This is the first report documenting the immunological significance of the COX-2 pathway in gliomagenesis in mice. In the current study, treatment with ASA or celecoxib inhibited systemic PGE2 production and delayed glioma development. This was especially true when the treatment was initiated shortly after the glioma-induction, suggesting significant involvement of the COX-2 pathway in the early stage of gliomagenesis. We also demonstrated a pivotal role of MDSCs and the MDSC-attracting chemokine CCL2 in the COX-2-mediated gliomagenesis.
The role of PGE2
in MDSC development has been reviewed (23
). In particular, Ep2
(one of PGE2
receptors)-deficient mice bearing 4T1 mammary carcinoma demonstrated a decrease of MDSC development and delayed tumor growth compared with WT mice (5
). In the current study, we demonstrated for the first time that COX-2 blockade by NSAIDs as well as genetic deletion of Cox-2
suppress glioma development in mice primarily by inhibiting MDSC accumulation in the TME (–). We also observed a negative correlation between MDSCs and degranulating (CD107a+
T-cells in the TME ( and ), suggesting that MDSCs suppress the effector function of CD8+
T-cells in the TME and thereby facilitate glioma progression.
Although the ASA treatment inhibited systemic PGE2
levels regardless of the timing to be initiated, it did not provide any therapeutic benefit when it was initiated after the tumor had already been established ( and Supplementary Fig. S1
). Similarly, while homozygous Cox-2−/−
mice demonstrated remarkably prolonged survival compared with Cox-2+/+
<0.0478), heterozygous Cox-2+/−
mice demonstrated only modest, albeit significant, prolongation of survival and reduction in plasma PGE2
levels compared with Cox-2+/+
mice (). Furthermore, the heterozygous Cox-2+/−
mice did not demonstrate significant differences in chemokine expressions and BIL profiles compared with Cox-2+/+
mice (). These data, together with remarkable suppression of PGE2
levels by the ASA treatment (), imply that a substantial threshold of PGE2
suppression has to be achieved to observe the clinical benefit of this strategy. These observations also suggest that ASA administration in humans might be only beneficial in prophylactic settings but not in patients with clinically diagnosed gliomas. Consistent with our interpretation, a recent study has demonstrated that long-term use of ASA reduces cancer risks in human (24
With regard to the effects of ASA on each of the two distinct MDSC populations (Ly6Ghi
gMDSCs and Ly6G−
mMDSCs), the ASA treatment preferentially reduced gMDSCs than mMDSCs in the organs tested (). Taken together with a previous study demonstrating that under inflammatory conditions, expansion of gMDSCs occurs at a greater degree than that of mMDSCs (25
), gMDSCs may be more susceptible than mMDSCs to PGE2
-mediated expansion and NSAID-mediated suppression. With regard to their functional differences, several studies have collectively indicated that gMDSCs preferentially express arginase-1 through the STAT3 signaling whereas mMDSCs express iNOS through the STAT1 signaling (7
). Of interest, this proposed paradigm contrasts to our data demonstrating that gMDSCs also express iNOS that can be inhibited by ASA (). Therefore, it is possible that our observations are unique to gliomas. Further studies are warranted to elucidate the mechanisms underlying the suppressive effects of each of MDSC populations in both human and mouse cancers located in a variety of organs.
CCL2 is known to be one of the primary chemokines attracting MDSCs towards TME (8
), which is consistent with our observation that genetic depletion of Ccl2
induced a significant decrease in MDSC accumulation in the TME (). In detail, both gMDSCs and mMDSCs express the receptor CCR2 although its expression levels have been shown to be higher on mMDSCs than gMDSCs (26
), suggesting that the CCL2-CCR2 chemokine-receptor axis can mediate the migration of both gMDSCs and mMDSCs.
Simultaneously, our data showed that CCL2 blockade (and resulting inhibition of MDSCs) led to increases of Cxcl10
expression and CD8+
T-cell infiltration in the TME ( and ). This contrasts to a previous study demonstrating that CCL2 mediates accumulation of activated T-cells in a glioma xenograft (27
). We think that this difference reflects the immunocompetent status of mice in our spontaneous tumor model versus the xenograft model that requires immune deficient hosts. Given that CCR2, a cognate receptor for CCL2, is expressed in a wide range of immune cells including monocytes, MDSCs, Tregs, and effector T-cells (28
), the CCL2-CCR2 axis operates in all cell populations that express the ligand CCL2 and/or the receptor CCR2 in our syngeneic glioma model. However, CCL2 in the TME is likely to recruit primarily MDSCs (8
). Then, MDSCs inhibit the type-1 functions of DCs (29
), including DC production of CXCL10 (18
). Taken together, we propose that CCL2-attracted MDSCs inhibit CXCL10 production in the TME through suppression of DCs.
COX-2 and PGE2
promote de novo
induction of Tregs (32
). Consistent with this, the ASA treatment inhibited Treg accumulation in the TME in our model (Supplementary Fig. S4
). In this regard, we have previously attempted to address a role of Tregs in gliomagenesis by mAb-mediated depletion (10
) and observed no significant effects of Treg-depletion on glioma development. Therefore, we rather focused on the roles of MDSCs in the current study. To elucidate a role of Tregs further, we are currently incorporating a Treg-attracting chemokine CCL22 in the SB-
mediated glioma induction system; this system will allow us to address specific effects of CCL22 in the TME and glioma-infiltrating Tregs attracted by CCL22. Furthermore, it has been shown that glioma cell-derived CCL2 promotes Treg migration (33
). We will elucidate the impact of these chemokines on Treg development and accumulation in future studies.
Collectively, the findings in the current study reveal the important roles of the COX-2 pathway in gliomagenesis through an increase in MDSC development and accumulation as well as a decrease in CXCL10-mediated CD8+
T-cell infiltration in the TME. The data also support development of immunoprevention strategies that could be implemented in people with an increased risk for glioma development. To this end, it will be critical to gain a better understanding of the etiology and risk factors for glioma development in humans. Although risk factors for gliomas are still largely unknown, patients with LGGs are known to be at extremely high risks for developing HGGs (34
). Based on our data demonstrating that early-stage gliomas on day 21 resemble human LGGs (Supplementary Fig. S1
) and that celecoxib treatment starting on day 21 is effective (), patients with LGGs may benefit from celecoxib treatment to reduce the risk for malignant progression with HGGs.
With regard to novel risk factors for glioma, we recently reported that single-nucleotide polymorphisms (SNPs) in IFNAR1
correlate with altered overall survival of patients with WHO grade 2–3 gliomas (10
). Others have reported that SNPs in IL-4R
correlate with survival of glioma patients (35
). Although these data do not directly dictate risk factors for glioma development, it might be helpful to reveal functional significance of these SNPs in order to identify individuals who have a risk of gliomas and are likely to benefit from future clinical trials with NSAID-based immunoprevention. Based on the current study, future studies evaluating SNPs in Cox-2, Ccl2 as well as Cxcl10
in relation to glioma risks and prognosis are warranted.