A basic problem in cancer research is identifying the cells responsible for tumour formation. Within the cancer stem cell model, there is a small subset of cells capable of initiating and sustaining growth of a neoplastic clone. Tumour stem cells are probably long-lived cells that accumulate cancer-inducing mutations. Furthermore, they have the unique ability to self-renew and, through differentiation, to generate mature non-tumourigenic cancer cells of all lineages. These mature cells appear to constitute the bulk of cancer cells within a tumour.
If the cancer stem cell hypothesis is correct, we have to reconsider treatment regimens that eradicate the bulk of cancer cells, but may not target the cell of origin. These cells are thought to be refractory to classical chemotherapy and responsible for metastasis and relapse. Further characterization of the stem cell population is required to identify potential targets for prospective therapies [26
With this study, we are the first to characterize a large panel of colon carcinoma cell lines and their corresponding xenografts for simultaneous expression of several stem cell markers and thereby identify different cell fractions.
The cell surface antigens examined showed a distinct expression pattern within both colon carcinoma cell line and xenograft panels such that different cell fractions could be distinguished. In general, the cell lines expressed CD133, CD44, CD24, CDCP1 and CXCR4 at higher levels than the xenografts. The cancer stem cell hypothesis suggests that the heterogeneity in a tumour results from ongoing differentiation and the majority of tumour cells lose their proliferative potential during this process of maturation [1
]. This could be a reason why the antigens are expressed at a lower level in vivo
. During xenograft development, tumour cells lose their stem cell characteristics and also distinct cell surface antigens. It is already well known for haematological malignancies like multiple myeloma, acute myeloid leukaemia or acute lymphocytic leukaemia that differentiation is driven by cell surface antigens [29
]. Aside from differentiation, lower expression of these surface markers might be explained by the influence of the tumour microenvironment. Tumour cells growing subcutaneously in nude mice are exposed to a completely different environment than tumour cells in vitro
. It is possible that expression of these antigens would have been different if transplanted orthotopically, as it is unclear whether xenotransplantation accurately reflects human stem cell biology or whether the transplanted cells are human cancer cells that have adapted to the mouse environment [30
]. The appropriate microenvironment is essential for maintenance of "stemness" as has been shown for hematopoietic stem cells [31
], brain tumour stem cells [32
] and, more recently, colon cancer stem cells. Vermeulen et al. showed that factors secreted by myofibroblasts also restore the cancer stem cell phenotype in more differentiated colon carcinoma cells. Thus, it appears that "stemness" of colon carcinoma cells is a dynamic quality that can be influenced by the microenvironment [33
]. On the other hand, one could also argue that in vitro
culture conditions select for the maintenance of stem cell properties. In our experiments, we could show that colon cancer cells show very different phenotypes in vitro
versus in vivo
, thus suggesting a close relationship between the microenvironment and the existence of different cell types. This knowledge is also crucial for testing new drugs under in vitro
versus in vivo
conditions. [Of note, the procedure for preparing single cell suspensions from the xenografts had no statistically relevant influence on antigen expression (see Additional file 1
: Figure S7).]
It is not yet known to what extent single antigens are responsible for stem cell maintenance, thus we do not know the functional relevance of these cell surface markers. CD133-expressing colon cancer cells produce interleukin 4 (IL-4) as an autocrine growth factor. IL-4 promotes the induction of anti-apoptotic genes, thus promoting cell survival. Administration of a neutralizing IL-4 antibody improved the efficacy of conventional chemotherapy [34
]. These data suggest that elimination of CD133-expressing cells could prove beneficial in the treatment of colorectal carcinoma. Others have described the more tumourigenic capacity of CD133-expressing cells compared to cells that do not express this antigen [7
]. Also, different Wnt factors affect proliferation and differentiation in CD133-expressing cells [35
]. The Wnt signalling cascade has emerged as an important regulator of normal and malignant stem cells in intestinal, hematopoietic and epidermal systems [36
]. Since CD133 has thus been characterized as a putative stem cell marker, we aimed to further clarify its exact role via additional functional analysis.
Our data show that CD133 is not expressed by every colorectal tumour cell. In our experiments, tumours that grew out of a CD133-expressing cell population showed enhanced growth behaviour compared to the control group. We could not, however, clearly identify the role of CD133 in this process. Maybe this marker is crucial for the development of the tumour. Alternatively, perhaps it is a marker that does not initiate but rather enhances tumour growth, for example, by means of better tumour vascularization. CD133-expressing progenitor cells in the kidney contributed to better tumour vascularization by differentiating into endothelial cells [37
]. Beier et al. demonstrated that glioblastoma cancer stem cells can be either CD133+ or CD133-, suggesting that this marker is not limiting for "stemness" [38
]. This fits with our results indicating that tumours also grew out of CD133-negative cells. CD133 knock down experiments would be useful to further explore the functional relevance of this cell surface marker.
CXCR4 is said to play a key role in tumour progression and metastasis in colon cancer [39
]. In our study, the mean CXCR4 expression within the xenograft panel was 0.85% with notable expression of > 1% in one of fifteen models. Expression may have been upregulated had cells been injected in a more appropriate microenvironment, e.g., via orthotopic implantation [40
Our study is limited in that the relationship between cell surface antigens and cell properties, like growth behaviour, remains to be further elucidated. In haematological malignancies, identification of cells with different growth capacities is based on cell surface antigens and these exhibit functional heterogeneity [42
]. Flow cytometry is a widespread and reliable method to detect antigens on single cells [43
]. We analyzed cell surface antigens on single cell suspensions derived from xenografted tumours from our colon carcinoma cell line panel. Our studies revealed that some xenograft cells express more than one of the five surface markers we studied and significant correlations between different markers emerged. CDCP1 is highly expressed in lung and colon cancers, where it is phosphorylated by Src family kinases and involved in anchorage independence of cancer cells [16
]. These properties are important for tumour progression and metastasis. The correlation we observed between CDCP1 expression and disease stage of donor patients is in line with the results of Uekita et al. who described a correlation between expression and phosphorylation levels of CDCP1 with the invasive potential of scirrhous gastric cancers [45
]. These data point to the relevance of this marker as a potential therapeutic target for modulating cancer metastasis.
In addition to correlations between markers and clinical parameters, we found small subsets of double- and triple-positive cells in our colon cancer panel which could comprise a stem cell population and should be analyzed in more detail.