In recent years, some malignancies have provided evidence supporting the notion that tumors are organized in a hierarchy of heterogeneous cell populations.1–3 Cancers are composed of heterogeneous cell populations ranging from highly proliferative immature cells to more differentiated cells of various cell lineages. The capability to sustain tumor formation and growth resides exclusively in a small proportion of tumor cells, termed cancer stem cells (CSCs) or tumor-initiating cells.4 Recent advances in stem cell research have facilitated the demonstration of the existence of CSCs in acute myeloid leukemia,1 breast cancer,5 and most recently, in brain tumors,6 prostate cancer,7 lung cancer,8 pancreatic cancer,9 melanomas,10 and retinoblastoma.11 Demonstration of CSC existence is accomplished through an experimental strategy that combines the sorting of tumor cell subpopulations, identified on the basis of differing expression of surface markers, with functional transplantation into appropriate animal models. These studies have shown that tumor-initiating cells are responsible for tumor formation and progression. The tumor clone is heterogeneous with respect to proliferation and differentiation. Interestingly, these CSCs share with other stem cell types, the key feature of self-renewal.
CD133 is a 5-transmembrane glycoprotein with a molecular weight of 117 kDa. It localized to membrane protrusions or microvilli. Antibodies to CD133 have been used to enrich for human hematopoietic stem cells,12 endothelial cells,13 neurons, and glial cells.14 CD133 is also expressed by the intestine-derived epithelial cell line Caco-2 in which it is downregulated upon differentiation.15 CD133 is believed to be the human ortholog of mouse Prominin, a protein expressed on the apical surface of neuroepithelial cells as well as several other embryonic epithelia and on brush border membranes of adult kidney proximal tubules.16 CD133 has also been found expressed on endothelial precursor cells and fetal neural stem cells as well as human prostatic epithelial stem cells.17–21 Recently, it was identified as stem cell marker of many tumors. In 2003, Singh et al22 showed that only a rare cell in human brain tumors expresses the neural lineage marker CD133. After purification, these CD133+ markers had self-renewing and differentiating ability in vivo. They could differentiate in culture into tumor cells that phenotypically resembled the tumor from the patient. Therefore, CD133 is believed to be a marker for stem cells in human brain tumors. In 2005, Collins et al19 identified the phenotypes in prostate cancer that expressed the prostate epithelial stem cell markers CD44+/a2b1hi/CD133+. They are the cause of tumor initiation and progression. In vivo tumor incidence of this subpopulation was greater despite injecting fewer than 500 cells. Other differentiated phenotypes, such as a2b1low/CD44+ (committed basal cells) and CD44−/CD57+ (secretory luminal cells), formed only rare tumors, even with an input of 106 cells. CD44+/a2b1hi/CD133+ is believed to be the maker of stem cell in prostate cancer.
Our preliminary experiment showed CD133 expressed lowly in Hep-2 cell line. CD133 positive cells possess a marked capacity for self-renewal, extensive proliferation, and multilineal differentiation potency in vitro. We supposed CD133 as a candidate of CSC in laryngeal carcinoma. In this study, the expression of CD133 was detected in a Hep-2 cell line. The result showed that CD133 positive cells were a minority in the tumor cell population. Applied with the magnetic cell sorting (MACS) technology, we reported the results of purifying CD133(+) cells from a Hep-2 cell line. Three-type cells' tumor-forming ability was examined in vivo to identify the marker of CSCs in Hep-2 cell line.
- Top of page
- MATERIALS AND METHODS
There is increasing evidence that cancers contain a small subset of their own stemlike cells called CSCs.1, 2 CSCs can self-renew to generate additional CSCs and also differentiate to generate phenotypically diverse cancer cells with limited proliferative potential.23, 24 This theory was first proved in leukemia. A subfraction of cells in acute myeloblastic leukemia (AML) resembled normal hematopoietic stem cells based upon morphological and immunohistochemical characteristics. It was found that this subset of cells, but not the rest of the tumor cells, could form AML when xenotransplanted into immunodeficient mice. The corresponding secondary AML in mice possessed histopathological characteristics similar to the primary tumor.1 Later, in solid breast cancer, the existence of CSCs was also proven in breast cancer. In this study, the authors used markers associated with normal ductal stem cells to positively (CD44) and negatively (CD24) sort cells. When small numbers of CD44 cells were injected into immunodeficient mice, tumors were formed at very high frequency, whereas the stem cell–negative fraction did not form tumors. The secondary tumors formed by CD44(+) cells were histologically similar to the primary tumors and also contained a subpopulation of CD44(+) and CD24(−) CSCs capable of forming tumors in other mice.5, 25 Studies have also shown that subpopulations of tumor cells from brain26 and prostate27 have stem-cell characteristics. Recent research also suggested that many cancer cell lines also contain CSCs. Using Hoechst 33342 staining and flow cytometry, Kondo et al28 examined a number of established cancer cell lines, including rat and human glioma cell lines and human neuroblastoma cell lines. These lines, all of which have been maintained in culture for decades, contained a small number of side population cells. They demonstrated that the side population cells, but not the non-side population cells, self-renewed in culture, were resistant to the anticancer drug mitoxantrone and formed tumors when transplanted in vivo. Our prophase investigations have shown only a small proportion of cells in Hep-2 cell line expressed CD133. CD133(+) cells possess a marked capacity for self-renewal, extensive proliferation, and multilineal differentiation potency in vitro. We supposed CD133 as a candidate of CSC in laryngeal carcinoma.29
In this study, a Hep-2 cell line of laryngeal cancer was also used. The quantitative presence of CD133-positive cells was analyzed by flow cytometry. About 3.15% ± 0.83% of the cells were found to be CD133(+) cells. The MiniMACS separation system was used to isolate different cells. We purified CD133(+) cells using the CD133 cell isolation kit. Flow cytometry was used to evaluate the efficiency of sorting. Purity of the CD133(+) population reached 90.26% ± 7.61% after isolation. A subsequent culture also demonstrated that the isolation kit did not influence cytoactivity of purified cells.
Cell growth activity and morphology in vivo could affect tumor-forming ability. H&E staining of CD133(+) cells and unsorted Hep-2 cells before animal experimentation showed that both groups contained the same malignant cells. All cells had a neoplastic appearance with large nuclei and prominent nucleoli. It suggested that CD133(+) cells were not nontumorous or heteromorphism stem cells. It is a subpopulation of Hep-2 cell line.
Traditionally, the heterogenic transplantation of cancer cells was carried out in nude mice with some immunologic function. This function could possibly influence the detection of CSCs in a solid tumor. The SCID mouse is characterized by an absence of functional T cells and B cells.31 It has therefore become the ideal animal model for CSC studies. We injected 5 × 105 sorted CD133(+), CD133(−) cells, and unsorted Hep-2 cells into SCID mouse, with each group having 20 injection sites. The results showed that CD133(+) cells possessed a strong ability to form tumors in vivo (p <.05). A later pathologic study demonstrated that the tumor came from human and was consistent with malignant tumor appearance.
Our study suggested that a hierarchy exists in Hep-2 cell line, because CD133(+) cells had a strong ability to form tumors compared with other Hep-2 cell subpopulations. In other words, a small subset of cells is enriched for clonogenic capacity. Compared with CD133(−) cells and unsorted cells, CD133(+) cells encompassed a small fraction of the cell line (3.15% ± 0.83%). Neither phase of cell cycle nor nontumor cell or migrating normal stem cells affected or increased proliferation ability. Integrated with our vitro experiment, only a small proportion (<5%) of cells in the Hep-2 cell line expressed CD133. CD133(+) cells possess a marked capacity for self-renewal, extensive proliferation, and multilineal differentiation potency in vitro. In this study, they also possessed the stronger tumor-forming ability in vivo. We have sufficient evidence to conclude that CD133 is one of the markers for CSCs in human laryngeal tumors, the Hep-2 cell line.
The identification of CSCs is an exciting area of cancer medicine. Not only does it allow a better understanding of tumor formation but it also allows the development of therapies that target the cell causing the cancer. Once the CSCs are isolated, we can study the properties of these cells and analyze their gene expression profiles using DNA/oligonucleotide microarrays, reverse transcription polymerase chain reaction, and cDNA subtraction methods. The signaling pathways, which required to maintain CSCs, could be identified. It would be the most effective way to discover new drugs for treating cancer. Purified CSCs could replace cell lines as an ideal study tool of laryngeal carcinoma. This new cancer model has significant implications for the design of future studies aimed at improving our ability to diagnose cancer and identify individuals risk for metastasis.32
Our study provides evidence for the origin of the Hep-2 cell line from CD133(+) enriched cells. Although CD133(−) cells were found to have poor reproductive activity, they did not completely loose the ability to reproduce. CSCs still exist in CD133(−) cells. This indicates that CD133 is not the exclusive marker of Hep-2 cell line stem cells. Recently, it was reported that CD44 as a marker for CSCs in primary head and neck cancer.33 There may be coexpressions with other special molecular markers. Further experiments are required to identify other markers of CSCs in Hep-2 cell line.
In our study, it is a pity that the phenotype of CSCs in primary laryngeal cancer was not involved. More research is needed to determine whether CD133 was also expressed in laryngeal squamous cancer and whether it was 1 of the makers of tumor-initiating cells.