The Biology of Metastasis to a Sanctuary Site

Experimental Model Systems of Brain Metastasis

Rodent model systems have been reported for brain metastases in melanoma (reviewed in ref. 14), lung carcinoma (15, 16), and breast carcinoma (17–24). A model of metastasis to the choroid of the eye was reported in which tumor cells also invaded the brain (25). Table 1 lists the salient characteristics of several prominent models; important facets of the BCM2 breast cancer brain metastasis model are presented in Fig. 1 .

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Fig. 1.

Development and use of a new human cell model for studies on breast cancer brain metastasis. Top, it is generally accepted that cancer cells reach the brain from the circulation. Circulating tumor cells were isolated from a patient with breast cancer with metastatic disease, and established in culture (named BCM2). Tumor cells were injected i.v. into immunodeficient mice, and metastases to the brain and bone were isolated, established in tissue culture, and reinjected into mice. Bottom, when genetically tagged with firefly luciferase and followed by noninvasive bioluminescence imaging, the brain-homing breast cancer cell variant shows a pronounced ability to colonize the brain of immunodeficient mice and extend down the spine, as often seen in patients with breast cancer with advanced metastatic disease in the brain. The histology of brain lesions from BCM2 brain variant cells (top) shows invasive metastatic growth with compression of the adjacent, still unaffected brain tissue, and compares with the histology of metastatic brain lesions from a breast cancer patient (bottom).

Metastatic murine or human cell lines are often the starting point in the development of these models. In contrast, the BCM2 brain line was established from circulating tumor cells from a patient with stage IV breast cancer (Fig. 1). To isolate a brain tropic line, cell lines were subjected to multiple sequential rounds of in vivo selection in mouse models by harvesting the initial brain lesions that developed after injection. These tumor cells were established as ex vivo cultures, re-injected into a new host, and again harvested from resulting brain metastases. The characteristics of several lines have been reported after sequential rounds of selection, including a progressively higher incidence of brain metastases, fewer metastases to other organs, and/or shortened host survival. Routes of injection included the tail vein, the left ventricle of the heart, or the internal carotid artery. The latter routes present technical challenges but favor brain colonization because the cerebral microcirculation is the first capillary bed encountered. Another in vivo model with some relevance to brain metastasis is the stereotactic implantation of tumor cells into the brain, although this procedure eliminates many steps of the metastatic process and causes trauma to the brain that may affect tumor cell growth. In vitro models include cocultures of tumor cells and brain-derived cells (26, 27), transendothelial cell invasion using cultured brain microvascular endothelial cells (28–31), and ex vivo brain slice invasion assays (32).

Quantification of brain metastases is typically done histologically, in which serial sections of the brain are examined. Standardization is needed so that preclinical efficacy experiments can be easily interpreted. In some experiments, the incidence of brain metastases is noted. However, given the technical difficulty of intracardiac or intracarotid artery injection in the mouse model, it is sometimes unclear if an animal without tumor signals in the brain received the full dose of tumor cells and failed to develop brain metastases, or if the injection “missed.” The use of luciferase-tagged tumor cells and noninvasive bioluminescence imaging of the animals immediately after injection and at later time points can help to clarify this issue.

For quantification as well as qualitative analysis of brain metastatic burden, histological examination is important because it not only reveals the extent of metastatic growth but also the size and shape of metastatic brain lesions. The number of brain metastases in step sections is therefore a reliable indicator, but again, questions still arise: does one score micrometastases, consisting of only a few cells, the same way as large “cannonball” metastases? Because patients are diagnosed with large metastases, and we do not fully understand the implications of micrometastases, it may be judicious to tally the two types of lesions separately. And what size cutoff determines micrometastasis? These models are aided by coupling with improved imaging techniques such as bioluminescence or fluorescence imaging of luciferase or fluorescent protein–labeled tumor cells, respectively. Additionally, superparamagnetic or micron-sized iron oxide particles, which are detected in magnetic resonance imaging scans, may provide insights (33–35). In a study of enhanced green fluorescent protein–labeled human breast carcinoma cells, imaging of dissected brains gave only semiquantitative results because a fluorescent signal could represent a solitary large brain metastasis or a collection of micrometastases upon histologic analysis (36). These data underscore the need for a complete histologic examination.

Although the establishment of a handful of model systems represents a significant accomplishment, additional models are needed to represent the heterogeneity of brain metastasis observed in the clinic. Almost no comparison has been made between the molecular characteristics of model systems and those of clinical brain metastasis specimens.

The Complex Roles of Angiogenesis and Permeability

Angiogenesis is an important aspect of tumor metastasis that has been reported in several brain metastasis models. Multiple facets of the vascular system have been investigated, including angiogenesis, the formation of new blood vessels, the utilization (or co-option) of existing blood vessels, and the permeability of the vascular system. The conclusions of the reported studies tend to vary by model but may describe the heterogeneity found in human disease.

The Fidler laboratory examined several aspects of angiogenesis in model systems including the brain. The brain is a highly vascularized organ. Capillary densities in brain metastases were actually lower than those of the surrounding brain (ref. 37; reviewed in ref. 38). An assay of vessel permeability was reported in which sodium fluorescein was injected into mice with experimental brain metastases of human KM12C tumor cells. For micrometastases smaller than 0.1 mm², the blood-tumor barrier was intact; however, the blood-tumor barrier leaked in larger (>0.4 mm²) metastases but still excluded therapeutic drugs (38).

Recent studies have examined the roles of vascular endothelial growth factor (VEGF), which influences both angiogenesis and blood vessel permeability (reviewed in refs. 39, 40). The 121 amino acid splice form of VEGF is soluble, whereas the other 145, 165, and 189 amino acid splice forms are extracellular matrix–bound, but are released and activated in the presence of proteases (41). Brain-colonizing sublines of the human MDA-MB-231 breast carcinoma cell line produced greater amounts of VEGF and another angiogenic factor, IL-8, in vitro than the parental cell line. The VEGF receptor inhibitor, PTK787, reduced the tumor burden, capillary density, and mitotic rate of brain metastases resulting from carotid artery injection of tumor cells in vivo (42). Similarly, antisense VEGF-165 transfectants of PC14PE6 lung adenocarcinoma and KM12SM colon carcinoma cells exhibited a decreased incidence of brain metastases following intracarotid injection. Halofuginone, a type Iα collagen inhibitor, reduced the size and vascular density of melanoma cells intracranially implanted into the rat brain (43).

Different outcomes were observed in other model systems, which may affect the efficacy of antiangiogenic therapies in this site. Transfection of VEGF-121 or -165 into the H226 human lung squamous carcinoma cell line exerted no effects on angiogenesis or brain metastasis following intracarotid injection (44). Mel57 human melanoma cells produced little endogenous VEGF but established infiltrative brain metastases in mice by co-opting existing peritumoral vessels (45), indicating that the preexisting vasculature can contribute to metastatic growth. Interestingly, the blood-brain barrier remained intact in these co-opted vessels. When the VEGF-121, -165, and -189 splice forms were transfected into Mel57 cells side-by-side, the three isoform transfectants induced comparable endothelial cell proliferation in vitro. The VEGF-121 transfectants failed to initiate angiogenesis but induced the dilation and permeability of existing peritumoral vessels in vivo. In contrast, VEGF-165 transfectants initiated angiogenesis, disrupted the blood-brain barrier, increased permeability, and produced the most marked metastatic growth by imaging (46). These findings indicate that different VEGF forms can serve different functions in the context of the brain. Analysis of VEGF expression and vascularization patterns in a small cohort of human clinical brain metastases suggested that conclusions from the model systems were applicable to human metastases (47). Likewise, the effect of antiangiogenic agents varied in the brain. Treatment of the VEGF-165 transfectants with the maximum tolerated doses of the VEGF receptor inhibitor ZD6474 resulted in a diminution of the VEGF-165 phenotype including restoration of the blood-brain barrier and a reduction in the size of metastases on contrast-enhanced magnetic resonance imaging (48). However, histologic analyses showed continued metastatic progression via co-option of existing vessels (37, 48). The co-option process was thought to be sufficient to sustain tumor viability as no hypoxia or necrosis was observed in this study. In contrast, treatment with a lower dose of inhibitor reduced new vessel formation but left the blood-tumor barrier permeable. The authors propose that lower dose “antiangiogenic” therapy may facilitate the delivery of other chemotherapeutics with a clinical benefit, despite the lack of a classical antiangiogenic effect (48).

In conclusion, factors including concentrations of angiogenic factors, the mix of angiogenesis factors (such as splice forms of VEGF), the level of antiangiogenic drug, and the availability of an existing vessel network for co-option may all contribute to antiangiogenesis drug responsiveness in brain metastases. Because bevicuzimab is a large monoclonal antibody, its effectiveness in the brain is expected to be poor. Studies of clinically relevant VEGF receptor tyrosine kinase inhibitors and other small molecule angiogenesis inhibitors, conducted at doses that are achievable in humans, will be important for further understanding this complex situation. These studies can not only address angiogenesis but the effects of altered blood vessel permeability on the delivery of other drugs to brain metastases.

Mechanistic Insights

Molecular Characterization of Brain Metastases

In addition to the use of model systems, human brain metastatic tissue is available in limited quantities from craniotomies and autopsies, and several molecular analyses have been reported. At a gross level, gains in chromosomes 15q and 20q were identified in brain metastases of prostate cancer (62). Increased p21 expression was reported in breast cancer brain metastases as compared with primary tumors (63). Although p21 has a classical role as a cell cycle inhibitor, it is also correlated with innate and chemotherapy-induced resistance by an undefined mechanism. Reduced expression of the metastasis suppressor genes Kiss1, Kai1, Brms1, and Mkk4 was reported in breast cancer brain metastases as compared with unlinked primary tumors (64). These data constitute ideal leads for testing in model systems.

Alterations in the DNA methylation of several genes were reported in brain metastases, including p16, DAP-kinase, thrombospondin-1, retinoblastoma 1, O⁶-methylguanine DNA methyltransferase, glutathione-S-transferase P1, p14, cyclin D2, retinoic acid receptor B, twist, and Hin-1 (65, 66). It is not known whether these findings are specific to the brain as opposed to other sites of metastasis, but suggest the hypothesis that drugs regulating gene expression such as HDAC inhibitors or DNA methyltransferase inhibitors may have efficacy in the brain.

The Issue of Dormancy in Brain Metastases

Micrometastases are rarely discussed in the literature regarding human brain metastasis. From a clinical standpoint, patients are diagnosed when brain metastases are sufficiently large to be observed with imaging, containing millions of tumor cells. However, these large metastases emanated from a micrometastasis, and the processes controlling their outgrowth are germane to metastasis prevention studies. The elimination of occult micrometastases serves as the rationale behind giving whole brain radiation therapy. Recently, Baumert et al. (67) reported autopsy results from 45 patients with brain metastases who underwent gamma knife radiosurgery. In 48 of 76 metastases, infiltration of tumor cells in small nests, i.e., micrometastases, was apparent in the area surrounding the border of the larger brain metastases. These lesions seem similar to those seen in the MDA-MB-231BR animal model.⁵

Another important question is whether all micrometastases are progressively growing, or alternatively, whether some enter periods of tumor dormancy. Certain cancers such as those of the breast and prostate recur years after the primary diagnosis in some patients, suggesting that the tumor cells can lie dormant in a distant location for long periods of time (68). The forces that induce dormancy or reawaken these tumor cells are largely unknown, but their identification could lead to new forms of therapy.

The Chambers laboratory labeled 231BR human breast carcinoma cells with micron-sized iron oxide particles and followed their fate in vivo using magnetic resonance imaging (33). If the tumor cells successively divided, the particles were apportioned between daughter cells and an imaging signal was lost. Single tumor cell “voids” were detectable 24 h after intracardiac injection and the fate of these cells was followed by repeated magnetic resonance imaging scanning for 1 month. Approximately 94% of tumor cells initially seeding the brain were not detectable by the end point of the experiment. Another 1.6% of tumor cells proliferated to form macroscopic metastases, detectable by a transfected enhanced green fluorescent protein label, whereas 4.5% of tumor cells were apparently dormant, retaining the micron-sized iron oxide particle labels for the duration of the experiment (33). These data provide the first in vivo evidence for dormancy in a model of brain metastasis of breast cancer. Approximately a 3:1 ratio of dormant to colonizing tumor cells indicates that a potent subpopulation of cells exists that should be the focus of molecular and preclinical research to generate information aimed at successfully targeting those potentially relapse-initiating cells.

Conclusions and Future Perspectives

The development of model systems for brain metastasis represents an important advance in this field. Additional model systems representing each cancer histologic type are needed so that the heterogeneity inherent in the patient population can be modeled. The current models have limitations, many of which can be addressed by further experimentation. These include a lack of validation against clinical brain metastases from craniotomies or autopsies, to ensure that the pathways active in the model are present in the human situation. Another potential weakness is the failure of models to incorporate the standard treatment, radiation therapy. Ideally, animals could receive a form of radiotherapy such as a dose approximating gamma knife radiosurgery, so that responses in this setting could be determined. Improvements are needed in the quantification of brain metastases in model systems. Imaging, although promising, fails to distinguish a collection of micrometastases from larger metastases, leaving us with tedious microscopic examinations as end points. Finally, almost nothing has been reported on cognitive function in animal models of brain metastases. Cognitive function is severely affected by brain metastases and the complications of their treatment. One could envision maze, agility, and learning tests, now applied in other fields, being incorporated as end points.

It is remarkable that, given our abilities in mouse models, a comprehensive data set has not been published of drug permeability into the normal brain, brain tissue around metastases (which may have altered permeability), large metastases, and micrometastases. Steadily, brain permeability is given increasing consideration in drug development. Given the fact that brain metastases represents an unmet medical need, it would make sense to prioritize the testing of new therapeutics in a brain metastasis model.

New strategies for the delivery of drugs to brain metastases are also a high priority. Strategies such as neural stem cell homing (69, 70), immune approaches (71), and novel delivery vehicles (72) are emerging and represent high-risk, high-impact avenues of exploration.