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Malone et al. J Cancer Metastasis Treat 2021;7:40 https://dx.doi.org/10.20517/2394-4722.2021.37 Page 9 of 18

markers that form a continuum between the two states. In the presence of M1-like activated microglia,
[83]
breast tumor cells are phagocytosed soon after infiltrating the brain . M2-like differentiated microglia are
thought to play a crucial role in the formation of the pre-metastatic niche by suppressing their innate
immune function and thus allowing circulating tumor cells to enter the brain unchallenged and unharmed.
The activation of M2-like microglia has been reported to promote breast cancer cell invasion in a Wnt-
[84]
dependent manner . In breast-to-brain metastasis, M2 polarization is also triggered by the loss of
expression of the long noncoding RNA X-inactive specific transcript (XIST) which is a major regulator of
X-chromosome inactivation [85,86] . The effect that the breast tumor secretome may have on the activation of
microglia is not extensively studied, nor is it known whether it is a stable or dynamic secretome, changing
over time and during the different phases of metastasis. In a series of publications in the last year,
bioinformatic approaches using both RNASeq and CYTOF approaches have started analyzing the immune
microenvironment of BCBM. The majority of these studies have not yet provided information on the early
changes in the CNS during the formation of the pre-metastatic/metastatic niche but do give new views on
the distinct tumor microenvironment around these metastases once established [73,87,88] . Overall samples from
[73]
brain metastases present an abundance of molecules regulating myeloid and lymphocytic cell signaling ,
including CSF2, IL23A and the pattern recognition receptor MARCO. Among microglia specific markers,
prominently genes of the IL-6 signaling pathway were upregulated, normally known for suppressing
systemic immune responses, as well as TREM1 and CXCL5. Interestingly, CXCL8 was also upregulated in
microglia around brain metastases, which functions to chemoattract neutrophils . Another chemokine
[73]
[89]
receptor, CX3CR1, was downregulated in the CNS around metastatic lesions . In an effort to look at
newly-formed metastases in the brain, Schulz et al. examined immune cell changes around smaller vs.
[90]
bigger tumors and reported that the infiltrating tumor cells “educate” the local immune cell environment,
with microglia displaying only changes in few genes when in proximity with small or large metastatic
lesions in the H2030-BrM model. A limitation of this analysis, however, was the use of non-immune-
competent mice.

The function of astrocytes in the normal, physiological brain is to provide structural and functional support
to the neurons, to regulate extrasynaptic neurotransmitter levels and to modulate specific steps in synapse
[91]
formation and plasticity . It has been speculated that astrocytes affect brain metastasis progression through
crosstalk with cancer cells via paracrine pathways, since they are shown to surround metastatic lesions in
the brain and their endfeet are prominent at the blood-brain barrier [52,74,92,93] . Because of this physical
proximity, astrocytes interact with cells upon extravasation and have been reported to induce Fas-
dependent death in both lung- and breast-derived cells that have infiltrated the brain [94-96] . Astrocytes can
also release plasminogen activators (PAs), primarily urokinase PA (uPA). PA-mediated plasmin generation
leads to the proteolysis of L1 cell adhesion molecule (L1CAM) which is expressed by metastatic cells to
promote spreading along brain capillaries and vascular co-option [97,98] . Both breast- and lung-derived brain
metastatic cells express high levels of serpins that target PA, primarily Neuroserpin and Serpin B2,
effectively neutralizing the plasmin-mediated antit-metastatic effects of astrocytes and thus promoting the
formation of the pre-metastatic niche . Activation of Notch signaling also becomes possible as a result of
[96]
the proximity of astrocytes [99,100] . IL-1β, secreted from tumor cells, activates Jagged-1 (JAG1) expression in
astrocytes; JAG1 then interacts with tumor cells to significantly stimulate Notch signaling [99,101,102] . The bi-
directional signaling mediated by the Notch pathway drives the formation of a permissive metastatic niche
and eventually a tumor microenvironment. The secretion of heparanase and neurotrophins by activated
astrocytes near sites of tumor cell arrest, extravasation, and invasion into the brain are also thought to
initiate and promote metastatic growth through conditioning of the premetastatic niche [93,103,104] . Heparanase
is an enzyme that degrades heparan sulfate proteoglycan, a major constituent of endothelial basement
membranes. It is linked to increased metastasis in multiple cancer types, as degradation of the proteoglycans
is thought to “loosen” the extracellular matrix and facilitate angiogenic responses through the release of