Improvement in modern primary cancer therapy ironically has led to an increased incidence of brain metastasis. Despite its clinical importance, mechanisms underlying brain metastasis are still unclear. As the brain lacks lymphatics, hematogenous dissemination is the most common pathway for brain metastasis. The brain has a unique vasculature called the blood-brain barrier that is formed of continuous, non-fenestrated endothelium linked by tight junctions and augmented by interaction with astrocytes. This vasculature also limits penetration by therapeutic or diagnostic agents. The brain parenchyma is also unique because its extracellular matrix contains little collagen or laminin. In many respects brain metastasis is mediated by interactions between tumor cells and the host environment in ways analogous to metastasis to other organs. However the details of the molecular mechanisms are partly different in the brain due to the blood-brain barrier and the distinctive paucity of the usual extracellular matrix components.

Introduction

Modern cancer therapies provide improved results mainly in primary cancer. Metastatic disease is rarely amenable to local therapies such as surgery or radiation and its presence is often a reason for treatment failure. Ironically improvement in cancer therapy recently correlates with an increased incidence of brain metastasis because brain metastasis is generally observed later in the course of the disease. Clinically in adults, 30% of cancers found in the brain originated from primary tumors from the lung, breast or skin.^1,2 These tumors are especially resistant to systemic therapies because of the obstacle to drug delivery imposed by the blood-brain barrier.³ Interestingly brain metastases are often HER2 positive perhaps because therapy directed against Her2 may be effective at other sites but leave brain metastases unaffected.^4,5 Inhibition of COX2, HBEGF or ST6GALNCA5 in breast cancer lead to suppression of the blood-brain barrier penetration by one human breast cancer cell line cells.⁶ LEF1 or HOXB9 were expressed in tumor cells that metastasized from the lung to the brain via activation of the WNT/TCF pathway.⁷ Despite the clinical importance of brain metastases, much of the experimentation and literature involving tumor formation in the brain has studied primary tumors. Hence much of the literature that will be cited in this review includes literature pertaining to glioma as well as that for metastasis.

During the formation of metastasis tumor cells have extensive interactions with the host microenvironment. The host vasculature interacts with tumor cells during metastasis through the processes of transit from primary colony, arrest within the vessels and finally extravasation and colony formation. If tumor cells failed to interact with vessels, the metastasis could not succeed. The brain has a unique vasculature which is formed of continuous, non-fenestrated endothelium with linked by tight junctions. In addition these vessels are supported by astrocytes providing a cellular structure for the vasculature that is unique to the brain. This structure, the blood-brain barrier (BBB) is responsible for the limitations on the permeability to circulating macromolecules and drugs into the brain parenchyma and for the reduced immune response to foreign material in the brain.⁸ In contrast to the brain, the liver and lung are the most common sites for cancer metastasis. They are both mainly accessed by tumor cells in the portal of venous circulations respectively, whereas circulating tumor cells to the brain arrive via the arterial blood supply. The liver contains the first capillary bed encountered by tumor cells within the portal circulation.⁹ The liver has a distinctive microvasculature including sinusoids with junctions lacking continuity because of fenestrae interrupting the usual vascular tight junctions.¹⁰ These fenestrae are small compared with tumor cells with most being approximately 100 nm and virtually none over 200 nm in diameter. The lung is the first capillary bed encountered by circulating tumor cells in the venous circulation and is composed of a network of microvessels and capillaries.

Clinical and experimental cancer research has revealed molecular signatures and cellular phenomenon peculiar to primary cancers that have metastasized to the liver, lung, and colon. HER2 expression is associated with brain metastases and in murine models its inhibition reduces the growth of brain metastases.^11,12 Carbohydrate surface expression on tumor cells has also been implicated in brain metastasis.⁶ However the details of brain metastasis are still unclear and the more and better understanding of microenvironment in the brain has been asked to go forward in the therapy for brain metastasis.

Tumor Cell Invasion to the Brain

Hematogenous dissemination is the most common pathway for brain metastasis as the brain lacks of lymphatics.¹³ Metastases are found at the anatomic "watershed area" where the distal capillaries in small diameter from superficial arteries trap the circulating tumor cells.¹⁴ Approximately 80% of metastasized tumors are found at cerebral hemispheres, 15% in the cerebellum, and 5% in the brainstem proportioned to the blood supply.² Brain parenchyma itself has limited content of extracellular matrix (ECM).^15,16 The extracellular area filled with ECM is approximately 20% of total volume of the mature brain is almost entirely associated with the vasculature.¹⁷ The BBB has a basal lamina-like ECM including collagen IV and laminin.¹⁷ Since interaction and degradation of ECM are pivotal for tumor cells to adhere, in the brain this must involve the vasculature.

Integrins, a class of heterodimeric proteins, play key roles in tumor cell adhesion to ECM and signaling between tumor cells and vasculature.^3,12 The involvement of some integrins such as αvβ5, α3β1 and β1 has been reported during experimental brain metastasis.¹⁸ Expression of activated αvβ5 was observed in metastatic human breast cancer and the expression promoted metastasis to the brain in mouse model.¹⁸ Based on observation that expression of integrin subunit α3 mediated metastasis of tumor cell to the brain, Yoshimasu, et. al., suggested that α3β1, only known heterodimer of α3 would decide incidence of brain metastasis.¹⁹ Carbonell et.al., also reported that blockage or loss of β1 prevented tumor cell adhesion to vascular basement membrane and growth in the brain.²⁰

In extracellular proteolysis that is critical for tumor cell invasion, two enzyme groups of matrix metalloproteases (MMPs) and urokinase plasminogen activator (uPA) are well characterized. MMPs are a family of zinc endopeptidases of the metzincin superfamily of metalloproteinases.²¹ There have been identified 25 homologs and 3 psuedo-MMP genes in humans.²² Roles of MMPs are not only related with degradation the ECM but also activation of cell surface molecules and contribution to the migration of cells. Thus MMPs can partly mediate progression of metastasis. The activity and expression of MMPs are regulated by gene expression, pro-enzyme activation, cytokines, growth factors etc. Several studies suggested that downregulation of MMPs such MMP-2, MMP-9, and MT1-MMP inhibited invasion of glioma cells in the brain. In human glioma, upregulation of both MT1-MMP and MMP-2 was observed in invasive area but not in the central region of the glioma tissues.²³ Kamino, et. al., showed that MMP-2 mediated human glioma invasion and the mediation is dependent Wnt-5a signaling pathway.²⁴ MMP-2 activation is significantly dependent on MT1-MMP clustering. Active MT1-MMP exists near the centrosome and moves to the cell surface via microtubules.²¹ Using human glioma cell (U1242 MG cells) in xenograft model, MMP-9 played a critical in EGFR/Ras/MEK and PI3K/AKT-dependent invasion and colony formation. In addition, suppression of MMP-9 expression using shRNA inhibited invasion and proliferation of glioma cell.²⁵

Another critical proteolytic enzyme for degradation of ECM is urokinase (uPA), a serine protease.²⁶ Large amount of uPA was observed in malignant and metastatic glioma but not in low grade glioma.²⁷ Binding of uPA secreted from tumor cells to the uPA receptor activates plasnomigen/ plasmin cascade and leads to degradation of ECM. Le, et. al., indicated that uPA-plasmin cascade activated conversion of pro-MMP-2 to MMP-2 and facilitated tumor cell invasion.²⁸ Other study showed that uPA mediated MMP-9 activation either. Blocking uPA with an uPA inhibitor directly inhibited MMP-9 activation and invasion of human glioblastoma to fibronectin.²⁹ In addition to roles in MMP activation, uPA affects survival and migration of tumor cell itself. When uPA in human gliobalstoma cells was downregulated with antisense uPA, migration of the cells was inhibited through PI3k/Akt signaling.³⁰ Gondi, et. al., observed that downregulation of uPA/uPA receptor in human glioma cells activated caspase-8 leading to apoptosis of the cells.³¹

Perivascular Growth of Early Brain Metastasis

The growth and proliferation of tumor cells is dependent on blood supply. Until beginning of angiogenesis or vasculogenesis, tumor cells utilize pre-existing vessels in different mechanisms of co-option or vascular remodeling of intussusception during brain metastasis.² Many studies of brain metastasis have described early growth of tumor cells along pre-existing brain vessels.^32-34 Kienast, et. al., showed that after extravasation human melanoma cells (MDA-MB-435) grew in perivascular area of the brain of mouse using mutiphoton microscopy.³⁵ In analyses of human clinical specimens, Carbonell, et. al., showed that approximately 98% of metastatic brain colonies from primary tumor of varied origin (breast, lung, skin) were vascular-associated.²⁰ This vascular utilization by tumor cells results in alteration of tight junction morphology in the brain vasculature. Liebner, et. al., showed that tumor growth induced dysregulation of claudin-1, tight junctional protein at the BBB, and suggested that the alteration would be correlated with severe edema observed in patient with brain tumor.³⁶

Carbonell, et. al., demonstrated that the association of tumor cells (breast carcinoma, melanoma, mammary carcinoma) with the vasculature in the brain was not merely coincidental, but was essential for their adherence to brain parenchyma and also for proliferation. In addition this work demonstrated in mouse models and in ex vivo work using brain slices that invasion of brain parenchyma occurred using the ECM of the vasculature as conduits for cell migration.²⁰ This is likely to be different from lymphoma and glioma that are able to invade through brain parenchyma without using vascular tracks although some data suggests that glioma also uses the vascular tree for invasion.^37-40

These findings suggest the neural elements of the brain parenchyma would not provide a sufficient substrate for metastatic tumor growth, and implicate that utilization of the existing vasculature would be a key procedure for brain metastasis.

Angiogenesis

Angiogenesis, new vascular formation from pre-existing vessels is essential for progression of metastasis.² It should be noted that angiogenesis in the brain in response to tumors is not typical brain vasculature with intact BBB but is a chaotic, disorganized leaky vasculature like tumor vasculature in other locations. Glioma is an exception with a special pathologically characteristic vascular pattern called palisading.^41,42 Recently this has been shown to be derived not from host.^43,44

A variety of growth factors, cytokines, gene expression, specific proteins play roles in new vascular formations.⁴⁵ These pro-angiogenic factors systemically interact with each other rather than functioning in isolation. A large number of growth factors including vascular endothelial growth factor (VEGF), transforming growth factor (TGF), epidermal growth factor (EGF), fibloblast growth factor (FGF) have been reported as angiogenic.^46-50 VEGF directly recruits endothelial cells and stimulates their proliferation. Overall expression of VEGF is thought to be the predominant pro-angiogenic growth factor in tumor angiogenesis.⁴⁶ The VEGF family is composed of VEGF-A, -B, -C, -D, -E and placenta growth factor (PIGF). VEGF-A has binding activity to both VEGFR-1 and VEGFR-2, whereas PIGF and VEGF-B only bind to VEGFR-1. VEGF-C and D have binding affinity to VEGFR-3 and play critical roles in lymphanogioenesis. Thus VEGF-A among the family members is the most important for tumor angiogenesis and usually called VEGF.⁵¹

There are two predominant mechanisms attributed to upregulation of VEGF in metastases. The first reason is hypoxia. When the oxygen supply to tumor cells is not adequate for their growth and survival, angiogenesis is triggered by the tumor. This pathological condition is hypoxia and it may be a major contributor to angiogenesis in brain tumors. There is substantial evidence that the major transcriptional pathway from hypoxia to angiogenesis is mediated mainly by hypoxia inducible factor HIF-1.⁵² Recently Du, et. al., suggested that HIF-1 played a role in recruitment of bone marrow derived myeloid cells as well as endothelial progenitor cells. The recruited cells were able to initiate angiogenesis in part by increasing VEGF concentration.⁵³ VEGF is a "two-edge sword" in vascular maintenance and disruption especially under hypoxia. Under mild hypoxia, increase of VEGF enhanced survival of endothelial cells in the area, whereas the increase of VEGF caused disruption of the BBB under severe hypoxia.^54,55 In an experimental brain metastasis model sprouting angiogenesis was triggered by VEGF made by the tumor cell, but in the absence of VEGF the tumor cells were able to utilize the endogenous vasculature.^34,56 As was noted above in brain metastases tumor cells preferentially associate with the vasculature, allowing the cells to maintain a vasculature without angiogenesis.

Other growth factors also can mediate VEGF expression. In glioblastoma multiforme (GBM), Goldman, et.al., observed that activation of EGF receptor on GBM cells by EGF resulted in enhancement of VEGF secretion.⁴⁷ Platelet-derived growth factor (PDGF) may be able to enhance transcription and secretion of VEGF by endothelial cells which express β-PDGF receptor. PI3K activation has been attributed as a signaling pathway mediating this mechanism.⁵⁷ Guo, et. al., showed that PDGF-B in some human glioma cells enhanced intracranial glioma frormation by stimulating VEGF expression of endothelial cells. PDGD-B also attracted pericytes leading to activation of intratumoral angiogenesis.⁵⁸

In seminal work in a glioma model, Holash, et. al., noted that vascular remodeling correlated with expression of angiopoietin-2 (Ang-2), a ligand for the endothelial receptor Tie2.³⁸ The endothelial receptor Tie-2 has four ligands, Ang-1-4, of which Ang-1 and -2 are the most studied.⁵⁹ Ang-1 inhibits vascular permeability and helps maintain vascular integrity. In contrast Ang2 enhances vascular permeability and destabilizes vessels. Thus in many, but not all situations the functions supported by Ang-1 are counteracted by Ang-2.⁵⁹ Additionally, the presence or absence of VEGF modulates Ang-2 function. In the retina VEGF counteracts the destructive action of Ang-2.⁶⁰

Although Ang-1 and -2 are known to be implicated in the maintenance of vasculature, the angiopoietins (Ang-1, -2, -4) have been implicated in angiogenesis in glioma models. The suggestion has been that Ang-2 first led to destabilization of the vessels. The impaired vascular function then resulted in induction of VEGF. In contrast to the action of Ang-2 alone, Ang-2 in the presence of VEGF then leads to vascular stabilization. Thus the binding of Ang-2 to its receptor Tie2 results in destabilasation of vasculature, and leads to VEGF-dependent angiogenesis.³⁸ Chopp, et. al., showed that upregulated of VEGF/VEGFR and Ang/Tie-2 mediated angiogenesis was a factor in ischemic brain.⁵⁵ Recently the roles of Ang-4 were emphasized in angiogenesis by human GBM cells. Brunckhorst, et. al., reported that upregulation of Ang-4 in GBM cells differentiate and function as endothelial cells, and that Ang-4 promoted angiogenesis via activation of Erk1/2.⁶¹ Thus angiopoietins could be notable therapeutic targets for angiogenesis during brain metastasis.

Many links between inflammation and cancer progression have recently been uncovered. Intratumoral angiogenesis is enhanced by myeloid cells which were recruited to tumor lesion via alteration in concentration of some cytokines. Tumor necrosis factor (TNF)-α, interleukin (IL)-6 and -8 would be mostly observed cytokines in the brain metastasis.

Fajardo, et. al., suggested that function of TNF-α in angiogenesis probably depends on its concentration. In mouse models, low levels of TNF-α stimulated angiogenesis but higher concentrations were inhibitory.⁶² However there are not many reports showing direct molecular pathway connections between TNF-α and angiogenesis. In one of the reports, Zhang, et. al., indicated that TNF-α induced a phosphorylation of VEGFR-2 and endothelial/epithelial tyrosine kinase (EtK) to lead to activation of PI3k-Akt angiogenic signaling.⁶³ TNF-α indirectly promoted angiogenesis in conjunction with other mediators of angiogenesis. For example, VEGF expression by human glioma cells was enhanced by TNF-α partly mediated through the transcription factor Sp-1 (c-Fos).^49,64 Yoshino, et. al., indicated that human glioma cells secreted high level of VEGF through activation of p38 MAPK/JNK depending on TNF-α.⁶⁵

Since interleukin-6 (IL-6) expression was reported as a specific characteristic of GBM and that its expression level was proportional to the degree of malignancy in glioma, there have been many studies on the role of IL-6.^66,67 Clinically IL-6 expression was observed in severe edematous areas, with newly formed vessels and infiltrating inflammation cells in surgically resected glioblastomas.⁶⁸ IL-6 induced transcriptional activation of VEGF in astrocytes and VEGF secretion by human glioblastoma cells depended on interaction between the transcoption factors STAT3 and Sp1.⁶⁹ Blocking IL-6 using a lentiviral vector in human glioma cells, Saidi, et. al., found that IL-6 expression functioned in survival, proliferation and invasiveness of glioma cells and that these functions were VEGF-dependent.⁷⁰ IL-6 also stimulated endothelial progenitor cell proliferation, migration and tube formation.⁷¹

Interleukin-8 (IL-8, CXCL8) is a chemokine which secreted by human cell types responding to growth factors or inflammation cytokines.⁷² Wakabayashi, et. al., demonstrated that there could be two different paracrine pathways in tubular mophogenesis of vascular endothelial cells stimulated by human glioma cells.⁷³ One was dependent on VEGF and bFGF and the other on IL-8. It is still unclear how glioma cells decide the pathway. IL-8 expression was enhanced in oxygen-deprived cells surrounding necrosis and upregulation of IL-8 indirectly promoted angiogenesis by recruitment of myeloid cells.⁷⁴ IL-8 expression in glioma cells can be upregulated by TNF-α.⁷⁵ IL-8 can be produced by brain endothelial cells in proportion to VEGF level either. However IL-8 production was only observed in brain endothelial cells associated with tumor but not by normal brain endothelial cells.⁷⁶ There is a strong link between IL-8 expression and MMP-2 activity in angiogenesis and metastasis. In melanoma, IL-8 expression enhanced MMP-2 activity leading to increased tumor growth and metastasis.⁷⁷ Yoo et al also showed that inhibition of IL-8 using short hairpin RNA resulted in suppression of angiogenesis and tumor growth by non-small cancer cells and breast cancer cells.⁷⁸

Conclusion

Although brain metastasis is mediated by interactions between tumor cells and the host environments just like metastasis to other organs, the details of the process and the molecular mechanisms involved may be in part organ dependent. In addition, individual impact of metastasis procedures including attachment, migration, extravasation, colony formation and angiogenesis on successful metastasis would be different in the brain mainly due to BBB, a unique vascular structure of the brain and due to the absence of ECM except in the perivascular region.

The differences observed between brain metastasis and those to other organs suggest that we should reconsider two conventional hypotheses regarding dependence of metastasis on the host. After examination of breast cancer patients, Paget postulated that the microenvironment of each organ would determine the destiny of metastatic tumor cells. However Ewing, et. al., suggested that the anatomy of the drainage of tumor cells from the circulation system including blood and lymphatic supplies to the primary tumor would determine the site where metastasis occurred.⁸ In general this model remains correct for many but not all lymph node metastases. Further with hematogenous metastasis the liver and lung are good models for Ewings suggestion. These organs are the two most common sites for cancer metastasis. Also as we noted above, the liver is the vascular bed encountered by within the portal circulation and the lung is the first capillary bed in the venous circulation. Thus the development of liver metastases from colon carcinoma is understandable from the anatomy of the circulation. However since these pioneers put forward their hypotheses new information has emerged to temper our formulations. Now we know that metastasis is a strikingly inefficient procedure. Although millions of tumor cells may enter circulation from a primary tumor, the number of metastases detected in any given patient is vastly fewer than the potential from the number of cells in the circulation.^79-81 Thus, neither of these hypotheses is suitable to explain the presence of metastasis clinically or experimentally, but these hypotheses have to be systemically integrated.

It is also noteworthy that primary cancers metastasizing to the brain generally originate from the lung, breast and skin. The organ dependence leads to the hypothesis that tumor cells from the organs will express specific genes relevant for survival and proliferation in the brain microenvironment. Although the more understanding and researches are asked to define genes and detail the mechanism, homology in the gene expression between tumor cells and host would be a valuable target in brain metastasis.

Increased incidence of brain metastasis is likely to be the result from improved cancer therapy. Although we are far from complete understanding of cancer, diagnosis and therapy in cancer are improving with incremental and continuing advances especially over the last decade. Many molecular pathways and cellular interactions between tumor cells and the host have been described in different organs. Based on all these accomplishments at clinic, lab and industry, we can expect that brain metastasis will be better understood and that this information will improve detection and therapy.

References

1.

Fidler IJ, Balasubramanian K, Lin Q, Kim SW, Kim SJ. The brain microenvironment and cancer metastasis. Mol Cells. 2010;30:93–8. http://dx.doi.org/10.1007/s10059-010-0133-9 . [PubMed: 20799011]

2.

Eichler AF, Chung E, Kodack DP, Loeffler JS, Fukumura D, Jain RK. The biology of brain metastases-translation to new therapies. Nat Rev Clin Oncol. 2011;8:344–56. [PMC free article: PMC3259742] [PubMed: 21487419]

3.

Nathoo N, Chahlavi A, Barnett GH, Toms SA. Pathobiology of brain metastases. J Clin Pathol. 2005;58:237–42. http://dx.doi.org/10.1136/jcp.2003.013623 . [PMC free article: PMC1770599] [PubMed: 15735152]

4.

Anders C, Carey LA. Understanding and treating triple-negative breast cancer. Oncology (Williston Park). 2008;22:1233–9. discussion 9-40, 43. [PMC free article: PMC2868264] [PubMed: 18980022]

5.

Musolino A, Ciccolallo L, Panebianco M, Fontana E, Zanoni D, Bozzetti C, et al. Multifactorial central nervous system recurrence susceptibility in patients with HER2-positive breast cancer: epidemiological and clinical data from a population-based cancer registry study. Cancer. 2011;117:1837–46. http://dx.doi.org/10.1002/cncr.25771 . [PubMed: 21509760]

6.

Bos PD, Zhang XH, Nadal C, Shu W, Gomis RR, Nguyen DX, et al. Genes that mediate breast cancer metastasis to the brain. Nature. 2009;459:1005–9. http://dx.doi.org/10.1038/ nature08021 . [PMC free article: PMC2698953] [PubMed: 19421193]

7.

Nguyen DX, Chiang AC, Zhang XH, Kim JY, Kris MG, Ladanyi M, et al. WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell. 2009;138:51–62. http://dx.doi.org/10.1016/j.cell.2009.04.030 . [PMC free article: PMC2742946] [PubMed: 19576624]

8.

Fidler IJ. The role of the organ microenvironment in brain metastasis. Semin Cancer Biol. 2011;21:10712. http://dx.doi.org/10.1016/j.semcancer.2010.12.009 . [PubMed: 21167939]

9.

Weiss L, Grundmann E, Torhorst J, Hartveit F, Moberg I, Eder M, et al. Haematogenous metastatic patterns in colonic carcinoma: an analysis of 1541 necropsies. J Pathol. 1986;150:195–203. http://dx.doi.org/10.1002/path.1711500308 . [PubMed: 3806280]

10.

Wisse E, De Zanger RB, Charels K, Van Der Smissen P, McCuskey RS. The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse. Hepatology. 1985;5:683–92. http://dx.doi.org/10.1002/hep.1840050427 . [PubMed: 3926620]

11.

Gril B, Palmieri D, Bronder JL, Herring JM, Vega-Valle E, Feigenbaum L, et al. Effect of lapatinib on the outgrowth of metastatic breast cancer cells to the brain. J Natl Cancer Inst. 2008;100:1092–103. http://dx.doi.org/10.1093/jnci/djn216 . [PMC free article: PMC2575427] [PubMed: 18664652]

12.

Gril B, Evans L, Palmieri D, Steeg PS. Translational research in brain metastasis is identifying molecular pathways that may lead to the development of new therapeutic strategies. Eur J Cancer. 2010;46:120410. http://dx.doi.org/10.1016/j.ejca.2010.02.033 . [PMC free article: PMC2858326] [PubMed: 20303257]

13.

Tomasello G, Bedard PL, de Azambuja E, Lossignol D, Devriendt D, Piccart-Gebhart MJ. Brain metastases in HER2-positive breast cancer: the evolving role of lapatinib. Crit Rev Oncol Hematol. 2010;75:110–21. http://dx.doi.org/10.1016/j.critrevonc.2009.11.003 . [PubMed: 20004109]

14.

Delattre JY, Krol G, Thaler HT, Posner JB. Distribution of brain metastases. Arch Neurol. 1988;45:7414. http://dx.doi.org/10.1001/archneur.1988.00520310047016 . [PubMed: 3390029]

15.

Rutka JT, Apodaca G, Stern R, Rosenblum M. The extracellular matrix of the central and peripheral nervous systems: structure and function. J Neurosurg. 1988;69:155–70. http://dx.doi. org/10.3171/jns.1988.69.2.0155 . [PubMed: 3292716]

16.

Bonneh-Barkay D, Wiley CA. Brain extracellular matrix in neurodegeneration. Brain Pathol. 2009;19:573–85. http://dx.doi.org/10.1111/j.1750-3639.2008.00195.x . [PMC free article: PMC2742568] [PubMed: 18662234]

17.

Dityatev A, Seidenbecher CI, Schachner M. Compartmentalization from the outside: the extracellular matrix and functional microdomains in the brain. Trends Neurosci. 2010;33:503–12. http://dx.doi.org/10.1016/j.tins.2010.08.003 . [PubMed: 20832873]

18.

Felding-Habermann B, OToole TE, Smith JW, Fransvea E, Ruggeri ZM, Ginsberg MH, et al. Integrin activation controls metastasis in human breast cancer. Proc Natl Acad Sci U S A. 2001;98:1853–8. http://dx.doi.org/10.1073/pnas.98.4.1853 . [PMC free article: PMC29346] [PubMed: 11172040]

19.

Yoshimasu T, Sakurai T, Oura S, Hirai I, Tanino H, Kokawa Y, et al. Increased expression of integrin alpha3beta1 in highly brain metastatic subclone of a human non-small cell lung cancer cell line. Cancer Sci. 2004;95:142–8. http://dx.doi.org/10.1111/j.1349-7006.2004.tb03195.x . [PubMed: 14965364]

20.

Carbonell WS, Ansorge O, Sibson N, Muschel R. The vascular basement membrane as soil in brain metastasis. PLoS One. 2009;4:e5857. http://dx.doi.org/10.1371/journal. pone.0005857 . [PMC free article: PMC2689678] [PubMed: 19516901]

21.

Ghajar CM, George SC, Putnam AJ. Matrix metalloproteinase control of capillary morphogenesis. Crit Rev Eukaryot Gene Expr. 2008;18:251–78. [PMC free article: PMC2905732] [PubMed: 18540825]

22.

Rao JS. Molecular mechanisms of glioma invasiveness: the role of proteases. Nat Rev Cancer. 2003;3:489–501. http://dx.doi.org/10.1038/nrc1121 . [PubMed: 12835669]

23.

Guo P, Imanishi Y, Cackowski FC, Jarzynka MJ, Tao HQ, Nishikawa R, et al. Up-regulation of angiopoietin-2, matrix metalloprotease-2, membrane type 1 metalloprotease, and laminin 5 gamma 2 correlates with the invasiveness of human glioma. Am J Pathol. 2005;166:877–90. http://dx.doi.org/10.1016/S0002-9440(10)62308-5 . [PMC free article: PMC1602359] [PubMed: 15743799]

24.

Kamino M, Kishida M, Kibe T, Ikoma K, Iijima M, Hirano H, et al. Wnt-5a signaling is correlated with infiltrative activity in human glioma by inducing cellular migration and MMP-2. Cancer Sci. 2011;102:540–8. http://dx.doi.org/10.1111/j.1349-7006.2010.01815.x . [PubMed: 21205070]

25.

Zhao Y, Xiao A, diPierro CG, Carpenter JE, Abdel-Fattah R, Redpath GT, et al. An extensive invasive intracranial human glioblastoma xenograft model: role of high level matrix metalloproteinase 9. Am J Pathol. 2010;176:3032–49. http://dx.doi.org/10.2353/ajpath.2010.090571 . [PMC free article: PMC2877863] [PubMed: 20413683]

26.

Vassalli JD, Sappino AP, Belin D. The plasminogen activator/plasmin system. J Clin Invest. 1991;88:1067–72. http://dx.doi.org/10.1172/JCI115405 . [PMC free article: PMC295552] [PubMed: 1833420]

27.

Bindal AK, Hammoud M, Shi WM, Wu SZ, Sawaya R, Rao JS. Prognostic significance of proteolytic enzymes in human brain tumors. J Neurooncol. 1994;22:101–10. http://dx.doi. org/10.1007/BF01052886 . [PubMed: 7538161]

28.

Le DM, Besson A, Fogg DK, Choi KS, Waisman DM, Goodyer CG, et al. Exploitation of astrocytes by glioma cells to facilitate invasiveness: a mechanism involving matrix metalloproteinase-2 and the urokinase-type plasminogen activator-plasmin cascade. J Neurosci. 2003;23:4034–43. [PMC free article: PMC6741112] [PubMed: 12764090]

29.

Zhao Y, Lyons CE Jr, Xiao A, Templeton DJ, Sang QA, Brew K, et al. Urokinase directly activates matrix metalloproteinases-9: a potential role in glioblastoma invasion. Biochem Biophys Res Commun. 2008;369:1215–20. http://dx.doi.org/10.1016/j.bbrc.2008.03.038 . [PMC free article: PMC2562868] [PubMed: 18355442]

30.

Chandrasekar N, Mohanam S, Gujrati M, Olivero WC, Dinh DH, Rao JS. Downregulation of uPA inhibits migration and PI3k/Akt signaling in glioblastoma cells. Oncogene. 2003;22:392–400. http://dx.doi.org/10.1038/sj.onc.1206164 . [PubMed: 12545160]

31.

Gondi CS, Kandhukuri N, Dinh DH, Gujrati M, Rao JS. Down-regulation of uPAR and uPA activates caspase-mediated apoptosis and inhibits the PI3K/AKT pathway. Int J Oncol. 2007;31:19–27. [PMC free article: PMC1905845] [PubMed: 17549401]

32.

Fitzgerald DP, Palmieri D, Hua E, Hargrave E, Herring JM, Qian Y, et al. Reactive glia are recruited by highly proliferative brain metastases of breast cancer and promote tumor cell colonization. Clin Exp Metastasis. 2008;25:799–810. http://dx.doi.org/10.1007/s10585-008-9193-z . [PMC free article: PMC2679391] [PubMed: 18649117]

33.

Ksters B, Leenders WP, Wesseling P, Smits D, Verrijp K, Ruiter DJ, et al. Vascular endothelial growth factor-A(165) induces progression of melanoma brain metastases without induction of sprouting angiogenesis. Cancer Res. 2002;62:341–5. [PubMed: 11809675]

34.

Leenders WP, Ksters B, Verrijp K, Maass C, Wesseling P, Heerschap A, et al. Antiangiogenic therapy of cerebral melanoma metastases results in sustained tumor progression via vessel co-option. Clin Cancer Res. 2004;10:6222–30. http://dx.doi.org/10.1158/1078-0432.CCR-04-0823 . [PubMed: 15448011]

35.

Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J, et al. Real-time imaging reveals the single steps of brain metastasis formation. Nat Med. 2010;16:116–22. http://dx.doi.org/10.1038/nm.2072 . [PubMed: 20023634]

36.

Liebner S, Fischmann A, Rascher G, Duffner F, Grote EH, Kalbacher H, et al. Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme. Acta Neuropathol. 2000;100:323–31. http://dx.doi.org/10.1007/s004010000180 . [PubMed: 10965803]

37.

Hoelzinger DB, Demuth T, Berens ME. Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment. J Natl Cancer Inst. 2007;99:1583–93. http:// dx.doi.org/10.1093/jnci/djm187 . [PubMed: 17971532]

38.

Holash J, Maisonpierre PC, Compton D, Boland P, Alexander CR, Zagzag D, et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science. 1999;284:1994–8. http://dx.doi.org/10.1126/science.284.5422.1994 . [PubMed: 10373119]

39.

Hu B, Guo P, Fang Q, Tao HQ, Wang D, Nagane M, et al. Angiopoietin-2 induces human glioma invasion through the activation of matrix metalloprotease-2. Proc Natl Acad Sci U S A. 2003;100:89049. http://dx.doi.org/10.1073/pnas.1533394100 . [PMC free article: PMC166411] [PubMed: 12861074]

40.

Kobayashi Z, Tsuchiya K, Machida A, Goto J, Yokota O, Miake H, et al. Metastatic CNS lymphoma presenting with periventricular dissemination - MRI and neuropathological findings in an autopsy case. J Neurol Sci. 2009;277:109–13. http://dx.doi.org/10.1016/j.jns.2008.10.029 . [PubMed: 19041988]

41.

Brat DJ, Mapstone TB. Malignant glioma physiology: cellular response to hypoxia and its role in tumor progression. Ann Intern Med. 2003;138:659–68. [PubMed: 12693889]

42.

Rong Y, Durden DL, Van Meir EG, Brat DJ. Pseudopalisading necrosis in glioblastoma: a familiar morphologic feature that links vascular pathology, hypoxia, and angiogenesis. J Neuropathol Exp Neurol. 2006;65:529–39. http://dx.doi.org/10.1097/00005072-200606000-00001 . [PubMed: 16783163]

43.

Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A, et al. Glioblastoma stem-like cells give rise to tumour endothelium. Nature. 2010;468:829–33. http://dx.doi. org/10.1038/nature09624 . [PubMed: 21102433]

44.

Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Cenci T, et al. Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature. 2010;468:824–8. http://dx.doi.org/10.1038/nature09557 . [PubMed: 21102434]

45.

Carmeliet P, Jain RK. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov. 2011;10:417–27. http://dx.doi.org/10.1038/nrd3455 . [PubMed: 21629292]

46.

Plate KH, Breier G, Weich HA, Risau W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature. 1992;359:845–8. http://dx.doi. org/10.1038/359845a0 . [PubMed: 1279432]

47.

Goldman CK, Kim J, Wong WL, King V, Brock T, Gillespie GY. Epidermal growth factor stimulates vascular endothelial growth factor production by human malignant glioma cells: a model of glioblastoma multiforme pathophysiology. Mol Biol Cell. 1993;4:121–33. [PMC free article: PMC300905] [PubMed: 7680247]

48.

Ke LD, Shi YX, Im SA, Chen X, Yung WK. The relevance of cell proliferation, vascular endothelial growth factor, and basic fibroblast growth factor production to angiogenesis and tumorigenicity in human glioma cell lines. Clin Cancer Res. 2000;6:2562–72. [PubMed: 10873113]

49.

Stiles JD, Ostrow PT, Balos LL, Greenberg SJ, Plunkett R, Grand W, et al. Correlation of endothelin-1 and transforming growth factor beta 1 with malignancy and vascularity in human gliomas. J Neuropathol Exp Neurol. 1997;56:435–9. http://dx.doi.org/10.1097/00005072-199704000-00012 . [PubMed: 9100674]

50.

Platten M, Wick W, Weller M. Malignant glioma biology: role for TGF-beta in growth, motility, angiogenesis, and immune escape. Microsc Res Tech. 2001;52:401–10. http://dx.doi. org/10.1002/1097-0029(20010215)52:4>401::AID-JEMT1025<3.0.CO;2-C . [PubMed: 11170299]

51.

Shibuya M. Brain angiogenesis in developmental and pathological processes: therapeutic aspects of vascular endothelial growth factor. FEBS J. 2009;276:4636–43. http://dx.doi. org/10.1111/j.1742-4658.2009.07175.x . [PubMed: 19664071]

52.

Fan X, Heijnen CJ, van der Kooij MA, Groenendaal F, van Bel F. The role and regulation of hypoxia-inducible factor-1alpha expression in brain development and neonatal hypoxic-ischemic brain injury. Brain Res Rev. 2009;62:99–108. http://dx.doi.org/10.1016/j.brainresrev.2009.09.006 . [PubMed: 19786048]

53.

Du R, Lu KV, Petritsch C, Liu P, Ganss R, Passegu E, et al. HIF1alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell. 2008;13:206–20. http://dx.doi.org/10.1016/j.ccr.2008.01.034 . [PMC free article: PMC2643426] [PubMed: 18328425]

54.

Nanka O, Valsek P, Dvorkov M, Grim M. Experimental hypoxia and embryonic angiogenesis. Dev Dyn. 2006;235:723–33. http://dx.doi.org/10.1002/dvdy.20689 . [PubMed: 16444736]

55.

Zhang ZG, Zhang L, Tsang W, Soltanian-Zadeh H, Morris D, Zhang R, et al. Correlation of VEGF and angiopoietin expression with disruption of blood-brain barrier and angiogenesis after focal cerebral ischemia. J Cereb Blood Flow Metab. 2002;22:379–92. http://dx.doi. org/10.1097/00004647-200204000-00002 . [PubMed: 11919509]

56.

Leenders WP, Ksters B, de Waal RM. Vessel co-option: how tumors obtain blood supply in the absence of sprouting angiogenesis. Endothelium. 2002;9:83–7. http://dx.doi. org/10.1080/10623320212006 . [PubMed: 12200959]

57.

Wang D, Huang HJ, Kazlauskas A, Cavenee WK. Induction of vascular endothelial growth factor expression in endothelial cells by platelet-derived growth factor through the activation of phosphatidylinositol 3-kinase. Cancer Res. 1999;59:1464–72. [PubMed: 10197615]

58.

Guo P, Hu B, Gu W, Xu L, Wang D, Huang HJ, et al. Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment. Am J Pathol. 2003;162:1083–93. http://dx.doi. org/10.1016/S0002-9440(10)63905-3 . [PMC free article: PMC1851242] [PubMed: 12651601]

59.

Fiedler U, Augustin HG. Angiopoietins: a link between angiogenesis and inflammation. Trends Immunol. 2006;27:552–8. http://dx.doi.org/10.1016/j.it.2006.10.004 . [PubMed: 17045842]

60.

Lobov IB, Brooks PC, Lang RA. Angiopoietin-2 displays VEGF-dependent modulation of capillary structure and endothelial cell survival in vivo. Proc Natl Acad Sci U S A. 2002;99:11205–10. http://dx.doi.org/10.1073/pnas.172161899 . [PMC free article: PMC123234] [PubMed: 12163646]

61.

Brunckhorst MK, Wang H, Lu R, Yu Q. Angiopoietin-4 promotes glioblastoma progression by enhancing tumor cell viability and angiogenesis. Cancer Res. 2010;70:7283–93. http://dx.doi. org/10.1158/0008-5472.CAN-09-4125 . [PMC free article: PMC2940950] [PubMed: 20823154]

62.

Fajardo LF, Kwan HH, Kowalski J, Prionas SD, Allison AC. Dual role of tumor necrosis factor-alpha in angiogenesis. Am J Pathol. 1992;140:539–44. [PMC free article: PMC1886157] [PubMed: 1372154]

63.

Zhang R, Xu Y, Ekman N, Wu Z, Wu J, Alitalo K, et al. Etk/Bmx transactivates vascular endothelial growth factor 2 and recruits phosphatidylinositol 3-kinase to mediate the tumor necrosis factor-induced angiogenic pathway. J Biol Chem. 2003;278:51267–76. http://dx.doi.org/10.1074/ jbc.M310678200 . [PubMed: 14532277]

64.

Ryuto M, Ono M, Izumi H, Yoshida S, Weich HA, Kohno K, et al. Induction of vascular endothelial growth factor by tumor necrosis factor alpha in human glioma cells. Possible roles of SP-1. J Biol Chem. 1996;271:28220–8. http://dx.doi.org/10.1074/jbc.271.45.28220 . [PubMed: 8910439]

65.

Yoshino Y, Aoyagi M, Tamaki M, Duan L, Morimoto T, Ohno K. Activation of p38 MAPK and/or JNK contributes to increased levels of VEGF secretion in human malignant glioma cells. Int J Oncol. 2006;29:981–7. [PubMed: 16964394]

66.

Van Meir E, Sawamura Y, Diserens AC, Hamou MF, de Tribolet N. Human glioblastoma cells release interleukin 6 in vivo and in vitro. Cancer Res. 1990;50:6683–8. [PubMed: 2208133]

67.

Rolhion C, Penault-Llorca F, Kmny JL, Lemaire JJ, Jullien C, Labit-Bouvier C, et al. Interleukin-6 overexpression as a marker of malignancy in human gliomas. J Neurosurg. 2001;94:97–101. http://dx.doi.org/10.3171/jns.2001.94.1.0097 . [PubMed: 11147905]

68.

Chang CY, Li MC, Liao SL, Huang YL, Shen CC, Pan HC. Prognostic and clinical implication of IL-6 expression in glioblastoma multiforme. J Clin Neurosci. 2005;12:930–3. http:// dx.doi.org/10.1016/j.jocn.2004.11.017 . [PubMed: 16326273]

69.

Loeffler S, Fayard B, Weis J, Weissenberger J. Interleukin-6 induces transcriptional activation of vascular endothelial growth factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in glioblastoma cells via direct interaction between STAT3 and Sp1. Int J Cancer. 2005;115:202–13. http://dx.doi.org/10.1002/ijc.20871 . [PubMed: 15688401]

70.

Saidi A, Hagedorn M, Allain N, Verpelli C, Sala C, Bello L, et al. Combined targeting of interleukin-6 and vascular endothelial growth factor potently inhibits glioma growth and invasiveness. Int J Cancer. 2009;125:1054–64. http://dx.doi.org/10.1002/ijc.24380 . [PubMed: 19431143]

71.

Fan Y, Ye J, Shen F, Zhu Y, Yeghiazarians Y, Zhu W, et al. Interleukin-6 stimulates circulating blood-derived endothelial progenitor cell angiogenesis in vitro. J Cereb Blood Flow Metab. 2008;28:90–8. http://dx.doi.org/10.1038/sj.jcbfm.9600509 . [PMC free article: PMC2581498] [PubMed: 17519976]

72.

Brat DJ, Bellail AC, Van Meir EG. The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis. Neuro Oncol. 2005;7:122–33. http://dx.doi.org/10.1215/ S1152851704001061 . [PMC free article: PMC1871893] [PubMed: 15831231]

73.

Wakabayashi Y, Shono T, Isono M, Hori S, Matsushima K, Ono M, et al. Dual pathways of tubular morphogenesis of vascular endothelial cells by human glioma cells: vascular endothelial growth factor/ basic fibroblast growth factor and interleukin-8. Jpn J Cancer Res. 1995;86:1189–97. http://dx.doi.org/10.1111/j.1349-7006.1995.tb03314.x . [PMC free article: PMC5920670] [PubMed: 8636009]

74.

Desbaillets I, Diserens AC, Tribolet N, Hamou MF, Van Meir EG. Upregulation of interleukin 8 by oxygen-deprived cells in glioblastoma suggests a role in leukocyte activation, chemotaxis, and angiogenesis. J Exp Med. 1997;186:1201–12. http://dx.doi.org/10.1084/jem.186.8.1201 . [PMC free article: PMC2199083] [PubMed: 9334359]

75.

Nabors LB, Suswam E, Huang Y, Yang X, Johnson MJ, King PH. Tumor necrosis factor alpha induces angiogenic factor up-regulation in malignant glioma cells: a role for RNA stabilization and HuR. Cancer Res. 2003;63:4181–7. [PubMed: 12874024]

76.

Charalambous C, Pen LB, Su YS, Milan J, Chen TC, Hofman FM. Interleukin-8 differentially regulates migration of tumor-associated and normal human brain endothelial cells. Cancer Res. 2005;65:1034754. http://dx.doi.org/10.1158/0008-5472.CAN-05-0949 . [PubMed: 16288024]

77.

Luca M, Huang S, Gershenwald JE, Singh RK, Reich R, Bar-Eli M. Expression of interleukin-8 by human melanoma cells up-regulates MMP-2 activity and increases tumor growth and metastasis. Am J Pathol. 1997;151:1105–13. [PMC free article: PMC1858026] [PubMed: 9327744]

78.

Yoo JY, Kim JH, Kim J, Huang JH, Zhang SN, Kang YA, et al. Short hairpin RNA-expressing oncolytic adenovirus-mediated inhibition of IL-8: effects on antiangiogenesis and tumor growth inhibition. Gene Ther. 2008;15:635–51. http://dx.doi.org/10.1038/gt.2008.3 . [PubMed: 18273054]

79.

Bockhorn M, Jain RK, Munn LL. Active versus passive mechanisms in metastasis: do cancer cells crawl into vessels, or are they pushed? Lancet Oncol. 2007;8:444–8. http://dx.doi. org/10.1016/S1470-2045(07)70140-7 . [PMC free article: PMC2712886] [PubMed: 17466902]

80.

Butler TP, Gullino PM. Quantitation of cell shedding into efferent blood of mammary adenocarcinoma. Cancer Res. 1975;35:512–6. [PubMed: 1090362]

81.

Liotta LA, Kleinerman J, Saidel GM. Quantitative relationships of intravascular tumor cells, tumor vessels, and pulmonary metastases following tumor implantation. Cancer Res. 1974;34:997–1004. [PubMed: 4841969]