The major cause of death from cancer is due to metastases that are resistant to therapy. Primary neoplasms and metastases contain multiple cell populations with different characteristics such as growth rate, cell surface receptors, antigenicity-immunogenicity, hormone receptors, adhesion molecule profiles, production of extracellular matrix proteins, sensitivity to cytotoxic drugs, angiogenic potential, invasiveness, and metastatic potential. This biologic heterogeneity is due to the genetic instability of tumor cells in general and metastatic cells in particular. The biologic heterogeneity of cells is also demonstrated in the production of organ-specific metastasis. Selected clones in a primary neoplasm can give rise to secondary metastases in specific organ sites, such as the brain.1,2
In the United States, as many as 170,000 new cases of brain metastases occur each year, which is 10 times the number of patients diagnosed with malignant primary brain tumors.3 More than 40% of cancer patients develop brain metastasis; specifically, nearly 50% of patients with lung cancer, more than 25% of patients with breast cancer, and 20% of patients with melanoma.4–6 The incidence of brain metastasis may be increasing as cancer patients are living longer as a result of improved treatment and also because the incidence of lung cancer and melanoma continues to rise.7 The progressive growth of metastasis in the brain is frequently associated with the terminal stage of the disease. The therapeutic approach for patients with brain metastases is dependent on the number and location of metastases, on the biology of the primary tumor, and on the extent of systemic disease.8 The median survival for untreated patients is 1 to 2 months, which may be extended to 6 months with conventional radiotherapy and chemotherapy.9 The challenge for therapy of brain metastases can be met only once we gain a better understanding of the pathogenesis of metastasis in general and brain metastasis in particular.
THE PATHOGENESIS OF METASTASIS
The process of metastasis is highly selective and consists of a series of sequential, interrelated steps that must all be completed if clinically relevant lesions are produced. At the primary site, tumor cells must proliferate and induce angiogenesis (establishment of new vasculature) if the tumor mass is to become larger than 1 mm3 in diameter.2 Tumor cells must invade host stroma and gain entrance into the lymphatics or vascular channels. Single cells or clumps of cells can circulate to reach distant organs. These tumor emboli must survive the turbulence of the circulation and immune and nonimmune defenses to be arrested in the capillary beds of receptive organs (“niche”). The tumor cells can grow within the vessels or extravasate into the organ parenchyma where the cells have to proliferate to establish micrometastasis. When these lesions increase in size, new cells can enter into the circulation to produce metastasis of metastases.1,2
Tumor cells can spread by 2 major routes. The first involves spread by direct extension in which a tumor growing in a body cavity releases cells or fragments that can seed serosal and/or mucosal surfaces and develop into new growths. Two examples are lung mediastinal tumors that enter the pleural cavity and malignant ovarian tumors that shed cells into the peritoneal cavity. Primary tumors of the central nervous system are highly invasive but rarely produce metastases in organs outside the nervous system. The mode of their spread appears to be by direct extension or via the cerebrospinal fluid. The second route of spread is via the lymphatic and hematogenous compartments of the circulatory system.1
Clinical observations have suggested that carcinomas frequently spread and grow in the lymphatic system, whereas malignant tumors of mesenchymal origin spread more frequently via the hematogenous route10; however, these 2 categories are rather arbitrary because the blood and lymph systems are intimately interlinked. The thin-walled venules, like the lymphatic channels, offer little resistance to penetration by tumor cells and thus provide a common pathway for the entry of tumor cells into the circulation. In contrast, the arteries, the walls of which contain elastic and collagen fibers, are rarely invaded by tumor cells. After infiltrating the vessels, tumor cells can detach and be carried away passively in the bloodstream or remain localized and proliferate at the site of vessel invasion. Frequently, a thrombus will form around actively growing tumor cells that have penetrated the circulatory system. Detachment and embolization of tumor cells, regardless of whether their transport is via the lymphatics or blood, are probably continuous processes.11,12 Most malignant tumors have a well-established blood supply with multiple thin-walled vessels. A sudden change in venous pressure, such as that occurring during a cough, has been shown to lead to momentary blood turbulence and the release of a shower of emboli.13 Similarly, diagnostic procedures and surgical trauma may cause a sudden increase in the number of tumor cells released into the circulation.14
During circulatory transport, tumor cells can undergo a variety of interactions, including aggregation with other tumor cells,15 platelets,16–18 lymphocytes, and other host cells.19,20 Some tumor cells are thromboplastic and elicit fibrin formation either during their circulation or soon after their arrest in capillary beds.21–24 If blood-borne tumor cells form homotypic or heterotypic aggregates, their arrest in the microcirculation is increased.15,25 The rates at which tumor cells or their cell emboli pass through capillary beds are not related to cell or embolic size but instead appear to be related to their deformability during transcapillary transport.26,27 Under certain conditions, the adhesion of tumor cells to endothelium in susceptible organs could lead to vessel wall damage and the subsequent accumulation of neutrophils. Because leukocytes commonly pass through the endothelium, tumor cells could enter the extravascular tissues by following pathways set by leukocytes that have traversed the vessel wall.23 Alternatively, platelets can aggregate at the site of tumor cell lodgment and release mediators that could contribute to vascular spasm, increased endothelial permeability, and, perhaps, increased motility of tumor cells.28 The process of extravasation may be facilitated by a specific tumor cell product(s) that is chemotactic to other tumor cells but not to normal cells.29 When malignant cells reach the extravascular environment, they continue to proliferate and induce vascularization.
METASTASIS AS A SELECTIVE PROCESS
Several years ago, we investigated the fate of circulating tumor emboli following the intravenous injection of [125I]-5-iodo-2′deoxyuridine-labeled tumor cells.30 This technique allows us to determine the distribution and in vivo survival of tumor cells in syngeneic recipients. Injected mice were killed at different times, and their organs were collected and processed to determine the number of viable tumor cells. The majority of the injected tumor cells were arrested initially in the lungs. Tumor cell death began shortly thereafter. By 24 hours after injection, only 1% of the injected tumor cells had survived in host organs, and by day 14, at which time tumor colonies were visible in the lungs, less than 0.1% of the original cells injected had survived.32 The fact that the vast majority of the circulating emboli died and only a small minority survived to yield metastases questioned whether the survival was random or selective for unique metastatic cells.30
To answer this question, we injected B16 melanoma cells intravenously into syngeneic C57BL/6 mice and harvested pulmonary tumor colonies 3 weeks later. The tumor cells were adapted to tissue culture. Cultured cells were reinjected into new syngeneic animals, and 3 weeks later, a new group of lung tumor colonies was removed and cultured to yield another line. The process was repeated consecutive times. With each succeeding cycle in vivo, the ability of the selected B16 lines to implant, survive, and form lung tumors increased.31
BIOLOGIC-METASTATIC HETEROGENEITY OF NEOPLASMS
What remained unanswered was the question of whether unique selected metastatic cells preexisted in the tumor cell population or whether they arose during metastasis by a process of adaptation to local environmental conditions. To distinguish between these possibilities, we performed an experiment similar in design to the classic fluctuation test devised by Luria and Delbrück32 to distinguish between selection and adaptation in the origin of bacterial mutants. In our original study, a cell suspension of the B16 melanoma parent line was divided into 2 parts. One portion was used for intravenous injection into syngeneic mice. The other portion was used to isolate multiple clones, which were then injected intravenously into groups of mice. Three weeks after tumor cell injection, the number of lung metastases in each recipient mouse was counted. While mice injected with parental cells exhibited a similar number of experimental lung metastasis, cells from different clones produced a significantly variable number of metastases ranging from none to multiple.33
The results clearly demonstrated that the B16 melanoma is heterogeneous and that a few highly metastatic tumor cell variants preexisted in the parental population. Control subcloning experiments demonstrated that the variability among the clones was not generated during the cloning procedure.33 The extreme degree of heterogeneity observed with the B16 melanoma was demonstrated in experiments using a fibrosarcoma induced in a C3H mouse by chronic UV irradiation34 and a UV-B–induced melanoma originating in a C3H mouse.35 Collectively, these experiments presented the first definitive proof that neoplasms are heterogeneous for production of metastasis.
THE CONTRIBUTION OF HOST FACTORS TO METASTASIS
Metastasis of tumor cells is not a random event because the pattern of metastasis by different primary neoplasms is predictable.1,2 This conclusion is not new. In 1889, Stephen Paget36 questioned whether the organ distribution of metastases produced by different human neoplasms was due to chance and analyzed more than 1000 autopsy records of women with breast cancer. His research documented a nonrandom pattern of metastasis, suggesting that the process was not due to chance but rather that certain tumor cells (the “seed”) had a specific affinity for the milieu of certain organs (the “soil”). Metastases resulted only when the seed and soil were compatible.
Paget’s “seed and soil” theory was challenged by J. Ewing,37 who hypothesized that metastatic dissemination occurs by purely mechanical factors that are a result of the anatomical structure of the vascular system. These explanations have been evoked separately or together to explain the metastatic site preference of certain types of neoplasms; that is, common regional metastatic involvement could be attributed to anatomical or mechanical considerations, such as efferent venous circulation or lymphatic drainage to regional lymph nodes, whereas metastases in distant organs (from numerous types of cancers) are indeed site-specific.38
Early experimental data supporting the “seed and soil” hypothesis of Paget were derived from studies on the preferential invasion and growth of mouse melanoma metastases in specific organs.39 In these studies, fragments of lung, ovary, and kidney tissues were grafted into the subcutaneous space or muscle of syngeneic mice. Three weeks later, radiolabeled melanoma cells were injected intravenously.40 Although tumor cells reached the vasculature of all organs, metastases developed only in the orthotopic and grafted lungs and ovaries, but not in the orthotopic or grafted kidneys.40 The introduction of peritoneovenous shunts for palliation of malignant ascites provided an opportunity to study some of the factors affecting metastatic propagation in patients with malignant ascites draining into the venous circulation, with the resulting entry of viable tumor cells into the jugular veins.41 Good palliation with minimal complications was reported for 29 patients with ovarian cancer. The autopsy findings in 15 patients substantiated the clinical observations that the shunts do not significantly increase the risk of metastasis. In fact, despite continuous entry of millions of tumor cells into the circulation, metastases in the lung (the first capillary bed encountered) were rare.41
A current definition of the seed-and-soil hypothesis consists of 3 principles. First, neoplasms are biologically heterogeneous and contain subpopulations of cells with different proliferative, angiogenic, invasive, and metastatic properties.42,43 Second, the process of metastasis is selective for cells that complete all the steps of the process.43 In fact, metastases can have a clonal origin, and different metastases can originate from the proliferation of different single cells.31,32,44,45 Third, the outcome of metastasis depends on multiple interactions (“crosstalk”) of metastatic cells with homeostatic mechanisms, which the tumor cells can use.46 The seed-and-soil hypothesis is now widely accepted. The seed has been renamed as the initiating cells, progenitor cell, cancer stem cell, or metastatic cell, and the soil is named the stroma, host factor, niche, or the organ/tissue microenvironment. Regardless of the terminology, it is clear that the development and survival of metastases are dependent on the continuous interplay between tumor cells and the microenvironment of specific receptive organs.2
Of tumors found in the brain, more than 30% are metastatic lesions produced by cancers such as lung, breast, melanoma, colon, and renal.3–9 The progressive growth of metastases in the brain is often associated with the terminal stage of the disease. The treatment of choice for a solitary metastasis is surgical excision plus radiation.9 For multiple metastases in the brain and meningeal disease, radiation and/or chemotherapy are used. After surgery alone, the median survival time ranges from 4 to 6 months; with surgery and radiation, the median survival time may exceed 6 months. A major reason for these poor results is the recurrent growth of tumors at the site of resected lesions, as well as the development of multiple metastases in other areas of the brain.9
EXPERIMENTAL MODELS TO STUDY BRAIN METASTASIS
To better understand the biology of cancer metastasis to the brain, we developed a murine model in which brain metastases are produced by the injection of metastatic cells into the internal carotid artery of anesthetized mice, resulting in a high incidence of brain lesions and low incidence of visceral lesions.47,48
Mice anesthetized by intraperitoneal injection of pentobarbitol sodium are restrained on a cork board on the back and placed under a dissecting microscope. The carotid artery is prepared for an injection distal to the point of division into the internal and external carotid arteries. A ligature of 5-0 silk suture is placed in the distal part of the common carotid artery. A second ligature is placed and tied loosely proximal to the injection site. The artery is nicked with a pair of microscissors, and a less than 30-gauge glass cannula is inserted into the lumen. Tumor cells are injected slowly, and the cannula is removed. The second ligature is tightened, and the skin is closed by sutures. This technique examines the last steps of the metastatic process: release of tumor cells into the circulation, arrest in capillaries, penetration and extravasation into the brain through the blood-brain barrier (BBB), and continuous growth of cells in the brain tissue.47,48
THE ORIGIN OF BRAIN METASTASES
Clinical observations have suggested that brain metastases produced by many solid tumors occur late in the disease49 and have raised the question of whether brain metastases are produced by tumor cells shed from lymph node or visceral metastases, that is, metastasis of metastases. For the pathogenesis of brain metastasis, this is an important question. If brain metastasis occurs by the metastasis of metastases, then aggressive, prophylactic resection of lymph node or visceral metastases may reduce the risk of development of fatal brain lesions. On the other hand, if brain metastasis occurs by the direct spread of specialized metastatic cells from the primary tumors, then prophylactic dissection of extracranial metastases may not prevent brain metastasis from occurring.
We studied the correlation between the formation of experimental brain metastasis and the malignant growth potential of 7 human melanoma cell lines, isolated from lymph node metastases or from brain metastases, and the potential of 3 variants of the mouse K-1735 melanoma.50 Growth rates in different concentrations of fetal bovine serum and colony-forming efficiency in semisolid agarose were measured. Tumorigenicity and lung metastasis formation were determined in nude mice (for the human melanoma cell lines) or in syngeneic mice (for the K-1735 variants). The ability to form brain metastasis was tested by injection of cells into the carotid artery. A high colony-forming efficiency in agarose, especially at concentrations of agarose greater than 0.6%,51,52 corresponded with high tumor take rates, rapid tumor growth rates, and metastatic colonization of the lungs of the recipient mice.50 For the human melanomas, the lymph node metastasis–derived cells were more tumorigenic and produced a high number of experimental lung metastases than the brain metastasis–derived cells. In the K-1735 mouse melanoma, the tumorigenic and metastatic behavior of the cells after intravenous and subcutaneous injection corresponded with growth in agarose cultures. However, for growth in the brain after intracarotid injection, the different melanoma cell lines showed similar frequencies of tumor take, regardless of tumorigenicity in other sites of recipient mice. Collectively, the results show that the brain metastasis–derived cell lines were not more malignant than the lymph node or lung metastasis–derived cells.50
PATTERN OF BRAIN METASTASIS PRODUCED BY DIFFERENT HUMAN CARCINOMAS
Human tumor cells from carcinomas of the lung, breast, kidney, and colon were injected into athymic mice either by a direct intracerebral route or into the internal carotid artery. All carcinoma cells invaded through the BBB and produced progressively growing lesions in the brain parenchyma. Subsequent to direct injection, all the human carcinoma cell lines grew in the brain of nude mice, thus demonstrating that if carcinoma cells can reach the brain parenchyma they can proliferate there. Subsequent to injection into the internal carotid artery, there was more tumor growth in the parenchyma than in other regions of the brain. The seeding of tumor cells in the brain parenchyma reveals that cells from all of the injected carcinomas crossed the BBB. Discrete colony formation by the colon carcinomas was seen. Notable is the growth by extension via the corpus callosum to the left hemisphere subsequent to injection of tumor cells into the right hemisphere, demonstrating a possible mechanism of secondary metastasis within the brain, leading to undetected satellite micrometastases at the time of diagnosis and surgery that could give rise to early recurrences of tumors close to the surgical cavity.44,48
PATTERN OF MELANOMA METASTASIS TO THE BRAIN
Malignant melanoma will produce metastases in the brain of most patients.52,53 Of these brain metastases, 49% are intraparenchymal, 22% are leptomeningeal, and 29% are dural.54,55 In the brain, the leptomeninges and ventricles form a contiguous compartment filled with cerebrospinal fluid produced by the choroid plexus in the ventricles. The brain parenchyma is composed of neurons, various glial cells, and endothelial cells that perform all central nervous system functions. Therefore, the brain parenchyma and the leptomeninges/ventricles system represent 2 distinct microenvironments in the central nervous system. Human melanoma often produces leptomeningeal or parenchymal brain metastasis.56 Metastatic tumor growth in the leptomeninges confers a particularly bad prognosis for patients because of its distribution and inaccessibility to surgical resection.57 In experimental models of brain metastasis, tumor growth at either site results in quick morbidity and mortality of mice. We reported that 2 murine melanomas, B16-BL6 and K-1735 C4, injected into the internal carotid artery of mice produce site-specific experimental brain metastasis. The B16 melanoma cells produce metastasis in the leptomeninges and ventricles, whereas the K-1735 melanoma cells produce metastasis only in the brain parenchyma.58
Subsequent to the injection of radiolabeled murine melanoma cells into the internal carotid artery, we found that most cells were trapped in the vasculature of the brain. For both the murine melanomas, only a few cells reached the meninges. The K-1735 cells failed to proliferate at this site, whereas the B16 cells did so.58 These data confirmed that initial tumor cell arrest in the microvasculature does not correlate with the subsequent development of progressively growing lesions.30
Transforming growth factor β (TGF-β) growth factor has been implicated in tumor interactions with the brain microenvironment. Our experiments revealed that TGF-β2, an isoform of the TGF-β cytokine family, is highly expressed only by the K-1735 C4 melanoma cells. When the analyses were expanded to somatic hybrid cells from cell-cell fusion between B16-BL6 and K-1735 C4 melanoma cells, we found that the TGF-β2 expression was strictly correlated with the in vivo phenotype of site-specific brain metastasis.59 Melanoma cells that grew only in the brain parenchyma expressed a high level of TGF-β2, whereas melanoma cells that grew only in the leptomeninges and ventricles did not produce a detectable amount of the TGF-β2 ligand or expressed a low level of the mRNA transcript.59
The biological behavior of different human melanoma cell lines and cells isolated from fresh surgical specimens of cutaneous melanoma, lymph node metastases, and brain metastases was determined subsequent to direct intracisternal implantation in nude mice.60 Melanoma cells isolated from cutaneous lesions or lymph node metastases produced leptomeningeal disease but did not invade the brain parenchyma, whereas cells isolated from brain metastases produced leptomeningeal disease and infiltrative intraparenchymal lesions. The ability of human melanoma cells to grow in the brain parenchyma was also shown to inversely correlate with their sensitivity to the antiproliferative effects of TGF-β2.60
VASCULAR SUPPLY TO BRAIN METASTASES
Many investigators have suggested that the mean vessel density (MVD) within or at the periphery of neoplasms correlates with the aggressiveness of the disease. This generalization, however, does not extend to brain metastases. Circulating tumor cells that reach the brain vasculature are initially aligned along existing blood vessels by intussusceptive vascular expansion. Enlargement of the tumor lesions is associated with dilation (ectasia) of blood vessels. Murine melanoma fibrosarcoma, human colon carcinoma, and human lung adenocarcinoma cells produced well-demarcated lesions in the brain parenchyma of nude mice.61 These metastases contain few but large blood vessels with dilated lumens. The lumen of blood vessels on the periphery of these experimental brain metastases was also dilated. The MVD within these lesions was 15 to 20 times lower than the MVD in the surrounding uninvolved brain parenchyma or brain parenchyma of normal uninjected nude mice. The experimental brain metastases produced by colon carcinoma, breast carcinoma, or lung carcinoma cells contained blood vessels with dilated lumens, and large metastases contained large blood vessels with transverse bridges and multilumen structures that were lined with CD31+ endothelial cells.61 The formation of multilumen vessels is thought to be a form of vascular remodeling from a vessel with large lumen to smaller size vessels by a process called nonsprouting angiogenesis; that is, new blood vessels are formed by the “splitting” of preexisting dilated blood vessels.62
We observed dilation of blood vessel lumen in both experimental brain metastases and surgical specimens of human lung, breast, and colon cancer brain metastases. This dilation was associated with the division of endothelial cells. We base this conclusion on the data showing that BrdU+, CD31+ cells were located within the walls of the vessel among nondividing endothelial cells. The observed vessel dilation, that is, angioectasia, therefore did not occur merely by stretching of the blood vessel wall but rather as a consequence of endothelial cell division within the wall of the blood vessel.62
The progressive growth of experimental brain tumors and experimental brain metastases63 is dependent on expression of vascular endothelial growth factor (VEGF)/vascular permeability factor. We base this conclusion on the data from experiments where different cancer cell lines were injected into the carotid artery of nude mice.64 The intracarotid injection of human colon carcinoma and lung adenocarcinoma cells produced large, fast-growing parenchymal brain metastases in nude mice,64 whereas the intracarotid injection of human lung squamous cell carcinoma and human renal cell carcinoma cells produced only a few slow-growing lesions in the brain of nude mice. Rapidly progressing brain metastases contained many enlarged blood vessels with transluminal bridges of endothelial cell processes. The expression of VEGF mRNA and protein by the tumor cells directly correlated with nonsprouting angiogenesis and growth of brain metastasis. Causal evidence for the essential role of VEGF in these processes was provided by transfecting human lung and human colon carcinoma cells with antisense-VEGF165 gene, which significantly decreased the incidence of brain metastasis and enlarged blood vessels. In contrast, transfection of human lung squamous carcinoma cells with sense-VEGF121 or sense-VEGF165 neither enhanced nor inhibited formation of brain metastases.64 These results indicated that VEGF expression is necessary but not sufficient for the production of brain metastasis.65–67
DIFFUSION OF OXYGEN DETERMINES THE LOCATION OF DIVIDING AND APOPTOTIC TUMOR CELLS
The diffusion coefficient of oxygen within tissues is on the order of 150 to 200 μm.68–70 Since cell viability is dependent on oxygen, we determined whether the proximity of tumor cells to blood vessels correlated with DNA synthesis by double-labeling tissue sections for BrdU (cell division) and CD31 reactivity (endothelium). The spatial distribution of BrdU+ nuclei of CD31− cells relative to the nearest blood vessel was determined using the Euclidean distance map.71 Because actively synthesizing endothelial cells stained for both CD31 and BrdU, they did not affect the analysis. In autochthonous human lung cancer, brain metastasis dividing cells were located mostly within 75 μm of the nearest vessel.61 The distance of apoptotic cells from the nearest blood vessel was determined by an end-labeling assay.72 Apoptotic cells (TUNEL+) in autochthonous human lung cancer brain metastases were mostly located 160 to 170 μm from the nearest blood vessel.61 Hence, the location of both dividing and apoptotic tumor cells within clinical specimen of brain metastases correlates with the diffusion coefficient of oxygen within tissues.68–70
BRAIN METASTASIS AND THE BLOOD-BRAIN BARRIER
The microvasculature of the brain parenchyma is lined by a continuous, nonfenestrated endothelium with tight junctions and little pinocytic vesicle activity.73–76 This structure, designated as the BBB, limits the entrance of circulating macromolecules into the brain parenchyma. The BBB and the lack of a lymphatic system are responsible for maintaining the brain as an immunologically privileged site77 and for protecting the brain against the entry of most drugs and invasion by microorganisms.78 The BBB does not prevent the invasion of the brain parenchyma by circulating metastatic cells. Although some but not all neoplastic cells can affect the integrity of this structure,79–81 the integrity of the BBB is altered in primary brain tumors and in metastases.78–86
Primary brain tumors and brain metastases are resistant to treatment by most chemotherapeutic drugs,87–89 and this resistance has been attributed to the inability of drugs to cross the BBB.87–89 However, because this structure is morphologically, biochemically, and functionally heterogeneous in different regions of the brain,90–94 its relationship to the failure to treat brain metastases with chemotherapeutic drugs is questionable. We investigated the functional viability of the BBB in the experimental brain metastasis system. Eight different human tumor cell lines were inoculated into the internal carotid artery of nude mice. The tumors produced lesions in different regions of the brain, and the pattern of the lesions varied from diffuse growths to solitary lesions with well-defined margins. Of several molecular tracers used to study the permeability of the BBB, we chose sodium fluorescein. Despite its low molecular weight (molecular weight, 376), this hydrosoluble molecule is excluded from the brain by an intact BBB.94,95 Sodium fluorescein is not sensitive to minor or transient changes in BBB permeability, and unlike horseradish peroxidase, it is not transported into brain tissue by nonspecific endocytosis.96
Before studying the function of the BBB in brain lesions, we ruled out that the procedure of intracarotid injection of tumor cells, which is followed by ligation of the artery, or the entry of a bolus of tumor cells into the brain damages the endothelial cells of the cerebromicrovessels and thus changes the permeability of the BBB. We found that solitary well-defined lesions had a lower density of blood vessels than normal brain tissue. The BBB is known to become permeable in ischemic regions of the brain where increased endothelial pinocytosis, opening of the interendothelial tight junctions, and damage to endothelial cells have all been observed.97 We found that degeneration and central necrosis often occurred in large (>0.5 mm in diameter, 0.2 mm2) brain metastases. In these lesions, therefore, damage to endothelial cells compromises the integrity of the BBB.96
We have reported that the BBB was intact in established experimental mouse UV-2237 fibrosarcoma brain metastases as well as in 2 human melanoma cell lines.98 The common characteristic to these mouse and human experimental brain metastases was their diffuse pattern of growth. The permeability of the BBB in these lesions was not increased as compared with normal brain tissue unless the tumor cell clusters coalesced to form tumor masses exceeding 0.25 mm2. Because the BBB is not intact in experimental brain metastases that exceed 0.25 mm2, the resistance to chemotherapy must be due to other mechanisms. A growing tumor mass may disturb this interaction especially if it depends on contact between astrocytes and endothelial cells. In any event, the normal brain tissue interspersed among the small tumor clusters or surrounding small tumor lesions might be responsible for the normal function of the BBB.
TUMORICIDAL ACTIVITY OF MURINE MICROGLIAL CELLS
Throughout the body, the connective tissue not only plays a tissue-supportive role but also embeds host-defense cells that are capable of phagocytosing foreign materials, microorganisms, and dead cells. In the absence of connective tissue in the brain, the surveillance function is performed by cells that are similar to macrophages in other organs.99 In humans, under normal conditions, microglia are small cells evenly distributed within the white and gray matter that make up about 5% of the glial populations in the white matter of the corpus callosum.100,101 In mice, more microglia are localized in the gray matter than white, particularly in the hippocampus, olfactory telencephalon, basal ganglia, and substantia nigra. The proportion of the resident microglia populations in the adult mouse brain varies from 5% in the cortex and corpus callosum, to 12% in the substantia nigra.100 These so-called “resting” microglia were once thought to be largely inactive; however, recent work describes their active patrol of the healthy brain, using their motile processes to constantly survey the microenvironment.101,102 Under pathological conditions, such as injury or inflammation, the nucleus and cytoplasm enlarge in microglia, and they acquire the ability to divide as they become mobile and phagocytose materials.100–102
Microglia are generally considered to be brain macrophages that are derived from monocytes and colonized by way of circulating monocyte invasion into the brain upon pathological developments such as apoptosis of neurons.99,103 Microglia and macrophages share molecular markers such as MAC1, F4/80, and FcIgG1/2b.104,105 Microglia are considered to be the most important immune effector cells in the central nervous system, but the function of microglia in brain metastasis is not well described. Immunohistochemical analysis of brain metastases in patients reveals expression of activated microglia surrounding areas of tumor burden, and presence of both macrophages and microglia is widely reported in both experimental and clinical human brain metastasis.106–110 We determined the in vitro function of microglia by examining the ability of lipopolysaccharide + interferon γ–activated microglia to induce cytotoxicity in cancer cells.111 Using the C8-B4 microglial cell line, we examined the effects of lipopolysaccharide + interferon γ–activated microglia on tumor cells and observed microglial-induced cytotoxicity in murine melanoma cells that was comparable to that induced by macrophages. Tumor cells that survive after being cultured once with activated microglia do not exhibit any acquired resistance to a second round of microglial-induced cytotoxicity. In addition, activated microglia were not cytotoxic to nontumorigenic cells. Upon activation, microglia can produce a variety of neuroactive molecules, such as cytokines and chemokines, including interleukin 1 (IL-1), IL-6, tumor necrosis factor α, and TGF-β, as well as radicals, such as nitric oxide (NO) and superoxide. A major diffusible mediator that can produce death in adjacent tumor cells is NO, which is regulated by the activity of inducible NO synthase.112,113 Previous work from our laboratory revealed that tumoricidal activity of macrophages is mediated via NO release.114,115 Nitric oxide is thought to induce death of neurons in close proximity to microglia.116 Furthermore, data suggest that microglia exert cytotoxic effects on colon cancer cells via NO.117 Indeed, we observed significantly higher levels of nitrate, a stable breakdown product of NO, in both activated microglia and macrophages. Inhibition of inducible NO synthase activity in either microglia or macrophages nearly completely abrogated tumoricidal effects on tumor cells.111,118
ASTROCYTES PROTECT TUMOR CELLS FROM CHEMOTHERAPY
The resistance of tumor cells growing in the brain parenchyma to chemotherapy has been attributed to the inability of circulating chemotherapeutic drugs to penetrate the BBB.119,120 Recent data, however, revealed that tumor cells growing in the brain parenchyma release vascular permeability factor–VEGF, leading to increased vessel permeability.120–122 Moreover, clinical observations demonstrate that brain metastases in patients are often diagnosed as lesions surrounded by edema and exhibit leakiness of contrast material. These data rule out the BBB as a sole mechanism of drug resistance. Overexpression of P-glycoprotein by tumor cells growing in the brain microenvironment123 has also been implicated in tumor cell resistance to chemotherapy. However, clinical trials using inhibitors of P-glycoprotein expression failed to reverse the resistance of tumor cells to chemotherapeutic drugs.124 Collectively, these data raise the possibility that an alternate mechanism can be responsible for drug resistance of brain metastasis.
Histological examinations of clinical specimens of human brain metastases and experimental murine brain metastases reveal that the lesions are surrounded and infiltrated by reactivated astrocytes that express glial fibrillary acidic protein. Astrocytes contribute to cerebral homeostasis by supporting the BBB,125,126 modulating cerebral blood flow,127,128 regulating the response of the central nervous system to inflammation and participating in synaptic transmission,129 cytosolic calcium-mediated astrocytic-neuronal signaling,130 and neurovascular coupling”.131 Astrocytes have also been shown to control extracellular homeostasis, such as ion concentration, glucose level, acid-base balance, and supply of metabolic substances to neurons.128,132 Astrocytes also support immune defense in the brain and protect neuronal cells from waste products133,134 and damage from hypoxia.135 Thus, astrocytes protect neurons from alterations in homeostasis. Because metastases develop when tumor cells exploit or usurp the homeostatic mechanisms of their host,1,2 we wished to determine whether tumor cells can exploit the cytoprotective properties of astrocytes for protection from apoptosis induced by chemotherapeutic drugs. We have recently reported that, in culture, reactive mouse astrocytes can protect melanoma cells from chemotherapy by sequestering intracellular calcium through gap junction communication channels, suggesting that tumor cells growing in the brain can harness the protective effects of reactive astrocytes for their survival.136
To study this possibility in greater detail, we explored whether a direct contact of tumor cells with astrocytes also increases expression of survival genes in the tumor cells.137 First, we evaluated the effect of different chemotherapeutics on tumor cells cultured alone, with astrocytes, or with fibroblasts and then assayed gene expression in the tumor cells. We also verified the functional correlation of genes identified with protection of tumor cells from taxol by knocking down genes using siRNAs. We found that astrocytes (but not fibroblasts) in direct cell-to-cell contact with tumor cells protect the tumor cells from apoptosis induced by different chemotherapeutic drugs and that this protection is directly correlated with up-regulation of several survival genes in the tumor cells.137
These findings raise the intriguing possibility that in addition to maintaining homeostasis138 regulating neuronal activity to so-called “neuron-astrocyte metabolic coupling”139 and protecting neurons from apoptosis produced by hydrogen peroxide,140 ethanol,133 and copper-catalyzed cysteine, astrocytes can also protect tumor cells from chemotherapeutic drugs.137 The murine astrocytes used in our studies expressed glial fibrillary acidic protein, the major intermediate filament protein in the central nervous system of adults.140–142 Under a variety of conditions, these murine astrocytes protected different human tumor cells from cytotoxicity mediated by P-glycoprotein–associated and P-glycoprotein–dissociated chemotherapeutic drugs. We also demonstrated that the protection of tumor cells from chemotherapy was contact-dependent because the effect was abrogated when tumor cells and astrocytes were separated by a semipermeable membrane or when tumor cell–astrocyte cultures were treated with CBX, a specific inhibitor of gap junction communications.143,144 The protection was unique to astrocytes because fibroblasts (or other tumor cells) were unable to provide protection.137 The protection of tumor cells by astrocytes required constant contact. Tumor cells that were first cocultured with astrocytes and then cocultured with fibroblasts were no longer protected from chemotherapy. In contrast, tumor cells initially cocultured with fibroblasts (sensitive) and then with astrocytes became more resistant to the chemotherapy. These results suggested that the protection of tumor cells from chemotherapeutic agents depended on constant interaction with astrocytes. We have previously reported that the protective effects by astrocytes are due to sequestration of calcium from the cytoplasm of tumor cells.136 Calcium has been shown to play a causal role in cell death.145 While the sequestration of calcium through gap junction communication can explain the mechanism by which astrocytes protect tumor cells from chemotherapy, it does not rule out additional mechanisms, such as up-regulation of survival genes in tumor cells, as shown in the current study.
The direct cell-to-cell contact between astrocytes and tumor cells altered the pattern of gene expression in both tumor cells and astrocytes. The use of murine astrocytes (and murine fibroblasts as negative control) and human tumor cells (breast, lung) allowed us to identify the altered genes in the tumor cells. Among many genes, we found significant up-regulation of multiple genes that regulate cell survival. Once again, the overexpression of these genes was dependent on continuous contact between astrocytes and tumor cells. For functional validation, the expression of BCL2L1, TWIST1, and GSTA5 was confirmed in clinical specimens of breast and lung cancer brain metastases. A study to investigate the correlation of gene expression and induction of drug resistance was performed with siRNAs. The role of those genes in protection against taxol was determined by coculturing astrocytes with MDA-MB-231 cells. This protection could be reversed only when the tumor cells were transfected with mixed siRNA targeting all 3 genes.137 These data demonstrate the biologic redundancy of these survival pathways that can be used by cells to resist cytotoxic drugs leading to apoptosis. The up-regulation of phosphorylated AKT and phosphorylated mitogen-activated protein kinase (MAPK) subsequent to coculturing of tumor cells with astrocytes, but not with 3T3 fibroblasts, supports the conclusion that activation of these pathways leads tumor cells to resistance, antiapoptosis, and survival when exposed to chemotherapeutic agents. Knocking down the 3 survival genes did not prevent AKT and MAPK pathways from being activated, whereas blockade of AKT and MAPK pathway activation inhibited the up-regulation of BCL2L1, TWIST1, and GSTA5 expression. Hence, the activation of AKT and MAPK pathways is upstream leading to the up-regulation of expression of survival genes in tumor cells contacting astrocytes. In any case, the increased expression of GSTA5, TWIST1, and BCL2L1 in tumor cells required constant contact with astrocytes. We base this conclusion on the data showing that high expression of these survival genes in tumor cells was reduced once the tumor cells were transferred to cultures of fibroblasts. Immunohistochemical staining of clinical specimen of lung cancer and breast cancer metastases to the brain clearly demonstrate the expression of GSTA5, TWIST1, and BCL2L1 genes in tumor cells but not in adjacent normal brain tissue. Collectively, these data clearly demonstrate that host cells in the organ microenvironment influence the behavior of tumor cells and reinforce the contention that the organ microenvironment must also be taken into consideration during the design of therapy. Indeed, tumor cells are known to exploit their host for growth and survival, and the role of astrocytes in the induction of drug resistance of brain metastases provides a new concept for the design of therapy for the most fatal aspect of cancer.137
More than 40% of patients with lung cancer and breast cancer develop brain metastasis. With improved local control and therapy of metastasis to visceral organs, the morbidity and mortality due to late diagnosed brain metastasis are projected to rise. The median survival for untreated patients is 1 to 2 months, which may be extended to 6 months with radiotherapy and chemotherapy. Despite improvements in diagnosis, surgical techniques, general patient care, and local and systemic adjuvant therapies, most deaths from cancer result from metastases that are resistant to conventional therapies. The process of cancer metastasis is sequential and selective and incorporates stochastic elements. Thus, the growth of metastases represents the endpoint of many events that few tumor cells survive. Primary tumors consist of multiple subpopulations of cells with invasive and metastatic properties, with the potential to form a metastasis in a process dependent on the interplay of tumor cells with host factors. Metastatic cells are genetically unstable with diverse karyotypes, growth rates, cell-surface properties, antigenicities, immunogenicities, marker enzymes, and sensitivity to various therapeutic agents resulting in biological heterogeneity. The finding that different metastases can originate from multiple progenitor cells contributes to the biological diversity and provides additional challenges to therapeutic intervention. Furthermore, even within a solitary metastasis of clonal origin, heterogeneity can develop rapidly. Immunohistochemical and morphometric analyses demonstrate that the density of blood vessels within experimental metastases in brains of nude mice or clinical specimen of human lung cancer brain metastases is lower than that in the adjacent tumor-free brain parenchyma. However, brain metastasis–associated blood vessels are dilated and contain numerous dividing endothelial cells. Tumor cells within brain tumors and brain metastasis produce VEGF, which induces permeability in adjacent vessels. The BBB in lesions that are larger than 0.25 mm in diameter is leaky. Primary brain tumors and metastases are resistant to chemotherapeutic drugs. The venerable seed-and-soil hypothesis suggests that the outcome of metastasis depends on the interaction between unique tumor cells and the specific organ 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. Activated astrocytes surround and infiltrate brain metastases. While a physiological role of astrocytes is to protect against neurotoxicity, activated astrocytes also protect tumor cells against chemotherapeutic drugs. 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. 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.
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