon cancer cell lines were dissociated with 0.25% trypsin and 0.02% ethylenediamine tetraacetic acid (EDTA) and then 1 × 106 cells were seeded in 10-cm plastic flasks (BD FALCON, CA, USA) at day 0. At day 1, sodium butyrate (NaBT; Wako, Osaka, Japan) was added at concentration of 0, 3, 5, and 8 mM and incubated for 72 h.12–14 The change in alkaline phosphatase13 level and activity13 was detected by enzyme-linked immunosorbent assay (ELISA) with SensoLyte™ pNPP alkaline phosphatase assay kit (AnaSpec, CA, USA) according to the manufacturer’s protocol.
Transplantation of Human Colon Cancer Cells into NOD/SCID Mice
Colon cancer cells were suspended in a 1:1 mixture of media and Basement Membrane Matrix High Concentration (BD Biosciences, CA, USA) and injected subcutaneously into the flank of NOD/SCID mice (5 weeks of age) with anesthesia. After 12–16 weeks all mice were sacrificed by cervical dislocation, and tumors were removed and analyzed by flow cytometry or histology. All of the xenograft lines were originally implanted into NOD/SCID mouse subcutaneously and never cultivated and expanded in vitro. To study the tumorigenecity of CD44+ and CD133+ cells, 1 × 104 cells from each population were injected subcutaneously into NOD/SCID mice. Tumorigenecity was evaluated at 6 weeks after NOD/SCID transplantation.
Colon cancer tissues were cut into small fragments, further minced with a sterile scalpel, and washed twice with Dulbecco’s modified Eagle medium (DMEM)/10% FBS with 100 μg/ml penicillin G and 100 unit/ml streptomycin (Invitrogen Corporation, CA, USA), then placed in DMEM/10% FBS with 2 mg/ml collagenase A (Roche Diagnostics, Basel, Switzerland) solution. The mixture was incubated at 37°C for up to 2 h to allow complete digestion. Every 15 min, the solution was mixed through a 10-ml pipette to encourage dissociation. Cells were filtered through 40-μm nylon mesh and washed twice, and then cell fragments and debris were eliminated by using Ficoll (GE Healthcare, Buckinghamshire, UK) density gradient centrifugation. Cells were stained for flow cytometry or following transplantation into NOD/SCID mouse.
Flow Cytometric Analysis and Cell Sorting
To characterize colon cancer initiating cells, the following antibodies were used: allophycocyanin (APC)-conjugated anti-human CD133/1 (clone AC133, mouse IgG2a, Miltenyi-Biotec, Bergisch Gladbach, Germany), phycoerythrin (PE)-conjugated anti-human CD44 (clone G44-26, mouse IgG2b, BD Pharmingen, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-human CD326 (EpCAM; clone HEA-125, mouse IgG1, Miltenyi-Biotec, Bergisch Gladbach, Germany).
To isolate human cells from mouse xenografts, biotinylated anti-mouse H-2Kd (clone SF1-1.1, mouse IgG2a, BD Pharmingen, CA, USA) and biotinylated anti-mouse CD45 (clone 30-F11, mouse IgG2b, BD Pharmingen, CA, USA) were used. Streptavidine-APC-Cy7 (BD Pharmingen, CA, USA) was used as secondary antibody. Doublet cells were eliminated by forward scatter-hight/forward scatter-width (FSC-H/FSC-W) and side scatter-hight/side scatter-width (SSC-H/SSC-W). Dead and dying cells were eliminated with 7-amino actinomycin D (7-AAD) (BD Pharmingen, CA, USA). FcR blocking was applied with FcR blocking reagent (Miltenyi-Biotec, Bergisch Gladbach, Germany).
Samples were analyzed and sorted with FACSVantage SE flow cytometers (Becton Dickinson, CA, USA) and data were analyzed with Diva software (Becton Dickinson, CA, USA).
NaBT-Induced Differentiation and Changes of Cell Surface Markers
NaBT-induced differentiation and changes of cell surface markers
Cell Surface marker
HT29 untreated (%)
HT29 NaBT treated (%)
Caco2 untreated (%)
Caco2 NaBT treated (%)
Flow Cytometric Analysis in Primary Colon Cancer
Flow cytometric analysis in primary colon cancer
Expression of cell surface marker (%)
lymph node metastasis (Sigmoid colon)
CK070326 NOD/SCID xeno*
CK070606 NOD/SCID xeno‡
CK070426 NOD/SCID xeno*
CK070514 NOD/SCID xeno‡
CK070611 NOD/SCID xeno‡
CK070317 NOD/SCID xeno‡
Tumorigenicity in NOD/SCID Mouse
Tumorigenicity of CD133 and CD44 in NOD/SCID mouse
Tumor size (cm)
0.8 ± 0.14
0.83 ± 0.28
Tumorigenicity of each combined populations in NOD/SCID mouse
Tumor size (cm)
1.2 ± 0.35
This study demonstrated that the CD133+ population varied from 0.3% to 82.0% with a mean of 35.5% in colon tumors. CD133+ cells showed significantly higher tumorigenicity in NOD/SCID mice than did CD133− cells. This result was very similar to the reports by O’Brien et al. and Ricci-Vitiani et al.9,10 On the other hand, the CD44+ population varied from 11.5% to 58.4% with a mean of 30.0%. CD44+ cells showed significantly higher tumorigenicity in NOD/SCID mice than did CD44− cells. Thus this study confirmed that CD133+ or CD44+ is an adequate marker for tumor initiating cells of human colon cancer.
Cancer initiating cells or cancer stem cells are considered to be immature cells, and they may occupy a small percentage of the tumor. It is difficult to identify appropriate markers that can detect such cells. One conceivable method is to use NaBT, which can induce differentiation in the cell.12–17 Human colon cancer cell lines, HT29 and Caco2, were treated with NaBT, and the results demonstrated that both CD133+ and CD44+ cells decreased dramatically. The rate of decrease was much greater in CD44 than CD133, suggesting that CD44 may be more suitable as a marker of immature cells than CD133.
As mentioned above, CD133 and CD44 are adequate markers of cancer initiating cells of human colon cancer, however, there are few studies that described the association between the two markers. Dalerba et al. described that most CD44+ cells were CD133+. They thus suggested that CD44 may be more appropriate as a cancer initiating cell marker than CD133.11 This suggestion may be partly supported by our experiment using NaBT, which showed that CD44 may be a more suitable marker of immature cells than CD133.
Our combined analysis demonstrated that there are two different groups. One group of five tumors consisted of four populations including both positive (CD133+CD44+), both negative (CD133−CD44−), and one positive (CD133+CD44− or CD133−CD44+). The other group of five tumors consisted of three populations, with CD133−CD44+ lacking. In our study, CD133+ cells were detected more or less in all ten clinical samples.
We studied the tumor formation ability of the three populations CD133−CD44−, CD133+CD44−, and CD133+CD44+, among which only the CD133+CD44+ population exhibited tumorigenicity. It was interesting that the CD133+CD44− population did not exhibit tumorigenicity. This result suggests that it would be much better to use the combined markers to identify cancer initiating cells of human colon cancer than either CD133 or CDE44 as a single marker. Our study is alsio interesting because it clarifies the significance of the combined use of the two major markers. We did not study the tumorigenicity of CD133−CD44+ population because sufficient numbers of this cell population could not be captured. Our study demonstrated that CD133+ or CD44+ cells show tumorigenicity. On the other hand, CD133− or CD44− cells did not show tumorigenicity. The CD133+CD44− population also did not show tumorigenicity. Thus it would be imagined that the CD133−CD44+ population may show no tumorigenicity. However, further study is needed to confirm this.
In summary, this study indicated that the CD133+CD44+ population may be real cancer initiating cells of human colon cancer.
This study was supported by CREST of Japan, Science and Technology Agency; a Grant-in-Aid for Scientific Research (S) (17109013) from the Japan Society for the Promotion of Science; a Grant-in-Aid for Scientific Research on Priority Areas (17015032) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a Grant for The Third Term Comprehensive Ten-year Strategy for Cancer Control, and a Grant for Cancer Research from the Ministry of Health, Labor and Welfare of Japan.
The authors would like to thank Miss T. Shimooka, Miss Y. Nakagawa, and Miss M. Kasagi for their excellent technical assistance.