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ORIGINAL ARTICLE
Year : 2012  |  Volume : 8  |  Issue : 3  |  Page : 399-403

Cell cycle analysis of the CD133 + and CD133 - cells isolated from human colorectal cancer


1 Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Gasteroentrology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
4 Department of Biostatistics and Epidemiology, School of Public Health, Isfahan University of Medical Sciences, Isfahan, Iran
5 Department of Surgery, Alzahra University Hospital, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Web Publication17-Nov-2012

Correspondence Address:
Marjan Gharagozloo
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.103520

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 > Abstract 

Aim: The CD133 antigen has been identified as a putative stem cell marker in colorectal cancer tissues. The aim of this study was to investigate the cell cycle state of CD133 + and CD133 - cells, isolated from primary human colorectal tumors.
Materials and Methods: After mechanical and enzymatic dissociation of the tumor samples, CD133 + and CD133 - subsets were identified and separated by magnetic cell sorting. Flow cytometric analysis was performed to compare the cell cycle of both CD133 + and CD133 - cells isolated from primary and liver metastatic cancer cells.
Results: The results indicated that CD133 + cells isolated from both primary and liver metastatic colorectal cancers were found in higher percentage in the G0/G1 phases. However, the CD133 - cells isolated from primary colorectal cancers were predominantly found in the S and G2/M phases. Surprisingly, the CD133 - cells isolated from liver metastatic colorectal cancers were mostly found in the G0/G1 phase.
Conclusion: The present study provides evidence that CD133 + cells are in a quiescent state in colorectal cancer, representing a mechanism that would at least partially explain chemotherapy resistance and tumor recurrence in post-therapy patients.

Keywords: Cancer stem cells, CD133, cell cycle, colorectal cancer


How to cite this article:
Gharagozloo M, Mirzaei HR, Bagherpour B, Rezaei A, Kalantari H, Sanei MH, Hosseini M, Mohajeri G, Tabatabai A, Hashemi M. Cell cycle analysis of the CD133 + and CD133 - cells isolated from human colorectal cancer. J Can Res Ther 2012;8:399-403

How to cite this URL:
Gharagozloo M, Mirzaei HR, Bagherpour B, Rezaei A, Kalantari H, Sanei MH, Hosseini M, Mohajeri G, Tabatabai A, Hashemi M. Cell cycle analysis of the CD133 + and CD133 - cells isolated from human colorectal cancer. J Can Res Ther [serial online] 2012 [cited 2019 Nov 13];8:399-403. Available from: http://www.cancerjournal.net/text.asp?2012/8/3/399/103520


 > Introduction Top


Colorectal cancer is one of the most common causes of cancer-related deaths in the world. [1] Rarely, it is diagnosed at an early stage and the available treatment, including surgery, chemotherapy, and/ or radiation therapy, are often not successful in completely eradicating the tumor. [2],[3] For many years, colorectal cancer has been explained as a histopathological sequence of events caused by the gradual alteration in the expression of oncogenes and tumor suppressor genes. [4],[5],[6] It has now emerged that a growing colorectal tumor is a heterogeneous mix of mostly differentiated primary cancer cells and cancer stem cells (CSCs), a rare population that has stem-like properties, including the capacity for self-renewal and a multipotency to differentiate. [2],[7],[8]

The existence of CSCs was established in human acute myeloid leukemia (AML). [9] Following that, CSCs were also detected in the breast, brain, and prostate cancers, as well as in melanoma and ovarian carcinoma. [10],[11],[12],[13],[14]

In recent times, CD133 has been extensively used as a marker for the identification of CSCs, from normal and cancerous tissues. Two independent studies identified CD133 as a marker for stem cells in colorectal carcinoma. [15],[16] These studies suggest that colorectal carcinoma is organized in a hierarchical fashion, in which the CD133 + tumor cells act as stem-like cells, and thereby, give rise to more differentiated tumor cells. [16] In fact, within the tumor, the differentiated primary cancer cells exhibit the characteristic of rapid proliferation, while the cancer stem cells are much slower at dividing, making them more resistant to conventional chemotherapeutic drugs. [7],[8] Following conventional treatment, such as chemotherapy, the majority of the tumor is eliminated; however, the CSCs can survive and differentiate. [17],[18] Thereby, the chemotherapy-resistant CSCs population causes a tumor relapse and eventually metastasis of the primary tumor mass. Therefore, a better understanding of CSCs is essential for understanding the biological and clinical consequences of the existing chemotherapy regimens and for designing new therapies to improve patient outcome. [8]

The current cell cycle data of colorectal CSCs have been limited to cell line studies, [19],[20] as currently, little is known about the cell cycle of cancer stem cells separated from tumor tissues. The present study is aimed at investigating the cell cycle status of both CD133 + and CD133 - cell populations isolated from human primary and liver metastatic colorectal cancers.


 > Materials and Methods Top


Colorectal cancer specimens were obtained from consenting patients undergoing a colon resection for colon adenocarcinoma. Histological diagnosis was based on the microscopic features of the carcinoma cells determining the histological type and grade. Tissue specimens were collected in serum-free Dulbecco's modified Eagle's medium (DMEM) containing 25 units/ml of penicillin and 25 μg/ml of streptomycin, and were kept at 4°C during the transfer to the laboratory. The cancer tissues were extensively washed in phosphate-buffered saline containing antibiotics penicillin (500 IU/ml), streptomycin (500 μg/ml), and amphotericin B (1.25 μg/ml). In order to prepare single-cell suspension, the tumor tissues were mechanically fragmented and digested by collagenase (1.5 mg/ml) and hyaluronidase (20 μg/ml) in DMEM/F12 containing Fetal Calf Serum (FCS) 2.5% and antibiotics, at 37°C, on a shaker, for 60 minutes. The cell suspensions were filtered through cell strainers (100 μM). The contaminating red blood cells were removed by incubation with ammonium chloride solution for 5 minutes, at 4°C. Subsequently, the cells were subjected to magnetic bead separation. All materials were obtained from Sigma, USA.

Human colorectal cancer cells were magnetically labeled and separated using a CD133 Cell Isolation Kit (Miltenyi Biotec, Germany). Magnetic cell separation was performed on tumor cell populations obtained from the enzymatic dissociation of colon cancer specimens using microbeads conjugated with CD133/1 (AC133, mouse IgG 1 , cell isolation kit, Miltenyi) and separated by a double passage, applying the eluted cells to a new positive selection column. Briefly, to isolate CD133 + cells using CD133 - conjugated super paramagnetic microbeads and Mini-MACS columns (Miltenyi Biotec GmbH, Germany), tumor single-cell suspensions were incubated with CD133 antibody conjugated to microbeads for 30 minutes at 4 o C. After incubation, the cells were washed in MACS buffer (Miltenyi Biotec GmbH, Germany) and centrifuged at ×300 g for 10 minutes. The cells were passed through a MiniMACS column retained in a magnetic field, and the column was washed with MACS buffer to remove the unbound cells. The eluent contained CD133 - cells, which were collected and used in subsequent experiments. The CD133 + cells were recovered by releasing the magnetic field and flushing the cells from the column. After magnetic sorting, both cell populations were counted on a neubaur counting chamber, and cell viability was assessed using trypan blue exclusion. The quality of sorting was controlled by flow cytometry with an antibody against CD133/2 (293C3-PE, Miltenyi) on both the CD133 + and CD133 - cell populations.

For cell cycle analysis, both CD133 + and CD133 - cells were suspended in 0.5 ml cold hypotonic solution containing 50 μg/ ml PI, 0.1% Triton ×-100, 0.1% sodium citrate solution, and 100 μg/ml RNase. The tubes were placed in the dark at 4°C. Analysis by flow cytometry was performed after a minimum of 30 minutes incubation time, within two hours of hypotonic treatment. Flow cytometric data were analyzed using WinMDI version 2.9. Cell cycle analysis of DNA histograms was performed using the Cylchred program.

Statistical analysis

To compare variables in both the CD133 + and CD133 - tumor cell subsets, the Mann-Whitney Test was performed. All differences were deemed significant at the level of P < 0.05. Confidence interval of 95% was considered for all tests. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 18.0 software (Chicago, Inc., USA).


 > Results Top


The present study has enrolled 20 patients, of age 45 years or older (mean age, 63.25 years; range, 45-79). Patient description and tumor characteristics are shown in [Table 1]. The cell cycle results of CD133 + and CD133 - tumor cells, isolated from primary and metastatic colon cancers are presented in [Table 2].
Table 1: Patient description and tumor characteristics

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Table 2: Cell cycle state of CD133+ and CD133- tumor cells

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A significant accumulation of cells in the G1 phase was observed in the CD133 + cells isolated from both primary and liver metastatic colon cancers. However, a higher percentage of CD133 - cells were accumulated in the S and G2/M phases, with a decrease in the G1 phase, demonstrating the different cell cycle pattern of CD133 + and CD133 - tumor cells [Figure 1]. Interestingly, in contrast to the CD133 - cells isolated from the primary colon cancer tissues, the CD133 - cells isolated from the liver metastatic colon cancers were in a higher percentage in the G1 phase of the cell cycle [Figure 2]. No significant association was identified between the tumor stage or grade and the cell cycle patterns of CD133 + and CD133 - cells.
Figure 1: Cell cycle distribution of CD133+ and CD133- tumor cells, isolated from primary and metastatic colon tumors

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Figure 2: Representative fl ow cytometric histograms of cell cycle analysis of CD133- and CD133+ cells isolated from primary or metastatic liver colon tumors

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 > Discussion Top


Stem cells are quiescent cells and localized at very specific niches in the organ. [21] Some prominent aspects of stem cells are slow cycling or fast cycling during homeostasis in vivo, low complexity of intracellular structure, small size with poor differentiation, and a high proliferative ability after injury or placement. [22],[23] Colon Cancer Stem Cells (CSCs) are shown to utilize characteristics commonly found in stem cell populations, albeit with aberrant regulation. [7] These cells can be isolated based on their phenotypes, cell culture methods, and their ability for dye elimination. [11],[24],[25],[26],[27],[28] Some cell surface markers are commonly used to identify and isolate cancer stem cells. In this study, we have used the CD133 antigen as a CSC marker in order to identify, isolate, and further investigate their cell cycle state within human colon cancer tissues.

We reported for the first time the cell cycle status of CD133 + and CD133 - cell subsets isolated from human colorectal cancer. The results reported here clearly showed that CD133 + cells were quiescent compared to CD133 - cells. This observation was in agreement with the previous stem cell theory, demonstrating the slow-cycling nature of stem cells. [22],[23] In contrast, two recent independent studies demonstrated that CD133 + cells from the colon cancer cell lines were more proliferative than CD133- cells. [19],[20] An explanation for this discrepancy was that it might be due to the perfect environment of the cell culture conditions, in which cells with stemness properties would grow and divide to produce progenitor cells; otherwise they would be lost in a few passages.

In comparison with CD133 + cells, a similar quiescent state was surprisingly observed in CD133- cells isolated from liver metastatic colon cancer, probably due to the transformation of CD133 + cells to CD133 - cell subsets or vice versa, in a metastatic state. This result was consistent with an earlier observation by Shmelkov et al., [29] that CD133 + tumor cells might give rise to a CD133 - cell subset during the metastatic transition. [28] It was also shown that prolonged culture of a pure CD133 + population resulted in the re-emergence of CD133 - cells in a HT29 colon cancer cell line. [30] Such a dynamic evoked the possibility of interconversion (plasticity) between CD133 + and CD133 - cell subsets. This equilibrium could be switched in one direction or another by tumor microenvironment cues. [30],[31]

A more recent study by Kemper et al.[32] has indicated that such a plasticity between two cell subsets is unlikely. They have shown the failure of the current commercial anti-CD133 antibodies in the detection of the glycosylated form of CD133 epitopes, such as the AC133 epitope. As the cell's glycosylation state changes upon its differentiation and/or malignant transformation, decreased glycosylation, possibly due to epitope masking by the differential folding of the CD133 protein, is correlated with the reduced detection of the AC133 epitope, on cell differentiation. [33] Therefore, differential glycosylation leads to alteration of the CD133 tertiary structure or localization of CD133 on the membrane, which prevents the antibodies from accessing their epitopes. [32]

On the other hand, another more recent study by Gupta et al. shows that there is a dynamic relationship among the subpopulations of human breast cancer for a given phenotypic state, over time. Using the Markov model, they have demonstrated that cancer cells can transit between phenotypic states stochastically. These findings demonstrate that the cancers are heterogeneous, while a phenotypic equilibrium probably exists in the populations of cancer cells. [34] Our result, together with the findings described above, indicate that CD133, as a colon CSC marker, must be used with caution and CD133 must not be used as a single CSC marker of colorectal cancer.

Collectively, our data indicate that CD133 + cells render a quiescent cell population in primary and liver metastatic colon tumors. Moreover, depending upon the aberrant niche microenvironments, there is a plasticity of CD133 expression in metastatic liver tumors, which at least partially explain chemotherapy resistance and tumor recurrence after therapy. To better understand the biological characteristics of CD133 + cells in primary colorectal cancer samples, more characterizations, such as, other antigen (e.g., CD29, CD44, and CD90 antigens) expression levels, cell proliferation, soft agar assay, and differential cytotoxicities of chemotherapeutic drugs against CD133 + and CD133 - cancer cell populations, will be needed, to support this conclusion. Future research must focus on better understanding and targeting of quiescent CSC populations, specifically identifying the regulators and factors that separate CSCs from normal stem cells.


 > Acknowledgments Top


The patients were selected from referrals to the Alzahra Hospital of the Isfahan University of Medical Sciences. This study was supported by grant 188120 from the Isfahan University of Medical Sciences. We wish to acknowledge Manizheh Narimani for her valuable assistance. The authors deeply appreciate all the patients and nurses who were involved in this study.

 
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    Figures

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    Tables

  [Table 1], [Table 2]


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