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

M1/M2: an outdated paradigm
Within the GBM TME, microglia and macrophages have classically been subdivided into M1 and M2
phenotypes to characterize them as either having anti-tumor or tumor-promoting (pro-tumor) properties,
[44]
respectively. The M1/M2 dichotomy was first discussed by Mills et al. as a way to distinguish the
+
phenotypic predilections of macrophages from the perspective of Th1 and Th2 lineages in CD4 T cells;
they proposed that M1 refer to macrophages with Th1 backgrounds that tended to produce inflammatory
induced nitric-oxide species (iNOS), while M2 would refer to Th2 derived macrophages that produced more
cell-division stimulating polyamines, like ornithine. In short, the original M1/M2 terminology sought
[44]
to extrapolate the same phenotypic dichotomy assigned to T-helper cells (Th1/Th2) to macrophages .
However, later research elucidated further phenotypic pathways for macrophages related to other cytokines
and factors (e.g., IL-10, TGF-B) that made the extrapolation from the initial binary Th1/Th2 characterization
[45]
[46]
less robust . As a result, Mantovani et al. proposed the conversion of the M1/M2 dichotomy into a
continuum, with M1 and M2 representing two opposite poles of immune function.
In this vein, M1 macrophages, or classically activated macrophages, are typically noted as inducing
prototypic inflammatory (pro-inflammatory) responses, while M2 macrophages, or alternatively activated
macrophages, are those with antagonism of normal inflammatory (anti-inflammatory) responses [47,48] .
More specifically, M2 has been further divided into subtypes; M2a correlates to Th2 responses, type II
inflammation, and pathogen elimination. M2b correlates to Th2 activation and immunoregulation. Finally,
[46]
M2c correlates to immunoregulation, matrix deposition, and tissue remodeling . Ultimately, the popularity
of using the M1/M2 paradigm in studies came from its simplicity; by mirroring the nomenclature used
for the Th1/Th2 phenotypes, M1/M2 provided a simple and easy to conceptualize model to understand
[46]
immunosuppressed myeloid populations .
However, while the M1/M2 framework was designed to be a simplified operational concept that provided
[48]
foundational language to a rapidly growing field , it has since been used erroneously in much of the
literature as a solid classification scheme for macrophages, and to an increasingly greater extent, microglia.
From a generalized view, using M1/M2 as a classification system is problematic for a variety of reasons.
First, the vast majority of data supporting the system comes from in vitro studies that have not been reliably
[49]
recapitulated in vivo . These concepts rarely translate to systemic models, as in vitro systems have limited
engagement with larger systemic variables beyond characteristics of cell maturation, adhesion, and cytokine
[50]
production . More specifically, macrophages in vitro versus macrophages in vivo have been documented to
[49]
have different morphologies, functions, and expression of specific cellular markers .
Beyond experimental inconsistencies, the M1/M2 phenotypes are also outdated in the sense that their
original formulation predated the significant new body of genomics research that has emerged in the last 15
years. Genome-wide microarray analysis of both glioma-associated microglia and macrophages in GL261
[17]
murine gliomas by Szulzewsky et al. have shown that both cell types have expression profiles that do not
[51]
fit within any previously documented M1/M2 classification scheme . Indeed, these myeloid populations
only had partial overlap with previously documented M1/M2 phenotypes, with 59.6% of genes that were
significantly upregulated (261/438 analyzed) not characterized as either M1 or M2; this indicates that there
[17]
is far more complexity than the M1/M2 label can provide, at least from a genomics standpoint . Of note,
some of the genes identified outside of the classic M1/M2 phenotype were associated with angiogenesis (Vegfa,
[17]
Hgf), suppression of immunity (Arg1, Tgfb3), and tumor invasion (Mmp2, Mmp14, Ctgf) in mouse models .
In the context of this review, the M1/M2 classification is further problematic when applied to microglia.
The original nomenclature for the M1/M2 classification came from studying macrophages and a significant
portion of the current literature has merely transposed this M1/M2 nomenclature to microglia without
[45]
respect to the differences between these two myeloid populations . As discussed previously, however, it