
==== Front
Cancer Biol Ther
Cancer Biol Ther
Cancer Biology & Therapy
1538-4047
1555-8576
Taylor & Francis

39238191
10.1080/15384047.2024.2398285
2398285
Version of Record
Review Article
Review
Analysis of neuroglia and immune cells in the tumor microenvironment of breast cancer brain metastasis
H. MO ET AL.
CANCER BIOLOGY & THERAPY
Mo Haixin a
Zhang Xin a b c
https://orcid.org/0000-0001-6219-4304
Ren Liangliang a
a Clinical Experimental Center, Jiangmen Engineering Technology Research Center of Clinical Biobank and Translational Research, Jiangmen Central Hospital , Jiangmen, China
b Dongguan Key Laboratory of Medical Bioactive Molecular Developmental and Translational Research, Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University , Dongguan, China
c Collaborative Innovation Center for Antitumor Active Substance Research and Development, Guangdong Medical University , Zhanjiang, Guangdong, China
CONTACT Liangliang Ren renll@mail3.sysu.edu.cn Clinical Experimental Center, Jiangmen Engineering Technology Research Center of Clinical Biobank and Translational Research, Jiangmen Central Hospital, Jiangmen, China
5 9 2024
2024
5 9 2024
25 1 2398285Integra05 9 2024
Integra05 9 2024
20 5 2024
15 8 2024
26 8 2024
© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Breast cancer stands as the most prevalent cancer diagnosed worldwide, often leading to brain metastasis, a challenging complication characterized by high mortality rates and a grim prognosis. Understanding the intricate mechanisms governing breast cancer brain metastasis (BCBM) remains an ongoing challenge. The unique microenvironment in the brain fosters an ideal setting for the colonization of breast cancer cells. The tumor microenvironment (TME) in brain metastases plays a pivotal role in the initiation and progression of BCBM, shaping the landscape for targeted therapeutic interventions. Current research primarily concentrates on unraveling the complexities of the TME in BCBM, with a particular emphasis on neuroglia and immune cells, such as microglia, monocyte-derived macrophages (MDMs), astrocytes and T cells. This comprehensive review delves deeply into these elements within the TME of BCBM, shedding light on their interplay, mechanisms, and potential as therapeutic targets to combat BCBM.

KEYWORDS

Breast cancer
brain metastasis
tumor microenvironment
neuroglia
immune cells
National Natural Science Foundation of China 10.13039/501100001809 82103582 National Natural Science Foundation of China 10.13039/501100001809 81802918 China Postdoctoral Science Foundation Grant 2019M660206 Science and Technology Project of Guangdong Province 2019A1515011565 Science and Technology Project of Guangdong Province 2021A1515012432 Science and Technology Project of Jiangmen 2020030103140008978 Medical Science Foundation of Jiangmen Central Hospital J202001 This study was supported by the grants from the National Natural Science Foundation of China [82103582], the National Natural Science Foundation of China [81802918], the China Postdoctoral Science Foundation Grant [2019M660206], the Science and Technology Project of Guangdong Province [2019A1515011565, 2018A030310007], the Science and Technology Project of Guangdong Province [2021A1515012432], the Science and Technology Project of Jiangmen [2020030103140008978, 2019030102430012905], and the Medical Science Foundation of Jiangmen Central Hospital [J202001].
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pmcIntroduction

In recent years, breast cancer has emerged as the most prevalent cancer diagnosed globally,1 ranking as the second leading cause of cancer-related fatalities among women.2 The primary culprits behind breast cancer-related mortality are cancer recurrence and metastasis. Brain metastasis represents a frequent complication of breast cancer, affecting approximately 15–30% of metastatic breast cancer patients.3 During the progression of breast cancer brain metastasis (BCBM), cells carrying metastatic potential traverse the blood-brain barrier (BBB) and intricately interact within the brain microenvironment to acclimate to its conditions, ultimately culminating in the formation of metastases. Notwithstanding these discoveries, the mechanisms driving BCBM remain incompletely understood, posing significant challenges in treating BCBM characterized by high mortality rates and unfavorable prognoses.4,5

Given the substantial impact of the tumor microenvironment (TME) within brain metastases on the progression and subsequent treatment outcomes of BCBM, investigating the TME in BCBM holds promise for identifying novel therapeutic targets. Presently, research endeavors focused on BCBM TME have concentrated on neuroglia and immune cells, particularly microglia, monocyte-derived macrophages (MDMs), astrocytes and T cells. These cellular components play pivotal roles in the initiation and advancement of BCBM. This article aims to scrutinize the roles of brain microenvironment cells in BCBM, intending to unveil strategies for treating brain metastases in breast cancer.

Microglia can foster tumor cell invasion, adhesion and colonization within the brain. MDMs facilitate cancer cell invasion, migration and adhesion via cytokine release and associated signaling pathways. Astrocytes exhibit a dual role, initially impeding tumor cells during the early stages of brain metastasis, while later promoting BCBM through cytokines, various signaling pathways, cancer-related genes and chemotherapy resistance. Diverse T cell subtypes perform distinct functions, and Helper T cells and cytotoxic T cells are instrumental in promoting immunity, and regulatory T (Treg) cells play a suppressive role in immune regulation, collectively influencing cancer development.

Tumor-associated macrophages in the TME of BCBM

In the central nervous system (CNS), tumor-associated macrophages (TAMs) encompass microglia and MDMs.6 Autopsy specimens from breast cancer brain metastases reveal the activation and aggregation of TAMs near the metastatic foci.7 TAMs exhibit two phenotypes: pro-inflammatory (iNOS+, COX2+) and anti-inflammatory (Arg1+, MRC1+). Pro-inflammatory TAMs, also known as M1 macrophages/microglia, exert cytotoxic effects on tumor cells, while anti-inflammatory TAMs, termed M2 macrophages/microglia, promote tumor cell growth and angiogenesis. The balanced coupling of pro-inflammatory and anti-inflammatory TAMs response in the early stages of metastasis might favor the establishment of metastatic foci.8 With the development of gene detection, especially single-cell sequencing technology, more and more studies have found that traditional M1/M2 typing cannot fully reflect the state of TAMs in vivo microenvironment. The researchers obtained two types of MAMs (APOE+ and S100A8+) from fresh tissue from brain metastases. These two types of macrophages differ in function as well as in molecular expression profile. APOE+ MAMs is similar to TAMs and highly expresses complement C1Q chains, SPP1 and HLA-related molecules, while S100A8+ subgroup is similar to inflammatory FCN1+ myeloid-derived suppressor cells with high expression of S100A family, CXCL8, FCN1 and low expression of HLA-related genes. Further analysis showed that these two subgroups represented cell states that were gradually differentiated phenotypically, and that gene expression was also changing during cell differentiation. In general, there were continuity and differentiation relationships between the two MAMs subgroup.9,10 Moreover, there exist discernible differences in TAMs phenotypes between meningeal and parenchymal brain metastases. Researchers comparing TAMs phenotypes in breast cancer affecting these two locations have observed that cancer cells in the brain parenchyma secrete more Lymphotoxin-β compared to the meninges. Lymphotoxin-β directly participates in the M2 polarization of TAMs within the brain parenchyma. Thus, relative to meningeal TAMs, those in the brain parenchyma exhibit a greater propensity toward M2 polarization.11 This difference may stem from the distinct monocyte subsets giving rise to TAMs in the meninges versus brain parenchyma. Additionally, TAMs phenotypes might also be associated with site-specific cancer cell characteristics of metastasis and organ-specific matrix.11 Consequently, tailored therapeutic strategies are essential for different brain metastatic sites.

Regarding the role of TAMs in cancer cells, the traditional view implicates TAMs’ recruitment to the tumor vicinity for cytotoxic action against cancer cells. However, as research on TAMs advances, scientists have uncovered that TAMs primarily influence BCBM by exerting cytotoxicity, releasing cytokines and participating in multiple signaling pathways. For example, through animal experiments and cell studies, researchers confirmed that TAMs facilitate breast cancer cell adhesion to brain capillaries and penetration into the brain parenchyma via the Lnc-BM/JAK2/STAT3/ICAM1 signaling pathway.12 Specifically, lncRNA associated with BCBM (Lnc-BM) regulates Janus kinase-2 (JAK2) conformation by promoting JAK2 from a “closed” (autoinhibited) to an “open” (activated) structure, activating its activity and promoting brain metastatic cancer cells to express signal transducer and activator of transcription 3 (STAT3) target genes, thereby creating a tumor microenvironment conducive to BCBM.12 In conclusion, TAMs are multifunctional cells, with their diverse phenotypes playing distinct roles within the TME.13

Microglia

Microglia are intrinsic cells of the brain, which play a role in regulating brain development and maintaining neuronal networks, they are also inherent immune cells of the CNS, originated from the yolk sac and distributed in brain parenchyma.14–16 Possessing a physiological phagocytic function, microglia play a significant role in neurological disorders.17 Depending on their biological functional state, microglia can exhibit diverse morphologies.18 In the normal adult brain, microglia present a branched resting state, while reactive microglia demonstrate spherical, rod-shaped, polymorphic, or amoeboid states.18

Studies indicate that during BCBM, microglia continuously infiltrate the vicinity of metastatic lesions, correlating positively with tumor load.8 Through the Wnt signaling pathway, they promote the invasion and colonization of breast cancer cells into brain tissue.19 Experimental observation showed that after co-culture of BV-2 cells with MDA-MB-231 and MCF-7 cells (breast cancer cells), the proliferation and migration ability of breast cancer cells were enhanced in a concentration-dependent manner, and the contents of protein, nucleic acid, lipid and carbohydrate of tumor cells were changed.20 This effect may be due to the fact that BV-2 cells affect the metabolism of tumor cells, thus enhancing the proliferation and migration ability of tumor cells.20 However, recent studies have found that microglia have pro-inflammatory and tumor suppressive effects.21 In animal experiments, microglia-deficient mice were found to have increased cancer cell metastasis, low survival rate and low natural killer and T cell responses.21 In addition, it was found that microglia could support T cell responses, and microglia and T cells synergistically exerted anti-tumor effects.21

Given the intricate nature of interactions between microglia and breast cancer cells within the microenvironment of BCBM and the heterogeneous response of microglia to cancer cells,22 the role of microglia in either promoting or inhibiting cancer cells is complex. To date, scientists have yet to thoroughly elucidate these mechanisms.

MDMs

In the CNS, aside from the resident microglia, the majority of macrophages are essentially MDMs. Under physiological and pathological conditions, the brain recruits monocytes into cerebral vasculature, which subsequently infiltrate the brain parenchyma, becoming MDMs.23 These MDMs assist in tissue remodeling and regeneration in pathological states.23,24 In a comparative analysis between surgical samples from primary breast cancer and BCBM patients, researchers observed a decrease in tumor-inhibiting components (CD8+ T cells, M1 macrophages – pro-inflammatory) and an increase in immune-suppressive components (M2 macrophages – anti-inflammatory) in BCBM.25 This observation closely correlates with the clinical prognosis of BCBM patients, similar to findings from TAM-related studies.25

Additionally, using microfluidic 3D cell culture techniques, researchers found that macrophages enhance the migratory capacity of breast cancer cells by releasing tumor necrosis factor-alpha (TNFα) and transforming growth factor β1 (TGFβ1).26 TNFα and TGFβ1 released by macrophages promote cancer cell NF-κB nuclear translocation, upregulating MMP1 expression and leading to increased persistence of cancer cell migration.26 Furthermore, TGFβ1 released by macrophages increases cancer cell migration speed by upregulating membrane type1-matrix metalloproteinase (MT1-MMP) expression.26 Moreover, scientists have found that the TGFβ1 signal upregulates colony-stimulating factor 1 receptor (CSF1R) in primary tumor cells and activates the CSF1/CSF1R autocrine signaling pathway, promoting invasion and migration of breast cancer cells.27 Apart from promoting breast cancer cell migration through TNFα and TGFβ1 release, macrophages also induce breast cancer cells to form elongated protrusions and enhance invasion by releasing epidermal growth factor (EGF).28 This mechanism forms a positive feedback loop, where colony-stimulating factor 1 (CSF1) produced by breast cancer cells stimulates macrophages to release EGF, which in turn enhances breast cancer cell CSF1 expression.28 In addition, it has been found that macrophages-induced invasion is related to the release of tumor microvesicles (T-MV), which are ingested by macrophages after being released by tumor cells.29

In summary, MDMs primarily facilitate breast cancer metastasis by releasing cytokines that promote tumor development and associated signaling pathways.

Astrocytes in the TME of BCBM

As the most abundant neuroglia in the CNS, astrocytes play an important role in maintaining the homeostasis of brain tissue.30,31 When diseases occur, normal reactive astrocytes utilize their adaptive functions to protect neural tissues and maintain neural function.32 As an component of the innate immunity in the CNS, reactive astrocytes have the capacity to release various cytokines and chemokines, exerting either promoting or inhibitory effects on tumors.32,33 In the early stages of brain metastasis, astrocytes can inhibit tumor cells, but in later stages, they tend to support tumor growth.34 Studies dating back to 2008 revealed the presence of numerous reactive neuroglia, including astrocytes and microglia, in surgically resected brain metastases from breast cancer patients,35 and reactive neuroglia play an important role in the progression of BCBM.

The facilitation of BCBM by astrocytes is intricately linked to cytokine signaling. Studies have demonstrated that astrocytes promote the progression of BCBM by engaging the Notch signaling.36 Specifically, breast tumor cells that metastasize to the brain activate surrounding astrocytes through the secretion of IL-1β.36 This activation subsequently triggers an increase in the expression of JAG1 within the astrocytes.36 These activated astrocytes then interact with cancer stem-like cells (CSCs), leading to the activation of the Notch signaling within the CSCs.36 The activation of Notch signaling elevates the levels of HES5 protein, ultimately fostering self-renewal among CSCs and facilitating metastatic growth.36 In vitro experiments conducted by researchers revealed that IL-1β, released by brain-metastatic breast tumor cells, plays a dual role. It not only drives the progression of brain metastasis in breast cancer through the Notch signaling but also can cross induced pluripotent stem cell-derived brain-specific microvascular endothelial-like cell layer, and stimulates astrocytes to produce chemokines (CCL2, CCL5, CCL7, CCL8, CXCL1, CXCL5).37 This stimulation accelerates the migration of breast cancer cells across the BBB, intensifying the chemotaxis and aggressiveness of these cancer cells.37 Furthermore, studies have found that the cytokine TGF-β2 secreted by astrocytes activates Smad3 through TGF-βR, forming the Smad3/Smad4 complex.38 This complex upregulates angiopoietin-like 4 (ANGPTL4) expression in breast cancer cells, which may promote tumor development by disrupting vascular endothelial junctions or conferring anoikis resistance in tumors.38 The authors also found a possible positive feedback loop between breast cancer cells and astrocytes, in which astrocytes increase the expression of TGF-β2 in response to IL-1β and TNF-α produced by tumor cells, thereby further promoting tumor growth.38 Scientists have also discovered that S1P3 is expressed in reactive astrocytes around brain metastatic lesions.39 Interestingly, reactive astrocytes regulate the blood-tumor barrier (BTB) in brain metastasis through S1P3 signaling, and overexpression of S1P3 leads to increased IL6/CCL2 secretion by astrocytes and increased BTB permeability.39 The researchers placed MDA-MB-231 breast cancer cells in astrocyte-conditioned medium, and found that cancer cells have increased migration rate, altered cell morphology (enlargement and elongation), and changes in actin cytoskeleton organization.40 This could be related to the astrocytes-secreted matrix metalloproteinases (MMP-2, MMP-9) that involved in tumor invasion and migration.40,41

Furthermore, direct contacts between CSCs and astrocytes promote the activation of the PCDH7-PLCβ-Ca2+ signaling pathway, thereby facilitating the colonization of breast cancer cells in the brain.42 Simultaneously, the co-culture of MDA-MB-231 breast cancer cells with astrocytes revealed an upregulation of pro-proliferative genes (CCND1, BCL2L2, CCNA1) and a downregulation of pro-apoptotic genes (CASP8, LATS2, FAF1, PDCD2) within cancer cells.42 Additionally, upon entry of human epidermal growth factor receptor 2 (HER2+) breast cancer cells into brain tissue, astrocytes increased the production of brain-derived neurotrophic factor (BDNF), which activated the PI3K/AKT pathway.43 This, in turn, elevated the levels of TrkB protein in brain metastatic breast cancer cells, promoting the formation of TrkB and HER2 heterodimers and supporting the survival of breast cancer cells within the brain.43

Researchers have conducted a series of studies on the role of astrocytes in enhancing cancer cell chemotherapy resistance. The binding of brain-metastatic breast cancer cells to astrocytes via Cx43gap junctions using PCDH7 has been documented.44 This interaction results in the transmission of cGAMP to astrocytes, thereby triggering the activation of the cGAS-STING pathway and inducing the release of inflammatory cytokines IFNα and TNF by astrocytes.44 Consequently, this leads to STAT1 and NF-κB pathways are activated, conferring growth advantages and augmenting the resistance of brain-metastatic breast cancer cells to chemotherapy.44 The specific mechanism of chemotherapy resistance is mainly astrocytes release IFNα or TNF, activate the STAT1 and NF-κB signaling pathway of breast cancer cells, and further enhance the transcription level of tumor pro-survival factors (cyclin D1, Bcl-xL and IAPs). Thus enhancing chemotherapy resistance of tumor cells.44,45 Besides the activation of the STAT1 and NF-κB pathways mentioned earlier that can increase chemotherapy resistance, further studies have found that continuous co-culture of breast cancer cells with astrocytes leads to an upregulation of cancer cell survival genes (GSTA5, BCL2L1, TWIST1) by activating AKT and MAPK signaling pathways, and the extent of upregulation positively correlates with increased chemotherapy resistance.46 Additionally, there is research suggesting a link between astrocyte-induced resistance to doxorubicin chemotherapy and the secretion of high levels of IL-6.47 Moreover, other studies have indicated that astrocytes’ inhibition of chemotherapy-induced cytotoxicity in cancer cells is associated with endothelin-dependent signaling mechanisms.48

Based on current literature reports, astrocytes exhibit a multifaceted role in promoting BCBM through a range of mechanisms encompassing cytokines, signaling pathways, cancer-related genes and chemotherapy resistance. As to whether other aspects of its mechanism are involved, further research and exploration by scientists are needed.

T cells in the TME of BCBM

T cells are employed by the body to mount immune responses against cancer, infections, and various pathological conditions. T cells are functionally categorized into helper T cells (Th, CD4+), cytotoxic T cells (CTL, CD8+) and Treg cells. CD4+ and CD8+T cells are instrumental in promoting immunity, while Treg cells play a suppressive role in immune regulation.49 Within the TME, the interplay of immune responses mediated by CD4+ and CD8+T cells, alongside the immunosuppression mediated by Treg cells, collectively regulates tumor progression. Research indicates an elevation in the presence of immune cells, including CD68+ (macrophages/monocytes), CD3+ (CD4+, CD8+), and Cytotoxic Lymphocytes, within breast cancer tissues in contrast to both normal breast tissues and benign pathological tissues.50 Conventionally, it was believed that T cells do not penetrate the brain parenchyma under normal physiological conditions but reside solely in the meninges, where these meningeal T cells might contribute significantly to brain function.51 However, deeper investigations have revealed that T cells infiltrate the brain parenchyma in pathological states.52

Comparative analysis of surgical samples from patients with primary breast cancer and brain metastases revealed fewer tumor-infiltrating lymphocytes in brain metastases, correlating closely with poor patient prognosis.53 Levels of CD4+, CD8+, and FOXP3+ cells were higher in primary breast cancer than in brain metastases.53 Using advanced technologies like GeoMx Digital Spatial Profiler and NanoString’s nCounter PanCancer IO 360™ assay, researchers examined the TME in HER2+ breast cancer patients at primary and metastatic sites.54 They observed increased expression of genes linked to immune activation and trafficking in primary lesions compared to metastatic ones.54 Moreover, primary lesions exhibited higher levels of immune cells and immune-related proteins such as CD3 and CD8, suggesting higher immunogenicity in primary lesions of HER2+ breast cancer patients.54 Similarly, another study evaluating tumor-infiltrating lymphocytes in primary breast cancer and brain metastases found fewer tumor-infiltrating lymphocytes and greater immune inertia in brain metastases compared to primary breast cancer.55 Researchers further investigated mitochondrial arginase 2 (Arg2), expressed by T cells, as it regulates CD8+T cell function.55,56 Overexpression of Arg2 was found to hinder tumor adaptive immune response by depleting extracellular arginine.55 Arg2-positive breast cancer patients had worse brain metastasis-free survival (BMFS) and overall survival (OS) compared with Arg2-negative breast cancer patients, suggesting a potential link between Arg2 and BCBM progression.55

A deeper exploration of the relationship between T cells and BCBM progression revealed that T cells in co-culture with breast cancer cells enhanced the latter’s ability to breach the BBB and facilitate brain metastasis.57 This may be due to the increased expression of Guanylate-Binding Protein 1 (GBP1) in breast cancer cells after co-culture of breast cancer cells with T lymphocytes.57 Overall, T cells play an indispensable role in BCBM, with different T cell subsets exhibiting distinct functions.

Targeting the TME inhibit the progression of BCBM

Absolutely, the brain, being a crucial organ, faces substantial challenges with BCBM, which presents high mortality rates. Hence, it’s pivotal to explore rational and effective treatment approaches to mitigate BCBM-associated mortality. Thorough exploration of the TME in BCBM has unveiled a range of treatment strategies targeting TME.

Targeting TAMs

TAMs represent a critical therapeutic target in BCBM. Targeting TAMs stands as a promising therapeutic approach to impede BCBM progression. Anti-inflammatory TAMs can promote brain metastasis, so inhibiting anti-inflammatory TAMs or reprogramming anti-inflammatory TAMs to pro-inflammatory TAMs might delay progression of brain metastasis.8,58 Mannosylated clodronate liposomes serve as a tool for macrophage clearance that can cross the BBB, effectively absorbed by M2 (MRC1-expressing TAMs) via binding MRC1.8,59 The internalized clodronate induces intracellular iron depletion and prompts M2 apoptosis, consequently depleting M2.8,58 Additionally, CSF1R inhibitors (M279) demonstrate their efficacy by curtailing M2, but M279 may have difficulty crossing the BBB.60,61 In a mouse model of BCBM, researchers utilized the CSF1R inhibitor (BLZ945) that can cross the BBB and observed its anti-tumor effects.62,63 The concurrent inhibition of CSF1R (BLZ945) and STAT5 (AC4–130) exhibits a synergistic anti-tumor impact, possibly normalizing TAMs phenotype.62 Several CSF1R-targeting drugs are presently in development and clinical trial phases.60 Given that the CSF1/CSF1R signaling pathway promotes breast cancer cell invasion, blocking CSF1 and CSF1R emerges as a promising cancer treatment strategy.64 Moreover, researchers believe that PI3K regulates metastasis-promoting TAMs, and the PI3K Class I inhibitor (BKM120) that penetrates the BBB restrains TAMs’ facilitation of breast cancer cell invasion into brain parenchyma.65,66

Given that microglia facilitate BCBM via Wnt signaling pathway activation, the use of Wnt inhibitors such as DKK-2, DKK-1, and secreted Frizzled-like protein-1 has shown efficacy in suppressing the pro-invasive effects of microglia.19 Additionally, the administration of STAT6 inhibitor (AS1517499) that can cross the BBB has demonstrated the ability to reduce Arg1 expression in microglia, thereby suppressing the anti-inflammatory phenotype of microglia and inhibiting brain tumor development.60,67 As mentioned above, microglia can up-regulate macrophage CSF1, CCL5, CXCL9, CXCL10, and regulate the response of NK, NKT and T cells to BCBM to promote the anti-tumor immune microenvironment. Mice deficient in microglia that knocked out the FIRE gene had reduced NK and T cell responses and attenuated tumor regression. It suggests the potential of microglia as a target for immunotherapy. Tamoxifen therapy is a first-line treatment for patients with ER+ breast cancer and is primarily targeted at ER+ breast cancer patients by competitively binding ER and blocking estrogen’s tumor-promoting effects. In addition, preclinical studies have found that estrogen can skew polarity of M2 microglia leading to brain metastasis, while tamoxifen can block this effect, enhance the phagocytosis of microglia, and inhibit BCBM.68 The HSP47 inhibitor Col003 inhibited the polarization of microglia to M2 phenotype, significantly increased the number of cytotoxic CD8+ T cells in brain metastases, inhibited the number of CD4+ Treg cells, restored anti-tumor immunity, and enhanced the efficacy of anti-PD-L1. Col003 can cross the BBB, which can be used as a potential strategy for the combined treatment of brain metastases.69

Targeting the release of TNFα and TGFβ1 by macrophages to promote cancer cell migration and simultaneously blocking both is a pivotal strategy in inhibiting cancer cell metastasis.26 Since macrophages promote cancer cell invasion through the CSF1/EGF paracrine loop, blocking either the CSF1R or the EGF receptor (AFS98, Iressa) can inhibit cancer development.28 Experiments have shown that Iressa can cross the BBB.70 Moreover, researchers have discovered that by employing liposome and nanotechnology modifications, zoledronic acid can effectively traverse the BBB.29,71 It subsequently demonstrates an inhibitory effect on tumor-promoting macrophages through interfering with endocytosis of T-MV and disruption of communication between cancer cells and macrophages.29

In summary, the therapeutic regimens targeting TAMs exert antitumor effects by inhibiting anti-inflammatory phenotypes or changing to pro-inflammatory, inhibiting related signaling pathways, blocking cytokines, affecting the information transmission between cancer cells and macrophages.

Targeting reactive astrocytes

Because brain-metastatic breast cancer cells induce astrocytes to secrete chemokines by secreting IL-1β, thus promoting the migration of breast cancer cells, and blocking IL-1β may prevent breast cancer metastasis to the brain.37 However, the effectiveness of IL-1β blockers in preventing BCBM needs to be further verified in animal experiments and clinical trials in the future.37 Reactive astrocytes contribute to tumor progression by releasing MMP-2 and MMP-9, consequently, MMP inhibitors (ONO-4817, Marimastat, Batimastat) demonstrate partial efficacy in curtailing tumor cell invasion and migration, and Marimastat has been reported to cross the BBB.41,72

As the contact between CSCs and astrocytes promoted BCBM by activating PCDH7-PLCβ-Ca2+ signaling pathway, some researchers found that edelfosine, as a PLC inhibitor that can pass the BBB, treated metastatic tumor of brain by affecting cell proliferation and signaling between astrocytes and cancer cells through animal experiments.42 Notably, edelfosine selectively induces apoptosis in cancer cells without harming healthy cells due to its membrane-targeting mechanism.42,73 An astrocytic subpopulation expressing phosphorylated platelet-derived growth factor receptor β (at tyrosine 751; p751-PDGFRβ) near perivascular brain metastases significantly influences metastatic progression.74 Pazopanib, a multi-target tyrosine kinase inhibitor that can cross the BBB, holds promise in preventing brain metastases by reducing the number of p751-PDGFRβ+ astrocytes.74,75 Tamoxifen, an ER inhibitor, has shown potential in crossing the BBB, potentially impacting breast cancer cell resistance to doxorubicin, possibly through the IL-6/JAK2/STAT3 pathway.47 Additionally, brain endothelial cells and astrocytes possess a mechanism to resist the impact of chemotherapy on cancer cells via an endothelial-dependent signaling pathway. The endothelin receptor (ETAR, ETBR) antagonist macitentan effectively disrupts this signaling pathway, thereby augmenting the sensitivity of tumor cells to chemotherapy drugs. When combined with paclitaxel, macitentan demonstrates efficacy in eliminating tumor cells within brain metastases in mice. This effect might stem from macitentan’s ability to enhance apoptosis in tumor-associated endothelial cells potentially leading to BBB disruption. However, the precise mechanism necessitates further investigation for clarification.76

In summary, therapeutic strategies targeting reactive astrocytes predominantly focus on restraining BCBM by blocking cytokines and relevant signaling pathways.

Targeting T cells

The research indicates a decreased CD8+ T cell infiltration in BCBM patients compared to primary breast cancer cases,25 with T cell immune functionality closely linked to BCBM patient prognosis. Chimeric antigen receptor (CAR) T cell therapy stands as a common targeted T cells treatment. This therapeutic approach fuses antibody-antigen specificity with T cell effector function, holding potential to ameliorate cancer prognosis.77 At present, CAR T cell therapy is widely used in hematological malignancies, but it develops slowly in solid tumors.78 In the past, researchers found that CAR-T cells have anti-tumor activity by targeting over a dozen TNBC antigens through preclinical studies.77 In addition, CAR T cell therapy has shown some efficacy in glioblastoma (GBM) and brain metastases. The study reported regression of intracranial and spinal tumors and increased cytokines and immune cells in the cerebrospinal fluid in a patient with recurrent multifocal GBM after treatment with CAR-T cells targeting the tumor-associated antigen interleukin-13 receptor alpha 2 (IL13Rα2).79 In addition, the study have confirmed the therapeutic effect of HER2-BBζ CAR T cells on HER2+ breast cancer metastasis to the brain by local tumor or regional intraventricular infusion.80 Notably, given the potential association between Arg2 expression in T cells and BCBM progression, research has shown that the adoptive transfer of Arg2−/− OT-I CD8+ T cells exhibits superior inhibition of tumor growth compared to OT-I CD8+ T cells.56 Furthermore, conventional chemotherapy not only diminishes Treg cell activity but also triggers CD4+ and CD8+ T cell activation, thereby fortifying immune responses.53,81 Correspondingly, radioimmunotherapy can foster the infiltration of CD4+ and CD8+ T cells while curbing the immunosuppressive effects attributed to Foxp3+ Treg cell infiltration.82

Conclusion and outlook

In addition to astrocytes, microglia, MDMs and T cells, which play a key role in BCBM, the neuroglia and immune cells in the TME also include ependymal cells, oligodendrocytes, B cells, NK cells and dendritic cells (DCs). There are few relevant studies on these cells in BCBM. There is no relevant report on ependymal cells in BCBM. In recent years, it has been found that ependymal cell health is a potential therapeutic target for GBM. The specific mechanism is that GBM cells increase the interaction between cerebrospinal fluid and tumor cells by disrupting the ependymal cell barrier, which may lead to increased malignancy of GBM.83 It has been reported that oligodendrocyte progenitor cells inhibit the growth of HER2+ breast leptomeningeal carcinomatosis, thereby limiting its spread beyond the leptomeninges.84 In addition, some researchers have found that there are a small number of B lymphocytes in brain metastases, and the expression level of GPR171 is related to B cells, suggesting that GPR171 may promote BCBM by inducing B cells.7,85 NK-92 is an NK cell line that has anti-tumor efficacy, but it is unable to cross the BBB. Some researchers have found that focused ultrasound can improve the targeting of immune cell therapy of brain metastases.86 In addition, some studies have focused on CAR-NK cells, which is a potential treatment strategy for BCBM.87,88 DCs are antigen-presenting cells that induce anti-tumor immunity by stimulating tumor-specific T lymphocytes. Interestingly, some researchers have cloned DC-CLM from DCs, which is a cadherin-like molecule that inhibits breast cancer cell growth.89

The mechanisms underlying breast cancer metastasis to the brain are notably intricate and intricately linked to the TME. Cells in this microenvironment – namely, neuroglia (astrocytes and microglia) and immune cells (MDMs, T cells) – play pivotal roles either promoting or inhibiting the progression of BCBM. These effects are associated with the distinct phenotypes and subsets of immune cells and the different stages of tumor development. Specifically, microglia facilitate BCBM through the Wnt signaling pathway and Lnc-BM/JAK2/STAT3/ICAM1 signaling pathway. MDMs promotes BCBM by releasing cytokines and associated signaling pathways. Astrocytes contribute to BCBM and chemotherapy resistance through cytokines and associated signaling pathways. The mechanism of T cells promote tumor development is related to Arg2 and GBP1 (Figure 1 and Table 1). It’s worth noting here that although there is no literature specifically addressing the effect of microglia on the permeability of the BBB when cancer develops brain metastases. But the literature confirms that during the initial inflammation, microglia migrate to the BBB and protect the integrity of the BBB, then microglia change to a reactive phenotype that engulfs BBB components.90 The breakdown of the BBB leads to leakage of systemic substances into the brain parenchyma, which eventually leads to generalized neuroinflammation.90 Moreover, it has been reported that brain metastasis is accompanied by systemic immune‐inflammation perturbations.91 So it can be speculated that when BCBM occurs, microglia may destroy the integrity of the BBB, leading to a large number of cancer cells into the brain, but further experiments are needed to verify this idea. Figure 1. The tumor microenvironment of breast cancer brain metastasis consists of neuroglia and immune cells. (A) Activation of the wnt signaling pathway by microglia promotes breast cancer cell invasion and colonization into brain tissue. Microglia promote breast cancer cell adhesion to brain capillaries through the lnc-BM/JAK2/STAT3/ICAM1 signaling pathway. (B) MDMs release TNFα, TGFβ1 and EGF, promoting cancer cell migration and invasion. Through the lnc-BM/JAK2/STAT3/ICAM1 signaling pathway, MDMs facilitate breast cancer cell adhesion to brain capillaries. MDMs-induced invasion is related to the release of T-MV. (C) Astrocytes contribute to BCBM by involving Notch signaling in CSCs. They confer growth advantages to brain metastatic cancer cells, enhance chemotherapy resistance by activating STAT1 and nf-κB pathways. Astrocytes increase the production of BDNF, favoring breast cancer cell survival in the brain. Astrocytes accelerate the migration rate of cancer cells in the BBB and enhance the invasiveness of cancer cells by secreting chemokines. TGF-β2 secreted by astrocytes may disrupt vascular endothelial junctions or confer anoikis resistance in tumors. Astrocytes increase BTB permeability via S1P3 signaling. Additionally, astrocytes secrete MMP-2 and MMP-9, enhancing cancer cell invasion and migration. CSCs contact with astrocytes activates the PCDH7-PLCβ-Ca2+ signaling pathway, facilitating breast cancer cell colonization in the brain. Astrocytes up-regulate the expression of cancer cell survival genes (GSTA5, BCL2L1, TWIST1) by activating AKT and MAPK signaling pathways, and the extent of upregulation positively correlates with increased chemotherapy resistance. Elevated IL-6 levels and endothelin-dependent signaling contribute to astrocyte-induced chemoresistance. (D) Overexpression of Arg2 in T cells suppressed tumor adaptive immune response. Breast cancer cells pass the BBB is associated with GBP1. The figure was generated by Adobe Illustrator.

Table 1. Mechanisms of brain metastasis from breast cancer based on neuroglia and immune cells.

Cell types	Mechanism	References	
Microglia	Wnt signaling: invasion, colonization	19	
Lnc-BM/JAK2/STAT3/ICAM1: adhesion	12	
MDMs	TNFα, TGFβ1: migration	26	
CSF1/EGF paracrine loop: invasion	28	
Lnc-BM/JAK2/STAT3/ICAM1: adhesion	12	
T-MV: invasion	29	
Astrocytes	Notch signaling: proliferation	36	
STAT1 and NF-κB pathways: proliferation, chemoresistance	44	
BDNF: proliferation	43	
Chemokines(CCL2, CCL5, CCL7, CCL8, CXCL1, CXCL5): cross the BBB, invasion	37	
TGF-β2/ANGPTL4 axes: crossing the BBB, anoikis resistance	38	
S1P3: crossing the BTB	39	
MMP-2, MMP-9: invasion, migration	40	
PCDH7-PLCβ-Ca2+: colonization	42	
AKT and MAPK signaling pathway (GSTA5, BCL2L1, TWIST1): chemoresistance	46	
IL-6: chemoresistance	47	
Endothelin-dependent signaling mechanism（ETAR, ETBR）: chemoresistance	48	
T cells	Arg2: suppression of the adaptive immune response	55	
GBP1: crossing the BBB	57	
Lnc-BM, lncRNA associated with BCBM; JAK2, Janus kinase-2; STAT3, signal transducer and activator of transcription 3; TNFα,tumor necrosis factor-alpha; TGFβ1,transforming growth factor β1; CSF1, colony-stimulating factor 1; EGF, epidermal growth factor; T-MV, tumor microvesicles; STAT1, signal transducer and activator of transcription 1;BDNF,brain-derived neurotrophic factor; BBB, blood-brain barrier; ANGPTL4, angiopoietin-like 4; Arg2, arginase 2; GBP1,Guanylate-Binding Protein 1.

Although both microglia and MDMs belong to TAMs, they have some differences in cell surface markers, morphology, and function. TAMs in brain metastases are mainly MDMs.92 Researchers often use specific cell surface markers and advanced tools to distinguish MDMs from microglia.93 In addition, microglia and MDMs have some morphological differences. Microglia are fixed cells with many branches and large volume, while MDMs have few branches and small volume and strong migration ability.93 The function of microglia and MDMs may be closely related to their morphology, and further studies are needed to determine how the morphological differences between MDMs and microglia affect their function and dynamics in physiological and pathological conditions.93 In physiological state, both microglia and MDMs, as immune cells, have phagocytic functions, but microglia are more inclined to regulate brain development and maintain neuronal networks, while MDMs are more prominent in their immune function.14,15,17,23 When tumors occur, microglia and MDMs have both pro-tumor and anti-tumor effects. Interestingly, microglia can proliferate to a limited extent, whereas MDMs cannot.94

Scientists have proposed an array of treatment strategies targeting the characteristics of neuroglia and immune cells within the TME (Table 2). However, most of these strategies are still in the research and experimental phases. Given the high incidence and poor prognosis of breast cancer metastasis to the brain, effective treatment methods remain scarce. Thus, urgent acceleration of research into the mechanisms and treatment modalities of BCBM is imperative. Future scientific endeavors will delve deeper into exploring and elucidating the mechanisms and treatment modalities concerning breast cancer metastasis to the brain, with a keen emphasis on the research and development of treatment strategies focusing on the TME in brain metastasis.Table 2. Information on drugs that target neuroglia and immune cells.

Cell types	Therapeutic drugs	Effects of drugs	Whether drugs can cross the BBB	References	
TAMs	Mannosylated clodronate liposomes	Depletes M2 (MRC1-expressing TAMs)	√	8,59	
CSF1R inhibitors (M279、BLZ945)	Curtail M2	M279-×、BLZ945-√	60–63	
STAT5 inhibitors (AC4-130)	The concurrent inhibition of BLZ945 and AC4-130 exhibits a synergistic anti-tumor impact, possibly normalizing TAMs phenotype	Uncertain	62	
PI3K Class I inhibitor (BKM120)	Restrains TAMs’ facilitation of breast cancer cell invasion into brain parenchyma	√	65,66	
Wnt inhibitors (DKK-2, DKK-1, secreted Frizzled-like protein-1)	Suppress the pro-invasive effects of microglia	Uncertain	19	
STAT6 inhibitor (AS1517499)	Reduces Arg1 expression in microglia, suppresses the anti-inflammatory phenotype of microglia	√	60,67	
ER inhibitor (tamoxifen)	Enhance the phagocytosis of microglia	√	47,68	
HSP47 inhibitor (Col003)	Inhibits the polarization of microglia to M2 phenotype	√	69	
Blocking either the CSF1R or the EGF receptor (AFS98, Iressa)	Inhibit macrophage-induced invasion	AFS98-uncertain, Iressa-√	28,70	
Zoledronic acid	Inhibits tumor-promoting macrophages	Uncertain	29,71	
Astrocytes	Blocking IL-1β (IL-1β neutralizing antibody)	Inhibits breast cancer cell migration	Uncertain	37	
MMP inhibitors (ONO-4817, Marimastat, Batimastat)	Inhibit tumor cell invasion and migration	ONO-4817-uncertain, Marimastat-√, Batimastat-uncertain	41,72	
PLC inhibitor (edelfosine)	Affects tumor cell proliferation and signaling between astrocytes and cancer cells	√	42	
Multi-target tyrosine kinase inhibitor (pazopanib)	Reduces the number of p751-PDGFRβ+ astrocytes	√	74,75	
ER inhibitor (tamoxifen)	Reduces doxorubicin resistance	√	47	
Dual inhibition of double ETAR, ETBR (macitentan)	Upregulates the sensitivity of metastatic tumor of brain to paclitaxel	Uncertain	76	
TAMs, tumor-associated macrophages; CSF1R, colony-stimulating factor 1 receptor.

Acknowledgments

Thanks to JH for providing the translation of the article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Abbreviations

BCBM breast cancer brain metastasis

BBB blood-brain barrier

TME tumor microenvironment

MDMs monocyte-derived macrophages

Treg regulatory T

CNS central nervous system

TAMs tumor-associated macrophages

Lnc-BM lncRNA associated with BCBM

JAK2 Janus kinase-2

STAT3 signal transducer and activator of transcription 3

TNFα tumor necrosis factor-alpha

TGFβ1 transforming growth factor β1

MT1-MMP membrane type1-matrix metalloproteinase

CSF1R colony-stimulating factor 1 receptor

EGF epidermal growth factor

CSF1 colony-stimulating factor 1

T-MV tumor microvesicles

CSCs cancer stem-like cells

ANGPTL4 angiopoietin-like 4

BTB blood-tumor barrier

HER2 growth factor receptor 2

BDNF brain-derived neurotrophic factor

STAT1 signal transducer and activator of transcription 1

Arg2 arginase 2

GBP1 Guanylate-Binding Protein 1

CAR chimeric antigen receptor

GBM glioblastoma

IL13Rα2 interleukin-13 receptor alpha 2

DCs dendritic cells

Author contributions

HM authored the article and created the charts, while XZ and LR revised the manuscript and figures. All authors made substantive contributions to this work.

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.
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