Emerging Principles of Brain Immunology and Immune Checkpoint Blockade in Brain Metastases

Jawad Fares; Ilya Ulasov; Peter Timashev; Maciej S. Lesniak

Disclosures

Brain. 2021;144(4):1046-1066. 

In This Article

The Immunology of Brain Metastases

Brain metastases exhibit one of the most diverse immune cell landscapes with substantial infiltration of T cells and neutrophils. In addition, a variety of immune agents, namely microglia and macrophages, lymphocytes and astrocytes complete the immune microenvironment of metastatic tumour cells.

Tumour-associated Myeloid Cells and Microglia

Cells of myeloid origin are characterized by high plasticity and the ability to assimilate signals from cytokines, chemokines and growth factors. Their activation can promote cellular invasion, angiogenesis, metastasis and immune suppression.[24,25] It has been estimated that cells of myeloid origin comprise up to 32.7% of intratumoural cells in brain metastases.[26] Nevertheless, recent studies show that while tumour-associated myeloid cells and microglia (TAMs) can be significantly increased in gliomas compared with non-tumour tissue, lymphocytes are more prevalent in brain metastases.[27]

Myeloid cells arise from two distinct sources, including the periphery (bone marrow-derived macrophages; CD49D+) or the yolk sac (microglia; CD49D−).[28–30] They tend to accumulate in settings of higher brain tumour grade and engage in significant bidirectional crosstalk with the tumour cells.[31–33]

Microglia are a central component of the brain immune microenvironment.[34] Preclinical observations proposed a role for microglia in the extravasation stage of the metastatic process.[35] In the case of inflammation, myeloid cells are recruited from circulating monocytes.[36] The immune role of microglia involves the presentation of antigens, cytotoxic activity via nitric oxide and superoxide, and phagocytic capacity.[37] In brain metastatic settings, microglia show restricted upregulation of interleukin (IL)-6 that exerts immunosuppressive effects on T cells and mediates resistance to immune checkpoint blockade.[38] In addition, microglia limit the expression of TREM1 receptors, which modulate pro-inflammatory responses during neuroinflammation.[39,40] Microglia-released chemokines, such as C-X-C motif chemokine 5 (CXCL5) and CXCL8, are increased in the setting of brain metastases; these chemokines recruit immunosuppressed neutrophils into the metastatic niche.[27]

Microglia express a variety of proteins that play conflicting roles in immune induction and suppression. The expression of high mobility group box 1 protein (HMGB1) facilitates antigen presentation and activation of the adaptive immune system. However, TAMs can also express immunosuppressive proteins like PD-L1,[41,42] usually triggered by inflammation and/or necrosis.[43] These biological properties allow TAMs to regulate and preserve the immune balance, preventing any swelling and, ultimately, damage within the confined area of the skull. Nevertheless, metastatic tumour cells hijack the immunosuppressive ability of TAMs to evade the immune system and promote their own survival.[44] TAMs also secrete nitric oxide and drain the tumour microenvironment in the brain of amino acids that are essential for the activation of cytotoxic T cells, which leads to the blockade of immune signalling pathways, such as IL-2 signalling, an important stimulatory pathway for the immune system.[44,45] Moreover, the production of reactive oxygen species leads to the degradation of proteins, lipids and nucleic acids, pushing T cells to commit to apoptosis.[44] The coexistence of reactive oxygen species and nitric oxide within the same microenvironment further leads to the formation of peroxynitrites, leading to the nitrosylation of T-cell receptors. This disturbs the mechanism of interaction of T cells with tumour cells and may contribute to metastatic immune resistance.[45]

Microglia have also been shown to express neurotrophin (NT)-3 to regulate immune cellular activation. NT-3, similar to other neurotrophins, plays a major role in stimulating and controlling neurogenesis.[46] In metastatic settings, NT-3-expressing microglia are hijacked to assist in the formation of brain metastases.[47,48] In addition, the secretion of IL-10 and transforming growth factor beta (TGFβ) by myeloid cells and microglia induces the activity of M2 TAMs and regulatory T cells, which are known to suppress immune responses.[45] The role of microglia extends, in some cases, to guiding the invasion of metastatic cells into the brain.[44,49] Imaging studies have shown that a wall of microglial cells is situated at the border between the metastatic cells and the brain parenchyma.[50]

Macrophages are generally known to modulate between polarizing states: M1 and M2. While the M1 state is pro-inflammatory, inducing a Th1 response against foreign pathogens and cancer cells, the M2 state contributes to immunosuppression and cellular repair. Although understanding of macrophage polarization remains limited in the setting of brain metastasis, TAMs generally presume an M2 composition, causing immunosuppression and assisting tumour cells in evading immunity.[44] Nevertheless, polarization of macrophages differs between different regions of the brain. In parenchymal metastases, the release of cytokines such as lymphotoxin β and the increase in NF-κB1 activity demonstrate a direct involvement in the M2 polarization of macrophages.[51] Subsequently, this results in the secretion of growth factors, remodelling of the extracellular matrix (ECM), and angiogenesis.[52] The release of TGFβ, matrix metalloproteinases (MMP) 2, MMP9, cathelicidin, cathepsins and scavenger receptor class A (SRA) have been reported to play a major role in pro-metastatic ECM remodelling, whereas the secretion of vascular endothelial growth factor A (VEGFA) and platelet-derived growth factor (PDGF) contribute to angiogenic formations.[25,52]

Upon settling in the brain parenchyma, metastatic tumour cells release factors to recruit TAMs to the tumour microenvironment (Figure 1). Factors like VEGFA, chemokine ligand (CCL) 2, CCL5, CCL9, CCL18 and colony stimulating factor 1 (CSF1) have been reported to be released in such settings.[25,52,53] One of the therapeutic strategies used to target TAMs is the colony stimulating factor 1 receptor (CSF1R) inhibitors that inhibit the release of pro-survival factors by the TAMs. Inhibition of CSF1R either depletes or depolarizes TAMs.[31,32]

Figure 1.

The interplay between metastatic tumour cells and TAMs in the brain microenvironment. (1) Upon entry into the brain parenchyma, tumour cells release cytokines and chemokines, such as CSF1, GM-CSF, MCP-1, HGF, SDF-1 and CX3CL1, to recruit myeloid cells from the periphery and brain-resident microglia into the tumour niche. (2) Myeloid cells derived from the bone marrow and the yolk sac enter the brain and microglia migrate towards the tumour cells. (3) TAMs release growth factors, such as EFG, IL-6, TGF-β, IL-1β and other proteases. (4) This promotes tumour survival and growth. Created with BioRender.com.

Unlike microglia, myeloid cells are poor APCs. They express isoforms of human leukocyte antigen (HLA)-G and HLA-E, in addition to the major histocompatibility complex (MHC) type I molecules that prevent the lysis of natural killer (NK) cells and T cells.[25] Nonetheless, their immunosuppressive ability is illustrated through the co-expression of inhibitory molecules, such as PD-L1 and PD-L2, and the release of inhibitory chemokines, such as IL-10, TGFβ, CCL5, CCL20 and CCL22, which prevent the activation of the adaptive immune response in various cancers.[25,44,54]

T Cells and NK Cells

Analysis of brain metastases confirmed a significantly higher proportion of lymphocytes, with melanoma brain metastases exhibiting the most lymphocytic infiltrates and CD8+ T cells predominating other lymphocytic fractions.[27] Yet, the level of inflammation around metastatic tumours is different from one patient to another and between cancer types, as the concentration of T cells around brain metastatic cells can vary from nil to very high.[55,56] The discrepancy in CD8+ T-cell density can be associated with the time at which brain metastases occurred.[57] Another explanation for this variation can be seen in the protein expression of metastatic cells. For example, high PD-L1 expression eases immune evasion and allows the metastatic cell to escape checkpoint by T cells (Figure 2).[56] Moreover, expression of forkhead box p3 (FOXP3) as well as programmed cell death protein 1 (PD-1) by regulatory T cells favours immune suppression in the metastatic tumour microenvironment.[56,58]

Figure 2.

T-cell regulation through an immune checkpoint. Top: The expression of CTLA4 and PD-1 on T cells serves as an immune checkpoint through which tumour cells can bind and deactivate T cells. Suppression of effector T-cell responses provides metastatic cancer cells with the opportunity to proliferate. Bottom: Immune checkpoint inhibitors, such as anti-CTLA4, anti-PD-L1 and anti-PD-1 antibodies, prevent tumour cells from applying the brakes to T-cell activation, which subsequently leads to T-cell activation, immune attack and tumour cell death. Created with BioRender.com.

In terms of clinical outcomes and survival, it has been demonstrated that patients with increased immune responses and cytotoxic T-cell infiltration of metastatic tumours show better prognoses.[58–60] Melanoma brain metastases exhibited higher T-cell infiltration than breast cancer brain metastases.[61] This may be the reason behind the success of some immunotherapeutic regimens in improving survival in patients with metastatic melanoma to the brain.[62,63] Furthermore, the infiltration of brain metastases with high levels of effector CD3+, cytotoxic CD8+ or memory CD45RO+ T cells has been shown to improve survival.[61] In addition, increased T-cell trafficking into the brain metastatic areas decreases the integrity of white matter tracts, providing a method to identify immunologically active microenvironments in the brain using diffusion tensor MRI.[64]

The blood–brain barrier also plays a role in limiting the infiltration of cytotoxic T cells. Angiogenesis and neovascularization are key hallmarks of metastasis.[65] Nonetheless, the magnitude of angiogenic potential differs between cancer types.

Aside from expressing inhibitory proteins and receptors, such as cytotoxic T-lymphocyte associated protein 4 (CTLA4), PD-1, lymphocyte-activation gene 3 (LAG3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), inducible T-cell costimulator (ICOS), and T cell immunoreceptor with Ig and ITIM domains (TGIT), regulatory T cells can also secrete large volumes of immunosuppressive cytokines. IL-10, IL-35 and TGFβ are reported to be released by regulatory T cells in the tumour microenvironment.[66] Moreover, regulatory T cells have been documented to drain the tumour microenvironment of immune-stimulatory cytokines, like IL-2, deactivating the antitumour Th1 immune response. More recently, it was shown that T-cell receptors that exhibit pan-cancer cell recognition via MHC class I-related proteins lacked detectable reactivity in melanoma. Providing the T cells with the MC.7.G5 T-cell receptors rendered them capable of killing autologous melanoma.[67]

NK cells also partake in the intratumoural immune response in the brain tumour microenvironment.[68] They have been shown to be present in metastatic brain tumours, gliomas and craniopharyngiomas.[69,70] Like cytotoxic T cells, NK cell functioning is often diminished in patients with brain tumours due to the release of anti-inflammatory molecules, such as TGFβ, by tumour cells. In addition, tumours that secrete inflammatory mediators like cyclooxygenases (COX) and prostaglandin E2 suppress NK cell antitumour activity.[71]

Neutrophils

Despite being the most abundant circulating immune cells, the function of neutrophils in the immune microenvironment of brain metastases remains unclear. Yet, recent advances have elucidated some of the mechanisms that govern neutrophil recruitment and activation in the brain metastatic microenvironment. Neutrophils can be recruited to the brain microenvironment through the secretion of CXCL8 chemoattractant by TAMs. The upregulation of ITGA3 and CXCL17 by brain metastatic cells further increases neutrophil abundance at the scene.[27] The overexpression of the adenosine receptor ADORA2A suppresses the pro-inflammatory phenotype of neutrophils.[27,72] Moreover, overexpression of CD177 in brain metastatic cells affects neutrophil migration and activation and suppresses T-cell proliferation.[27] In addition, the upregulation of MET is associated with the recruitment of immunosuppressive neutrophils to the immune microenvironment in brain metastases.[27,73]

Recently, it has been shown that brain metastatic cells overexpress an epigenetic modifying protein, enhancer of zeste homolog 2 (EZH2), to stimulate signalling pathways that suppress recruited neutrophils.[74] In the setting of brain metastases, EZH2 phosphorylation changes its function from being a methyltransferase to a transcription factor that increases c-JUN expression.[74] This leads to the secretion of pro-tumorigenic inflammatory cytokines, including G-CSF, which recruits Arg1+ and PD-L1+ immunosuppressive neutrophils into the brain to drive metastasis outgrowth.[74] Blocking the influx of this subset of neutrophils by G-CSF-blocking antibodies or immune checkpoint blockade therapies combined with Src inhibitors impeded brain metastasis in multiple mouse models.[74] Furthermore, neutrophils can play a role in priming the brain metastatic niche. It has been shown that S100 calcium-binding protein A (S100A)-8 and S100A9 are upregulated in the pre-metastatic niche in the brain, leading to recruitment of neutrophils, which support subsequent metastatic seeding and colonization (Figure 3).[75]

Figure 3.

Role of neutrophils in brain metastases. Left: In the setting of brain metastases, a high neutrophil-to-lymphocyte ratio is associated with poor prognostic outcomes. Middle: In the tumour microenvironment, neutrophils are recruited through increased release of S100A8 and S100A9 proteins by tumour cells. Right: The increased expression of CD117, ADORA2A, ITGA3 and MET by brain metastatic cells leads to neutrophilic suppression in the immune microenvironment. The phosphorylation of EZH2 protein increases the expression of c-JUN, which leads to the release of G-CSF. G-CSF, in turn, leads to the recruitment of neutrophils that overexpress PD-L1 and Arg1. Furthermore, tumour-associated myeloid cells and microglia release chemokines, such as CXCL8, to suppress neutrophils in the tumour microenvironment. Created with BioRender.com.

Neutrophils have a prognostic value in the setting of brain metastases. A high ratio of neutrophils to lymphocytes in the peripheral blood is associated with reduced survival time, even after surgical resection of brain metastases.[76] Furthermore, in patients treated with stereotactic radiosurgery, the post-treatment neutrophil-to-lymphocyte ratio was associated with poor overall survival.[77] Nevertheless, in patients with non-small cell lung cancer (NSCLC) who harbour EGFR mutations, a neutrophil-to-lymphocyte ratio ≤2.99 was associated with prolonged survival.[78] Therefore, the neutrophil-to-lymphocyte ratio could serve as a useful prognostic biomarker in specific patients with brain metastases.

Astrocytes

Astrocytes are some of the most abundant cell types that are unique to the CNS and play important roles in mediating tissue-specific communication in the brain. Therefore, it is likely that metastases that preferentially grow within the brain must find ways to adapt and favourably interact with these unfamiliar cellular players.[8] Brain metastatic cells are known to express high levels of IL-1β upon interaction with astrocytes. This leads to the activation of the Notch signalling pathway, which increases stemness of cancer stem cells and drives their growth in the tumour microenvironment.[79] Moreover, it was shown that cancer cells within established brain metastases form functional gap junctions with astrocytes in the adjacent microenvironment, thereby creating a conduit for bidirectional communication to support outgrowth.[80,81] Through these gap junctions, cancer cells reprogram astrocytes by providing cGAMP to induce a pro-inflammatory program, characterized by the production of interferon alpha (IFNα) and tumour necrosis factor alpha (TNFα).[80,81] In turn, these cytokines support outgrowth of metastases by activating signal transducer and activator of transcription (STAT) 1 and nuclear factor-κB (NF-κB) signalling within cancer cells (Figure 4). By interfering with the formation of gap junctions through pharmacological regimens, this heterotypic signalling loop can be blocked, thus mitigating brain metastasis outgrowth.[81] In addition, astrocytes possess the capability to release factors that can remodel the ECM, such as MMPs and heparanase, which contribute to the invasion of metastatic cells across the blood–brain barrier and facilitate subsequent metastatic colonization.[82,83]

Figure 4.

Interaction of astrocytes with brain metastatic cancer cells. Metastatic cancer cells release cGAMP to reprogram astrocyte behaviour and increase STAT3 signalling. In turn, astrocytes form gap junctions with metastatic cancer cells in the brain to exchange stimulatory signals, such as TNF and IFNα, which activate STAT1 and NF-κB pro-tumorigenic signalling. Created with BioRender.com.

Brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by STAT3 in a subpopulation of reactive astrocytes surrounding metastatic lesions.[84] These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. Blocking STAT3 signalling in reactive astrocytes reduces experimental brain metastasis from different primary tumour sources, even at advanced stages of colonization.[84,85] A safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumour effects in patients with advanced systemic disease that included brain metastasis.[84]

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