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Page 6 of 14 Choi et al. Neuroimmunol Neuroinflammation 2018;5:42 I http://dx.doi.org/10.20517/2347-8659.2018.47

was also only recently that we have come to recognize the numerous differences between microglia and
macrophages in terms of structure, function, and expression of tumor-related pathways [17,18] . As a result, while
the M1/M2 nomenclature for macrophages has issues with oversimplification, the use of said classification
scheme for microglia may be simply inaccurate. Importantly, a variety of genome-wide expression studies
[18]
of microglia in a variety of disease state models, including generalized inflammatory states , amyotrophic
[54]
[53]
[52]
lateral sclerosis (ALS) , autoimmune encephalomyelitis , Alzheimer’s disease , traumatic brain injury ,
[49]
[55]
and GBM all showed no clear evidence of true M1/M2 differentiation among microglia and instead mostly
showed that they simultaneously express both M1 and M2 phenotypic markers.
Furthermore, it is difficult to classify microglia along the M1/M2 continuum, especially in the context of
GBM, since activated microglia in the TME have several more functions that do not fit into the classic
functional categories associated with M1/M2 in macrophages. For example, while the M2 phenotype for
macrophages has largely been regarded as being immunosuppressed - specifically in the context of increased
trophic polyamines - the M2 subtype in microglia may actually have a more active pro-tumor role that
supports gliomagenesis and invasion [22,46,47,56-60] .

As a result, we recognize the equivocal and limited nature of using M1/M2 as a classification scheme
and will be utilizing a more flexible paradigm to organize our discussion of microglia and macrophages,
specifically within the context of the TME in GBM. To address these differences as well as move away from
the M1/M2 nomenclature, we focus on specific markers and pathways present on microglia or macrophages
and designate these molecular targets as having either anti-tumor or pro-tumor/immunosuppressive
characteristics [Figure 2].

Therapeutic strategies involving glioma-associated microglia and macrophages in the GBM TME
Several preclinical and clinical studies have already examined the efficacy of targeting glioma-associated
microglia and macrophages for anti-tumor therapy [Table 1]. In general, there are two main strategies for
treatment: inhibition of tumor-promoting microglia and macrophages [Figure 2C and D] or upregulation
of anti-tumor receptors and cytokines in microglia and macrophages [Figure 2A and B] [61,62] . It should be
noted that while there are some receptors and resultant cascades that are present in both microglia and
macrophages, there are also a variety of signaling pathways unique to either microglia or macrophages,
which lends credence to the idea that these cell types have distinct roles in the TME.

Activation of the intrinsic anti-tumor properties of microglia and macrophages
First, while much of the scientific literature corroborates a story of microglia and macrophages having
mostly a pro-tumor or immunosuppressive role in the GBM TME, there is some evidence that microglia and
macrophages have intrinsic anti-tumor properties as well. An in vitro study by Hwang et al. demonstrated
[63]
that microglia conditioned culture medium (MCM) promotes apoptosis of glioma cells, with additional
cytotoxic effect when exposing microglial cells to lipopolysaccharides (LPS) or IFNγ. Moreover, this effect
was glioma-specific, without unwanted astrocyte cytotoxicity. Proteonomic analysis of the MCM revealed
LPS- and IFNγ-related proteins along with markedly elevated expression of cathepsin proteases - particularly
cathepsin B. When cathepsin B was suppressed, glioma-apoptosis was no longer observed, indicating this
[63]
protein’s importance in microglial anti-tumor function .

[64]
A similar study by Kees et al. examined toll-like receptor 3 (TLR3) and its agonist, polyinosinic-
polycytidylic acid [poly(I:C)], on both microglia and macrophages. The activation of TLR3 on these
myeloid cells resulted in the secretion of glioma specific toxic soluble factors in co-culture with GBM
cells [Figure 2A and B]. Of note, a similar phenomenon involving poly(I:C)-induced TLR3 activation in
[65]
dendritic cells demonstrated by Garzon-Muvdi et al. showed anti-tumor effect. Likewise, TLR9 has also