The cancer stem cell hypothesis has gained a great deal of attention in recent years, with reports describing such cells in many different types of human cancer. But to date, there have been few studies of TPCs in animal models of the disease. In the current studies we identify TPCs in the most commonly used animal model of medulloblastoma, the Ptc mutant mouse. The cells we describe differ from previously identified brain tumor stem cells in that they do not express the stem cell marker CD133 and cannot form neurospheres. Instead, they express the neural progenitor marker Math1 and can be enriched based on their expression of the carbohydrate antigen CD15. Transplantation of Math1+CD15+ cells is sufficient to give rise to tumors in 100% of hosts, and these tumors resemble the original tumors in terms of histology and cellular heterogeneity. Based on these findings, we conclude that the Math1+CD15+ compartment contains TPCs.
The nature of the cells that initiate and propagate medulloblastoma has been the subject of debate for many years (Eberhart, 2007
; Katsetos et al., 2003
; Read et al., 2006
). The fact that these tumors are frequently found on the surface of the cerebellum and express markers associated with the granule lineage has led many investigators to speculate that these tumors arise from GNPs (Kadin et al., 1970
; Miyata et al., 1998
). On the other hand, the demonstration that many medulloblastomas express stem cell markers, form neurospheres and are propagated by CD133+ cells has raised the possibility that these tumors may arise from, and be propagated by, neural stem cells (Hemmati et al., 2003
; Singh et al., 2003
; Singh et al., 2004
). Recent studies by our lab and others (Schuller et al., 2008
; Yang et al., 2008
) have shed light on the cell of origin for medulloblastoma, demonstrating that Hh pathway activation in either stem cells or GNPs can lead to medulloblastoma. But while these studies provided insight into the normal cells that can give rise to tumors (the cells of origin), they did not identify the cells are critical for propagation of tumors once they are established (the TPCs). In the current study we show that tumors from Ptc+/-
mice are propagated by GNP-like cells, and in particular, by a subset of these cells that expresses CD15.
CD15 is expressed on both progenitors and stem cells in the embryonic and adult central nervous system (Capela and Temple, 2002
; Capela and Temple, 2006
). In light of this, one possible interpretation of our findings is that CD15+ cells in Ptc+/-
tumors might represent neural stem cell-like cells. However, several observations argue against this notion. First, the fact that CD15 is expressed on a subset of GNPs in the neonatal cerebellum (Figure S2
and (Marani and Tetteroo, 1983
)) indicates that CD15 expression is not exclusive to stem cells. The fact that the CD15+ cells in our tumors co-express Math1, are unable to form neurospheres and show no evidence of multi-lineage differentiation also suggests that these are not stem-like cells. Thus, we believe that the CD15+ cells we have isolated are progenitor-like cells with a unique capacity for tumor propagation.
CD15+ cells propagate tumors following transplantation, and when they represent a small subset of the cells in a tumor, isolating them significantly enriches for tumor propagating capacity. However, it is important to note that CD15+ cells sometimes represent a fairly large fraction of the tumor. The high percentage of cells expressing a marker of TPCs is not unprecedented: in human brain tumors, CD133 has been reported to be found on up to 40% of cells (Singh et al., 2003
; Singh et al., 2004
). Whether this means that a large percentage of cells in the tumor are capable of tumor propagation (Kelly et al., 2007
), or whether the true TPCs represent a subset of the CD133+ or CD15+ cells, will need to be examined by sub-fractionating each population with additional markers.
To investigate the molecular basis for the tumor-propagating capacity of CD15+ cells, we performed microarray analysis on CD15+ and CD15− cells from Ptc+/-
tumors. This analysis revealed that CD15+ cells have a distinct gene expression profile characterized by increased expression of genes associated with proliferation and self-renewal and decreased expression of genes involved in apoptosis and differentiation. Consistent with this expression profile, we have also observed that CD15+ cells are more proliferative than CD15− cells, both in vitro and in vivo. This property distinguishes CD15+ cells from the tumor-propagating cells found in human leukemia, which are more quiescent than the bulk of the tumor (Bonnet and Dick, 1997
; Holyoake et al., 1999
). On the other hand, CD133+ cells from human brain tumors have been reported to be more proliferative than their CD133− counterparts (Singh et al., 2004
), perhaps indicating a distinction between TPCs in the nervous and hematopoietic systems. Interestingly, we have found that CD15+ cells in the neonatal cerebellum also have higher proliferative capacity or higher intrinsic Hh responsiveness than CD15− cells. It is possible that the tumors in Ptc
mutant mice arise from these cells and retain this proliferative capacity. Alternatively, it is possible that tumor cells acquire these properties, and concomitantly acquire expression of CD15. Further studies will be necessary to determine the relationship between normal and neoplastic CD15+ cells and the mechanisms that regulate CD15 expression.
Our observation that CD15+ cells recapitulate the heterogeneity within the original tumor, and in particular, generate CD15+ as well as CD15− cells, suggests that these cells may sit at the top of a hierarchy of differentiation within the tumor. Although our data indicate that many CD15− cells represent tumor cells (based on expression of Math1 and loss of Ptc
), these cells appear to be less proliferative than their CD15+ counterparts. However, as shown in and Figure S5
, these cells are not completely quiescent. Indeed, since CD15− cells are frequently much more abundant than CD15+ cells, they may contribute substantially to the growth of tumors in vivo. In the context of a rapidly growing tumor, it may be critical to eliminate both the tumor-propagating cells and their more differentiated progeny in order to effectively eradicate the tumor.
The identification of CD15 as a marker for tumor-propagating cells in a mouse model of medulloblastoma raises the question of whether it is relevant for human medulloblastoma as well. Our observation that CD15 is expressed in a subset of human medulloblastomas is consistent with this possibility. Since we identified CD15 as a marker of TPCs in tumors from Ptc+/-
mice, which are thought to be genetically similar to human nodular/desmoplastic medulloblastomas (Goodrich et al., 1997
; Pietsch et al., 1997
; Pomeroy et al., 2002
), we expected that CD15 expression might be more common in this subtype of tumor. However, our immunohistochemical and flow cytometric analysis suggest that CD15 expression is not restricted to such tumors, and can occur in a variety of medulloblastoma subtypes. These findings indicate that tumor morphology and the presence of Hh pathway mutations may not be critical determinants of CD15 expression, and suggest that the role of CD15 should be investigated in all medulloblastoma subtypes.
Support for the notion that CD15 may have significance in human medulloblastoma comes from our demonstration that a CD15+ gene expression signature can predict survival in medulloblastoma patients. One interpretation of this finding is that tumors with strong expression of the CD15+ signature contain a large percentage of CD15+ cells, and that having many such cells makes them particularly aggressive. Alternatively, it is possible that expression of the CD15+ signature does not reflect expression of CD15 itself, but rather the presence of properties (increased proliferative capacity, decreased tendency to differentiate or die) that are associated with CD15+ cells. In either case, the signature we have derived from our studies of TPCs in mice may be a valuable tool for predicting outcome in human medulloblastoma patients. Such tools are critical, since most medulloblastoma patients are treated with an aggressive regimen of radiation and chemotherapy; while this results in high cure rates, it is associated with severe side effects including cognitive deficits, endocrine disorders and an increased risk of secondary tumors. Stratification of patients based on predictors of outcome such as the CD15+ signature may allow clinicians to limit use of the most aggressive therapies to patients with poor prognosis, and to use less toxic but equally effective treatments for patients whose prognosis is more favorable.
In summary, our studies identify a population of TPCs in a mouse model of medulloblastoma. The fact that these cells express markers of progenitors and cannot form neurospheres indicates that TPCs need not resemble normal stem cells. Rather, some tumors may be propagated by cells that resemble neural progenitors. Identification of TPCs in a mouse model of human medulloblastoma will allow us to study the mechanisms by which they propagate tumors and test approaches to targeting them in vivo. Ultimately, such studies will be necessary to determine whether eradication of TPCs cells is critical for effective therapy of brain tumors.