Article

A = Perivascular=20 Niche for Brain Tumor Stem Cells

Christopher Calabrese,1 = Helen Poppleton,1 Mehmet Kocak,2 Twala L. Hogg,1 = Christine Fuller,3 Blair Hamner,4 Eun Young Oh,1 M. = Waleed Gaber,5 = David Finklestein,6 = Meredith Allen,1 Adrian Frank,1 Ildar = T. Bayazitov,1 Stanislav = S. Zakharenko,1 Amar Gajjar,7 = Andrew Davidoff,4 and Richard=20 J. Gilbertson1,7,

1 Department = of=20 Developmental Neurobiology, St. Jude Children's Research Hospital, 332 = North=20 Lauderdale Street, Memphis, TN 38105, USA2 Department = of=20 Biostatistics, St. Jude Children's Research Hospital, 332 North = Lauderdale=20 Street, Memphis, TN 38105, USA3 Department = of=20 Pathology, St. Jude Children's Research Hospital, 332 North Lauderdale = Street,=20 Memphis, TN 38105, USA4 Department = of Surgery,=20 St. Jude Children's Research Hospital, 332 North Lauderdale Street, = Memphis, TN=20 38105, USA
5 Department = of=20 Radiation Oncology, St. Jude Children's Research Hospital, 332 North = Lauderdale=20 Street, Memphis, TN 38105, USA6 The = Hartwell Center=20 for Bioinformatics and Biotechnology, St. Jude Children's Research = Hospital, 332=20 North Lauderdale Street, Memphis, TN 38105, USA7 Department = of=20 Oncology, St. Jude Children's Research Hospital, 332 North Lauderdale = Street,=20 Memphis, TN 38105, USA

Corresponding author
Richard J. Gilbertson
richard.gilbertson@stjude.o= rg

Summary

Cancers are=20 believed to arise from cancer stem cells (CSCs), but it is not known if = these=20 cells remain dependent upon the niche microenvironments that regulate = normal=20 stem cells. We show that endothelial cells interact closely with = self-renewing=20 brain tumor cells and secrete factors that maintain these cells in a = stem=20 cell-like state. Increasing the number of endothelial cells or blood = vessels in=20 orthotopic brain tumor xenografts expanded the fraction of self-renewing = cells=20 and accelerated the initiation and growth of tumors. Conversely, = depletion of=20 blood vessels from xenografts ablated self-renewing cells from tumors = and=20 arrested tumor growth. We propose that brain CSCs are maintained within = vascular=20 niches that are important targets for therapeutic approaches.

SIGNIFICANCE

We present evidence that brain tumors = orchestrate=20 vascular niches that maintain the CSC pool. Disruption of these niche=20 microenvironments ablates the fraction of self-renewing cells in brain = tumors=20 and arrests tumor growth. Our data identify a potential role for niche=20 microenvironments in the maintenance of brain CSCs and identify a = mechanism by=20 which antiangiogenic drugs inhibit brain tumor = growth.

Introduction

There is now compelling evidence that the = bulk of the=20 malignant cells in cancers are generated by rare fractions of = self-renewing,=20 multipotent, and tumor-initiating cells, termed cancer stem cells (CSCs) = (Al-Hajj et al., 2003, Lapidot et al., 1994, Singh et al., 2004). Whether CSCs arise from normal stem cells or more = differentiated=20 cells is not known. Nevertheless, CSCs resemble the normal stem or = progenitor=20 cells of the corresponding tissue of origin. For example, brain CSCs = express=20 CD133 and Nestin that mark neural stem and progenitor cells (Galli et = al.,=20 2004, Singh et al., 2004, Taylor et al., 2005), and we recently reported that CSCs isolated from = ependymomas are=20 remarkably similar to radial glia that are neural precursor cells = (Taylor et=20 al., 2005).

If tumors are derived entirely from CSCs, = then drugs=20 that kill these cells could prove highly effective treatments of cancer. = On the=20 other hand, the similarities between normal and malignant stem cells = predict=20 that such treatments may also possess significant toxicities. Recent = encouraging=20 data have provided proof of principle that selective targeting of CSCs = is=20 possible (Yilmaz et al., 2006). However, the development of anti-CSC therapies for = each type of=20 cancer is likely to require the identification of factors that maintain = CSCs,=20 but not normal stem cells, in each tissue.

One important difference between normal stem = cells and=20 CSCs might be the degree to which these cells are regulated by the = immediate=20 microenvironment. Stem cells of various tissues exist within protective = niches=20 that are composed of a number of differentiated cell types (Fuchs et = al., 2004, Moore et al., 2006). These mature cells provide direct cell contacts and = secreted=20 factors that maintain stem cells primarily in a quiescent state. For = example,=20 histologic and ex vivo cell culture studies of mouse tissues suggest = that neural=20 stem cells lie within a vascular niche in which endothelial cells = regulate stem=20 cell self-renewal (Louissaint et al., 2002, Palmer et al., 2000, Ramirez-Castillejo et al., 2006, Shen et al., 2004). Thus, CSCs might arise from normal stem cells that = have acquired=20 mutations that enable them to escape from niche control (Wodarz et al., = 2006). Alternatively, deregulation of extrinsic factors = within the=20 niche might lead to uncontrolled proliferation of stem cells and = tumorigenesis=20 (Clarke et al., 2006). If CSCs depend upon aberrant niche microenvironments, = then these=20 niches might represent targets for treatments of cancer.

Here, we demonstrate that=20 Nestin⁺/CD133⁺ cancer cells that include the CSC = fraction=20 are located next to capillaries in brain tumors. We show further that=20 endothelial cells interact selectively with = Nestin⁺/CD133⁺=20 brain cancer cells in culture and supply secreted factors that maintain = these=20 cells in a self-renewing and undifferentiated state. Increasing the = number of=20 endothelial cells or blood vessels in orthotopic brain tumor xenografts = expanded=20 the numbers of self-renewing Nestin⁺/CD133⁺ cancer = cells=20 and accelerated the initiation and growth of tumors. Conversely, = antiangiogenic=20 therapies depleted tumor blood vessels and associated self-renewing=20 Nestin⁺/CD133⁺ cancer cells from xenografts and = arrested=20 tumor growth. Thus, we propose that the brain tumor microvasculature = forms niche=20 microenvironments that maintain CSCs and that represent therapeutic = targets in=20 brain tumors.

Results

Nestin+ Brain Tumor Cells = Associate with=20 the Tumor Vasculature

Normal neural stem and precursor cells and = brain CSCs=20 express the major intermediate filament protein Nestin (Galli et al., = 2004, Hemmati et al., 2003, Palmer et al., 2000, Singh et al., 2004, Taylor et al., 2005). In the hippocampus, Nestin⁺ cells are = located close=20 to capillaries where endothelial cells are believed to regulate the = self-renewal=20 and differentiation of stem cell daughters (Palmer et al., 2000, Wurmser et al., 2004). As a first step to establish if brain CSCs are located = within a=20 vascular niche, we determined the incidence of Nestin⁺ cells, = and the=20 proximity of these cells to tumor capillaries in sections of = medulloblastoma (n=20 =3D 20), ependymoma (n =3D 23), oligodendroglioma (n =3D 20), and = glioblastoma (n =3D=20 10).

In keeping with the low incidence of CSCs = within brain=20 tumors (Galli et al., 2004, Singh et al., 2004, Taylor et al., 2005), between 0.15% and 0.22% of cells within tumors were=20 Nestin⁺ (>500 cells counted per tumor section; Figure 1A). = The=20 number of Nestin⁺ cells in brain tumors appeared to correlate = with=20 microvessel density (MVD): oligodendrogliomas and glioblastomas that had = the=20 highest MVD also contained the greatest numbers of Nestin⁺ = cells (Figure 1A). = Further,=20 Nestin⁺ cells were located significantly closer to tumor = capillaries=20 than Nestin^=E2=88=92 cells, independent of histologic = diagnosis (Figure 1A = and=20 1B).

Figure 1. Nestin+ Cells in Brain Tumors Are = Located=20 Next to Capillaries

(A) Coimmunofluorescence analysis of Nestin and CD34 expression in = sections=20 of a primary medulloblastoma (MB), ependymoma (EP), oligodendroglioma = (OLIG),=20 and glioblastoma (GBM). Arrows indicate Nestin+ cells. The microvessel = density=20 (MVD) and percentage Nestin⁺=20 tumor cells are shown for each tumor type.

(B) Graph reports the percentage of Nestin+ and Nestin=E2=88=92 cells that were = located in=20 incremental distances of 5 =CE=BCm from the nearest CD34+ endothelial cell in each = tumor type=20 (p < 0.005, p < 0.0005 for the average distance of Nestin+ versus Nestin=E2=88=92 cells from = CD34+ cells).

(C) Reconstruction in three dimensions of capillaries (CD34, red) and = Nestin⁺ tumor cells = (green) in=20 50 =CE=BCm sections of two gliomas.

(D) Nestin and CD133 coimmunofluorescence (left), concurrent=20 coimmunofluorescence (Nestin/CD34) and FISH (17p/17q) (middle), and=20 coimmunofluorescence (Nestin/Ki-67) staining (right) of primary = medulloblastoma.=20 Asterisks marks a capillary lumen.

(E) Hematoxylin and eosin staining (left), Nestin and GFP=20 coimmunofluorescence (middle), and Nestin and CD34 coimmunofluorescence = (right)=20 of orthotopic xenografts of GFP+ Daoy medulloblastoma = cells. Arrows=20 indicate Nestin⁺ = cancer cells.=20 All nuclei are stained with DAPI.

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Since individual tumor sections represent = three=20 dimensional tissues in only two dimensions, it is difficult using = standard=20 histology to determine the precise relationship between individual cells = and the=20 vasculature (Palmer et al., 2000). Therefore, we used multiphoton laser-scanning = microscopy to=20 reconstruct, in three dimensions, serial images taken through 50 =CE=BCm = sections of=20 four gliomas that were coimmunostained for CD34 and Nestin (=E2=88=BC70 = images per=20 section). This analysis showed clearly that Nestin⁺ tumor = cells=20 directly contact tumor capillaries (Figure 1C).<= /P>

Endothelial cells have been reported to = express Nestin=20 (Aihara et al., 2004). However, the morphology of Nestin⁺ cells in = brain=20 tumors was more typical of neural stem cells, and less than 0.1% of = these cells=20 coexpressed the endothelial marker CD34. Furthermore, 73.4% =C2=B1 20% = of=20 Nestin⁺ cells in tumor sections coexpressed the cell surface = protein=20 CD133, which marks quite specifically normal neural stem and precursor = cells and=20 brain CSCs (>800 cells counted per tumor; Figure 1D) = (Singh et=20 al., 2004, Taylor et al., 2005, Uchida et al., 2000).

Focusing on medulloblastoma, we next sought = to=20 determine if Nestin⁺ cells in brain tumors are cancer cells = or=20 entrapped normal neural stem and precursor cells. First, we performed=20 consecutive coimmunofluorescence (Nestin and CD34) and fluorescence in = situ=20 hybridization (FISH) analysis of sections of medulloblastomas that = we had=20 shown previously to contain an isochromosome of 17q (Thompson et al., = 2006). Over one-third of the = Nestin⁺-vessel-associated cells=20 in these tumors contained an isochromosome 17q, confirming that they are = cancer=20 cells (Figure 1D). = It is=20 likely that a greater proportion of Nestin⁺ cells in these = tumors are=20 cancer cells, since FISH underestimates the frequency of low-copy-number = cytogenetic alterations because portions of cell nuclei are lost during = tissue=20 sectioning. Indeed, Ki-67/Nestin coimmunofluorescence demonstrated that = 29.1% =C2=B1=20 11.6% of Nestin⁺ cells in these tumors are proliferating = (>500=20 cells counted per section; Figure 1D). = Less than=20 1% of cells in the normal human subventricular zone express Ki-67=20 (Quinones-Hinojosa et al., 2006, Sanai et al., 2004). Therefore, Nestin⁺ cells in medulloblastoma = appear to=20 possess aberrant proliferative capacity, suggesting further that they = are cancer=20 cells.

Finally, we prelabeled Daoy (Jacobsen et = al., 1985) and MEB-MED-8A (Pietsch et al., 1994) medulloblastoma cells with green fluorescence protein = (GFP) by=20 retroviral transduction and transplanted 1 =C3=97 10⁶ = GFP⁺=20 cells orthotopically into the brains of immunocompromised mice (n =3D 3 = mice per=20 cell line). Engrafted Daoy and MEB-MED-8A cells formed highly = infiltrative=20 primitive neuroectodermal tumors (Figure 1E = and data=20 not shown). CSCs have been identified in established brain tumor cell = lines and=20 are responsible for the in vivo malignancy of these cells (Kondo et al., = 2004). In keeping with our analysis of primary = medulloblastomas, 0.83%=20 and 1.3% of cells in Daoy and MEB-MED-8A xenografts, respectively,=20 expressed Nestin (>300 cells counted per tumor; Figure 1E), = and these=20 cells were located significantly closer to capillaries (Daoy, 32 =C2=B1 = 3.3 =CE=BCm;=20 MEB-MED-8A, 44 =C2=B1 5.6 =CE=BCm) than were Nestin^=E2=88=92 = cells (Daoy, 74 =C2=B1 2.5 =CE=BCm;=20 MEB-MED-8A, 80 =C2=B1 3.6 =CE=BCm; p < 0.0001 for both cell lines). = Nestin⁺=20 cells in these xenografts coexpressed GFP, confirming that they are = cancer cells=20 (Figure 1E). = Together,=20 these data indicate that the great majority of Nestin⁺ cells = in brain=20 tumors are vessel-associated cancer cells.

CD133+ Brain Tumor Cells = Interact=20 Physically with Endothelial Cells in Culture

To investigate further if brain CSCs = interact directly=20 with endothelial cells, we prepared single-cell suspensions from a = freshly=20 resected primary medulloblastoma (MB5) and ependymoma (EP1) that we = obtained=20 direct from the operating room and labeled these with a green = fluorescent dye=20 (PKH67). We then used antibody-based cell sorting to isolate = CD133⁺=20 cells from these brain tumors since this fraction is enriched for CSCs = (Singh et=20 al., 2004, Taylor et al., 2005). Subsequent karyotype analysis of these tumor cells = that we have=20 reported elsewhere confirmed that they are cancer cells (Taylor et al., = 2005). We similarly fractionated populations of Daoy = medulloblastoma=20 cells. We then mixed CD133⁺ or CD133^=E2=88=92 = tumor cells with=20 primary human endothelial cells (PHECs) in a 3D matrigel assay in which = the=20 endothelial cells form vascular tubes (n =3D 8 separate cultures per = cell=20 population).

PHECs formed vascular tubes within = 5=E2=80=937 hr independent=20 of the CD133 status of the cocultured tumor cells (Figure 2A). = CD133⁺ tumor cells, independent of origin, associated rapidly = with=20 endothelial vascular tubes, forming intimate contacts often along the = entire=20 length of an endothelial tube (Figure 2A = and 2B). In=20 contrast, significantly fewer CD133^=E2=88=92 tumor cells made = contacts with=20 endothelial cells and generally remained rounded and separate from the=20 vascular-like structures. To control for the possibility that = CD133^=E2=88=92=20 tumor cells are unable to form cell-cell contacts in 3D matrigel = cultures, we=20 repeated these experiments using primary human astrocytes (PHAs) instead = of=20 PHECs. Both CD133⁺ and CD133^=E2=88=92 tumor cells = interacted=20 readily, but not differently, with PHAs in these cocultures (Figure 2A = and 2B).=20 Thus, CD133⁺ brain tumor cells that contain the CSC fraction, = but not=20 CD133^=E2=88=92 cells, interact closely with vascular = endothelial cells in=20 culture.

Figure 2. CD133+ Brain Tumor Cells = Associate with=20 Endothelial Cells in 3D Matrigel Cultures

(A) Overlay of phase-contrast and autofluorescence photomicrographs = of=20 unlabeled endothelial cells or astrocytes that were cocultured with = CD133+ or CD133=E2=88=92 green = fluorescence-labeled primary=20 medulloblastoma (MB5) or ependymoma (EP1) cells.

(B) The number of green fluorescent (tumor cells) that contacted = directly an=20 unlabeled cell (endothelial or astrocyte) was determined by high-power=20 microscopic review of cocultures. The reviewer was blinded to CD133 = tumor cell=20 status. Graph reports the percentage of CD133+ and CD133=E2=88=92 tumor cells in = each coculture that=20 formed a direct contact with cocultured cells. Each bar is marked with = the total=20 number of tumor cells in each culture (p < 0.05, p < 0.005, p < 0.0005 reports the difference between CD133+ and CD133=E2=88=92 tumor cell = contacts with cocultured=20 cells).

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Endothelial Cells = Maintain=20 Self-Renewing and Undifferentiated Brain Tumor Cells

Endothelial cells secrete soluble factors = that=20 maintain the self-renewal and undifferentiated phenotype of normal = neural stem=20 cells (Ramirez-Castillejo et al., 2006, Shen et al., 2004). To test if endothelial-derived factors similarly = maintain=20 self-renewing brain tumor cells, we first passaged tumor spheres of=20 CD133⁺ cells that we isolated from a primary medulloblastoma = (MB1), a=20 primary ependymoma (EP1), and the Daoy medulloblastoma cell line, under=20 conditions that promote stem cell growth (Taylor et al., 2005). Tumor spheres derived from each of these cell = populations could=20 be passaged serially and expressed CD133 and Nestin, confirming that = these are=20 clonogenic, self-renewing stem-like cells (Figure 3A=E2= =80=933C). We=20 then transferred tertiary tumor spheres of these cells to the base of = culture=20 wells while the upper transwell compartment was seeded with PHECs or = control=20 cells (CD133^=E2=88=92 tumor cells, fibroblasts, or PHAs) (Figure 3D). = This=20 transwell system allows the exchange of diffusible factors, but not = cells,=20 between chambers. Cocultures were maintained for 2 weeks in=20 reduced-serum-containing medium.

Figure 3. Endothelial Cells Maintain = Self-Renewing and=20 Undifferentiated Brain Tumor Cells in Culture

(A=E2=80=93C) CD133 and Nestin immunofluorescence analysis of Daoy = (A)-, primary=20 medulloblastoma (MB1) (B)-, and primary ependymoma (EP1) (C)-derived = tertiary=20 tumor spheres.

(D) Cartoon depicting the experimental approach adopted to determine = the=20 ability of PHECs to maintain the self-renewal and undifferentiated = phenotype of=20 brain tumor cells.

(E) Comparison of the size (top images: DAPI staining of four = representative=20 tumor spheres; top graph: average total sphere area of tumor spheres in = the=20 appropriate cultures), maintenance (bottom graph: percentage of tertiary = tumor=20 spheres remaining after 120 hr of coculture), and self-renewal (bottom = figures=20 report the percentage of quaternary-forming tumor spheres derived from=20 cocultured tertiary spheres) of Daoy medulloblastoma cells and primary=20 medulloblastoma (MB1) and primary ependymoma (EP1) cells cocultured with = PHECs=20 (+) or control cells (=E2=88=92). Results are shown for differentiated = tumor cells as=20 controls. Similar results were obtained when PHAs or fibroblasts were = used as=20 controls (p < 0.05, p < 0.005, exact Wilcoxon test).

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Tumor spheres that were cocultured with = PHECs=20 increased in size and after 2 weeks were up to five times larger than = those=20 grown in the presence of control cells (Figure 3E). = Further,=20 significantly more PHEC-cocultured tumor spheres remained as intact=20 CD133⁺/Nestin⁺ tumor spheres compared to those = cultured=20 with control cells. Thus, PHECs maintained the proliferation of tumor = sphere=20 cells even under reduced-serum conditions that caused control cultured = spheres=20 to disaggregate, spread out over the culture surface, and lose = expression of=20 CD133 and Nestin (Figure 3E = and data=20 not shown).

To test if PHECs maintained also the = self-renewal=20 capacity of brain tumor cells, we disaggregated tertiary tumor spheres = that had=20 been cocultured for 2 weeks with either PHECs or control cells and = reseeded=20 suspensions of these cells in stem cell culture medium that was serum = free (Figure 3D). = Although=20 we were unable to recover any quaternary tumor spheres from cultures of = MB1=20 cells, both Daoy- and EP1-derived sphere cells that had been cocultured = with=20 PHECs were significantly more likely to generate quaternary tumor = spheres than=20 cells isolated from control cocultures (Figure 3E). = These=20 data identify striking similarities between normal neural stem cells and = self-renewing brain tumor cells and suggest that stem-like properties of = brain=20 CSCs are enhanced by endothelial-cell-secreted factors.

Endothelial Cells = Promote the=20 Propagation of Brain Tumors In Vivo

If endothelial-derived factors support the=20 self-renewal of brain CSCs, then we reasoned that endothelial cells = should=20 promote the propagation of brain tumors in vivo. To test this, we = injected 1 =C3=97=20 10⁶ unsorted GFP⁺ Daoy medulloblastoma cells = either alone=20 or in combination with red fluorescence dye (PKH26)-labeled PHECs, under = cranial=20 windows in the cerebral cortex of immunocompromised mice (n =E2=89=A5 3 = mice per cell=20 mix). Tumor cells (green) were readily distinguished from endothelial = cells=20 (red) using dual-color fluorescence microscopy (Figure 4A).<= /P>

Figure 4. Endothelial Cells Promote the Growth = of=20 Orthotopic Brain Tumor Xenografts

(A) The first column shows autofluorescence micrographs of mixed = suspensions=20 of GFP⁺ Daoy cells = (green) and=20 PHECs or PHAs (red) just prior to orthotopic injection. Numbers of PHECs = and=20 PHAs are in thousands. Subsequent columns show overlaid intravital red = and green=20 fluorescence photomicrographs of xenografts that were captured via the = cranial=20 window at the indicated time intervals following transplantation. = Numbers report=20 the level of green and red fluorescence in each xenograft at each time = point=20 (percentage of day 0 value).

(B) Coimmunofluorescence (Nestin and human-specific CD34) analysis of = the=20 orthotopic xenografts in (A) that were established by transplantation of = Daoy=20 cells alone (left) or Daoy cells plus 500 =C3=97 103 PHECs (right). Numbers at = the=20 bottom of each photomicrograph report the percentage of Nestin (green) = and human=20 CD34 (red)-expressing cells in each tumor (p < 0.005, exact Wilcoxon test).

(C) Map of the distribution of Nestin and human CD34-expressing cells = across=20 an entire section of a Daoy orthotopic xenograft that included 500 = =C3=97 103 PHECs. The map was = generated from a=20 composite review of high-power coimmunofluorescence = photomicrographs.

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Injections of GFP⁺ Daoy cells = alone=20 resulted in a steady increase in tumor fluorescence (green) that could = be=20 detected using intravital fluorescence microscopy via the cranial = window, and=20 that correlated closely with increasing tumor burden (Figure 4A = and Figure S1 = in the Supplemental = Data=20 available with this article online). Tumor fluorescence reached a peak = in these=20 mice at around 7 weeks postinjection, at which point the animals = succumbed to=20 their disease (mean peak fluorescence at 7 weeks; 526 =C2=B1 176 SD; n = =3D 6=20 independent animals; see Figure S1). = Remarkably, 1 =C3=97 10⁶ GFP⁺ Daoy medulloblastoma = cells that=20 were coinjected with 100,000 PHECs formed tumors much more rapidly, = reaching=20 peak tumor burden after only 4 weeks (Figure 4A). = This=20 acceleration of xenograft growth occurred in a cell-dose- and = cell-specific=20 manner: increasing numbers of coinjected PHECs produced a stepwise = increase in=20 tumor growth rate, but coinjection of PHAs did not affect xenograft = growth (Figure 4A). = Red=20 fluorescence declined steadily in PHEC/tumor cell xenografts, since the=20 half-life of PKH26 dye in vivo is =E2=88=BC2 weeks. Thus, the = increasing tumor=20 fluorescence in these xenografts resulted entirely from the expansion of = the=20 tumor cell population.

Xenografts that were established in the = presence of=20 PHECs contained up to 25 times more Nestin⁺/GFP⁺ = tumor=20 cells than did xenografts of Daoy cells alone (cells detected by = Nestin/GFP=20 coimmunofluorescence; Figure 4B). = PHECs=20 (differentiated from endogenous mouse endothelial cells using a human=20 CD34-specific antibody; Figure S2)=20 coclustered with Nestin⁺/GFP⁺ tumor cells=20 in xenografts (Figure 4C). = Importantly, PHECs remained separate from tumor capillaries and did not = increase=20 the MVD of xenografts relative to that observed in tumors formed by=20 GFP⁺ Daoy cells alone (data not shown). Together, these data = suggest=20 the hypothesis that endothelial cells contribute specific factors to the = tumor=20 microenvironment that expand the CSC pool and accelerate the initiation = and/or=20 growth of brain tumor xenografts.

To test more directly if endothelial cells = promote=20 tumor growth by supporting CSCs, and if freshly resected primary tumor = cells are=20 responsive to these signals, we repeated our cranial window transplant=20 experiments using 50,000 CD133⁺ or CD133^=E2=88=92 = cells that we=20 isolated from two primary medulloblastomas (MB4 and MB5). Primary = tumor=20 cells were obtained direct from the operating room, GFP-labeled by=20 lentiviral transduction, and then transplanted as described below after = less=20 than four passages in culture. Two out of every three primary = medulloblastomas=20 can be expected to generate tumors following orthotopic transplant of = 50,000=20 CD133⁺ tumor cells, but no significant tumor growth has been = reported=20 from injections of CD133^=E2=88=92 tumor cells (Singh et al., = 2004). As expected, CD133^=E2=88=92 primary tumor = cells grew only=20 transiently (MB4) or not at all (MB5) as transplants, even in the = presence of=20 coinjected PHECs (Figure 5). = In=20 contrast, CD133⁺ tumor cells formed fluorescent masses within = 7 days=20 of injection. The growth of CD133⁺ cell masses was = enhanced=20 dramatically by the presence of PHECs and resulted in the formation of a = rapidly=20 growing tumor in mice that were injected with CD133⁺ MB5 = cells. While=20 coinjection of PHECs also enhanced initially the growth of transplanted=20 CD133⁺ MB4 cells, the growth of these tumor cells was not = sustained.=20 Together, these data support the hypothesis that endothelial cells = contribute to=20 a niche microenvironment that promotes the initiation of brain tumors by = CSCs.

Figure 5. Endothelial Cells Promote the = Initiation of=20 Orthotopic Brain Tumor Xenografts by CD133+, but Not CD133=E2=88=92 Primary = Medulloblastoma Cells

Autofluorescence photomicrographs of the growth of orthotopic = xenografts of=20 50,000 GFP-labeled CD133⁺ or=20 CD133^=E2=88=92 cells = that we isolated=20 from two primary medulloblastomas (MB4 and MB5) and transplanted = orthotopically=20 under cranial windows into the cerebral cortex of nude mice. Tumor cells = were=20 transplanted alone or with 50,000 red fluorescence-labeled PHECs. The = first=20 column shows autofluorescence micrographs of cells immediately prior to=20 transplantation. Subsequent columns show overlaid intravital red and = green=20 fluorescence microscopy images of tumors captured via the cranial window = at the=20 indicated time intervals following implantation. Numbers report the = level of=20 green and red fluorescence in each xenograft at each time point = (percentage of=20 day 0 value).

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Depletion of Brain = Tumor Blood=20 Vessels Eradicates Self-Renewing Tumor Cells

Our finding that endothelial-derived factors = enhance=20 the initiation of brain tumors by CD133⁺ tumor cells suggests = that=20 antiangiogenic drugs could inhibit brain tumor growth, at least in part, = by=20 disrupting a CSC vascular niche. To test this, we investigated the = impact of=20 antiangiogenic therapies on the numbers of self-renewing tumor cells in, = and=20 growth of, medulloblastoma and glioma orthotopic xenografts.

Angiogenic signaling in tumors is often = initiated by=20 oncogenic cell signal pathways (Jain et al., 2006). Therefore, we first upregulated angiogenic signaling = in=20 GFP⁺ Daoy cells by transfection with the ERBB2 = oncogene=20 (Daoy^ERBB2). ERBB2 is overexpressed in aggressive forms of=20 medulloblastoma (Gajjar et al., 2004) and elicits a cell signal that upregulates vascular = endothelial=20 growth factor (VEGF) (Saucier et al., 2004). Daoy^ERBB2 cells secreted 60-fold more VEGF = than did=20 vector control (Daoy^V) cells, and treatment of these cells = with=20 Erlotinib=E2=80=94a selective inhibitor of the EGFR and ERBB2 tyrosine = kinases (Akita et=20 al., 2003)=E2=80=94or an anti-ERBB2 siRNA downregulated VEGF = expression (see Figures S3A and = S3B). We=20 then established orthotopic xenografts of 1 =C3=97 10⁶ = GFP⁺=20 Daoy^ERBB2 or Daoy^V cells under cranial windows in = the=20 cerebral cortex of nude mice. To target both the = =E2=80=9Cupstream=E2=80=9D and =E2=80=9Cdownstream=E2=80=9D=20 components of angiogenic signaling in this model, we treated = tumor-bearing=20 mice with either Erlotinib (50 mg/kg/2=C3=97 day for 5 days), which = abolishes ERBB2=20 signaling in vivo (Hernan et al., 2003); a single dose of 10 mg/kg of the anti-VEGF monoclonal = antibody=20 Bevacizumab; or vehicle-only control (n =3D 10 mice bearing each = xenograft for=20 each treatment). Tumors were then excised and analyzed as outlined in Figure 6A.

Figure 6. Antiangiogenic Therapy Depletes the = Tumor=20 Vasculature, Ablates Self-Renewing Tumor Cells, and Inhibits the Growth = of=20 Daoy^ERBB2 Orthotopic=20 Xenografts

(A) Cartoon depicting the experimental approach adopted to determine = whether=20 depletion of blood vessels from brain tumors ablates the CSC = fraction.

(B=E2=80=93G) Graphs (all error bars =3D mean =C2=B1 SE) report VEGF = expression levels (B),=20 microvessel density (C), self-renewal capacity (serial tumor = sphere-forming=20 assay) (D), growth (tumor fluorescence) (E), apoptosis (TUNEL) (F), and=20 proliferation (Ki67 labeling) (G) of vehicle (=E2=80=9CV=E2=80=9D)-, = Erlotinib (=E2=80=9CE=E2=80=9D)-, or=20 Bevacizumab (=E2=80=9CB=E2=80=9D)-treated DaoyERBB2 and DaoyV orthotopic xenografts = (p < 0.05, p < 0.005, p < 0.0005, exact Wilcoxon test).

(H) Coimmunofluorescence analysis of tumor spheres formed ex vivo by = cells=20 that were derived from vehicle-treated DaoyERBB2 and DaoyV xenografts.

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As expected, Daoy^ERBB2 xenografts = expressed=20 much higher levels of VEGF, contained a significantly higher MVD = (compare=20 vehicle-only treated tumors in Figure 6B = and 6C),=20 and grew more rapidly following transplantation (Figure S4) = than did=20 Daoy^V tumors. Also, single-cell suspensions of excised=20 Daoy^ERBB2 tumors generated five times more self-renewing=20 CD133⁺/Nestin⁺ tumor spheres than cells from=20 Daoy^V tumors (compare vehicle-only treated tumors in Figure 6D). = All tumor=20 sphere cells coexpressed GFP, confirming that they were derived from=20 transplanted tumor cells and not normal mouse cells (Figure 6H), = and=20 displayed evidence of multipotency when cultured under conditions that = promote=20 differentiation (Figure S5). = Importantly, ERBB2 signaling in Daoy cells does not impact directly cell = proliferation, apoptosis, or self-renewal (see Figures S6A to = S6D). Thus,=20 expansion of the tumor vasculature in Daoy^ERBB2 relative to=20 Daoy^V tumors was associated with an increased number of=20 self-renewing, multipotent cancer cells.

Both Erlotinib and Bevacizumab treatments = induced a=20 profound antiangiogenic response in Daoy^ERBB2 xenografts that = was=20 associated with significant tumor growth arrest (Figure 6B, = 6C, and=20 6E). Interestingly, despite these marked antiangiogenic and growth = inhibitory=20 effects, neither drug altered the level of apoptosis or necrosis in=20 Daoy^ERBB2 xenografts, and only Erlotinib decreased slightly = cell=20 proliferation in these tumors relative to vehicle treatment (Figure 6F = and 6G and=20 data not shown). However, both drugs resulted in a complete loss of=20 self-renewing GFP⁺/Nestin⁺/CD133⁺ tumor = cells=20 from single-cell suspensions of treated Daoy^ERBB2 xenografts = (Figure 6D). = In stark=20 contrast, since the ERBB2-VEGF axis in Daoy^V tumors is much = less=20 active than in Daoy^ERBB2 xenografts, neither Erlotinib nor=20 Bevacizumab affected the MVD in Daoy^V tumors (Figure 6C). = In=20 agreement with our hypothesis, these treatments also failed to alter the = number=20 of self-renewing GFP⁺/Nestin⁺/CD133⁺ = cells in=20 Daoy^V tumors (Figure 6D). = Although=20 both Erlotinib and Bevacizumab therapy produced a moderate increase in=20 Daoy^V tumor cell apoptosis that presumably resulted from = blockade of=20 endogenous EGFR/ERBB2 and VEGF signaling, this did not impact tumor = growth (Figure 6E = and 6F).=20 Importantly, neither Erlotinib nor Bevacizumab affect directly the = self-renewal,=20 proliferation, or apoptosis of Daoy cells in culture (see Figure S6E = and data=20 not shown). Together, these data provide strong support for the = hypothesis that=20 brain tumor growth depends critically upon the presence of an intact CSC = vascular niche and identifies this niche as a potential target for = treatments of=20 brain tumors. As a first step to determine if this therapeutic approach = might=20 have clinical benefit, we treated Daoy^ERBB2 and = Daoy^V=20 tumor-bearing mice for 2 weeks with 50 mg/kg/2=C3=97 day of Erlotinib (n = =3D 8 mice=20 with each tumor per treatment). Erlotinib prolonged significantly the = survival=20 of mice bearing Daoy^ERBB2 tumors (median survival of=20 Erlotinib-treated mice =3D 66 =C2=B1 3.4 days versus 45 =C2=B1 2.9 days = in vehicle-treated=20 mice; exact log rank p < 0.05), but not those harboring = Daoy^V=20 xenografts (median survival of Erlotinib-treated mice =3D 53 =C2=B1 2.8 = days versus 54=20 =C2=B1 3.1 days in vehicle-treated mice; log rank p =3D 0.73).

Finally, since ongoing clinical trials of = Bevacizumab=20 suggest that this drug has promising therapeutic activity against = glioblastoma=20 (Reardon et al., 2006), we investigated if Bevacizumab might deplete = self-renewing=20 cancer cells from this brain tumor. To do this, we treated with = Bevacizumab or=20 vehicle mice bearing orthotopic xenografts of U87 glioma cells that = express the=20 Luciferase reporter gene (U87G^Luc). Transplantation of 1 = =C3=97=20 10⁶ U87^Luc cells into the brains of = immunocompromised mice=20 resulted in the formation of brain tumors that could be detected readily = using=20 bioluminescence (Figure 7A). = As we=20 observed in Daoy^ERBB2 medulloblastoma xenografts, a single = dose of 10=20 mg/kg Bevacizumab, but not vehicle, reduced markedly the MVD and = growth of=20 U87^Luc tumors (n =3D 5 mice per treatment group; Figure 7A=E2= =80=937C).=20 Furthermore, Bevacizumab decreased profoundly the numbers of = vessel-associated=20 Nestin⁺ tumor cells in U87^Luc xenografts and the = number of=20 self-renewing GFP⁺/Nestin⁺/CD133⁺ tumor = cells=20 that could be recovered from these tumors, without impacting tumor cell=20 proliferation, apoptosis, or necrosis (Figure 7A = and 7D=E2=80=937G=20 and data not shown). Interestingly, residual Nestin⁺ cells in = Bevacizumab-treated glioma xenografts were not distributed randomly = through=20 tumors but were associated with persisting tumor vessels (Figure 7A). = Thus, we=20 propose that antiangiogenic drugs arrest brain tumor growth, at least in = part,=20 by disrupting a vascular niche microenvironment that is critical for the = maintenance of CSCs.

Figure 7. Bevacizumab Therapy Depletes the = Tumor=20 Vasculature, Ablates Self-Renewing Tumor Cells, and Inhibits the Growth = of=20 U87^Luc Glioma = Orthotopic=20 Xenografts

(A) Coimmunofluorescence analysis of Nestin and CD34 expression in = sections=20 of glioma xenografts 1 week following treatment with Bevacizumab or = vehicle.=20 Arrows point to Nestin⁺ cells.=20 Bioluminescence images of these tumors generated prior to excision are = shown=20 above.

(B=E2=80=93G) Graphs (all error bars =3D mean =C2=B1 SE) report tumor = growth=20 (bioluminescence) (B), microvessel density (C), percentage Nestin+ cells (D), self-renewal = capacity=20 (serial tumor sphere-forming assay) (E), apoptosis (TUNEL) (F), and=20 proliferation (Ki67 labeling) (G) of vehicle (=E2=80=9CV=E2=80=9D)- and = Bevacizumab=20 (=E2=80=9CB=E2=80=9D)-treated xenografts (p < 0.05, p < 0.005, exact Wilcoxon test).

View larger version: [In=20 this window] [In new window]

Discussion

Evidence suggests that normal neural stem = cells exist=20 in vascular niches, into which endothelial cells secrete factors that = regulate=20 neural stem cell function (Palmer et al., 2000, Ramirez-Castillejo et al., 2006, Shen et al., 2004). Here, we provide several lines of evidence that the = brain tumor=20 microvasculature forms a niche that is critical for the maintenance of=20 CSCs.

Since niches control stem cell function, it = may seem=20 counterintuitive that CSCs would be located within these regulatory=20 microenvironments. However, our data support the hypothesis that = vascular niches=20 in brain tumors are abnormal and contribute directly to the generation = of CSCs=20 and tumor growth. In the normal brain, neural stem cell niches are = restricted to=20 the hippocampus and subventricular zones where rates of cell = proliferation are=20 quite low (Sanai et al., 2005). In contrast, we found that a significant proportion of = vessel-associated Nestin⁺ tumor cells are proliferating, and = that=20 these cells are distributed throughout tumors, from all regions of the = cerebrum=20 and cerebellum. We show further that PHECs maintain self-renewing brain = tumor=20 cells in culture and promote the initiation and growth of orthotopic = brain tumor=20 xenografts. Increasing the number of capillaries in orthotopic brain = tumors also=20 expanded dramatically the number of self-renewing and multipotent cells=20 contained in these tumors. Therefore, we propose that the tumor = microvasculature=20 generates specific niche microenvironments that promote the formation = and/or=20 maintenance of brain CSCs. Interestingly, atypical ectopic perivascular = niches=20 were recently described around perivascular areas of the inflamed CNS = (Martino=20 et al., 2006). These entities behave as anatomically atypical, = although highly=20 specialized, ectopic niches that regulate long-term survival and = behavior of=20 transplanted neural stem cells. Thus, atypical and ectopic stem cell = niches=20 might play central roles in a number of nervous system diseases, = including brain=20 tumors.

If the recruitment of aberrant vascular = niches is an=20 important component in the progression of brain tumors, then this might = explain=20 why the most aggressive brain tumors are highly angiogenic (Folkerth, = 2004, Phillips et al., 2006, Plate et al., 1992). Intriguingly, protein ligands that are found within = the neural=20 stem cell niche have been shown to regulate both stem cell self-renewal = and=20 angiogenesis, suggesting further that these two processes are linked. = For=20 example, KIT ligand (also known as stem cell factor), which is a potent=20 glioma-derived proangiogenic factor (Sun et al., 2006), also promotes the migration, survival, and = proliferation of=20 neural progenitor cells (Erlandsson et al., 2004, Jin et al., 2002), and PEDF, which maintains neural stem cell = self-renewal, is a=20 potent regulator of angiogenesis (Pumiglia et al., 2006, Ramirez-Castillejo et al., 2006). Furthermore, very recent studies of orthotopic = glioblastoma=20 xenografts suggest that CSCs of this brain tumor secrete angiogenic = factors that=20 promote the recruitment and formation of tumor blood vessels (Bao et = al., 2006). It is noteworthy that VEGFR-1⁺ bone marrow = progenitor=20 cells were shown recently to form niches at the sites of lung and = melanoma tumor=20 metastasis (Kaplan et al., 2005), and that bone marrow endothelial niches are required = for the=20 homing and retention of normal hematopoietic and leukemic stem cells = (Kiel et=20 al., 2005, Sipkins et al., 2005). In the brain, the formation of multiple vascular CSC = niches,=20 each with the capacity to generate numerous tumor cells, might = facilitate=20 significantly brain tumor growth and invasion.

As well as regulating stem cell = proliferation and=20 cell-fate decisions, niches also play a protective role, shielding stem = cells=20 from environmental insults. Thus, vascular niches might protect brain = CSCs from=20 chemo- and radiotherapies, enabling these cells to reform a tumor mass = following=20 an initial clinical response. Evidence indicates that endothelial cells = can=20 protect stem cells and tumor cells from radiation damage (Garcia-Barros = et al.,=20 2003, Paris et al., 2001). For example, solid tumor xenografts grown in mice with = radiation-resistant endothelial cells are much less sensitive to = radiation=20 damage than tumors grown in wild-type mice (Garcia-Barros et al., 2003). Our data suggest a mechanism, at least in brain = tumors, by which=20 endothelial cells might promote tumor radioresistance. Further, studies = of the=20 hematopoietic stem cell niche suggest that niche microenvironments can = promote=20 cell survival signals in CSCs, enabling them to resist chemotherapies = (De Toni=20 et al., 2006, Dick et al., 2005).

If the notion that niches protect CSCs = proves correct,=20 then targeting these microenvironments could prove a highly effective = treatment=20 of cancer. We found that antiangiogenic therapies including Bevacizumab = deplete=20 tumor blood vessels and self-renewing cancer cells from orthotopic = models of=20 medulloblastoma and glioma, resulting in tumor growth arrest. Notably, = VEGF has=20 been shown to enhance the survival of neural stem cells (Wada et al., = 2006). Thus VEGF-specific inhibitors like Bevacizumab might = impact=20 brain tumor growth by targeting both a vascular niche and the associated = CSCs.=20 Exciting early data from clinical trials of Bevacizumab combined with = the=20 chemotherapeutic drug CPT-11 suggest that this combination is one the = most=20 effective treatments of glioblastoma identified to date (Reardon et al., = 2006). In light of our data, it is tempting to speculate that = the=20 activity of this drug combination results from targeting CSCs, as well = as the=20 disease bulk. This important mechanism of drug action should be studied = further=20 in ongoing preclinical and clinical trials of antiangiogenic drugs in = brain=20 tumors.

Experimental = Procedures

Tumor Samples and = Cell=20 Lines

Tumor samples were obtained under protocol = XPD01-092=20 (St. Jude Children's Research Hospital Institutional Review Board). = Tumors were=20 immediately disaggregated into single-cell suspensions as described in = detail=20 elsewhere (Taylor et al., 2005). Primary tumor cell cultures were maintained in = Neurobasal medium=20 (Invitrogen) containing 2 mM L-glutamine, N2 supplement (Invitrogen), = B27=20 supplement (Invitrogen), 20 ng/ml hrEGF (Invitrogen), 20 ng/ml hrbFGF=20 (Invitrogen), and 50 =CE=BCg/ml BSA. All experiments were conducted at = cell passage=20 <4. Previous cytogenetic studies showed that all cultured primary = tumor cells=20 were cancer cells (Taylor et al., 2005). PHECs, U87, and Daoy cells were obtained from the ATCC = (Manassas, VA). PHAs were from Cambrex, MD. PHECs were cultured in = endothelial=20 cell growth medium (EGM) containing 2% fetal bovine serum (GIBCO, USA). = PHAs=20 were cultured in astrocyte growth medium that contains 2% serum (AGM, = Cambrex).=20 Daoy cells were transfected with the complete coding region of = ERBB2=20 (Daoy^ERBB2) or vector control (Daoy^V), and U87 = glioma=20 cells with the luciferase reporter, as described (Hernan et al., 2003). GFP was expressed in Daoy^V and = Daoy^ERBB2=20 by transduction with MSCV-IRES-GFP retrovirus. Primary tumor cells were = labeled=20 green by GFP-lentiviral transduction. GFP⁺ tumor cells were = isolated=20 by fluorescence-activated cell sorting (FACS). Daoy and U87 cells were=20 maintained under standard culture conditions in DMEM containing 10% FCS = unless=20 otherwise specified.

Tumor Sphere = Initiation=20 Assays

Single-cell suspensions were made in = Neurobasal medium=20 in 96-well plates (diluted as 10,000 to 1 cells/well). The number of = tumor=20 spheres that formed subsequently per well was quantified after 14 days. = Tumor=20 spheres were then disaggregated and reseeded to evaluate self-renewal by = formation of secondary tumor spheres. Where indicated, this process was = repeated=20 to form tertiary and quaternary tumor spheres.

Coculture = Studies

For transwell coculture studies, tumor = spheres were=20 added in 500 =CE=BCl of culture medium to each well of a 24-well = coculture plate=20 (BD Biosciences). Transwell inserts containing 0.4 =CE=BCm diameter = membrane pores=20 that allow the passage of diffusible molecules, but not cells, were then = placed=20 into each well and seeded with 1 =C3=97 10⁴ PHECs or control = cells (see=20 text). Plates were then incubated under standard culture conditions in = medium=20 containing 2% fetal calf serum for 120 hr, and the number of remaining = tumor=20 spheres was counted using phase-contrast microscopy. The self-renewal of = tumor=20 sphere cells cocultured with PHECs or control cells was determined using = the=20 tumor sphere initiation assay described above. The differentiation of = tumor=20 sphere cells in cocultures was determined by seeding tumor spheres in = culture=20 wells on coated coverslips (poly-D-lysine/laminin) (BD Biosciences); = tumor cells=20 were then fixed with 4% paraformaldehyde and analyzed by = immunofluorescence=20 using previously published antibodies and techniques (Taylor et al., = 2005). Neurosphere size was measured by analyzing =C3=97200 = magnification=20 images of DAPI-stained tumor spheres and the ImageJ analysis tool (http://rsb.info.nih.g= ov/ij/).

For Matrigel coculture studies, = GFP⁺=20 Daoy^V or Daoy^ERBB2 cells or primary tumor cells = prelabeled=20 with PKH67 (Sigma) were sorted using CD133-antibody-coated magnetic = beads (100=20 =CE=BCl/10⁸ cells; Miltenyi Biotec) as described (Taylor et = al., 2005). Purity of CD133 populations was confirmed by CD133=20 immunofluorescence. Sorted tumor cells (6000 cells/ml) were cultured = =C2=B1 PHECs or=20 PHAs (10,000 cells/ml) in Matrigel-coated (35 =CE=BCl; BD BioScience) = 8-well culture=20 slides (BD BioScience) overnight at 37=C2=B0C. PHECs or PHAs were imaged = by=20 phase-contrast microscopy, and sorted cells expressing GFP or stained = with PKH67=20 were imaged using fluorescence microscopy.

Generation and = Treatment of=20 Orthotopic Transplants

Female CD-1 nu/nu mice (Charles River) aged = 6=E2=80=938 weeks=20 were anesthetized and placed into stereotactic apparatus equipped with a = Z axis=20 (Kopf instruments). A small portion of the scalp was removed, a window=20 (approximately 10 mm =C3=97 5 mm) was made in the skull using a dental = drill, and the=20 pial layer was removed. Tumor cells (1 =C3=97 10⁶ in = 5 =CE=BCl Matrigel)=20 were implanted with or without PHAs or PHECs into the cerebral surface = at a=20 depth of 0.5=E2=80=932 mm using a 25 =CE=BCl Hamilton syringe with an = unbeveled 30G needle.=20 The wound was then covered with a sterile glass window fixed in place = using=20 tissue adhesive. Immediately following wound closure, implanted animals = were=20 transferred to the intravital imaging system (Nikon, USA) and imaged to = obtain a=20 baseline measurement, and animals were reanesthetized as required for = subsequent=20 imaging. Data analysis was performed using MetaMorph Imaging software = (Universal=20 Imaging, Downington, PA). Measurements were restricted to a fixed region = within=20 each image throughout the course of the study and are presented as = maximum=20 fluorescence (Fmax) normalized to minimum = fluorescence=20 (Fmin), which compensated for potential = interday=20 variation in lamp intensity. U87^Luc xenografts were prepared = in a=20 similar manner through small cranial burr holes without cranial windows. = Tumor=20 burden was measured in U87^Luc tumor-bearing mice by = bioluminescence=20 using a Series IVIS Imaging System (Xenogen Corporation, Alameda, CA). = Mice=20 received an intraperitoneal injection of 15 mg/ml D-luciferin (Xenogen) = in=20 sterile PBS and were imaged 5 min later. All animals were imaged at a = range of=20 25 cm for 10 s. Acquired images were analyzed with the use of Living = Image=20 Software version 2.50 (Xenogen). Bioluminescence measurements were = recorded as=20 photons per second.

Erlotinib (OSI Pharmaceuticals) was = administered to=20 tumor-bearing mice per os (p.o.) in a vehicle of 10% DMSO, 10% Cremephor = in a 1% CMC solution. Animals were treated with Erlotinib (50 = mg/kg/twice=20 daily) or vehicle for 5 days. Treatment was repeated where indicated = following a=20 2 day break. Bevacizumab (Genentech Inc.) was administered by tail-vein=20 injection at a dose of 10 mg/kg. All animal procedures were conducted in = accordance with all appropriate regulatory standards under protocol #356 = (St.=20 Jude Children's Research Hospital Animal Care and Use = Committee).

Histologic, FISH, = and Gene=20 Expression Analysis

Dual-color FISH, immunofluorescence,=20 immunohistochemical, and TUNEL analyses of formalin-fixed, = paraffin-embedded=20 tumor samples, xenografts, or cultured tumor cells were performed using=20 immunoreagents, FISH probes, and methods as described previously (Taylor = et al.,=20 2005, Thompson et al., 2006). Nestin-specific antibodies included a monoclonal = antibody=20 (Chemicon cat. no. MAB5326) that recognizes the human but not the mouse = protein=20 and a polyclonal antibody (Abcam) that recognizes human, mouse, and rat = Nestin.=20 The distance between Nestin⁺ cells and CD34 endothelial cells = was=20 measured in dual-immunofluorescence-stained images of brain tumor = sections using=20 the line-measuring tool in ImageJ. In order to remove observer bias and = to allow=20 analysis of large numbers of tumor sections, immunohistochemical = staining of=20 xenografts, including assessment of MVD (CD34 staining), was scored = blind to the=20 xenograft and treatment using ImageJ software analysis. Briefly, images = were=20 processed at the same magnification (=C3=97200) by removal of background = hematoxylin=20 staining followed by monochromization. Monochrome images (giving binary = data:=20 black areas of immunohistochemical stain and white nonstaining areas) = were=20 analyzed using ImageJ software, which calculated the absolute values for = each=20 channel, resulting in determination of percentage of immunopositivity = per unit=20 area (constant =C3=97200 field). Two-photon microscopy was performed = using an Ultima=20 imaging system (Prairie Technologies, Middletown WI), a Ti:sapphire = Chameleon=20 Ultra XR femtosecond-pulsed laser (920 nm) (Coherent, Santa Clara, CA), = and a 63=20 =C3=97 0.9 NA water-immersion IR objective (Olympus, Center Valley, PA). = Stacks of=20 images were reconstructed in 3D using Imaris software (Bitplane, Saint = Paul,=20 MN). VEGF mRNA expression levels were determined in cultured cells and=20 xenografts by reverse-transcriptase real-time polymerase chain reaction = analysis=20 as described previously (Thompson et al., 2006).

Acknowledgments

This work was supported by the Sontag = Foundation=20 (R.J.G.), NCI grant CA096832 (R.J.G.), the V-Foundation for Cancer = Research=20 (R.J.G.), and the American Lebanese Syrian Associated Charities (ALSAC). = We=20 thank the Hartwell Center for Bioinformatics and Biotechnology and the = Animal=20 Resource Center at St. Jude Children's Research = Hospital.

Supplemental Data

Document S1. Six Supplemental Figures

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Article History

Received: June 22, 2006
Revised: October 3, 2006
Accepted: = November 28,=20 2006
Published: January 15, 2007

Tumor=20 stem cells derived from glioblastomas cultur...
Radial=20 glia cells are candidate stem cells of epen...
Molecular=20 subclasses of high-grade glioma predict ...

More=20 related articles...

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Table of=20 Contents
Summary=20
PDF=20 (2447K)
Suppleme= ntal=20 Data

Search Medline for

= Christopher=20 Calabrese
Richard=20 J. Gilbertson

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Table of=20 Contents
Summary=20
PDF=20 (2447K)
Suppleme= ntal=20 Data

Search Medline for

= Christopher=20 Calabrese
Richard=20 J. Gilbertson

Tumor=20 stem cells derived from glioblastomas cultur...
Radial=20 glia cells are candidate stem cells of epen...
Molecular=20 subclasses of high-grade glioma predict ...

More=20 related articles...

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Article

A = Perivascular=20 Niche for Brain Tumor Stem Cells

Summary

SIGNIFICANCE

Introduction

Results

Nestin+ Brain Tumor Cells = Associate with=20 the Tumor Vasculature

CD133+ Brain Tumor Cells = Interact=20 Physically with Endothelial Cells in Culture

Endothelial Cells = Maintain=20 Self-Renewing and Undifferentiated Brain Tumor Cells

Endothelial Cells = Promote the=20 Propagation of Brain Tumors In Vivo

Depletion of Brain = Tumor Blood=20 Vessels Eradicates Self-Renewing Tumor Cells

Discussion

Experimental = Procedures

Tumor Samples and = Cell=20 Lines

Tumor Sphere = Initiation=20 Assays

Coculture = Studies

Generation and = Treatment of=20 Orthotopic Transplants

Histologic, FISH, = and Gene=20 Expression Analysis

Acknowledgments

Supplemental Data

References

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