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Choi et al. Neuroimmunol Neuroinflammation 2018;5:42 I http://dx.doi.org/10.20517/2347-8659.2018.47 Page 9 of 14

which can bind to the receptor CD74 on glioma-associated microglia. Interestingly, they found a positive
correlation between CD74-positive glioma-associated microglia and patient survival, indicating anti-tumoral
characterization of this marker. This positive prognostic factor offers a potential area of exploration into
pathways involving CD74 to further elucidate candidate receptors or cytokines for encouraging microglial
recruitment for anti-GBM response.

Inhibition of pro-tumor functions of microglia and macrophages
There are also several strategies that aim to inhibit pro-tumor or reactivate immunosuppressive pathways
in microglia and macrophages. Interleukin 8 [IL-8 or chemokine (C-X-C motif) ligand 8, CXCL8] has been
implicated in several tumorigenic pathways, most pronouncedly via its binding CXCR1/2 on endothelial cells
and macrophages; this has been associated with tumor growth and chemoresistance, increased invasion,
and tumor angiogenesis [73,74] . Furthermore, increased presence of IL-8 has been found in the TME of GBM
[73]
along with upregulation of its receptors in macrophages and endothelial cells [Figure 2D and E] . A follow-
[75]
up study by Infanger et al. demonstrated similar findings, with IL-8 linked to maintenance and growth of
GBM cancer stem-like cells. In the same investigation, they found that CXCR2 silencing reversed the tumor-
promoting effects of endothelial cells in vivo, demonstrating the potential therapeutic benefit of inhibiting
IL-8 signaling for anti-tumor response.

Further work in understanding the TME and its impact on glioma-associated macrophages include studies
on BLZ945 and PLX3397: inhibitors of colony stimulating factor-1 receptor (CSF1R). Contrary to their
original hypothesis that CSF1R inhibition would lead to tumor inhibition through global depletion of
tumor-associated microglia and macrophages, they actually achieved tumor inhibition in xenograft mouse
models through enhanced survival and promotion of tumor-associated macrophages that demonstrated
[76]
antitumor properties [76,77] . Specifically, when Pyonteck et al. used BLZ945 to inhibit CSF1R in mice, they
reported shifts in the gene signatures of macrophage populations away from pro-tumor/immunosuppressive
phenotypes with consequent inhibition of GBM progression, in vivo. However, continued use of CSF1R
[78]
inhibitors resulted in acquired resistance to further CSF1R inhibition in GBM mouse models . Therefore,
[77]
a follow-up study by Yan et al. , focused on using a combinatorial approach with PLX3397 (another
potent CSF1R inhibitor) and the tyrosine kinase inhibitors dovitinib or vatalanib; this combination therapy
demonstrated significant and lasting reduction in tumor volume compared to PLX3397 alone, indicating
that these anti-tumor macrophages rendered glioma cells more sensitive to treatment.

Interestingly, while exposure to PLX3397 preserved macrophage density and resulted in a phenotypic
shift, non-glioma associated stromal microglia were almost fully depleted. Yet, while this data suggests the
preservation and redirection of tumor-associated macrophages to an anti-tumor phenotype coupled with
depletion of stromal microglia in surrounding tissues, neither study adequately characterized the true ratio
of microglia to macrophages in the surviving tumor-associated milieu, nor were they able to adequately
attribute the ratio of anti-tumor cells to that of peripheral macrophages [77,78] . As a result, while their results
suggest enhancement of anti-tumor tumor-associated macrophages alone, further characterization of
both populations of cells in the context of CSF1R inhibition is necessary to accept that assertion without
doubt. Regardless, the CSF1R pathway potentially indicates a promising therapeutic avenue for targeting
[77]
macrophages in the GBM TME .
[79]
Additionally, Cx3cr1 knock-out (KO) experiments by Feng et al. , gave further insight into the complexity
of tumor adaptive pathways involving both microglia and macrophages. The ligand for CX3C chemokine
receptor 1 (CX3CR1), CX3CL1 (fractalkine), is an important chemokine-signaling protein in the healthy
CNS that mediates inflammatory response of both microglia and macrophages, including properties of
adhesion and migration [Table 1]. When deleted, Cx3cr1 KO mice experienced increased gliomagenesis and
greater tumor burden [Figure 2A and B]. Interestingly though, there was no effect on microglial migration