Several pancreatic cancer cell lines and human tissue

Several pancreatic cancer cell lines and human tissue samples were surveyed for cell surface, intracellular, and functional markers. CD20, CD24, CD44, CD133, CD184 (CXCR4), CD326 (EpCam), Sox-2, and OCT3/4 were examined in established pancreatic cancer cell lines. The functional marker, i.e., SP, was also determined in these cell lines. Side population was a term originally used to define a group of hematopeotic stem UMI-77 that had the ability to efflux the fluorescent vital dye Hoechst 33342 (Goodell et al., 1996). These cells have been shown to be long-term repopulating cells found in both normal tissues and cancer cells. However, when compare to non-SP, the SP has higher expression of genes linked to regulation of stem cell function and has been described in a variety of cancers (Wu and Alman, 2008). Since previous reports also suggest that the SP is influenced by cell culture density, we examined SP under 2 different density conditions (Tavaluc et al., 2007). The cardinal aim was to define a marker or a combination of markers that were common to all cancers tested, and to test its therapeutic resistance and tumor-initiating capacity. Various logistic combinations of surface markers and SP were examined; the combination of SP plus surface markers was found to be a potentially better indicator for PCSC in terms of tumor-initiating capacity. Using these techniques, we prospectively identified a population of novel PCSC in the testing phase. In the validating phase, these cells were subjected to a xenotransplantation assay and found to have superior tumorigenicity compared to controls. Our findings potentially indicate that PCSC are a very heterogeneous population of cells in terms of markers, the response to chemotherapy, radiation, and hypoxia. Importantly, we show a novel class of putative PCSC or pancreatic cancer cells with greater tumor-initiating capacity that has not previously been described.
Results
Discussion Despite significant research, the identity of pancreatic cancer stem cells remains controversial (Hermann et al., 2009). While different groups have identified CD44+CD24+CD326+ cells as cancer stem cells, others suggest that CD44+CD 24- and CD 44+CD133+ are also cancer stem cells (Kallifatidis et al., 2009; Li et al., 2007). Although the identity remains questionable, detailed proteomic and therapeutic studies have already been conducted on different putative cancer stem cells (Dai et al., 2010; Mueller et al., 2009; Rausch et al., 2010). The value of these types of studies would be improved if an agreement could be established on the identity of pancreatic cancer stem cells. This study was designed to examine previously described cancer stem cell assays in pancreatic cancer. Specifically, we examined surface markers, intracellular markers, SP, and effects of radiation, hypoxia, and gemcitabine. In studying the combinations of surface markers and SP, we have identified a novel combination that may be more tumorigenic than previously described CD44+CD24+CD326+ cells. By xenotransplantation, we find that SP/CD44-CD24-CD326+ cells are highly tumorigenic in pancreatic cancers. In this study of PCSC, we prospectively defined the characteristics of the population we thought would be the best pancreatic cancer stem cells. Most researchers in this field would agree that cancer stem cells are a small subset of cancer cells that have the ability to self-renew and maintain a tumor (Clarke et al., 2006). In accordance with the cancer stem cells hypothesis, we searched for a rare population of cells, since cancer stem cells are thought to be a very small fraction of the bulk of the tumor. We also searched for a population that would be present in the majority of cell lines, i.e., consistency between cell lines. A putative cancer stem cell would also likely be resistant to treatments such as chemotherapy and radiation. Finally, a cancer stem cell also must demonstrate self-renewal by growth in an immunocompromised mouse model.