CD133 has been used as a marker of tumor-initiating cells in neural cancers and is also generally accepted as a CSC marker for colon cancer [10–12]. However, there are some reports suggesting that CD133+ cancer cells are not a true representation of CSCs in colon cancer [39, 40]. We found that CD133+ colon cancer cells isolated from the HCT116 cell line had a greater clonogenic and tumorigenic ability than CD133- cells irrespective of CXCR4 expression. The in vitro and in vivo assays lend credence to the viewpoint that CD133 could be a marker for colon cancer tumor-initiating cells.
In 2005, Brabletz et al. proposed the concept that there are two forms of CSCs in tumor progression, namely stationary CSCs and migratory CSCs . Hermann and colleagues published data supporting the existence of these two distinct subsets in CD133+ pancreatic CSCs. The CSCs co-expressing CXCR4 were cancer cells with a migratory and invasive phenotype in pancreatic cancer . In specimens from CRC patients,Pang et al. demonstrated the existence of migratory CSCs with the CD26 surface antigen as a marker . In this study, we determined that the percentage of CD133+CXCR4+ cancer cells in metastatic liver tumors was nearly eight times higher than that in primary colorectal tumors, indicating enrichment of this CSC subpopulation in metastatic liver tumors and their potential involvement in CRC metastasis to the liver. Transwell migration and invasion assay results indicated that the CD133+CXCR4+ subpopulation had higher migratory and invasive capacities in vitro. Consistent results were obtained by the standard tail vein metastatic assay in vivo. This indicated that CD133+CXCR4+ cancer cells are a subpopulation of CSCs with a metastatic phenotype. To evaluate the metastatic capacity of different subpopulations, we employed the tail vein metastasis model, which is also known as the experimental metastasis model. The limitation of this model lies in the fact that it cannot reflect the complete metastatic process as does the spontaneous metastasis model in which the tumor cells are injected into the liver and allowed to first form a primary tumor. The complete metastasis cascade includes the following steps: escape of cells from the primary tumor, entry of cells into the lymphatic or blood circulation (intravasation), survival and transport in circulation, escape of cells from circulation (extravasation), and growth of cells to form secondary tumors in a new organ environment . However, the tail vein metastasis model is able to mimic the extravasation of cancer cells from blood vessels in the target organ which is regarded as a critical step in the metastatic process. Therefore, as in many studies[17, 44, 45], it is sufficient to use this model for the comparison of metastatic capacity among different groups.
EMT results in morphological and molecular changes that occur when epithelial cells lose their characteristics and gain mesenchymal properties. The expression of mesenchymal markers, such as N-cadherin and vimentin, and the loss of E-cadherin are key molecular events of EMT. Transcription factors, such as Snail and Twist, bind to consensus E-box sequences in the E-cadherin gene promoter and down-regulate E-cadherin transcription [46, 47]. The association between EMT and CSC has been reported previously. Several studies have provided evidence showing that CSCs express EMT markers and that induction of EMT could convert epithelial cells into breast CSCs [27–30]. This demonstrates the essential role of EMT in CSCs acquiring invasive and metastatic phenotypes. We have proven our hypothesis that EMT is involved in the origin of migratory CSCs in colon cancer, using real-time RT-PCR to determine EMT-related gene expression. Pang et al. reported that EMT-like attributes contribute to the invasive phenotype and metastatic capacity of the migratory subpopulation in CRCs . This is in line with our findings that the corresponding alteration in mRNA expression levels of EMT-related genes and higher migratory and invasive capacities have been observed in CD133+CXCR4+ cancer cells. Furthermore, we found that treatment with SDF-1 could further induce the occurrence of EMT in CD133+CXCR4+ cancer cells. The above data indicate that the CD133+CXCR4+ subpopulation contributes to liver metastasis of colorectal cancer via EMT.
Consistent with our findings, Esther and colleagues demonstrated that transforming growth factor-β (TGF-β) induced the EMT process and de-differentiation in Fao rat hepatoma cells. This process coincided with upregulated CXCR4 expression and also sensitization of these cells to respond to SDF-1, which mediated migration . Similar results were observed in oral squamous cell carcinoma [26, 49]. However, the reason cancer cells that have undergone EMT have a higher expression of CXCR4 is far from clear. Exploring the origin of migratory CSCs warrants further research and requires integration of current tumor initiation and progression concepts, including CSC, EMT, accumulation of genetic alterations and the tumor environment as driving forces . A deeper understanding of these factors could provide further insights into tumor biology.
The CSC hypothesis suggests that CSCs are a minority population that has the potential to self-renew, differentiate and regenerate a phenocopy of the original tumor. They would seem the most probable candidates that are resistant to chemotherapy, and they have been investigated previously [3, 5, 50–52]. Novel treatments targeting CSCs may result in the complete eradication of tumor growth, and furthermore, based on the migratory CSC theory, if treatment targeting migratory CSCs can be developed, it might be possible to prevent tumor metastasis. We hypothesized that blockade of the SDF-1/CXCR4 axis might suppress colon cancer metastasis to the liver, with the knowledge that the liver secretes high amounts of SDF-1 . This is also in line with the theory that organs producing SDF-1 attract CXCR4+ tumor cells and form metastatic tumors analogous to the directed homing of leukocytes. In our study, a nude mouse hepatic metastasis model was employed, and the results indicated that chemical inhibition of CXCR4 with AMD3100 could inhibit colon cancer metastasis to the liver. The anti-metastasis effect caused by the blockade of the SDF-1/CXCR4 axis is supported by another report . This finding provides important clues for the development of a targeted therapy in the treatment of CRC.
To validate the above findings in in vitro experimental and in animal models, we carried out a prospective study to investigate whether CD133+CXCR4+ cancer cell content was associated with disease progression and prognosis. Statistical analysis showed that high CD133+CXCR4+ cell content is associated with poor 2-year survival of colorectal cancer patients. The clinical data provide evidence to support our hypothesis that double positive cancer cells might be involved in the metastatic process. Our data showed that cancer located in the rectum was associated with a high content of CD133+CXCR4+ cancer cell compared with colon cancer. This might be due to higher CXCR4 expression in rectal cancer than in colon cancer, suggesting that the percentage of CD133+CXCR4+ cancer cells in future studies should be investigated separately in colon and rectal cancer rather than in a mixed way.