Evidence suggests that in various cancers, a subpopulation of circulating tumor cells (CTCs) has the CSC phenotype during the initial metastasis process [95,96,97,98] through the activation of epithelial–mesenchymal transition (EMT), which endows them with inherent metastatic potential [99]. The activation of EMT facilitates the invasion and migration of cancer cells from the primary tumor site to intravasation and translocation to distant metastasis sites [99], whereas the activation of mesenchymal epithelial transition (MET) after extravasation and cell seeding may facilitate colonization [100]. EMT transcriptional factors and the mesenchymal marker vimentin are expressed at higher levels in CD44+/CD24-/low BCSC-like cells than in more differentiated epithelial CD44-/CD24+ cells [101]. In addition, the changes in ECM dynamics may contribute to the disruption of asymmetric stem cell division, leading to CSC overexpansion [102]. Hyaluronic acid, which constitutes the ECM structures, interacts with the cell surface protein CD44, enhancing CSC properties by activating the stem cell marker NANOG [103]. CSCs in TNBC cell lines display highly invasive properties with elevated expression of proinvasive genes, such as those for interleukin (IL)-1, IL-6, IL-8, and urokinase plasminogen activator (uPA) [86]. Together with the stimulation of the nuclear factor kappa-light-chain enhancer of activated B cell (NF-κB) signaling, stromal uPA activates matrix metalloproteinase-2 (MMP-2) production and becomes responsive on the surface of tumor cells to cleave any component of the ECM, thereby allowing a break through the basement membrane and facilitating the EMT process [104,105].

In patients with cancer, approximately 1 × 10⁶ cancer cells per 1 g of tumor enter the circulation daily. However, only a fraction of these cells survive and reach a distant niche [106]. It has been reported that tumor cells take a longer time to extravasate the brain because of the extra boundaries contributed by the BBB [107]. In lung cancer, it takes approximately 48 h to extravasate the brain, whereas it takes only 6 h to extravasate the liver. On the other hand, breast cancer cells require 2 to 7 days to extravasate the brain. Consequently, CTCs have a longer survival time within the cerebral vasculature compared with that in other metastatic sites [107]. Studies have revealed that the mutation or inactivation of cell cycle-regulating genes and apoptosis-inducing genes enable CSCs to escape apoptosis [108]. In addition to DNA repair systems, autophagy is one of the processes that is strongly associated with CSC physiology in TNBC cells, including tumorigenesis, differentiation, plasticity, migration, or invasion, as well as pharmacological, viral, and immunoresistance [109,110,111]. Autophagy is a cellular degradation process that is essential for promoting cellular adaptation to stressors, cytokine mediation, chromosome stability, stem cell microenvironment, and cellular homeostatic mechanism [109,112,113,114,115]. It has been determined that in triple-negative BCSC, the knock down of autophagy-related genes, such as ATG8/LC3 and ATG12, or pharmacological blockade of autophagy flux decreases the CD44+/CD24-/low CSC phenotype [116], probably through the IL6/STAT3/JAK2 pathways that contribute to the conversion of non-CSCs into CSCs [109]. The autophagy process can be induced upon the loss of integrin-mediated cell attachment to the surrounding ECM [117], and it further protects cells from detachment-induced cell death, termed anoikis [118]. Two key autophagy proteins, BECLIN1 and ATG4, are upregulated in mammospheres compared with adherent cells and are needed for their maintenance and expansion [119,120]. Upon EMT and autophagy induction, the process aids in the evasion from immune surveillance programs, such as T-cell-mediated lysis [121,122] and inhibit NK-mediated tumor cell killing by degrading granzyme B [123,124].

Therefore, to enter the brain parenchyma, cancer cells must pass through microcapillary walls by opening tight junctions [128]. These junctions can be destabilized by cancer cells through the expression of cytokines, chemokines, and inflammatory mediators [129,130]. However, it is not yet clear how circulating CSCs affect the function of the BBB. Current potential mechanisms of the extravasation strategy of tumor cells include docking, locking, trans-endothelial migration, and adhesion to the subendothelial matrix, which mimic the mechanisms used by immune cells [131]. The CXCL12/CXCR4/CXCR7 ligand receptor system is the vital axis that regulates neuro-glio-vascular interactions and vessel growth during human brain development. In a traumatic brain injury murine model, the CXCL12/CXCR4 axis was stimulated to promote neural stem cell migration through the stimulation of MMP-2 secretion [132,133]. Nevertheless, the CXCL12/CXCR4 axis promotes metastasis and invasion across a wide variety of solid tumors. For example, the CXCL12/CXCR4 autocrine-positive feedback mechanism controls the survival and proliferation of neural progenitor derived glioblastomas under hypoxic stress [134,135,136]. In breast cancer brain metastases, the CXCL12/CXCR4 axis takes part in the homing, motility, and progression of metastases to regulate the migration of CTCs through the BBB [105]. Notably, BCSCs have high CXCR4 expression and are defined as a pro-metastatic subpopulation, whereas the expression of CXCL12 defines regions with a higher risk of causing metastasis for breast cancer invasion [137,138].

Moreover, cancer cell-derived exosomes have been noted to have multiple roles in the events of metastasis, with a key role in pre-metastatic niche formation through vascular remodeling and modulation of cellular behaviors in the pre-metastatic site. Metastatic TNBC cells secrete miR-105-enriched exosomes to down-regulate ZO-1 protein expression in endothelial cells, disrupting the endothelial cell barrier and increasing vascular permeability, thereby facilitating invasion and migration [139]. Reciprocally, brain endothelial cells actively influence tumor cell extravasation and proliferation, which are mediated by bilateral cell surface receptors and adhesion molecules, such as integrins, selectins, chemokines, and the receptors of the immunoglobulin superfamily (ICAM-1, VCAM1) [140]. Activated αvβ3 integrin in circulating TNBC cells appeared to interact with platelets causing thrombus formation, which facilitates the arrest of tumor cells within the vasculature [141]. Furthermore, the activation of αvβ3-integrin assists in the intracerebral growth of TNBC cells through the continued upregulation of vascular endothelial growth factor (VEGF) [142]. An in vivo study injected brain-seeking TNBC cell lines into the murine carotid artery and demonstrated that the VEGF secreted from the cells destabilized the brain microvascular endothelial cells through activation of angiopoietin-2 (Ang2) expression, which further mediated BBB disruption (changes in the tight junction between ZO-1 and claudin-5). The interaction plays a critical role in the initial cascade of colonization of TNBC in the brain tissue, because the administration of neutralizing Ang-2 peptibody prevented changes to the BBB integrity and further inhibited the formation of TNBC cells colonization into the brain [143].

Evidence supports dynamic crosstalk between cancer cells and the brain microenvironment contributed by surrounding stromal cells, immune cells, and ECM, which are crucial for tumor growth after cell seeding in the brain [144]. Nonetheless, most CTCs are likely to die even after successfully arresting and extravasating the brain [145]. The adaptation period following dissemination is typically preceded by a period of dormancy in which cells are in a slow cell-cycling state; this is mediated by the expressions of stemness-associated transcription factors such as the SOX2, SOX9, and Wnt signaling pathways [146], which can last up to several decades [147]. Further exit from dormancy requires evasion from the immune surveillance mechanism and angiogenic switch to form micro-metastases [148]. Striking overlap exists between the behaviors of CSCs in tumors and the behavior of cancer cells in a dormant state, especially in the context of tumorigenesis after cell seeding; this suggests that the dormant extravasated tumor shows the CSC phenotype [149,150]. A study revealed that a combination of intracellular and extracellular signals within the tumor microenvironment, namely regulation of quiescence, alteration of angiogenic response, and modulation of immune surveillance govern the regulation of the dormant state [151].

Astrocytes have long been recognized as a key stromal component and immunomodulator of metastatic brain tumors which have tumor-killing or tumor-promoting effects, likely reflecting the fact that these cells exist as distinct subtypes with distinct functions [152,153,154]. Several astrocyte subtypes have been identified, termed A1 and A2 [155]. A1 astrocytes are regarded as proinflammatory astrocytes, which are induced by classically activated neuroinflammatory microglia through the secretion of IL-1a, TNF, and C1q. In the event of CNS injury and neurodegenerative disease, A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis, and phagocytosis, thereby inducing the death of neurons and oligodendrocytes [156]. A2 astrocytes are thought to promote tissue repair through the production of neurotrophic factors. It is thought that most tumor-associated astrocytes are likely to be of the A2 subtype, which highly expresses the phosphorylated (activated) form of the signal transducer and activator of transcription-3 (p-STAT3) pathway [153]. Notably, STAT3 signaling in tumor cell is a key signaling mechanism activated by cytokines and growth factor receptors that establish an immunosuppressive microenvironment during the early stages of breast carcinogenesis to promote tumor growth and metastases [157]. In addition, STAT3 expression in the BCSC-like subset of the TNBC cell line serves as a chemo-resistant marker [158]. Mechanistically, these p-STAT3+ reactive astrocytes block the access of immune cells, such as CD8+ cytotoxic T cell, to cancer cells through the upregulation of immunosuppressive molecules, such as programmed cell death–1 ligand 1 (PD-L1), VEGF-A, lipocalin-2, and tissue inhibitor of metalloproteinases 1 (TIMP-1). Furthermore, the crosstalk between microglia and reactive astrocytes contributes to the establishment of an immunosuppressive microenvironment, thereby facilitating the metastasis of breast cancer to the brain. The p-STAT3+ reactive astrocytes exhibit increased expression of the CD74 ligand, MIF (macrophage migration inhibitory factor), and increased binding to CD74+ microglia, which upregulate the expression of midkine (neurite growth-promoting factor 2 (NEGF2), a downstream target of the NF–κB signaling pathway that promotes the development of brain metastases. All pieces of evidence suggest the significance of the STAT3 pathway during the interplay between the TNBC CSCs and astrocytes contributing to the microenvironment.

In addition, the phosphoinositide 3-kinase (PI3K)-Akt pathway has been implicated as a regulator of brain metastasis. PI3K signaling promotes various cellular processes that include proliferation, survival, metabolism, and angiogenesis in response to extracellular signals that activate receptor tyrosine kinases or G-protein coupled receptors. Notably, a study revealed that PI3K signaling in human breast cancer brain metastasis samples was not only active in the metastatic cells but also in the CNS microenvironment. Furthermore, in vitro and in vivo demonstrations revealed that treatment with buparlisib (BKM120), a pan-PI3K class 1 inhibitor with excellent BBB penetrance, reduced the macrophage or microglia-induced invasion and glial-assisted infiltration of breast cancer cells into the brain parenchyma [159]. The Wnt pathway acts in a compensatory manner when the TNBC cell line is challenged with buparlisib. Effectively, the dual PI3K and Wnt pathway inhibitor works in synergy to promote cell viability and enhance antitumor efficacy in TNBC cell lines [160]. In addition, results demonstrate that rational combinations of the buparlisib with MEK1/2 inhibitor in mice bearing intracranial TNBC tumors (SUM149, MDA-MB-231Br, MDA-MB-468, or MDA-MB-436) improved survival for intracranial SUM149 and MDA-MB-231Br, but not MDA-MB-468 or MDA-MB-436, suggesting therapeutic potential for the treatment of some BM-TNBC [161]. Currently, a phase II clinical trial of BKM120 plus capecitabine is being conducted in patients with BM-TNBC (NCT02000882, Table 1).

Furthermore, TNBC cells manifest distinct gene expression patterns after metastasizing to different organs because the interplay with the site-specific extrinsic signals affects subsequent metastatic outgrowth. For example, mutations of phosphatase and tensin homolog (PTEN), a tumor suppressor gene and negative regulator of PI3K-Akt signaling, are rarely observed in the primary breast or lung cancers; however, a 21% increase in PTEN mutations was observed in brain metastatic tumors [162,163]. In a study with 111 tissue samples of breast cancer brain metastases, loss of PTEN protein expression was noted in 48.6% of all samples. Notably, it is significantly associated with the TNBC subtype (67.5%, p = 0.001) [164]. Previous studies have revealed that PTEN inhibition increased the expressions of the stemness and mesenchymal markers, OCT4 and SNAIL in TNBC cell lines [165], suggesting the role of PTEN suppression in CSC maintenance of brain metastasis. In addition, loss of PTEN expression in TNBC cells seemed to be the essential trait acquired from the brain-specific microenvironment, which led to an increased secretion of cytokine chemokine ligand 2 (CCL2, chemo-attractant during inflammation) and the recruitment of Iba1+ myeloid cells that enhance tumor cells proliferation and reduce apoptosis [163,166]. PTEN overexpression was evidenced to attenuate the invasiveness and migration of breast cancer cells as well as astrocyte activation [164]. Mechanistically, in the brain-specific microenvironment, reactive astrocytes suppress PTEN expression in TNBC cells with the intercellular transfer of PTEN-targeting microRNA (miR-19a), through exosomes secretion, whereas the blockade of astrocyte exosome secretion rescues PTEN loss and suppresses brain metastasis in vivo [163]. Notably, the PTEN level in TNBC cells is restored after the cells leave the brain microenvironment. All these studies suggest that the critical role of plasticity in PTEN suppression might contribute to the CSC phenotype in brain-adaptive TNBC cells, thereby indicating new opportunities for effective anti-metastatic therapies in BM-TNBC [165,167,168,169].