In most normal organ systems, such as the blood, CNS, and skin, cells are functionally and phenotypically organized according to a strict cellular hierarchy in which self-renewing stem cells give rise to short lived progenitors then terminally differentiated effector cells. The earliest studies in acute myeloid leukemia (AML) demonstrated that tumor cells resembling normal hematopoietic stem cells can self-renew and give rise to relatively differentiated and non-tumorigenic blasts (4
). Therefore, it has been generally assumed that cancers are organized in a hierarchical manner similar to normal tissues. However, several CSC populations have been identified in PDAC, and it is not clear how each of these fits into a specific hierarchy or are related to one another. One possibility is that all of the current markers recognize the same cell, but the vast majority of ALDH+
pancreatic tumor cells appear to lack CD44 and CD133. Therefore, it is likely that these antigens identify at least two, or even three, unique cell populations (6
). Alternatively, since each putative CSC marker enriches for cells with increased tumorigenic potential but fails to isolate pure populations of CSCs (i.e.
, every cell expressing a specific marker is not tumorigenic), it is possible that combining antigens will greatly increase CSC purity. However, this does not appear to be the case as the tumor initiating cell frequency of rare PDAC cells co-expressing CD44, CD24, and ALDH is not significantly greater than either ALDH+
). Moreover, c-Met is expressed, albeit at variable levels, on CD44+
, or ALDH+
cells, but increased tumorigenic potential is limited to CD44+
The significance of the various pancreatic CSC markers and the cells they identify clearly requires further clarification. If multiple CSC populations actually exist, an understanding of how they are related to one another will be important since clinically effective targeting likely requires the elimination of all self-renewing cells within the tumor. One possibility is that PDAC cells are organized in a hierarchical and linear manner with a single, phenotypically distinct CSC at the apex giving rise to the other CSC populations and ultimately non-clonogenic mature tumor cells (). It is also possible that each phenotypically distinct CSC population represents a specific cellular state of the same clonogenic cell that gives rise to mature tumor cells. Another possibility is that each CSC population is unrelated to another and parallel lines of mature tumor cell production exist. Finally, it is conceivable that a rigid hierarchy of unidirectional differentiation does not exist, but that the system is plastic with non-clonogenic cells giving rise to tumorigenic CSCs displaying a variety of phenotypes. In order to better understand how different CSCs are related to one another, studies examining the overlap between putative CSC populations and the cell types that arise from each specific CSC are needed.
Potential relationships between CSCs and mature tumor cells
Beyond the organization of phenotypically defined CSC populations, it is also unclear whether the various CSCs are functionally similar or distinct. Although tumor formation, histologic recapitulation, and self-renewal define CSCs, other properties, including relative drug resistance, invasion, migration, and metastatic potential have been ascribed to CSCs and may contribute to their clinical impact (28
). It is possible that certain CSC populations could be primarily responsible for tumor initiation and maintenance at the primary site of disease, whereas others could be responsible for tumor dissemination and growth at metastatic sites, such as the subpopulation of CD133+
CSCs expressing CXCR4 (7
). It is also possible different organs, such as the liver and lung, harbor different microenvironments with distinct endothelial or stromal cell types or extracellular matrix components that promote or inhibit tumor growth () (29
). Therefore, if metastatic dissemination depends on the interaction of CSCs with a particular niche, then different niches might call for unique CSCs. An evaluation of the tumor forming potential of specific CSCs at orthotropic and different metastatic sites may determine whether certain populations are better suited to grow within particular locations.
Potential functional relationships between CSCs
It is also possible that the phenotypes exhibited by CSCs are dictated by the external microenvironment. For example, pancreatic tumors are characterized by desmoplasia and dense fibrosis that may expose cells to relative hypoxia, and the hypoxic state has been found to alter the expression of the CSC marker CD133 in brain tumors (30
). In addition, several markers used to identify CSCs, such as ALDH and the side population assay, are indicative of drug resistance mechanisms and it is possible that their expression is induced in response to cellular damage. Finally, it is possible that the adaptive metabolic changes undertaken by tumor cells also modifies the expression of CSC makers, although such findings have yet to be reported (31
Recent studies have demonstrated a clear link between CSCs and the epithelial-to-mesenchymal transition (EMT) in solid tumors. Therefore, it is possible that CSCs represent a specific cellular state expressing multiple phenotypes. Reports using breast cancer models have demonstrated that the induction of EMT by TGF-β or the modulation of specific gene expression (e.g.
, induction of Twist
or repression of E-Cadherin
) results in increased expression of CD44 and tumorigenic potential (32
). In pancreatic cancer, ALDH+
cells appear have a gene expression profile consistent with EMT and increased invasive and migratory potential compared to bulk tumor cells and CD44+
). Moreover, studies examining ZEB1, an inducer of EMT, in pancreatic cancer cells have identified a direct link between EMT, increased tumorigenicity, and drug resistance (34
). Therefore, it is possible that a more “epithelial” or “mesenchymal” state is important in determining the functional properties of CSCs. The specific functional properties of different pancreatic CSCs are unclear, and the quantification of tumor formation, metastatic potential, and drug resistance is needed.