We recently demonstrated that the transcription factor Hes3^9,10,11 is a marker of established neural stem cell cultures from the fetal and adult rodent central nervous system^{12,13,14,15,16}. Factors that promote the expansion of these cells, including Notch ligands, Angiopoietin 2, insulin, as well as inhibitors of the Janus kinases (JAK) and p38MAP kinases, also increase the expression of Hes3. In the adult rodent and primate brains, Hes3 identifies a perivascular cell subpopulation that co-expresses other markers of immature cells, such as Sox2. In this report, we demonstrate that HES3 not only provides a novel identifying marker for the CSC population in GBMs, but is a key mediator of the number of these cells, thereby offering a potential therapeutic target.

To address the potential value of Hes3 as a target in cancer medicine, we used primary cultured cells that were isolated from GBM biopsies. These cells were maintained in vitro using standard protocols for the establishment of non-cancerous neural stem cells cultures^19,20,21. These cells can be expanded with the support of the mitogen Epidermal Growth Factor (EGF) or basic Fibroblast Growth Factor (bFGF)²¹. Our previous work has established that the same cells used in this study can be propagated in culture over long periods of time and many passages, express several markers of NSCs, including, Sox2 and nestin, as well as prominin, can be induced to differentiate into neurons, astrocytes, and oligodendrocytes, they phenocopy the tumor of origin in xenograph experiments, and they respond to many growth factors that NSCs also respond to^21,22.

We recently showed that cultured NSCs express Angiopoietin 2 (Ang2) as well as its receptor, Tie2, and that treatment with Ang2 increases cell number both in vitro and in vivo^14,15. Here we show that GBM cells also express Ang2 together with the more established marker Sox2 (Fig. 1c). NSCs express a small number of GM1+ gangliosides on their cell surface which can be identified by binding to a fluorophor-conjugated B subunit of cholera toxin¹⁶; in contrast, their differentiated progeny expresses many binding sites. In our cultured GBM cells (in the presence EGF or bFGF), a very small percentage of cells labeled with conjugated cholera toxin (Fig. 1d–f). In contrast, when cells where treated with serum (which induces the differentiation of NSCs), the number of cholera toxin binding sites greatly increased. These results further support that the cultured GBM cells used express markers commonly associated with neural stem cells and that they possess the potential to differentiate into cells representing the main cell types of the nervous system.

Our previous work with NSCs showed that Hes3 expression is opposed by the actions of JAK. Its activity can be assessed by measuring STAT3 phosphorylation on tyrosine residue 705 (STAT3-Tyr), which is downstream of JAK²³. In contrast, Hes3 expression is supported in conditions where STAT3 is phosphorylated on serine residue 727 (STAT3-Ser)¹². NSCs cultured in bFGF exhibit low Hes3 mRNA levels, but these can be increased by concomitant exposure to Ciliary Neurotrophic Factor (CNTF) and JAK inhibitor (JAK I)¹², a treatment which elevates STAT3-Ser and inhibits STAT3-Tyr (Suppl. Fig. 1a,b). Similar results can be obtained by low concentrations of CNTF²⁴ that also elevate STAT3-Ser but not STAT3-Tyr phosphorylation (Suppl. Fig. 1c). The potential of JAK I to promote the undifferentiated state in NSC cultures is also demonstrated by the fact that it inhibits the expression of the glial marker GFAP in the presence of bFGF (Suppl. Fig. 1d). Low CNTF concentrations or concomitant high concentrations of CNTF and JAK I increase the number of cultured NSCs in the presence of bFGF (Suppl. Fig. 1e).

We hypothesized that GBM cells cultured in the absence of serum and the presence of mitogen would induce the expression of Hes3 and, therefore, they would resemble, in that respect, the cells of their origin in the patient. We tested the following four conditions: (a) EGF, (b) EGF + JAK I, (c) bFGF, and (d) bFGF + JAK I. We found that the highest STAT3-Ser and lowest STAT3-Tyr phosphorylation state was in the FGF + JAK I condition (Fig. 2a). In these conditions, cells were able to proliferate; in fact, their number was greater than in the other conditions, after one week of treatment (Fig. 2b). We note that inclusion of the JAK inhibitor allowed cell cultures to reach a higher terminal cell density, as assessed by cell number counting following a 2 week treatment (Fig. 2c,d). Hes3 expression varied among the four conditions; the highest incidence of Hes3+ cells was in the bFGF + JAK I condition (Fig. 2e,f). Since the culture system presented here lacks serum to support cell growth, the presence of a mitogen (bFGF or EGF) is required. As a consequence, we studied the effects of treatments (e.g. JAK I) in the presence of these defined factors.

In this manuscript we applied findings that we generated during our previous work with non-cancerous NSCs^{12,13,14,15,16}. This work showed that NSCs utilize non-canonical signal transduction pathways to regulate their survival and numbers in vitro and in vivo. Key to these non-canonical pathways is the phosphorylation state of STAT3. When STAT3 is phosphorylated on serine 727 in the absence of detectable phosphorylation on tyrosine 705, cells survive and proliferate in a self-renewing manner. In contrast, when the tyrosine residue is also phosphorylated, NSCs differentiate with a preference towards glial fates^19,25,26. STAT3-Ser phosphorylation promotes the expression of the transcription factor Hes3, which we showed to be a novel biomarker of established NSCs. In addition, Hes3 identifies new pools of cells in various areas of the adult brain which can be placed in culture where they exhibit self-renewal and multi-potentiality, generating neurons, astrocytes, and oligodendrocytes.

We also showed that Hes3 is expressed in tumor tissue from glioblastoma patients, and that it co-localizes with the putative CSC marker prominin. Here, we extended our studies to show that (1) primary GBM cell cultures also express Hes3 under conditions that support STAT3-Ser phosphorylation and suppress STAT3-Tyr phosphorylation and that (2) Hes3 interference reduces the numbers of cells in culture.

These early results suggest that Hes3 is a new marker of GBM cells and a mediator of cell expansion. Future studies will address the relevance of Hes3 in cultures from different GBM subtypes (e.g., as defined by The Cancer Genome Atlas). It will also be important to address the mechanisms that regulate the cell number changes reported here. For example, our results help distinguish between cell expansion rates and maximal cell density. Specifically, whereas inclusion of the JAK I in the presence of EGF did not alter cell number during the early culture phase, it did allow cells to reach higher densities. Given that JAK is implicated in the canonical Notch signaling pathway and subsequent gliogenic differentiation²⁷, it is intriguing to speculate that non-canonical Notch signaling branches which are opposed by JAK¹² may be competing with canonical pathways and that Hes3 may be a critical regulator of the balance between these two branches.

Our previous work with non-cancerous NSCs showed that activation of the STAT3-Ser/Hes3 signaling pathway promotes cell survival without altering the cell cycle duration¹². Whether this applies to GBM cells, will have to be determined in future studies.

It is difficult to define a cancer stem cell population. The GBM-derived cells used in this work, however, do exhibit CSC properties, as suggested by their (1) expression of undifferentiated markers, (2) ability to differentiate into neuron and glia - like cells, and (3) ability to phenocopy the tumor of origin in immunocompromised mice. These results suggest that Hes3 may be relevant as a target for the elusive CSC population of GBM and, possibly other solid tumors such as breast, colon and pancreatic cancers^{28,29,30,31,32}. Preliminary results show that in spontaneously arising brain tumors in adult mouse null for the p53 tumor suppressor gene, Hes3+ cells are clearly seen both within the tumor mass (identified by morphology and staining for KI67), as well as outside the borders of the tumor (data not shown). Additional work will require validation in multiple in vitro and in vivo paradigms.