In this report, we provide evidence for the first time that CSCs exist in a mouse model of glioma, rendering support for the cancer stem cell hypothesis. We present several lines of evidence that, in the S100
glioma mouse model, CSCs are enriched in the side-population (SP) cells. SP cells exhibit increased self-renewal ability, as demonstrated by an increased percentage of secondary sphere formation () and generation of an increased number of SP cells upon culture (). Importantly, SP cells are more tumorigenic than non-SP cells (, Supplementary Table 3
While earlier studies suggested that CSCs are enriched in the CD133+ population in human gliomas, more recent studies indicate that not all glioma stem cells are CD133+. While we were able to show that CSCs exist in S100
gliomas, we were not able to purify CSCs using PROM1/CD133 expression. Emerging studies are consistent with this in showing that CD133 is not an obligate marker for CSC in gliomas; some CD133− cells have been shown to be tumorigenic and have the potential to give rise to CD133+ cells (11
). We do not yet know whether the S100
model is representative of human gliomas in which CSCs are CD133−, or whether CD133 antibodies that are available for mouse PROM1/CD133 do not have strong affinity for the glycosylated form of CD133 present on cancer cells.
Recent studies suggest molecular heterogeneity among CSCs (11
) suggesting that a single marker is unlikely to identify all CSCs, even within tumors of the same clinical grade from the same organ. We were unable to identify CSCs in S100ß-verbB;p53 gliomas using an immunophenotype (including ABCG2/BCRP1 or CD133 expression); hence, we used a cellular phenotype common to stem cells to sort for a specific subpopulation (side population) that enriches for CSCs. Staining for SP cells has been used to isolate bone marrow stem cells for many years (38
), and recently this technique has been adopted by many researchers to enrich for normal and cancer stem cells from multiple tissue types (28
). For example, Kondo et al. have shown that cancer-initiating cells of the C6 rat glioma cell line are enriched in the SP (28
), and others have previously shown that normal NSCs neurosphere cultures are enriched in the SP (29
), consistent with our observations (). However, some have reported increased cellular toxicity associated with Hoechst 33342 staining (a staining technique used to identify SP cells) questioning the usefulness of this technique to test for tumor initiation (40
). In our hands, we observe equivalent levels of cell death in FACS-sorted SP and non-SP cells, most likely from the sorting itself, suggesting that selective cell death is not the major reason for differential tumorigenic potential we observe with SP cells.
While the molecular heterogeneity of SP cells is unknown in this tumor model, the relative enrichment (10–20 fold) of CSCs in the SP compared to non-SP tumorsphere cells (, Supplementary Table 3
) is sufficient to enable identification of candidate genes that are differentially expressed in CSCs. By comparing purified populations of cells enriched in cancer stem vs. normal stem cells and in cancer stem vs. non-stem cancer cells, we identified a small number of genes whose expression distinguishes brain CSCs from neural stem and non-stem cancer cells. Notably, 23 of the 45 genes encode either secreted, membrane proteins or extracellular matrix components (), suggesting that a major distinguishing feature of the glioma SP cells we analyzed is their ability to interact with their microenvironment. The ability of cancer cells to establish themselves in a foreign cellular environment is an essential characteristic for successful metastasis and a defining characteristic of CSCs. Consistently, many genes on our list have known functions in mediating breast cancer metastasis. For example, a recent study has shown that FOXC2, a transcription factor on the list, is important in mediating breast cancer metastasis by regulating expression of genes that are involved in epithelial-mesenchymal transition (32
). Consistently, we observed higher levels of Snai2
, a transcription factor regulated by Foxc2
, in tumorspheres than normal neurospheres (Supplementary Table 5
). In addition, S100a4
was originally cloned as a gene that is differentially expressed in metastatic breast cancer cells, and it has been shown to have a causal role in mediating breast cancer metastasis (41
). Expression level of other genes on the list, such as S100A6, has been shown to correlate with pancreatic cancer prognosis (43
) and colon cancer invasion/metastasis (33
). These observations suggest an intriguing possibility that the same molecular pathways that regulate metastasis in neoplasms outside of the nervous system may also be involved in gliomas.
expression and function have been reported in some cell populations in the brain, this has not been previously reported in brain cancer cells. In rodent brains, S100a4
was reported to be expressed in a subset of white matter glial cells and reactive astrocytes after injury (45
). While we do observe S100A4+/GFAP+ reactive astrocytes in transplant-recipient mouse brains, we were able to specifically identify S100A4+ cancer cells, as the astrocytes were distinguishable from cancer cells by their normal cellular morphology and GFAP expression. (Supplementary Figure 2A
, and not shown). Similarly, S100a6
was reported to be expressed in the subventricular zone and ependymal layer of normal brain (46
), where Prom1
, and Nestin
(markers of NSCs) are also expressed. In human glioma samples, we observed that: 1) only a small subset of cancer cells express S100A4 and S100A6 (, ), and 2) many S100A4 and S100A6+ cells are associated with the stem cell niche in the brain, the endothelium (34
) (). In support of a link between these genes and CSCs, S100A4 and S100A6 expression has been reported to be associated with other stem cells (47
). We are currently testing the hypothesis that S100A4 and S100A6 may be new markers for CSCs by prospective sorting and transplantation of S100A4+ or S100A6+ cells in S100
gliomas and by examining co-expression of S100A4 and S100A6 with known stem cell markers in human gliomas.
Regardless of whether S100A4 and S100A6 expression identifies human glioma stem cells, our data indicate that the abundance of S100A4+ and S100A6+ cells in clinical glioma samples distinguishes undifferentiated, aggressive GBMs (grade IV) from grade III gliomas (p<0.001, ). Using 19 different primary human samples (triplicate plugs from each sample), we show that only grade IV GBM samples contain >8% S100A4+ cells. While a study involving a larger sample size is needed to further validate this finding, our pilot study provides a promising indication that the genes we identified by studying mouse gliomas may be useful in studying human gliomas. Considering the significant differences in the clinical outcomes and treatment regiments for GBM patients compared to those for patients with lower grade gliomas, a retrospective and/or a prospective study involving a larger set of samples to validate the use of S100A4 and S100A6 as biomarkers for GBMs and CSCs is a promising line of investigation.
While this manuscript was in revision, another group reported identification of a population of tumor-initiating cells in a mouse model of breast cancer (49
), supporting the existence of CSC-like cells in mouse models of solid tumors. Together with other studies using mouse models (19
), our study provides growing support for the cancer stem cell hypothesis. Therefore, in selected tumor models, murine cancer cells are organized in a hierarchy consistent with the cancer stem cell hypothesis, and that the understanding of the biology of CSCs (cell of origin, therapy resistance, and stem-niche interaction, for example) can be significantly advanced using these in vivo