TY - JOUR
AU - Fidler, Isaiah, J
AB - BACKGROUND It is estimated that at least 200 000 cases of brain metastases occur each year in the US, which is 10 times the number of patients diagnosed with primary brain tumors. Brain metastasis is associated with poor prognosis, neurological deterioration, diminished quality of life, and extremely short survival. Favorable interactions between tumor cells and cerebral microvascular endothelial cells encourage tumor growth in the central nervous system, while tumor cell interactions with astrocytes protect brain metastases from the cytotoxic effects of chemotherapy. CONTENT We review the pathogenesis of brain metastasis and emphasize the contributions of microvascular endothelial cells and astrocytes to disease progression and therapeutic resistance. Animal models used to study brain metastasis are also discussed. SUMMARY Brain metastasis has many unmet clinical needs. There are few clinically relevant tumor models and no targeted therapies specific for brain metastases, and the mean survival for untreated patients is 5 weeks. Improved clinical outcomes are dependent on an enhanced understanding of the metastasis-initiating population of cells and the identification of microenvironmental factors that encourage disease progression in the central nervous system. Approximately 200 000 cases of brain metastases occur in the US each year (1), and between 20% and 40% of patients with systemic cancers develop brain metastasis during the course of their disease (2). Brain metastasis is associated with poor prognosis, neurological deterioration, diminished quality of life, and extremely short median survival (3). Most brain metastases are the product of primary tumors that originate in the lung (40%–50%), breast (15%–20%), skin (5%–10%), or gastrointestinal tract (4%–6%) (4). Reports suggest that the incidence of brain metastasis may be increasing because of improved control of primary tumors, refinements in brain imaging techniques, and a rise in melanoma skin cancer (5–7). In general, the distribution of brain metastases parallels cerebral blood flow, with 80% of metastases located in the cerebral hemispheres, 15% in the cerebellum, and 5% in the brainstem (8). The majority of patients possess multiple lesions at the time of diagnosis (9), and nearly all will experience some degree of neurocognitive impairment during the course of their disease (10). The deterioration in neurological function is a consequence of the destruction or displacement of normal brain tissue, peritumoral edema, increased intracranial pressure, or vascular compromise (11). Current therapeutic approaches to brain metastases include surgery, whole brain radiotherapy, stereotactic radiosurgery, and chemotherapy. The median survival time for untreated patients with multiple metastases is 5 weeks (12), whereas multimodality therapy employing combinations of surgery, whole-brain radiotherapy, and stereotactic radiosurgery may extend overall survival to 3–18 months in a select population of patients (13). The recent improvement in local control of central nervous system (CNS)2 disease has stimulated efforts to increase the enrollment of brain metastasis patients in early-phase clinical trials (14). Additional improvements in clinical outcomes are dependent on an enhanced understanding of the molecular phenotype of cells that initiate brain metastasis and identification of microenvironmental factors that promote disease progression in the CNS. In this review, we discuss how the brain microenvironment contributes to metastasis and therapeutic resistance, with particular emphasis on blood vascular endothelial cells and astrocytes. Pathogenesis of Brain Metastasis To form a metastasis, a tumor cell must complete a sequential series of steps that begins with its detachment from the primary mass and invasion of the surrounding tissue. In normal tissues, epithelial cells are held in tight apposition to one another by adhesive proteins that serve to maintain the structural integrity of the tissue. Downregulation of 1 of these adhesion proteins, E-cadherin, has been shown to correlate with the metastatic potential of several tumor types, including those that metastasize to the brain (15, 16). The first barrier encountered by invading tumor cells is the basement membrane separating epithelial from connective tissue compartments. Invading tumor cells secrete proteolytic enzymes that degrade the epithelial basement membrane in a process that signifies the transition from a benign carcinoma in situ to a malignant invasive tumor (17). Migrating tumor cells then encounter the endothelial basement membrane of thin-walled blood vessels, which they must penetrate to enter the circulation. Alternatively, tumor cells may circulate in lymphatic channels that communicate with the systemic circulation. Tumor cells circulate as single-cell entities, homotypic emboli, or they form heterotypic associations with other cellular elements (e.g., platelets, neutrophils). Tumor cell retention in target organs may be due to size restriction, or it may be the consequence of selective molecular adhesive interactions with microvascular endothelial cells or the subendothelial basement membrane. The migration of tumor cells from the vascular compartment occurs by several mechanisms. Tumor cells that gain access to the underlying tissue parenchyma activate angiogenic programs in adjacent microvascular endothelial cells to recruit a new vascular supply. Angiogenic vessels attenuate metabolic pressures associated with unrestrained tumor cell division (18) and increase the probability of further tumor cell dissemination (19). Results of an analysis of the individual steps of metastasis with intravital time-lapse fluorescence microscopy revealed that postextravasation growth is the major rate-limiting step in metastasis (20). Metastasis is widely regarded as a selective process inasmuch as the formation of a secondary growth requires that a tumor cell successfully complete each of the abovementioned steps. Studies in which tumor cells were labeled with radioactive isotopes and injected into syngeneic mice emphasized that metastasis was a tremendously inefficient process (21). Within 24 h of tumor cell injection, 99% of the tumor cells were no longer viable. Moreover, <0.1% of injected tumor cells eventually formed metastases. Tumor cells that are successful in generating metastases are the progeny of a highly specialized subpopulation of cells that emerge from a genetically heterogeneous tumor (22). Experimental evidence supports the hypothesis that a metastasis originates from a single progenitor cell (23) and that increased metastatic potential is associated with increasing genetic instability (24). This enhanced genetic instability is thought to render metastatic clones more resistant to therapy. An expanding body of evidence suggests that the organ microenvironment also plays a critical role in dictating the outcome of metastasis and in determining tumor responsiveness to therapy (18, 25). Indeed, many of the first-line therapeutic regimens currently in use for the treatment of human cancers are designed to target cancer cells (i.e., chemotherapy) and also to modulate the tumor microenvironment (e.g., antiangiogenic therapy) (26). A recent study suggested that some brain-metastasizing cells actually bring cellular components from their original microenvironment with them during their transit to the CNS (27). However, the role of the codisseminating cells in brain metastasis, if any, remains unknown. In the following section, we review the most common in vivo and in vitro systems used in the laboratory study of brain metastasis. Brain Metastasis Models IN VIVO MODELS Investigators rely on 2 animal model systems to study brain metastasis in the whole animal: experimental and spontaneous models. To examine the cellular and molecular interactions that take place during the arrest and extravasation of tumor cells in the brain, many researchers rely on the experimental model in which tumor cells growing in culture or harvested from a dissociated tumor are directly injected into the arterial circulation of mice. The injection of tumor cells into the carotid artery provides several advantages as a technique to study brain metastases. Most notably, this approach produces a high incidence of brain lesions and a low frequency of visceral metastases (28). Another advantage of the experimental model is that it provides investigators with a means to study arrest and extravasation over a wide range of time intervals. However, despite these advantages, the approach suffers from several limitations. For example, studies employing the experimental model system routinely use tumor cells growing in culture, which may not have the same metastatic potential as cells derived from the primary lesion. Similarly, because the model bypasses the early stages of metastasis, it may allow growth in the brain of tumor cells that otherwise would not be able to metastasize to the CNS. Cultured tumor cells are routinely harvested for injection following exposure to enzymatic solutions that can alter the expression of cell surface adhesion molecules, some of which are responsible for mediating the patterns of tumor cell arrest. Indeed, there is evidence suggesting that trypsin, an agent frequently used for harvesting cultured cells, can actually redirect the distribution of metastases (29). Finally, when considering the number of tumor cells introduced into the circulation in experimental models, Glaves (30) concluded that it would take most primary tumors in excess of 24 h to release the same number of cells that are delivered in a single bolus in experimental metastasis studies. To overcome the limitations associated with the experimental brain metastasis model, several laboratories have adopted the use of spontaneous tumor models. This model system more closely approximates the dissemination of tumor cells that occurs in patients, because tumor cells are implanted in their natural environment and must be competent in each step of metastasis. However, to date there are relatively few animal tumor models that spontaneously metastasize to the brain. We previously reported (31) that B16-BL6 melanoma cells form brain metastases in approximately 25% of mice following the injection of tumor cells into the footpad of syngeneic C57BL mice. Indeed, the pattern of distribution of spontaneously metastasizing B16-BL6 melanoma cells is remarkably similar to that found in man (31, 32). Our experience with the B16-BL6 melanoma model suggests that surgical removal of the primary tumor provides sufficient time for metastasis formation in target organs, and that death is usually the result of respiratory failure secondary to extensive pulmonary metastases. Recently, Francia and colleagues (33) suppressed the growth of pulmonary metastases with antitumor agents and thus generated new tumor models of breast cancer and melanoma that were capable of spontaneous brain metastasis. To establish a brain metastasis melanoma variant, the investigators first injected WM239 melanoma cells into the subcutaneous space of nude mice and then allowed the tumors to develop before surgically removing them. Following a period of several months, the mice developed lung metastases, which were harvested and used to create the more aggressive 113/6–4L subline. The selection procedure was repeated using the 113/6–4L subline with the exception that mice were treated with a combination therapy of vinblastine and cyclophosphamide to control the pulmonary tumor burden. The combined treatment led to a significant improvement in overall survival and permitted the eventual emergence of brain metastases. The metastatic cells were then harvested and recycled in mice to establish a variant melanoma cell with enhanced potential for forming brain metastases. IN VITRO MODELS One major drawback of the in vivo models is that they are time-consuming because they require several rounds of tumor implantation and selection before variant cells with high metastatic potential can be isolated and analyzed. For example, a full 10 cycles of selection were needed to generate the B16-F10 melanoma cell line with enhanced ability to generate lung metastases (34), and no fewer than 6 selection cycles were required to establish the highly invasive B16-BL6 cell line (35). Moreover, the repeated recycling of tumor cells in experimental animals is not necessarily the most practical approach for examining tumor samples obtained from patients. Therefore, the development of an in vitro approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential would be advantageous. Agar assays are particularly useful tools to distinguish tumor cells from nontransformed cells, because cells that lack the ability to undergo anchorage-independent growth are unable to proliferate on an agar substrate (36). By plating tumor cells on increasing concentrations of agar, from 0.3% (soft agar) to 0.6% (hard agar), one can select for only those cells that were competent in forming metastases (36). Additional experiments confirmed that the growth capacity of human tumor cells on hard agar correlated with their metastatic potential in vivo (37). Plating GI-101A human breast cancer cells on 0.9% agar has recently been shown to select for the subpopulation of metastasis-initiating cells (38). The agar-selected cells, designated GI-AGR, were 5 times more invasive than the parental GI-101A cells. In addition, mice injected with GI-AGR cells had significantly more experimental brain metastasis and shorter overall survival than did mice injected with GI-101A cells. A comparative gene expression analysis between GI-AGR cells, parental GI-101A cells, and a population of GI-101A cells that were generated in vivo using several rounds of selection in the brains of nude mice (GI-BRN) suggested that the GI-AGR cells were markedly distinct from the parental cells, but shared an overlapping pattern of gene expression with the GI-BRN cells. The molecular phenotype of GI-AGR and GI-BRN cells was consistent with that of cancer stem cells and the basal subtype of breast cancer. Both the in vitro and in vivo selection processes were found to enrich for the population of breast cancer cells that coexpressed CD44 and CD133 (38). Ninety percent of the GI-AGR and GI-BRN breast cancer cells growing in cell culture conditions coexpressed CD44 and CD133, and both of these proteins were expressed in GI-101A experimental brain metastases. Moreover, we also found these proteins coexpressed in clinical samples of breast cancer brain metastasis. As previously mentioned, the organ microenvironment plays a critical role in determining the outcome of metastasis as well as the responsiveness of a tumor to therapy. In the next section, we discuss how brain endothelial cells and astrocytes contribute to the formation of metastases in the CNS and review the evidence implicating endothelial cells and astrocytes in therapeutic resistance. The Organ Microenvironment and Brain Metastasis ENDOTHELIAL CELLS The arrest of circulating tumor cells in the microcirculation of target tissues is generally regarded as one of the key rate-limiting steps in metastasis (39). Studies have shown that most brain metastases localize to the gray-white matter junctions, where the progressive narrowing of blood vessels trap tumor emboli (8). To date, very little information is available regarding the adhesive interactions that take place between tumor cells and brain microvascular endothelial cells. Previously, we (40) generated an extensive library of tissue-specific microvascular endothelial cell lines from H-2Kb-tsA58 transgenic mice to study how tumor cells interact with endothelial cells from different anatomic regions. We compared the adhesion of B16-F1 melanoma cells to lymphatic endothelial cells and brain endothelial cells, because information generated from a comparative gene expression analysis on these endothelial cells suggested that they exhibited marked differences in patterns of gene expression. Reports from investigations of experimental animal models indicated that hematogenous metastasis of melanoma cells was most likely mediated by the melanoma α4β1 integrin binding to the inducible endothelial cell glycoprotein, vascular cell adhesion molecule-1 (VCAM-1) (31, 41). The α4β1 integrin is absent in primary and dysplastic nevi, but its expression levels increase during the transition from radial growth phase to vertical growth phase (42, 43). Clinical studies documented a direct correlation between α4β1 integrin expression and the occurrence of metastasis, reduced disease-free survival, and decreased overall survival (44). We determined that VCAM-1 was constitutively expressed on monolayers of brain and lymphatic endothelial cells and that melanoma cell adhesion to lymphatic endothelial cells was mediated by the melanoma α4 integrin binding to constitutively expressed VCAM-1 (45). However, monoclonal antibody–blocking strategies targeting both the melanoma α4 integrin and endothelial cell VCAM-1 receptor had no effect on the adhesion of melanoma cells to brain endothelial cells. The results of these experiments suggested that melanoma cells may use an alternative receptor–ligand pair to promote their attachment to brain endothelial cells. To identify the molecular determinants that mediate breast cancer arrest and extravasation in the brain, Bos and coworkers (46) applied comparative genome expression analysis to experimental breast cancer brain metastasis models. The results of those experiments suggested that cyclooxygenase COX2, the epidermal growth factor (EGF) ligand HB-EGF, and the α2,6-sialyltransferase ST6GALNAC5, are critical mediators of cancer cell passage through the blood–brain barrier. COX2 was suspected of increasing the permeability of the blood–brain barrier, whereas autocrine activation of the breast cancer cell EGFR by HB-EGF was thought to promote cell invasiveness (46). The expression of ST6GALNAC5 [ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 5]3 in breast cancer cells was found to increase their adhesion to brain microvascular endothelial cells in vitro and enhance the formation of brain metastases in vivo. It remains unclear whether other types of tumors that metastasize to the brain rely on ST6GALNAC5 to facilitate their entry into the CNS. Once a tumor cell has spread to a distal organ, one of the primary determinants that govern its progression and survival is its proximity to a vascular blood supply (18). We examined the distribution of human lung cancer cells (47) that had formed brain metastases in nude mice and determined that tumor cell division takes place within 75 μm of the nearest blood vessel, and that tumor cells residing further than 150 μm from a blood vessel were destined for programed cell death (47). These measurements correspond with the diffusion distance of oxygen in tumors, which has been determined to be approximately 120 μm (48). Many tumors respond to declining oxygen tensions by producing factors that encourage neighboring endothelial cells to form new vascular networks (i.e., angiogenesis). The extent to which tumors are dependent on the process of angiogenesis varies among different types of tumors. For example, studies have shown that the neovascularization response observed during glioblastoma and renal cell carcinoma progression is significantly greater than the new blood vessel formation that accompanies the growth of lung tumors (49). That cancers of the lung do not elicit a more profound angiogenic response may be related to the immense surface area of the pulmonary vascular bed, which is several orders of magnitude greater than that of any other organ (50). In addition, there are reports that some tumors are capable of sustained growth in the absence of an angiogenic response. These tumors appear to meet their nutritional requirements by residing in close proximity to preexisting blood vessels, a process referred to as vessel cooption (51). The results generated from real-time imaging studies are beginning to provide new information regarding the different growth patterns of tumors in the brain. Failure to initiate angiogenesis was shown to lead to regression of lung adenocarcinoma cells residing in the brain, whereas the inability to locate vessels for cooption was found to be fatal for melanoma cells in the brain (52). Yano and coworkers (53) studied the molecular determinants that mediate angiogenesis in a variety of experimental brain metastasis models. Colon carcinoma (KM12SM) cells and lung adenocarcinoma (PC14-PE6) cells produced large, fast-growing parenchymal brain metastases, whereas lung squamous cell carcinoma (H226), renal cell carcinoma (SN12-PM6), and melanoma (TXM13) cells produced only a few slow-growing brain metastases. These investigators noted a direct correlation between tumor cell expression of vascular endothelial cell growth factor (VEGF) and angiogenesis and growth (53). Kim and colleagues (54) generated several breast cancer brain metastasis variant cell lines with increasing metastatic potential by repeated recycling of MDA-231 cells in nude mice. They found that tumors formed by the brain-metastasizing variants contained significantly more blood vessels compared to parental tumors and that the variant population produced much greater levels of VEGF. The majority of the angiogenic enhancing effects of VEGF are mediated by VEGF receptor 2 (VEGFR2), which belongs to a family of tyrosine kinase receptors whose expression is largely restricted to the vascular endothelium (55). Activation of VEGFR2 expressed on endothelial cells stimulates each of the processes required for new blood vessel formation and also signals for endothelial cell survival (56). Therefore, most therapeutic efforts directed toward inhibiting the pathological angiogenesis that accompanies tumor growth have focused on the VEGF-signaling pathway. Several therapeutic agents that block VEGF-induced signaling have received US Food and Drug Administration clearance and are currently available for use in the clinical setting, including bevacizumab, a chimeric monoclonal antibody that binds VEGF, and small-molecule inhibitors of the VEGFR2 tyrosine kinase (e.g., sorafenib and sunitinib). Recently, the addition of bevacizumab to paclitaxel–carboplatin therapy was demonstrated to significantly increase progression-free survival and overall survival in patients with advanced non–small cell lung cancer (NSCLC) (57). Unfortunately, bevacizumab therapy was also found to increase the risk of clinically significant bleeding (57). Concerns over intracranial bleeding resulted in a general exclusion of patients with brain metastases from receiving bevacizumab therapy. However, a recent retrospective analysis of more than 12 000 patients determined that the risk of cerebral hemorrhage is not disproportionately higher in patients with CNS metastases and suggested that the administration of bevacizumab should no longer be contraindicated based solely on the presence of brain metastasis (58). We examined the effects of simultaneously blocking VEGFR2, EGF receptor, and HER2 (human epidermal growth factor receptor 2) with the small molecule multitargeted tyrosine kinase inhibitor, AEE788, in experimental models of lung adenocarcinoma (PC14-PE6) brain metastases. In control treated animals, tumors were supported by a few large, dilated blood vessels and the tumor microenvironment contained modest levels of basic fibroblast growth factor (bFGF) (Fig. 1). However, in animals that had received 4 weeks of therapy with AEE788, the tumors were supplied by a greater number of small, irregular blood vessels and there was a dramatic increase in expression levels of bFGF. There were no significant differences in tumor size or overall survival between control and AEE788-treated animals. To identify a potential source of bFGF, we treated monolayers of PC14-PE6 lung cancer cells and brain microvascular endothelial cells with AEE788 and measured concentrations of bFGF protein using ELISA. We found that both the tumor cells and the brain endothelial cells significantly increased their synthesis and secretion of bFGF in the face of AEE788 treatment. bFGF is a potent endothelial cell mitogen, which controls the “angiogenic switch” of some tumors (59). These experiments suggested that when one angiogenic-signaling pathway is inhibited, both tumor cells and endothelial cells respond by producing alternative angiogenic factors. (A), Immunohistochemical staining for blood vessels (CD31) and bFGF in PC14-PE6 experimental brain tumor models that had been treated with vehicle (left panel) or the multi–tyrosine kinase inhibitor AEE788 (right panel) for a period of 28 days. Fig. 1. Open in new tabDownload slide The large, dilated tumor blood vessels observed in control tumors were replaced by an increased number of small, irregular blood vessels in the AEE788 treatment group. Concentrations of bFGF appeared to be increased in the AEE788 treatment group. (B) PC14-PE6 lung adenocarcinoma cells and brain microvascular endothelial cells were treated with either vehicle or 1 μmol/L AEE788 for 48 h, and expression levels of bFGF were measured by ELISA. Fig. 1. Open in new tabDownload slide The large, dilated tumor blood vessels observed in control tumors were replaced by an increased number of small, irregular blood vessels in the AEE788 treatment group. Concentrations of bFGF appeared to be increased in the AEE788 treatment group. (B) PC14-PE6 lung adenocarcinoma cells and brain microvascular endothelial cells were treated with either vehicle or 1 μmol/L AEE788 for 48 h, and expression levels of bFGF were measured by ELISA. The results of our study do not appear to be unique to tumors growing in the brain. Cascone and colleagues (60) recently reported similar findings in a H1975 NSCLC xenograft model. These investigators used cross-species hybridization of microarrays to identify patterns of mouse and human gene expression in tumors that had become resistant to bevacizumab therapy. Their findings suggested that prolonged administration of bevacizumab leads to the upregulation and activation of EGF-receptor and FGFR-signaling pathways in the tumor stroma, which restores the neovascularization response and allow tumors to progress. Dual targeting of the VEGF- and EGF-signaling pathways was able to significantly increase progression-free survival. The group also observed that the vast majority of gene expression changes associated with acquired resistance to bevacizumab occurred predominantly in stromal cells and not tumor cells. These studies emphasize the important contribution of the organ microenvironment in mediating resistance to therapy. Brain endothelial cells express a number of efflux proteins, which function to restrict the movement of toxic substances into the CNS compartment. P-glycoprotein belongs to the ATP-binding cassette superfamily of transporters and is a product of the multidrug-resistant gene (61). P-glycoprotein is expressed on the apical membrane of brain microvascular endothelial cells, where it mediates efflux of a broad range of xenobiotic compounds, including the chemotherapeutic agents vinblastine, methotrexate, vincristine, doxorubicin, and etoposide. Studies conducted in P-glycoprotein knockout mice were instrumental in demonstrating the contribution of P-glycoprotein in restricting the distribution of chemotherapeutic agents in the brain. For example, Gallo et al. (62) noted significantly higher concentrations of paclitaxel in the normal brain and melanoma brain metastases in P-glycoprotein knockout mice compared to the same regions in wild-type mice. Recent studies indicate that P-glycoprotein can also mediate the efflux of several small molecule tyrosine kinase inhibitors including, erlotinib, gefitinib, and imatinib (63). The blood–brain barrier consists of several different cell types, such as neurons, pericytes, astrocytes, and microvascular endothelial cells that work cooperatively to form a “neurovascular unit.” In the next section, we discuss the contribution of astrocytes to brain metastasis. ASTROCYTES Astrocytes play a critical role in maintaining cerebral homeostasis by providing support to the blood–brain barrier (64), regulating the CNS response to inflammation (65), modulating synaptic transmission (66), and serving as an intermediary in neuronal regulation of blood flow (67). Recent studies have also determined that astrocyte dysregulation plays a central role in the pathogenesis of several disease processes, including brain metastasis. One of the hallmarks of CNS pathologies is reactive astrogliosis, the process through which astrocytes undergo alterations in cellular morphology and patterns of gene expression (68). Two characteristic features of astrogliosis are upregulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), and hypertrophy of astrocyte cellular processes (69). Recently, we examined the kinetics of astrogliosis in experimental models of NSCLC (PC14-PE6) brain metastasis (70). Reactive astrocytes could be observed in small developing metastasis as early as 7 days following tumor cell injection (Fig. 2). The intensity in GFAP labeling of the metastases-associated astrocytes was much more pronounced than the labeling observed in similar regions of the brain of nontumor mice. As the tumor mass continued to develop, the magnitude of the astrogliosis appeared to increase, as indicated by the extensive overlap and interdigitation of astrocyte processes. At the same time point, an occasional CD68-positive cell could be observed localizing to tumors. The CD68-positive cells were determined to be of the macrophage lineage on the basis of their morphologic appearance. In the advanced phase of the disease, there was marked infiltration of the tumor mass by both reactive astrocytes and macrophage cells. A large-scale examination of human brain metastases also concluded that reactive astrogliosis and infiltration of tumors by CD68-positive cells are typical of the clinical disease process (71). Immunofluorescent staining of GFAP and CD68 in experimental PC14-PE6 lung adenocarcinoma brain metastases. Fig. 2. Open in new tabDownload slide Astrocytes are labeled with GFAP (blue) and macrophage cells are labeled with CD68 (red). (A), Activated astrocytes localize to small metastases (tumor cells indicated by arrow). The intensity in GFAP labeling of metastases-associated astrocytes was significantly more pronounced than the labeling observed in similar regions of the brain in nontumor mice. (B), Additional astrocytes become activated as the tumor mass expands and an occasional CD68-positive can be observed in the tumor microenvironment. (C), Four weeks following tumor cell injection, there is a marked increase in the number of reactive astrocytes localizing to tumor and trafficking of CD68 expressing cells to metastases is much more pronounced. Scale bar: 100 μm. Reproduced from Langley et al. (70) with permission from Spandidos Publications. Fig. 2. Open in new tabDownload slide Astrocytes are labeled with GFAP (blue) and macrophage cells are labeled with CD68 (red). (A), Activated astrocytes localize to small metastases (tumor cells indicated by arrow). The intensity in GFAP labeling of metastases-associated astrocytes was significantly more pronounced than the labeling observed in similar regions of the brain in nontumor mice. (B), Additional astrocytes become activated as the tumor mass expands and an occasional CD68-positive can be observed in the tumor microenvironment. (C), Four weeks following tumor cell injection, there is a marked increase in the number of reactive astrocytes localizing to tumor and trafficking of CD68 expressing cells to metastases is much more pronounced. Scale bar: 100 μm. Reproduced from Langley et al. (70) with permission from Spandidos Publications. Having determined that the pathology of the experimental model resembles that of the human disease, we next questioned whether astrocytes could affect intracellular signaling pathways that regulate cell division and survival of tumor cells. To address this question, we generated a conditionally immortal astrocyte cell line from the brains of H-2Kb-tsA58 transgenic mice (70). This mouse line is a superior source of immortalized cells in that each tissue harbors a temperature-sensitive SV40 large T antigen, which overcomes many of the limitations typically associated with the in vitro transfection process (e.g., different sites of gene integration, multiple copy number) (40). In addition, the thermolabile large T antigen allows the user to regulate the level of cell division. We found that astrocytes grown under the permissive 33 °C temperature expressed marked levels of the SV40 large T antigen and had a doubling time of 36 h. However, when the cells were transferred to a nonpermissive temperature of 37°C for a period of 72 h, SV40 large T-antigen expression was no longer detectable and cell division reached a plateau. We then established a transwell coculture system comprised of astrocytes and PC14-PE6 lung cancer cells and noted that extracellular signal-regulated kinase-1/2 was phosphorylated on tumor cells that had been cocultured with astrocytes for a period of 24 or 48 h. Recently, Lin and colleagues (72) used a similar coculture system to demonstrate that astrocytes protect tumor cells from the cytotoxic effects of chemotherapy. These investigators demonstrated that the protective effect was dependent on physical contact and gap-junction–mediated communication between astrocytes and tumor cells. Kim and colleagues (73) used gene expression profiling on human breast cancer cells and lung cancer cells that had been coincubated with astrocytes and found that direct contact between astrocytes and tumor cells led to the activation of survival genes in tumor cells (Fig. 3). The degree of upregulation of glutathione S-transferase alpha 5 (GSTA5), BCL2-like 1 (BCL2L1), and twist homolog 1 (Drosophila) (TWIST1) correlated with increased tumor cell resistance to a broad panel of chemotherapeutic agents. The group confirmed expression of GSTA5, BCL2L1, and TWIST1 in clinical samples of breast and lung cancer brain metastases. In addition, the protective effects were determined to be unique to astrocytes because fibroblasts (or other tumor cells) were unable to protect tumor cells. Scanning electron micrograph taken from an in vitro co-culture experiment depicting an astrocyte (A) surrounded by 4 human MDA-231 breast cancer cells. Fig. 3. Open in new tabDownload slide A single astrocyte may have direct contact with multiple tumor cells. Gap-junction–mediated communication between astrocytes and tumor cells leads to the upregulation of genes that promote tumor cell survival. The protein products of these genes protect tumor cells from several different chemotherapeutic agents and are present in clinical samples of breast and lung cancer brain metastases. Scale bar: 10 μm. Reproduced from Kim et al. (73) with permission from Neoplasia Press, Inc. Fig. 3. Open in new tabDownload slide A single astrocyte may have direct contact with multiple tumor cells. Gap-junction–mediated communication between astrocytes and tumor cells leads to the upregulation of genes that promote tumor cell survival. The protein products of these genes protect tumor cells from several different chemotherapeutic agents and are present in clinical samples of breast and lung cancer brain metastases. Scale bar: 10 μm. Reproduced from Kim et al. (73) with permission from Neoplasia Press, Inc. To further examine how the organ microenvironment influences cancer cells, Park and colleagues (74) applied competitive cross-species hybridization of microarray experiments to extract gene expression data of cancer and host cells when human cancer cells were engrafted into different organs of immunocompromised mice. Analyses of gene expression data sets showed that the brain microenvironment induces a change in gene expression by metastatic cells (74), similar to the data obtained by coculturing tumor cells with astrocytes (73). Interestingly, the analyses performed on 4 different cell lines that were implanted orthotopically or subcutaneously were clustered on the basis of the identity of the cancer cell line and were independent of the location of the transplant, with few exceptions. That is, the gene expression patterns of breast cancer cells implanted into the subcutaneous space were very similar to those for the same cells implanted into the mammary fat pad. In contrast, tumors from the 4 different cancer cell lines growing in the brain displayed marked differences in gene expression patterns compared to the same tumors implanted at either the subcutaneous or the orthotopic site. Indeed, the cancer cells that were growing in the brain no longer maintained their cell-line–specific gene expression program. Instead, these cells appeared to have acquired neuronal cell characteristics in that the most enriched gene sets among the upregulated genes involved neurological signaling. These characteristics are considered unique to cells of the neuronal lineage. Genes involved in neuropathic pain signaling, synaptic long-term potentiation, and axonal guidance signaling were among the significantly upregulated genes. Whether the acquisition of neuronal transcriptional patterns in tumor cells contributes to the chemoresistant nature of brain metastases remains unclear, as does the molecular mechanism responsible for the transcriptome reprogramming. Conclusions The outcome of brain metastasis is dependent on the interaction that takes place between tumor cells and nontransformed cells residing in the CNS microenvironment. Communication between tumor cells and endothelial cells appears to regulate tumor growth and survival, whereas the crosstalk between tumor cells and astrocytes plays an important role in determining the tumor response to therapy. The continued development of clinically relevant brain metastasis models will advance our understanding of these complex communication networks and should lead to the development of therapeutic strategies that improve clinical outcomes. 2 Nonstandard abbreviations: CNS central nervous system VCAM-1 vascular cell adhesion molecule-1 EGF epidermal growth factor VEGF vascular endothelial cell growth factor VEGFR2 VEGF receptor 2 NSCLC non–small cell lung cancer bFGF basic fibroblast growth factor GFAP glial fibrillary acidic protein. 3 Human genes: ST6GALNAC5 ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 GSTA5 glutathione S-transferase alpha 5 BCL2L1 BCL2-like 1 TWIST1 twist homolog 1 (Drosophila). " Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. " Authors' Disclosures or Potential Conflicts of Interest:Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: " Employment or Leadership: I.J. Fidler, MD Anderson Cancer Center. " Consultant or Advisory Role: I.J. Fidler, Actelion Pharma. " Stock Ownership: None declared. " Honoraria: None declared. " Research Funding: I.J. Fidler, NIH. " Expert Testimony: None declared. References 1. Gavrilovic IT , Posner JB. Brain metastases: epidemiology and pathophysiology . J Neurooncol 2005 ; 75 : 5 – 14 . 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TI - The Biology of Brain Metastasis
JF - Clinical Chemistry
DO - 10.1373/clinchem.2012.193342
DA - 2013-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-biology-of-brain-metastasis-NceHgXOisx
SP - 180
VL - 59
IS - 1
DP - DeepDyve
ER -