An accumulating body of evidence suggests that tumor heterogeneity exists in various types of cancers, including GBM 
. However, differential regulatory molecules and pathways specific to each tumor type are poorly understood. In agreement with a recent study by Anido et al. 
, we identified a subpopulation of GBM in which CD44 expression was upregulated (CD44high
GBM demonstrated correlation with poorer clinical prognosis. Xu et al. 
showed combined treatment of mouse intracranial tumors derived from a glioma cell line with CD44 antagonist and the current first line chemotherapy, temozolomide, prolonged survival of mice. Temozolomide is known to preferentially kill non-stem GBM cells 
. Collectively, these data raise a possibility that CD44-expressing GBM cells are relatively therapy resistant and likely a reasonable therapeutic target, especially in recurrent GBM tumors that survived over the current therapies. However, the case number of our comparison is still limited and a definite conclusion should be drawn with more accumulated data set in the future.
Here, we provide the first evidence for the presence of CD44v6 in BTSC derived from CD44high
GBM. Khan et al. 
suggested that CD44v6 regulates the aggressiveness of breast cancer cells. We found that both CD44v6 overexpression and OPN overexpression increased sphere forming ability of mouse intracranial tumor cells. In turn, knockdown of CD44v6 resulted in reduced growth of human BTSC derived from CD44high
GBM but not from CD44low
GBM in vitro
. On the other hand, the effect on serum-propagated cells from the matched CD44high
GBM was less prominent and not statistically significant. However, these data need to be carefully interpreted, as serum-propagated human GBM cells do express CD44v6 (). It is possible that targeting CD44v6 may reduce the growth of both BTSC and non-stem GBM cells with different potency. Future study is needed to address this question.
Interestingly, CD44v6 was not detected in normal mouse brains or neural progenitors (). The clear difference of CD44v6 expression between normal neural cells and glioma cells may indicate a potential therapeutic target molecule in GBM. The data in this study suggest that, in a subset of GBM, CD44v6 may preferentially target BTSC in GBM and such a treatment may not significantly affect the normal cells in the brain.
Several studies have demonstrated that elevated AKT expression in GBM correlates with poor clinical prognosis 
. Recently, Gallia et al. 
exhibited some data suggesting that inhibition of the AKT pathway eliminates the growth of GBM and GBM stem-like cells, implicating a role for AKT in BTSC survival and proliferation. Additionally, Eyler et al. 
provided evidence that treatment of BTSC with AKT inhibitors induces apoptosis, decreases motility and invasiveness of BTSC in vitro
, and inhibits tumor growth in vivo
in a xenograft model. In colon cancers, action of CD44v6 is likely mediated through the AKT pathway 
. Consistent with these findings, our data suggest that downstream targets of the CD44v6 action in BTSC include the AKT-mediated signaling pathway (). Knockdown of CD44v6 eliminated in vitro
growth of BTSC in CD44high
GBM (). In addition, a ligand for CD44v6, OPN, phosphorylated AKT in these cells (). These data may indicate that the activity of the AKT-mediated pathway may, at least in part, depend on the OPN-CD44v6 status. The experiments using PI3K/AKT inhibitors exhibited that various inhibition of AKT affected the neurosphere formation in CD44high
GBM cells, while CD44low
GBM cells appeared to be relatively less dependent on the AKT pathway (). Collectively, these results prompted a speculation that CD44v6-mediated AKT pathway plays a role in proliferation, specifically in CD44high
Another question still remains open. Both CD44high and CD44low GBM cells formed neurospheres without significant difference in their growth rate. Neurospheres derived from GBM157 (CD44low) had similar tumorigenic potential in comparison to CD44high neurosphere samples. These data suggest that CD44 and CD44v6 are not universally expressed by sphere-forming tumorigenic stem-like GBM cells. To determine what extent of GBMs are dependent on the CD44v6/AKT pathway and the mechanisms underlying the interaction between CD44v6 and AKT, future studies with larger numbers of GBM specimens and other ligands of CD44v6 will be required.
In conclusion, we identified that CD44high GBM relied on their variant form 6 for proliferation and conferred a shorter survival period on the patients. Our data suggested that the mechanism of the CD44v6 action on BTSC proliferation is mediated, at least in part, through its interactions with OPN and the subsequent activation of the AKT pathway. Collectively, targeting the CD44v6 pathway through inhibition of CD44v6 itself or its ligands appears to be a promising strategy for future therapeutic development for patients with CD44high GBM.