CD133 is considered a universal marker of organ-specific stem cells and tumor-initiating cells. As previous studies were based on the detection of CD133 by using commercially available antibodies, which may not recognize the full gamut of CD133 expression pattern, we sought to design a genetic model to monitor the expression of CD133 in normal adult murine epithelium and in colon cancers, using protumorigenic Il10–/–
deficient mice. In the present study, we have generated such a mouse model using the technology to introduce the reporter gene lacZ
in the CD133 locus, in which the endogenous alternative CD133 promoters (28
) control the expression of lacZ
. Unexpectedly, we discovered that CD133+
cells are not exclusively localized to stem cell niches. On the contrary, we detected CD133 in the majority, if not in all, of differentiated epithelial cells in organs with hollow ductal and nonductal luminal cavities, including the colon. We demonstrated that CD133 is expressed ubiquitously in the majority of the human primary colon tumor cells and in colon tumors emerging in Il10–/–CD133lacZ
mice. Notably, the majority of stromal and inflammatory cells were CD133–
in primary human tumors or murine Il10–/–CD133lacZ
-derived colonic tumors. In contrast, CD133+
cells coexpressing EpCAM were detected in human metastatic colon cancer, and both populations were capable of tumor initiation. Although both CD133+
cells isolated from metastatic colon adenocarcinomas can perform as cancer-initiating cells, the CD133–
subset results in the formation of more aggressively growing tumors. Cumulatively, our data show that CD133 expression is not restricted to organ-specific stem cells or to cancer-initiating cells in metastatic adenocarcinomas.
CD133 was originally shown to be primarily expressed on CD34+
hematopoietic stem and progenitor cells (1
). Subsequently, it was shown to be expressed on subsets of endothelial progenitor cells (4
). Using both the genetic model and immunohistochemical staining, we have discovered that in contrast to the hemangiogenic cells, CD133 expression in epithelial tissues is not restricted to stem or progenitor cells, but it is broadly distributed through the mature ciliated epithelial lining of luminal cavities. This is a surprising finding in relation to previously published data, which were obtained using immunohistochemical methods with commercially available antibodies. One explanation for this inconsistency could be hidden in the potential difference between the presence of mRNA and the CD133 protein in epithelial cells. Alternatively, this discrepancy may be due to the antibody affinity and different glycosylation and/or splice variants of CD133. However, our data demonstrate that CD133 immunohistochemical staining in both human and mouse tissues phenocopied the ubiquitous expression of CD133 in the transgenic reporter mouse model, strongly suggesting that previous studies might have underestimated the true spectrum of CD133 expression pattern in the adult tissues. These data suggest that CD133 expression is not restricted to organ-specific epithelial stem cells, but on the contrary, CD133 is expressed on differentiated epithelium. As such, CD133 mostly qualifies as a marker for identification of ductal and ciliated luminal epithelium but not as a specific marker of organ-specific stem cells.
Several studies have shown that CD133+
cells, but not CD133–
cells, derived from human colon carcinomas were the only cells that could initiate tumors in immunodeficient mice (22
). However, in our initial assessment, we found that 40% of the metastatic tumors were negative for CD133 expression, suggesting that CD133+
cells are not responsible for the growth of these tumors. On the contrary, in human primary colon cancer samples, we could not detect the EpCAM+
tumor cells. In fact, based on immunohistochemical analysis, the majority of primary colon cancer cells were CD133+
. This observation is also supported by the analysis of murine primary tumors in Il10–/–CD133lacZ
mice, in which all of the EpCAM+
tumor cells were also CD133+
. Most importantly, in both primary human colon tumors and colon tumors in Il10–/–CD133lacZ
mice, we were not able to detect a tumor epithelium that was CD133–
. Thus, the CD133–
population in primary tumors most likely consists of stromal, endothelial, and inflammatory cells but not tumorigenic cells of epithelial origin. Therefore, it is not surprising that in previous studies on primary colon tumors, only the CD133+
subset was shown to contain cancer stem cells. On the contrary, after metastatic transition, we identified a subset of EpCAM+
cells, suggesting that some epithelial cells within the primary tumor may downregulate the expression of CD133. Intriguingly, the CD133–
cells isolated from metastatic colon tumors were also capable of long-term tumor growth in a serial xenograft model. Of note, the injection of the CD133–
subset into immunodeficient mice resulted in more aggressive tumor growth, compared with growth of tumors that were initiated by the CD133+ fraction. These data suggest that CD133–
colon tumors might either be more genetically unstable malignant derivatives of CD133+
primary colon cancer cells or originate from an as of yet unrecognized CD133–
population of intestinal cells.
Further analysis revealed that CD133–
cells display other phenotypical characteristics of cancer stem cells. Metastatic CD133–
tumor cells are CD44+
, whereas CD133+
cells are CD44low
. The CD44+
cells were previously identified as cancer stem cells in human breast cancer (31
), and later CD44 was also suggested as a marker of colon cancer stem cells (32
). Therefore, our data indicate that the CD133–
fraction of colon cancer is more enriched for colon cancer-initiating cells. It is also remarkable that faster growing CD133–
metastatic colon cancer cells express CEA at a higher level, which is indicative of aggressiveness in colorectal cancer (30
). This also may explain high growth rate of CD133–
cells in vivo.
Cumulatively, our data show that in metastatic colon adenocarcinomas, tumor-initiating cells could be found in both CD133+ and CD133– fractions. Could this finding be due to the contribution of contaminating CD133+ cells to the growth of CD133– tumors? If this was true, then most likely, (a) CD133+ xenografts would grow faster and (b) serial transplantation of CD133– cells would result in the expansion of the CD133+ subset. On the contrary, our experiments demonstrated that CD133– cells not only maintained their tumor-initiating potential but also were endowed with the capacity to generate much larger tumors than CD133+ cells. We have also addressed the purity issue of the CD133– subset by immunohistochemistry, flow cytometry analysis, and quantitative PCR in serial xenografts. We could not detect any trace of CD133+ cells in serially transplanted tumors. These data suggest that it is unlikely that contaminating CD133+ cells were driving the growth of CD133– tumors.
As a few recent reports demonstrated that only CD133+
cells serve as tumor-initiating cells in colon cancer, it remains unclear why our findings are not fully consistent with previously published data (22
). An obvious explanation is that if all cancerous cells in primary colon tumors are CD133+
, then cancer stem cells would be contained only within this fraction. The emergence of the CD133–
subset could be indicative of the epithelial-mesenchymal transition and eventually lead to the formation of metastasis. Taking that into account, we predict that the serial transplantation of primary CD133+
tumors used in the aforementioned publications eventually would result in the formation of a CD133–
subset, which is also capable of tumorigenesis. It also should be pointed out that the concept of tumor-initiating cells is largely based on the capacity of a purified population of xenografted human tumor cells to evade the residual immune response of the murine NOD/SCID host and grow in an artificial microenvironment, in which specific cross-species compatible growth factors, elaborated by the murine stromal cells, could induce the proliferation of xenografted human cells. Thus, this xenograft assay by itself may fail to identify true cancer-initiating human cells that are not conducive to growth in a nonpermissive mouse microenvironment. Therefore, certain authentic human cancer-initiating cells may fail to form tumors in the incompatible murine microenvironment but would most likely grow in a genetically compatible human. Our data clearly demonstrate that more sensitive and reliable bioassays need to be devised to formally define a true cancer-initiating cell that will not be dependent on the immune status of the murine host or cross-species reactivity of protumorigenic growth factors. In the absence of such bioassays, one needs to appreciate the data demonstrating that NOD/SCID mice bred in various laboratory microenvironments, with different immune constitution, may permit the growth of different populations of human tumor xenografts, including CD133–
as well as CD133+
metastatic colon cancers.
Taken together, our findings demonstrate the following: first, CD133 protein is widely expressed on differentiated luminal and ductal epithelial cells in adult organs and therefore is not a specific marker of organ-specific stem and progenitor cells but rather a marker of differentiated ciliated luminal epithelial cells. Although organ-specific stem cells may express CD133, careful consideration must be taken in distinguishing them from CD133+ differentiated cells. As such, CD133 could be used for the identification of mature ciliated ductal and luminal epithelial cells. Second, the vast majority of primary colon cancer epithelial cells express CD133, while CD133– cells represent stromal elements of the tumor. Third, the expression of CD133 is downregulated in the subset of tumor epithelial cells after metastatic transition. Fourth, since CD133+ and CD133– subpopulations isolated from metastatic colon tumors are capable of long-term tumorigenesis, metastatic colon cancer-initiating cells could originate from CD133+ and/or CD133– cells.
Irrespective of whether tumor-initiating cells express CD133, most studies have overlooked what function CD133 protein may serve in directing tumor growth. Whether CD133 protein plays a role in supporting the growth of tumor is not known and is the subject of active scrutiny. However, our findings that CD133– cells are capable of tumorigenesis and form more aggressively growing tumors than CD133+ cells suggest that CD133 does not by itself exert a functional role in supporting tumor growth. These data are supported by the finding that colon tumors could also form in Il10–/–CD133–/– mice, again indicating that CD133 by itself may not be critical for tumor initiation. Nonetheless, identification of the function of CD133 may increase our understanding of its role in tumorigenesis.