From: Subject: Interleukin-23-Expressing Bone Marrow-Derived Neural Stem-Like Cells Exhibit Antitumor Activity against Intracranial Glioma -- Yuan et al. 66 (5): 2630 -- Cancer Research Date: Wed, 16 Jan 2008 18:50:50 +0100 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_0000_01C85870.B701F780" X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3198 This is a multi-part message in MIME format. ------=_NextPart_000_0000_01C85870.B701F780 Content-Type: text/html; charset="Windows-1252" Content-Transfer-Encoding: quoted-printable Content-Location: http://cancerres.aacrjournals.org/cgi/content/full/66/5/2630 Interleukin-23-Expressing Bone Marrow-Derived Neural = Stem-Like Cells Exhibit Antitumor Activity against Intracranial Glioma = -- Yuan et al. 66 (5): 2630 -- Cancer Research 3D"Cancer 3D"Ubiquitin 3D"
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[Cancer Research 66, 2630-2638, March 1, 2006]
=A9 = 2006 American = Association=20 for Cancer Research

Cell, Tumor, and Stem Cell = Biology

Interleukin-23=96Expressing Bone Marrow=96Derived Neural Stem-Like = Cells Exhibit=20 Antitumor Activity against Intracranial Glioma=20

Xiangpeng Yuan1, = Jinwei=20 Hu1, Maria L. Belladonna2, = Keith=20 L. Black1 and John S. Yu1 =

1 Maxine Dunitz Neurosurgical Institute,=20 Cedars-Sinai Medical Center, Los Angeles, California and 2 = Department=20 of Experimental Medicine, University of Perugia, Perugia, Italy

Requests for reprints: John S. Yu, Maxine = Dunitz=20 Neurosurgical Institute, Cedars-Sinai Medical Center, Suite 800 East, = 8631 W.=20 3rd Street, Los Angeles, CA 90048. Phone: 310-423-0845; = E-mail: yuj{at}cshs.org .


3D"   =20 Abstract
Top
3D"Abstract
Introduction
Materials and Methods
Results
Discussion
References
 
N= eural=20 progenitor-like cells have been isolated from bone marrow and = the=20 cells have the ability of tracking intracranial tumor. = However, the=20 capacity of the cells to deliver molecules for activating = immune=20 response against intracranial tumor and the identity of = cellular and=20 molecular factors that are involved in such immune responses = have yet=20 to be elucidated. Here, we isolated neural stem-like cells = from the=20 bone marrow of adult mice. The isolated cells were capable of = producing progenies of three lineages, neurons, astrocytes, = and=20 oligodendrocytes, in vitro and tracking glioma in = vivo.=20 By genetically manipulating bone marrow=96derived neural = stem-like=20 cells (BM-NSC) to express a recently discovered cytokine, = interleukin=20 (IL)-23, the cells showed protective effects in intracranial=20 tumor-bearing C57BL/6 mice. Depletion of subpopulation = lymphocytes=20 showed that CD8+ T cells were critical for the = antitumor=20 immunity of IL-23=96expressing BM-NSCs and that = CD4+ T cells=20 and natural killer (NK) cells participated in the activity.=20 Furthermore, the IL-23=96expressing BM-NSC-treated survivors = were=20 resistant to the same tumor rechallenge associated with = enhanced=20 IFN-3D{gamma}, but not IL-17, expression in the brain tissue. = Taken=20 together, these data suggest that IL-23=96expressing BM-NSCs = can=20 effectively induce antitumor immunity against intracranial = gliomas.=20 CD8+ T cells are critical for such antitumor = activity; in=20 addition, CD4+ T cells and NK cells are also = involved.=20 (Cancer Res 2006; 66(5): 2630-8)


3D"   =20 Introduction
Top
Abstract
3D"Introduction
Materials and Methods
Results
Discussion
References
 
M= alignant=20 gliomas represent a significant cause of tumor-related = mortality and=20 continue to be associated with poor prognosis despite = extensive=20 surgical excision and adjuvant radiotherapy and chemotherapy = (= 1, = 2).=20 Their resistance to treatment is related to the tumor cell's=20 exceptional migratory nature and ability to grow extensively = into=20 normal neural tissue away from the main tumor mass. These = cells=20 preclude complete surgical resection and eventually serve as = a source=20 for recurrent tumor growth. It is this characteristic that = also makes=20 it difficult for otherwise promising gene therapeutic = strategies and=20 other local interventions to efficiently access the tumor = cells (= 3).=20 Recent evidence indicated a new strategy for targeting tumor = cells=20 within the central nervous system by using neural stem cells = to=20 deliver therapeutic gene and/or their products efficiently to = treat=20 brain tumors (= 4=96= 6).=20

Bone marrow represents an attractive source for generating = neural=20 stem cells, which have an advantage over fetal neural stem = cells,=20 or allogeneic cell lines as a cellular vehicle for brain = tumor=20 therapy because of their autologous characteristic. The bone=20 marrow=96derived neural stem cells obviate the accessibility = and=20 ethical problems associated with fetal neural stem cells as = well as=20 potential immunologic incompatibility due to the requirement = for=20 allogeneic application (= 7=9613).=20 In the present study, we sought to isolate neural stem cells = from=20 murine bone marrow as a cellular vehicle for glioma therapy.=20 Using bone marrow=96derived neural stem-like cells (BM-NSC) = as=20 a vehicle, we studied the antiglioma function of a newly = discovered=20 cytokine, interleukin (IL)-23, which consists of a = heterodimer=20 of IL-12 p40 subunit and a novel protein, p19. It has been = shown=20 that IL-23 acts on memory T cells and dendritic cells = directly=20 to promote IL-12 and IFN-3D{gamma}=20 = production=20 in vitro (14,=20 15).=20 Recently, IL-23=96expressing tumor cell showed antitumor and=20 antimetastatic function (16).=20 We showed that neural stem-like cells can be generated from = adult=20 bone marrow and the generated BM-NSCs can track migratory = glioma=20 cells and deliver IL-23 in situ. IL-23=96expressing = BM-NSCs when=20 injected into brain are able to induce tumor-specific = antitumor=20 activity and protective immunity against intracranial = glioma.=20


3D"   =20 Materials and Methods
Top
Abstract
Introduction
3D"Materials and = Methods
Results
Discussion
References
 
<= STRONG>Cell=20 Cultures and Cell Lines
Whole bone marrow was harvested from = the=20 femurs of adult C57BL/6 mice as described previously (11).=20 Cells were plated on poly-D-lysine=96coated=20 24-well plates (BD Biosciences, San Jose, CA) and cultured in = serum-free DMEM/F-12 (Invitrogen, Gaithersburg, MD) = supplemented=20 with B27 (Invitrogen), 20 ng/mL basic fibroblast growth = factor=20 (Peprotech, Rocky Hill, NJ), and 20 ng/mL epidermal growth = factor=20 (Peprotech) along with antibiotics. The cells were plated at = a=20 density of 1 x 106 per = well. Fetal=20 neural stem cells were harvested from the frontoparietal = regions of=20 day 15 fetal C57BL/6 mice as described previously (17).=20 The cells were grown in culture under the same conditions as=20 mentioned above. Spheres, formed in both bone marrow cell and = fetal=20 neural stem cell culture, were processed for = immunocytochemistry=20 staining. Bone marrow=96derived spheres were subjected to = cell=20 differentiation by plating onto laminin-coated 24-well plates = in=20 growth medium devoid of growth factors containing retinoic = acid (1=20 =B5mol/L, Sigma, St. Louis, MO) and dibutyryl cyclic AMP (1 = mmol/L,=20 Sigma). Medium was replenished every 3 days. Cells were=20 differentiated for 7 to 14 days and processed for = immunocytochemistry=20 staining.

NIH-3T3 cells (murine fibroblasts) and GL26 cells (murine = glioma)=20 were grown in DMEM and RPMI 1640, respectively, supplemented=20 with 10% fetal bovine serum, 2 mmol/L L-glutamine,=20 and antibiotics (all the reagents from Invitrogen). Green = fluorescent=20 protein (GFP)=96expressing GL26 cells were derived from = stable=20 transfection of GL26 cells with pEGFP-N1 vector (BD = Biosciences) by=20 calcium phosphate precipitation method as described = previously (18).=20 The stable transfectants were selected with geneticin.

Adenoviral Vectors Construction and In vitro=20 Infection
The viral vector bearing the gene for murine = IL-"23=20 (AdIL-23) was constructed by subcloning the single-chain = IL-23 cDNA=20 (15)=20 into a shuttle vector pMH5 (Microbix Biosystems, Inc., = Toronto,=20 Ontario, Canada) downstream of the mCMV promoter. The = resultant=20 shuttle vector was cotransfected with pBHGE3 (Microbix = Biosystems)=20 into 293 cells (Microbix Biosystems). Recombinants were = subsequently=20 subjected to three rounds of plaque purifications. The = purified=20 viral vector was clarified by Southern hybridization and=20 immunocytochemistry to confirm the bearing of IL-23 = expression=20 cassette within the vector genome and the expression of IL-23 = from=20 the vector transduced cells, respectively.3=20 The viral vectors bearing the gene for LacZ (AdLacZ) or a = 2.4-kb=20 noncoding stuffer DNA (AdEmpty) were constructed the same way = as=20 AdIL-23. The construction of IL-12-bearing vector (AdIL-12) = was=20 described previously (= 6).=20 For in vitro gene transduction, BM-NSCs, fetal neural = stem=20 cells, and NIH-3T3 cells were infected with 100 = multiplicities of=20 infection of AdIL-23, AdEmpty, AdLacZ, and AdIL-12 as = indicated.=20 Twenty-four hours after infection, the cells were washed = thrice with=20 PBS to ensure that final intracranial injections or in = vitro=20 analysis were devoid of free viral particles. NIH-3T3 cells = were=20 treated by either mitomycin C (25 mg/mL, Sigma) or = irradiation=20 (5,000 rads) to induce growth arrest.

Animal Studies In vivo
Intracranial = tumor=20 implantation. C57BL/6 wild-type mice, athymic nude mice, = and CD4=20 T-cell knockout mice (6-8 weeks old, all from The Jackson = Laboratory,=20 Bar Harbor, ME) were anesthetized with i.p. ketamine and = medetomidine=20 and stereotactically implanted with 1 x 104 GL26 cells or = GFP-expressing GL26=20 glioma cells in 2.5 =B5L of 1.2% methylcellulose/PBS in the = right=20 striatum as described previously (= 6).=20

BM-NSC migration and transgene delivery. To determine = whether=20 BM-NSCs were able to track glioma cells, deliver transgenes = to=20 tumor targets, and generate progenies of different phenotype, = AdLacZ=20 and AdIL-23 infected BM-NSCs (2 x=20 105 cells) were peritumorally (1 mm lateral and 3 = mm=20 behind the tumor implantation site) and intratumorally (at = the tumor=20 implantation site) injected into the brain with a 7-day = established=20 glioma. Animals were euthanized on days 12, 24, 28, and 42 = after the=20 BM-NSC injection by intracordic perfusion-fixation with 4%=20 paraformaldehyde. Animal brains were cut into 40-=B5m coral = sections=20 and processed for immunohistochemistry staining.

Animal survival and brain tissue evaluation. For C57BL/6 = mice=20 survival experiments, the intracranial glioma-bearing mice = were=20 randomly divided into four groups 3 days after tumor = implantation=20 and treated with intratumoral injections of saline (3 =B5L,=20 n =3D 12), 2 x = 105=20 BM-NSCs infected with either AdEmpty (BM-NSC-E, n =3D = 18) or=20 AdIL-23 (BM-NSC-IL-23, n =3D 17), or NIH-3T3 cells = infected with=20 AdIL-23 (NIH-3T3-IL-23, n =3D 20) in 3 =B5L serum-free = medium.=20 Animals used for histologic evaluation were treated similarly = to the=20 survival experiment and euthanized 4 weeks after the BM-NSC=20 injections by intracordic perfusion-fixation as mentioned = above.=20

Depletion of CD4+ and CD8+ T cells and = natural=20 killer cells. For depletion with monoclonal antibodies = (mAb),=20 each mouse was injected i.p. with 0.5 mg rat anti-mouse CD8 = (53-6.7)=20 and anti-CD4 (GK1.5, both from American Type Culture = Collection,=20 Manassas, VA) mAb or normal rat IgG as control antibody in = 200=20 =B5L PBS 1 day before tumor implantation, once daily for the=20 following 3 consecutive days and then twice weekly. Natural = killer=20 (NK) cells were depleted by i.p. injection of 20 =B5L = rabbit=20 anti=96asialo GM1 antiserum (Wako Chemicals, Richmond, VA) = or=20 normal rabbit serum as control using the same schedule as = above.=20 Intracranial tumor-bearing mice with specific cell population=20 depletion and CD4 T-cell knockout mice with intracranial = tumor=20 implantation were treated with either BM-NSC-IL-23 or = BM-NSC-E=20 and were followed for survival.

Animal rechallenge. C57BL/6 wild-type mice that survived=20 intracranial tumor implantation due to BM-NSC-IL-23 treatment = were=20 rechallenged with GL26 cells or GL26 cells plus either = BM-NSC-IL-23=20 or BM-NSC-E. Age-matched naive mice were challenged as = control. After=20 the rechallenge/challenge, animals were followed for = survival.=20 Some animals were euthanized on days 1, 3, 5, 7, 10, and 14=20 after the rechallenge/challenge for reverse transcription PCR = (RT-PCR) analysis of the brain tissues. All of the animals = used=20 were done in strict accordance with Institutional Animal Care = and Use Committee guidelines in force at Cedars-Sinai Medical = Center.

ELISA and RT-PCR
Supernatants from BM-NSCs and = fetal=20 neural stem cells, which were infected with AdEmpty, AdIL-12, = or=20 AdIL-23 for 24 hours, were analyzed by a sandwich ELISA = specific for=20 p40 as described previously (15).=20 The cell pellets were subjected to total RNA extraction with = a RNeasy=20 Mini kit (Qiagen, Valencia, CA). Brain tissues from the=20 rechallenged/challenged animals as described above were = subjected to=20 total RNA extraction with a RNeasy Lipid Tissue Mini kit = (Qiagen).=20 The RNA was reverse transcripted by using a Bioscript kit = (Bioline,=20 Randolph, MA) and oligo(dT)12-18 primer = (Invitrogen). The=20 PCR was carried out in a 20 =B5L reaction mixture that = contained 1 =B5L=20 cDNA as template, specific oligonucleotide primer pairs (Table=20 1), and Accuzyme (Bioline). Each specific gene was = concurrently=20 amplified with internal control =DF-actin in the same = reaction=20 tube as described previously (19).=20 The amplified products were identified by agarose gel = electrophoresis=20 and ethidium bromide staining.


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  		Table 1. Oligonucleotides = for PCR =

 = ;
Relative=20 Quantitative Real-Time PCR
Total RNA isolation and cDNA = preparation=20 were done as described above. Detection of IFN-3D{gamma}=20 = mRNA level was=20 done by a real-time PCR assay using the iCycler iQ system = (Bio-Rad=20 Laboratories, Hercules, CA). A 12.5 =B5L reaction mixture = containing=20 1 =B5L cDNA and 200 nmol/L of each primer was mixed with = 12.5=20 =B5L of 2x iQ SYBR Green = Supermix (Bio-Rad=20 Laboratories). The reaction conditions were designed as = follows: 95=B0C=20 for 3 minutes to activate the iTaq DNA polymerase followed by = 40=20 cycles with 30 seconds at 95=B0C and 30 seconds at 60=B0C. = PCR=20 amplification of the endogenous =DF-actin was done for each = sample to=20 control for sample loading and to allow normalization between = samples. The threshold cycle (Ct; the cycle number at which = the=20 amount of amplified gene of interest reached a fixed = threshold) was=20 determined subsequently. Each data point was examined for = integrity=20 by analysis of the amplification plot and disassociation = curves. All=20 amplifications were conducted in triplicates. The relative=20 quantitation of IFN-3D{gamma}=20 = mRNA=20 expression was calculated by the comparative Ct method. The = relative=20 quantitation value of target, normalized to endogenous = control=20 =DF-actin and relative to a calibrator, is calculated as = follow:=20 fold increased =3D 2=96[3D{Delta}=20Ct = (survived=20 animal) =96 3D{Delta}=20Ct = (naive=20 animal)], where 3D{Delta}=20Ct = =3D Ct=20 (IFN-3D{gamma}) =96 Ct (=DF-actin).

Cytotoxicity Assays
Spleen cells were harvested = from each=20 mouse. The harvested cells (5 x=20 106/mL) were restimulated in vitro by coculture with=20 mitomycin C=96treated GL26 cells (5 x=20 105/mL) for 5 days and used as effector cells in a = lactate=20 dehydrogenase release assay. GL26 parental tumor cells or = p815 cells=20 (1 x 104/well) and = serial=20 dilutions of effector cells were incubated in a 96-well = U-bottomed=20 plate at 37=B0C for 5 hours. Supernatants then were analyzed = with=20 a cytotoxicity detection kit (Roche Applied Science, Indianapolis,=20 IN) according to the manufacturer's instructions. Results = were=20 expressed as the percentage of specific lysis.

H&E, Luxol Fast Blue, and Immunohistochemistry Staining = of Brain=20 Sections
The perfusion-fixed brains were cut into 10-=B5m = coronal=20 sections and stained with either H&E or luxol fast blue = as=20 per standard protocol. To characterize the brain tissue by=20 immunohistochemistry, free-floating 40-=B5m sections were = treated=20 with 10% donkey serum (Sigma) for 30 minutes at room = temperature and=20 then stained with primary antibodies for anti-=DF-galactosidase = protein=20 (mouse mAb, 1:1,000, Promega, Madison, WI), anti-p40 subunit = of=20 murine IL-23 (mouse mAb, 1:50, BD Biosciences), anti=96=DF-tubulin = III=20 (TuJ1, mouse mAb, 1:200, Chemicon, Temecula, CA), anti=96glial=20 fibrillary acidic protein (GFAP; rabbit polyclonal, 1:1,000,=20 Chemicon), anti-myelin/oligodendrocyte (mouse mAb, 1:1,000,=20 Chemicon), anti-F4/80 (rat mAb, 1:50, Serotec, Raleigh, NC),=20 anti-CD4 (rat mAb, 1:50, BD Biosciences), anti-CD8 (rat mAb,=20 1:100, BD Biosciences), and isotype control antibodies. The=20 primary antibodies were detected with either Texas = red=96conjugated=20 donkey anti-mouse, anti-rat IgG (1:200, The Jackson = Laboratory)=20 before mounting the sections or Vector Elite ABC kit (Vector=20 Laboratories, Burlingame, CA) and developed with = diaminobenzidine=20 (Sigma) and counterstained with hematoxylin before mounting = the=20 sections.


3D"   =20 Results
Top
Abstract
Introduction
Materials and Methods
3D"Results
Discussion
References
 
<= B>Neural=20 stem-like cells can be generated from adult mouse bone = marrow. We=20 harvested adult mouse whole bone marrow cells and cultured = the cells=20 under the conditions described previously that are permissive = for=20 neural stem cells (11,=20 20).=20 Free floating spheres were formed after 8 to 12 days of = culture.=20 These bone marrow=96derived spheres were morphologically=20 indistinguishable from the neurospheres of fetal brain neural = stem=20 cells and were stained positive for the neural stem cell = marker,=20 nestin (Fig.=20 1A). Upon being switched into differentiation = conditions (11),=20 the spheres attached and spread out. After 7 to 14 days of = in=20 vitro differentiation, we analyzed the culture for cells=20 expressing neural and glial markers. TuJ1-positive = (neuron-specific=20 =DF-tubulin III), GFAP-positive (astrocyte marker), and=20 myelin/oligodendrocyte-positive (oligodendrocyte marker) = cells were=20 clearly identified in the differentiated progenies of the = bone=20 marrow=96derived spheres (Fig.=20 1B).



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  		Figure 1. Generation of = neural=20 stem-like cells from adult mouse bone marrow. A, = neurospheres=20 were formed within the culture of adult mouse bone marrow = cells=20 (a). The formed spheres were self-renewing in the=20 free-floating pattern (b), morphologically = indistinguishable=20 from the neurospheres of fetal neural stem cells (c), = and=20 expressed a similar pattern of nestin (d) as that = expressed=20 by fetal neural stem cells (e). B, bone = marrow=96derived=20 spheres were able to differentiate into =DF-tubulin = III=96expressing=20 neurons (a), GFAP-expressing astrocytes (b), = and=20 myelin/oligodendrocyte marker=96expressing oligodendrocytes=20 (c). Bar, 50 =B5m. =

 = ;
BM-NSCs=20 can track intracranial tumors and express transgene both = in=20 vitro and in vivo. We sought to determine whether = bone=20 marrow=96derived sphere cells have migratory properties = similar=20 to that of fetal neural stem cells (= 4=96= 6).=20 To investigate their ability to migrate toward intracranial = gliomas,=20 the bone marrow=96derived sphere cells were transduced with = AdLacZ=20 and injected intratumorally or peritumorally (at a site = distant=20 from the tumor bed) into mouse brain harboring an established = glioma. The AdLacZ transduced cells migrated into the tumor=20 mass (Fig.=20 2A, a) or tumor "satellite," which was away from = the main=20 tumor mass (Fig.=20 2A, b). Some =DF-galactosidase-positive cells were = seen in=20 immediate proximity to and intermixed with invading tumor = "islands"=20 (Fig.=20 2A, c, arrow). Based on the above characteristics, = the=20 sphere cells were named BM-NSCs. To test the ability to = express=20 transgenes, the BM-NSCs were transduced with adenoviral = vectors=20 carrying either stuffer DNA, IL-12, or single-chain IL-23. In = parallel, fetal neural stem cells were also transduced as = controls.=20 After 24 hours, the transduced cells and conditioned medium = were=20 harvested for mRNA analysis and protein assay, respectively. = One=20 subunit of IL-23, p19, was detected as mRNA in both BM-NSC = and fetal=20 neural stem cell lysate by RT-PCR (Fig.=20 2B, a). Another subunit of IL-23 and of IL-12, = p40, was=20 detected in the protein format by ELISA in the conditioned = medium (Fig.=20 2B, b). The mRNA of p19 and the protein of p40 = were=20 similarly expressed by both BM-NSCs and fetal neural stem = cells after=20 the adenoviral vectors transduction. To test their ability to = delivery transgene in vivo, the BM-NSCs transduced = with=20 AdIL-23 were injected into tumor-bearing mice brain. The p40=20 expression was detected both in and around the tumor mass (Fig.=20 2C). To examine the phenotype of BM-NSCs after=20 intracranial implantation, brain sections were double stained = with anti-=DF-galactosidase or anti-p40 antibodies together = with=20 cell type=96specific markers. The implanted cells were = differentiated=20 into positive staining of TuJ1, myelin/oligodendrocyte, or = GFAP.=20 There were no F4/80 (specific for macrophage)=96positive = cells noted=20 (Fig.=20 3). Taken together, these data suggest that BM-NSCs have = the=20 ability to track glioma cells in vivo, to express = transgenes=20 both in vitro and in vivo after transduction with=20 adenoviral vectors, and to differentiate into the neural cell = types of the central nervous system.



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  		Figure 2. BM-NSCs = displayed strong=20 tropism for intracranial gliomas and were able to express = transgenes=20 after transduction with adenoviral vectors. A, = BM-NSCs=20 (red) distributed throughout the intracranial tumor = mass=20 (green) and intermingled among green tumor cells = after=20 peritumoral implantation into brain (a). BM-NSCs = migrated=20 away from the main tumor mass to a tumor satellite following = intratumoral implantation (b), and BM-NSCs were also = seen in=20 the immediate proximity to and intermixed with an invading = tumor=20 island deep into normal neural tissue (c). The = BM-NSCs were=20 identified by immunohistochemistry as = =DF-galactosidase-positive cells=20 (red) due to AdLacZ vector transduction before the = cells were=20 implanted. Tumor cells were GFP stably transfected = (green).=20 Arrowheads, tumor edge; arrow, tumor island. = Bar, 100=20 =B5m. B, BM-NSCs transduced by adenoviral vectors to = express=20 transgenes in vitro. BM-NSCs and fetal neural stem = cells were=20 transduced by AdEmpty (E), AdIL-23 (23), and = AdIL-12=20 (12), respectively. BM-NSCs were capable of = expressing=20 transgene with a similar pattern as that of fetal neural = stem cells,=20 which were verified with either RT-PCR to amplify the p19 = subunit of=20 IL-23 (a) or ELISA to detect the p40 subunit of IL-23 = and=20 IL-12 (b). Columns, mean of triplicate = determinations;=20 bars, SD. ND, not detected. C, BM-NSC, = as a=20 tumor-targeting cellular vehicle, delivered transgene into=20 intracranial tumor. After BM-NSCs were transduced with = AdIL-23, when=20 implanted into brain, the cells were able to deliver the = transgene=20 into glioma as identified by immunohistochemistry staining = of p40 in=20 both wild-type C57BL/6 mice (a and b) and = athymic nude=20 mice (c and d) intracranial tumor models. = Isotype=20 control antibody was stained negative for both wild-type = mice=20 (e and f) and athymic nude mice (g and=20 h). Bar, 100 =B5m. =

 = ;


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  		Figure 3. In vivo=20 differentiation of intracranial implantation of BM-NSCs. = BM-NSCs=20 transduced by AdIL-23 in vitro were intracranially = implanted=20 into mice bearing experimental brain tumor. The transplanted = BM-NSCs=20 (green) were identified within the brain (A, E, = I, and=20 M). The cells (red) showed neural = differentiation as=20 stained positive for Tuj1 (B-D), oligodendroglial=20 differentiation as stained by = myelin/oligodendrocyte-specific=20 antibody (F-H), and astrocytic differentiation as = identified=20 by GFAP antibody (J-L). Macrophage marker-positive = cell was=20 not found in the implanted BM-NSC (N-P). The sections = were=20 labeled also with 4',6-diamidino-2-phenylindole = (blue) to=20 identify nuclei (D, H, L, and P). Bar, 100 = =B5m. =

 = ;
Intracranial=20 tumor-bearing C57BL/6 mice show prolonged survival after=20 IL-23=96expressing BM-NSC treatment. To determine whether = the=20 IL-23=96expressing BM-NSCs provided a therapeutic benefit for = brain=20 tumor, we delivered BM-NSCs transduced with AdIL-23 or = AdEmpty or=20 delivered NIH-3T3 cells transduced with AdIL-23 into = established=20 intracranial gliomas in C57BL/6 mice. BM-NSC-IL-23=96treated = mice=20 showed significantly prolonged survival compared with mice = treated by=20 BM-NSC-E (Fig.=20 4A). Approximately 60% of the = BM-NSC-IL-23=96treated mice=20 survived beyond day 120 with tumor-free condition, which was = verified=20 by histology staining of the brain sections. Only 20% of the=20 NIH-3T3-IL-23=96treated mice survived. BM-NSC-E treatment was = not=20 protective and had the same "effect" as the saline control = treatment.=20 Statistical analysis revealed that the effects of both = BM-NSC-IL-23=20 and NIH-3T3-IL-23 were significantly different from that of = the=20 BM-NSC-E (P < 0.001, BM-NSC-IL-23 versus BM-NSC-E; = P =3D=20 0.0018, NIH-3T3-IL-23 versus BM-NSC-E, log rank). There was = also a=20 significant difference in effect between BM-NSC-IL-23 and=20 NIH-3T3-IL-23 (P =3D 0.0310, log rank).



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  		Figure 4. Effect of=20 IL-23=96expressing BM-NSCs on the experimental glioma in = C57BL/6 mice.=20 A, Kaplan-Meier survival curve of intracranial = glioma-bearing=20 C57BL/6 mice that were injected intracranially with = BM-NSC-IL-23,=20 NIH-3T3-IL-23, BM-NSC-E, or saline. Representative of three=20 independent experiments. B, intratumoral = CD8+ and=20 CD4+ T-cell infiltration 4 weeks after the = transduced=20 BM-NSCs were injected. BM-NSC-IL-23 treated mice showed = robust=20 infiltration of CD8+ (a and b) and=20 CD4+ (d and e) T cells within the = tumor=20 mass. BM-NSC-E=96treated mice showed negligible = CD8+=20 (c) and CD4+ (f) T-cell = infiltration.=20 C, histology staining of glioma-bearing mice brains = that were=20 treated with either BM-NSC-IL-23 (a and d) or = BM-NSC-E=20 (b and e). Age-matched naive mice were = included as=20 control (c and f). Both H&E (a-c) = and luxol=20 fast blue (myelin stain) showed no evidence of demyelination = (d-f). Bar, 100 =B5m. =

 = ;
Based=20 on the known ability of IL-23 to induce proliferation of = memory T=20 cells, we sought to assess whether the BM-NSC-IL-23 treatment = could=20 induce enhanced intratumoral T-cell infiltration. Stained by=20 immunohistochemistry, brain tumors from the mice treated with = BM-NSC-IL-23 showed robust infiltration of CD8+ = and=20 CD4+ T cells (Fig.=20 4B, a, b, d, and e). There was negligible=20 CD8+ and CD4+ T-cell infiltration, however, = within=20 tumors from mice treated with BM-NSC-E (Fig.=20 4B, c and f). The NIH-3T3-IL-23 treatment = also=20 resulted in intratumoral T-cell infiltration, but the = intensity of=20 that infiltration was significantly lower than that seen in = the=20 BM-NSC-IL-23=96treated tumors (data not shown). Analyzing the = brains=20 that were treated with transduced BM-NSCs revealed that there = was no=20 morphologic difference between BM-NSC-IL-23=96 and = BM-NSC-E=96treated=20 mice after either luxol fast blue staining, which was used to = identify neural demyelination, or H&E staining. Both = groups of=20 treated mice showed a similar staining pattern of brain = tissue=20 compared with that of naive control mice (Fig.=20 4C). These data indicated that IL-23=96expressing = BM-NSCs=20 have a strong protective effect against intracranial tumor = and that=20 there is no deleterious effect with morphology significance=20 associated with IL-23=96expressing BM-NSCs.

Involvement of tumor-specific CTL and CD8+ and = CD4+=20 T cells in the antitumor activity of IL-23=96expressing=20 BM-NSCs. To examine whether a tumor-specific immunity was = involved in the protective activity of IL-23=96expressing = BM-NSCs,=20 mice that survived intracranial tumor implantation for 12 = weeks=20 after BM-NSC-IL-23 treatment and age-matched naive mice were=20 subjected to CTL activity assay. CTL activity against = parental tumor=20 GL26 but not irrelevant tumor p815 was augmented in spleen=20 cells, which were obtained from mice that survived = intracranial=20 tumor implantation after BM-NSC-IL-23 treatment and were=20 restimulated in vitro with mitomycin C=96treated GL26 = for 5 days=20 (Fig.=20 5A, a). To determine whether IL-23 mainly contributed = to=20 this CTL activity, spleen cells from brain tumor-bearing mice = treated with either BM-NSC-IL-23 or BM-NSC-E were analyzed 4 = weeks=20 after treatment. As shown in Fig.=20 5A, GL26-specific CTL activity was detected only = in=20 BM-NSC-IL-23=96treated mice. To examine the involvement of = particular=20 lymphocytes in the BM-NSC-IL-23=96induced antitumor activity, = depleting=20 anti-CD4 or anti-CD8 mAb were injected before and after = BM-NSC-IL-23=20 treatment. Normal rat IgG at the same dose and schedule was = included=20 as control. Flow cytometry showed that the injection of mAb = depleted=20 the appropriate cell population by 95% (data not shown). = Depletion of=20 CD8+ T cells greatly impaired the protective = effect of=20 BM-NSC-IL-23 in intracranial tumor-bearing mice (P =3D = 0.0010,=20 CD8 depletion versus wild type, log rank). There was no = long-term=20 survival observed in the CD8+ T-cell=96depleted = mice (Fig.=20 5B). Similarly but less significantly, the = impairment of=20 the protective effect was also observed in the = CD4+=20 T-cell=96depleted mice (P =3D 0.0097, CD4 depletion = versus wild=20 type, log rank). When compared with BM-NSC-E, BM-NSC-IL-23 = showed=20 different protective activity on CD8+ versus=20 CD4+ T-cell=96depleted mice (Fig.=20 5B). In addition, on CD4+ T-cell = knockout=20 transgenic mice BM-NSC-IL-23 produced an effect comparable = with that=20 of produced on the CD4+ T-cell=96depleted = wild-type mice=20 (data not shown). These data suggest that BM-NSC-mediated = IL-23=20 delivery induces a potent tumor-specific protective immunity, = that=20 CD8+ T cells play critical role in the antitumor = activity=20 of BM-NSC-IL-23, and that CD4+ T cells are also = involved=20 in the process.



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  		Figure 5. Involvement of=20 tumor-specific CTL activity and CD8+ and = CD4+=20 T cells in the effect of BM-NSC-IL-23. A, = tumor-specific CTL=20 activity of spleen cells induced by BM-NSC-IL-23. Spleen = cells=20 obtained from mice that survived intracranial GL26 glioma=20 implantation for 12 weeks after BM-NSC-IL-23 treatment = (n =3D=20 3) or from age-matched naive mice (n =3D 3) were = restimulated=20 with mitomycin C=96treated GL26 cells for 5 days, and the = CTL activity=20 of the resultant effector cells was measured against = parental GL26=20 tumor target in a lactate dehydrogenase release assay. = Irrelevant=20 p815 was also used as a tumor target for the effector cells = that=20 were from BM-NSC-IL-23=96treated mice (a). Spleen = cells=20 obtained from glioma-bearing mice that were intracranially = injected=20 with either BM-NSC-IL-23 (n =3D 3) or BM-NSC-E = (n =3D 3)=20 for 4 weeks were restimulated with mitomycin C=96treated = GL26 cells=20 for 5 days, and the CTL activity of resultant effector cells = was=20 measured against parental GL26 tumor target in a lactate=20 dehydrogenase release assay. Irrelevant p815 was also used = as a=20 tumor target for the effector cells that were from=20 BM-NSC-IL-23=96treated mice (b). Points, mean = of three=20 mice; bars, SD. Representative of three independent=20 experiments. B, Kaplan-Meier survival curve of = intracranial=20 glioma-bearing C57BL/6 mice. Mice were randomly divided into = four=20 groups, in which one group was intracranially injected with = BM-NSC-E=20 plus i.p. injections of normal rat serum and the other three = groups=20 were intracranially injected with BM-NSC-IL-23 plus i.p. = injections=20 of either CD8+ T-cell depleting antibody, = CD4+=20 T-cell depleting antibody, or normal rat serum, = respectively.=20 Representative of three independent experiments. =

 = ;
NK=20 cells are involved in the antitumor immunity induced by = IL-23=96expressing=20 BM-NSCs. To examine the involvement of NK cells in the = antitumor=20 activity induced by BM-NSC-IL-23, the first series of = experiments=20 were carried out with athymic nude mice, in which T = lymphocytes=20 are absent. As shown in Fig.=20 6, no mice survived the intracranial GL26 glioma after = either=20 BM-NSC-IL-23 or BM-NSC-E treatment. However, there was a = significant=20 difference in survival time between the two treatments = (P =3D=20 0.0195, log rank). A longer survival rate was observed in = mice=20 treated with BM-NSC-IL-23 compared with those treated with = BM-NSC-E=20 (Fig.=20 6A). Furthermore, after depleting NK cells in the = athymic=20 mice by injecting an anti=96asialo GM1 antibody, no = significant=20 difference (P =3D 0.8801, log rank) in the survival = rate between=20 BM-NSC-IL-23 and BM-NSC-E treatments was observed (Fig.=20 6B). In C57BL/6 wild-type mice, anti=96asialo GM1 = antibody=20 injection, which depleted NK cells in the wild-type animals, = greatly=20 impaired the protective effect of BM-NSC-IL-23 against = intracranial=20 tumor. Only 20% of the mice survived intracranial glioma=20 implantation, but there was an 3D~=2060% = survival rate=20 after normal rabbit serum injection, which served as control = (Fig.=20 6C). The survival rate between the two groups was=20 significantly different (P =3D 0.0457, log rank). = These data=20 suggest that NK cells are involved in the antitumor activity = produced=20 by IL-23=96expressing BM-NSCs.



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  		Figure 6. Involvement of = NK cells=20 in the antitumor immunity induced by BM-NSC-IL-23. A, = Kaplan-Meier survival curve of intracranial glioma-bearing = athymic=20 nude mice that were intracranially injected with either = BM-NSC-IL-23=20 or BM-NSC-E. B, Kaplan-Meier survival curve of = intracranial=20 glioma-bearing athymic nude mice that were depleted of NK = cells with=20 an anti=96asialo GM1 antibody injection and intracranially = injected=20 with either BM-NSC-IL-23 or BM-NSC-E. C, Kaplan-Meier = survival curve of intracranial glioma-bearing C57BL/6 mice = that were=20 intracranially injected with BM-NSC-IL-23 plus i.p. = injection of=20 either anti=96asialo GM1 antibody for NK cell depletion or = normal rat=20 serum as control. Representative of three independent = experiments. =

 = ;
IL-23=96expressing=20 BM-NSCs induce long-term immune memory and enhanced IFN-=20 expression in brain. To examine whether a memory = protective=20 immunity was established in those animals that survived = intracranial=20 GL26 glioma after IL-23=96expressing BM-NSC treatment, the = surviving=20 C57BL/6 mice were subjected to rechallenge with intracranial=20 injection of parental GL26 glioma cells. All of the = rechallenged=20 animals survived beyond day 90 after the rechallenge and were = tumor-free upon sacrifice as verified by H&E staining of = the=20 brain sections. In contrast, age-matched naive mice were = uniformly=20 susceptible to GL26 glioma challenge and died of brain tumor = within=20 35 days of the challenge.

To study the possible molecules that may involve in the activity = of IL-23=96expressing BM-NSC in brain, we focused on IL-17 = and=20 IFN-3D{gamma}, because these molecules have been shown to play = important=20 roles for the dendritic cell and T-cell activation states, = which=20 were promoted by IL-23 (14,=20 15,=20 21).=20 The BM-NSC-IL-23=96treated survivors of the earlier = experiment were=20 rechallenged with parental GL26 cells alone or GL26 with = either=20 BM-NSC-IL-23 or BM-NSC-E. After the rechallenge, brain = tissues were=20 analyzed by RT-PCR. We were consistently unable to detect any = IL-17=20 expression. However, IFN-3D{gamma}=20 was = detected=20 from day 1 and reached the maximum level 5 to 7 days after = the=20 rechallenge (Fig.=20 7A). Then, the expression declined to an = undetectable=20 level 2 weeks after the rechallenge. The IFN-3D{gamma}=20 = expression was=20 always higher in the GL26 plus BM-NSC-IL-23 rechallenged = animals than=20 in the GL26 plus BM-NSC-E or the GL26 alone rechallenged = animals.=20 Age-matched naive animals did not have detectable IFN-=20 expression in their brains after GL26 plus BM-NSC-IL-23 or = GL26 alone=20 challenges (Fig.=20 7B). To further examine the IFN-3D{gamma}=20 = expression=20 level, we used relative quantitative real-time PCR to analyze = the=20 brain samples 7 days after tumor rechallenge. As shown in Table=20 2, IFN-3D{gamma}=20 = mRNA was=20 up-regulated in all rechallenged mice that survived the = earlier=20 experiment compared with the age-matched naive mice that were = challenged with GL26 plus BM-NSC-IL-23 or GL26 alone. We did = not find=20 IFN-3D{gamma} mRNA up-regulation in mice that survived the = earlier=20 experiment but without rechallenge. These data suggest that=20 IL-23=96expressing BM-NSCs used to treat intracranial gliomas = can=20 induce long-term antitumor memory that is associated with = enhanced=20 IFN-3D{gamma}, but not IL-17, up-regulation in the brain.



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  		Figure 7. RT-PCR assay of = cytokine=20 expression within brain after the rechallenges/challenges. = A,=20 IFN-3D{gamma}=20=20 expression was detected in the brain tissues of mice that = survived=20 earlier experiment and rechallenged with GL26 alone (lane = 4)=20 or GL26 with either BM-NSC-IL-23 (lane 2) or BM-NSC-E = (lane 3). IL-17 expression was not detected whether = GL26=20 alone (lane 8) or GL26 with either BM-NSC-IL-23 = (lane=20 6) or BM-NSC-E (lane 7) was used in the = rechallenges.=20 Neither IFN-3D{gamma}=20 nor=20 IL-17 expression was detected in age-matched naive mice = after the=20 GL26 plus BM-NSC-IL-23 challenge (lanes 5 and = 9).=20 B, IFN-3D{gamma}=20=20 expression profile in brain tissue of different animals and=20 different rechallenges/challenges. IFN-3D{gamma}=20=20 expression was detected in mice that survived earlier = experiments=20 and rechallenged with GL26 alone (lanes 4 and = 7) or=20 GL26 with either BM-NSC-IL-23 (lanes 2 and 5) = or=20 BM-NSC-E (lanes 3 and 6). IFN-3D{gamma}=20=20 expression was not detected in age-matched naive mice that = were=20 challenged with GL26 plus BM-NSC-IL-23 (lane 8) or = GL26 alone=20 (lane 9). Brain tissues harvested on day 7 after the=20 rechallenge/challenge. Representative of three independent=20 experiments. L, 1 kb plus DNA ladder. =

 = ;

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  		Table 2. Relative = IFN- mRNA up-regulation in brain tissue =

 = ;

3D"   =20 Discussion
Top
Abstract
Introduction
Materials and Methods
Results
3D"Discussion
References
 
I= n this=20 study, we have presented evidence that neural stem-like cells = can be=20 isolated from whole bone marrow of adult mice. The mice = BM-NSCs were=20 able to be propagated and differentiated into neural and = glial cells=20 in vitro and were able to track intracranial glioma = cells when=20 implanted into glioma-bearing brain. More importantly, these = cells=20 displayed the ability to track invading tumor islands that = had=20 infiltrated deep into normal neural tissue well away from the = main=20 tumor mass. By incorporating the tumor tropic properties of = NSCs with=20 a novel cytokine, IL-23, which acts on memory T cells and=20 dendritic cells (14,=20 15),=20 we found that IL-23=96expressing BM-NSCs when injected into = brain=20 showed antitumor activity against intracranial glioma. Mice = that=20 survived brain tumor implantation after BM-NSC-IL-23 = treatment=20 acquired a tumor-specific protective immunity. CD8+ = T=20 cells were crucial for this potent antitumor activity; in = addition,=20 CD4+ T cells and NK cells were also involved in the=20 induction of this antitumor activity. Furthermore, enhanced=20 IFN-3D{gamma}=20 = production in=20 the brain tissue was important for the BM-NSC-IL-23=96elicited = immune=20 memory response against subsequent parental tumor rechallenge. =

Effective eradication of primary or metastatic brain tumors = and=20 generation of a long-lasting immune response with an effective = gene=20 delivery system are important goals for cancer gene immunotherapy.=20 Most gene delivery strategies employ viral vectors to deliver = genes directly to tumor cells in vivo; however, the = limitation=20 of transgene distribution to extensive areas, especially the=20 invading tumor foci, has limited the efficacy of such = approaches.=20 The present study sought to take advantage of the migratory=20 ability of neural stem cells to track brain tumors and to = deliver=20 therapeutic molecules into expansive tumor regions. We = generated=20 neural stem-like cells from adult mice bone marrow. The = BM-NSCs=20 displayed the ability to track brain tumor in a mouse = intracranial=20 glioma model. After intratumoral or peritumoral implantation, = the BM-NSCs extensively migrated throughout the established=20 tumor mass. In addition, the BM-NSCs tracked into the = invading=20 tumor islands that had migrated away from the main tumor = mass.=20 This tracking characteristic has not been shown previously in = bone marrow=96derived neural progenitor-like cells (12).=20

By transducing the BM-NSCs with an adenoviral vector encoding = a=20 newly discovered cytokine, IL-23, we were able to deliver the = molecule into intracranial tumor sites. It has just been = shown that=20 IL-23=96expressing tumor cells produced antitumor activity = (16).=20 Delivering IL-23 by using BM-NSCs in the current study = resulted in a=20 protective effect in intracranial glioma-bearing mice with a=20 significant prolongation of survival. These results strongly = suggest=20 that IL-23=96expressing BM-NSCs produce antiglioma activity. = To verify=20 the potential role of IL-23 in this antiglioma activity, we = compared=20 the effect of IL-23=96transduced BM-NSCs with that of an = empty control=20 vector transduced BM-NSCs. All of the intracranial = glioma-bearing=20 mice died within 40 days after treatment with the empty = control=20 vector transduced BM-NSCs. To further verify the potential = benefit of=20 the migration of BM-NSC in producing antiglioma activity, we = compared=20 the survival benefit offered by BM-NSC-IL-23 to that = conferred by=20 IL-23 secretion by nonmigratory NIH-3T3 cells, which produce = similar=20 levels of IL-23 to BM-NSC-IL23 in vitro and in = vivo.=20 NIH-3T3-IL-23 treatment produced significantly less = protective effect=20 compared with BM-NSC-IL-23. Taken together, the antiglioma = activity=20 of IL-23=96expressing BM-NSCs may be a direct combined = consequence=20 of the ability of BM-NSCs to target migrating tumor cells and = the ability of IL-23 to activate immune responses against = tumor=20 cells.

It has been shown that IL-23 can act directly on dendritic cells = to promote tumor peptide presentation to T cells (15).=20 In addition, T-cell responses may be amplified by the potent = effect=20 of IL-23 on memory-activated T cells (14).=20 Our experiments in immunocompromised hosts and in animals = selectively=20 depleted of various lymphocyte populations suggest that = T-cell immune=20 responses are important for antitumor function of = IL-23=96expressing=20 BM-NSCs and that CD8+ T cells play a crucial role = in=20 BM-NSC-IL-23=96mediated antitumor activity, because the = immune=20 protective effect was greatly impaired in T-cell=96deficient = athymic=20 nude mice and in wild-type mice depleted of CD8+ T = cells.=20 In CD4 knockout mice and in wild-type mice depleted of=20 CD4+ T cells, we found that CD4+ T = cells were=20 also involved in the antitumor activity of BM-NSC-IL-23 = albeit to a=20 lesser extent than CD8+ T cells. In addition, in = both=20 athymic nude mice and wild-type mice, we observed the role of = NK=20 cells in the immune protective effect of BM-NSC-IL-23. NK = cell=20 depletion abolished and reduced the protective effect in = athymic nude=20 and wild-type mice, respectively. Lo et al. described the = role of=20 CD8+ T cells in the antitumor activity of = IL-23=96expressing=20 tumor cells in a colon adenocarcinoma model (16).=20 Our results confirmed that CD8+ T cells are crucial = to the=20 IL-23=96mediated antitumor activity. However, the finding = that=20 CD4+ T cells and NK cells were involved in the = IL-23=20 activity in our current study represents a significant = divergence=20 from the results forwarded by Lo et al. (16),=20 in which IL-23=96expressing tumor cells induced antitumor = activity=20 but did not require CD4+ T cells or NK cells. This may=20 possibly be explained by their use of a different tumor cell = line=20 (CT26) in their colon adenocarcinoma s.c. tumor model, which = is=20 distinct from the GL26 glioma we used in our study as an=20 intracranial brain tumor model. Alternatively, this = difference may be=20 attributable to the unique environment of the central nervous = system=20 with regard to antigen presentation by microglia or recruited = dendritic cells. Interestingly, when we used B16-F10 cells as = intracranial tumor model, we also observed the involvement of = NK=20 cells in the antitumor activity of IL-23 (data not shown). = The=20 variance in the cellular subsets crucial to an antitumor = effect=20 needs further delineation.

IL-23 can activate macrophages to produce proinflammatory = cytokines=20 that may contribute to autoimmune inflammation of the brain = (21=9624).=20 In the present study, we found that BM-NSC-IL-23=96treated = long-term=20 survivors showed enhanced IFN-3D{gamma}=20 = expression in=20 brain tissue upon tumor rechallenge. We were consistently=20 unable, however, to detect IL-17, a cytokine involved in the = IL-23=20 signaling pathway and which induces inflammation (21,=20 23,=20 24),=20 even when we delivered IL-23 with the tumor rechallenge. = These=20 findings were consistent with the results that elucidated = that mice=20 brains maintained normal morphologic characteristics after=20 BM-NSC-IL-23 treatment as shown by H&E and = demyelination-specific=20 staining. These data suggest that BM-NSC-IL-23 treatment used = in the=20 current study does not induce detectable inflammation in the = brain=20 either because the IL-23 level delivered by BM-NSCs is not = high=20 enough to do so or because the in vivo expression of = exogenous=20 IL-23 predominantly enhances long-lasting memory T-cell=20 immunity, such as antigen-specific CTL and IFN-3D{gamma}=20=96producing Th1=20 immune responses (25).=20 To understand this, it will be required to closely = investigate the=20 signal pathways that are involved in inflammation and in = protective=20 immune responses induced by exogenous IL-23 expression. =

In summary, in this study, we showed that neural stem-like cells = generated from adult bone marrow can be used as a targeting=20 vehicle to track migratory and invasive tumor cells within = the=20 central nervous system. In combination with the unique action = of IL-23 on tumoricidal potency, autologous neural stem-like=20 cell=96mediated tumor targeting immune therapy represents = an=20 attractive new treatment modality for malignant brain tumors. =


3D"   =20 Acknowledgments =
 
Grant support:=20 NIH grants 1K23 NS02232, 1R01 NS048959, and 1R21 NS048879 = (J.S.=20 Yu).

The costs of publication of this article were defrayed in part = by=20 the payment of page charges. This article must therefore be = hereby=20 marked advertisement in accordance with 18 U.S.C. = Section 1734=20 solely to indicate this fact.

We thank Drs. Sebastian Wachsmann-Hogiu and Daniel H. Farkas = for=20 their kind assistance in obtaining images of stained brain = sections,=20 Dr. Scot Macdonald for the critical review of the article and = helpful=20 comments, and Dr. Hong Zhou for discussions.


3D"   =20 Footnotes
 
Note: X. Yuan and J. Hu contributed = equally to=20 this work.

3 Yuan et al., unpublished = data.=20

Received 5/16/05. Revised 12/ 9/05. Accepted 12/27/05.


3D"   =20 References
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Abstract
Introduction
Materials and Methods
Results
Discussion
3D"References
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF=20 CONTENTS
Cancer = Research Clinical Cancer=20 Research
Cancer=20 Epidemiology Biomarkers & Prevention Molecular=20 Cancer Therapeutics
Molecular=20 Cancer Research Cell Growth=20 & Differentiation
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http://cancerres.aacrjournals.org/javascript/ajax/xmlhttprequest.js /*=0A= =0A= Cross-Browser XMLHttpRequest v1.2=0A= =3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D= =3D=3D=3D=3D=3D=3D=3D=3D=0A= =0A= Emulate Gecko 'XMLHttpRequest()' functionality in IE and Opera. Opera = requires=0A= the Sun Java Runtime Environment .=0A= =0A= by Andrew Gregory=0A= http://www.scss.com.au/family/andrew/webdesign/xmlhttprequest/=0A= =0A= This work is licensed under the Creative Commons Attribution License. To = view a=0A= copy of this license, visit = http://creativecommons.org/licenses/by-sa/2.5/ or=0A= send a letter to Creative Commons, 559 Nathan Abbott Way, Stanford, = California=0A= 94305, USA.=0A= =0A= Attribution: Leave my name and web address in this script intact.=0A= =0A= Not Supported in Opera=0A= ----------------------=0A= * user/password authentication=0A= * responseXML data member=0A= =0A= Not Fully Supported in Opera=0A= ----------------------------=0A= * async requests=0A= * abort()=0A= * getAllResponseHeaders(), getAllResponseHeader(header)=0A= =0A= */=0A= // IE support=0A= if (window.ActiveXObject && !window.XMLHttpRequest) {=0A= window.XMLHttpRequest =3D function() {=0A= var msxmls =3D new Array(=0A= 'Msxml2.XMLHTTP.5.0',=0A= 'Msxml2.XMLHTTP.4.0',=0A= 'Msxml2.XMLHTTP.3.0',=0A= 'Msxml2.XMLHTTP',=0A= 'Microsoft.XMLHTTP');=0A= for (var i =3D 0; i < msxmls.length; i++) {=0A= try {=0A= return new ActiveXObject(msxmls[i]);=0A= } catch (e) {=0A= }=0A= }=0A= return null;=0A= };=0A= }=0A= // Gecko support=0A= /* ;-) */=0A= // Opera support=0A= if (window.opera && !window.XMLHttpRequest) {=0A= window.XMLHttpRequest =3D function() {=0A= this.readyState =3D 0; // = 0=3Duninitialized,1=3Dloading,2=3Dloaded,3=3Dinteractive,4=3Dcomplete=0A= this.status =3D 0; // HTTP status codes=0A= this.statusText =3D '';=0A= this._headers =3D [];=0A= this._aborted =3D false;=0A= this._async =3D true;=0A= this._defaultCharset =3D 'ISO-8859-1';=0A= this._getCharset =3D function() {=0A= var charset =3D _defaultCharset;=0A= var contentType =3D = this.getResponseHeader('Content-type').toUpperCase();=0A= val =3D contentType.indexOf('CHARSET=3D');=0A= if (val !=3D -1) {=0A= charset =3D contentType.substring(val);=0A= }=0A= val =3D charset.indexOf(';');=0A= if (val !=3D -1) {=0A= charset =3D charset.substring(0, val);=0A= }=0A= val =3D charset.indexOf(',');=0A= if (val !=3D -1) {=0A= charset =3D charset.substring(0, val);=0A= }=0A= return charset;=0A= };=0A= this.abort =3D function() {=0A= this._aborted =3D true;=0A= };=0A= this.getAllResponseHeaders =3D function() {=0A= return this.getAllResponseHeader('*');=0A= };=0A= this.getAllResponseHeader =3D function(header) {=0A= var ret =3D '';=0A= for (var i =3D 0; i < this._headers.length; i++) {=0A= if (header =3D=3D '*' || this._headers[i].h =3D=3D header) {=0A= ret +=3D this._headers[i].h + ': ' + this._headers[i].v + '\n';=0A= }=0A= }=0A= return ret;=0A= };=0A= this.getResponseHeader =3D function(header) {=0A= var ret =3D getAllResponseHeader(header);=0A= var i =3D ret.indexOf('\n');=0A= if (i !=3D -1) {=0A= ret =3D ret.substring(0, i);=0A= }=0A= return ret;=0A= };=0A= this.setRequestHeader =3D function(header, value) {=0A= this._headers[this._headers.length] =3D {h:header, v:value};=0A= };=0A= this.open =3D function(method, url, async, user, password) {=0A= this.method =3D method;=0A= this.url =3D url;=0A= this._async =3D true;=0A= this._aborted =3D false;=0A= this._headers =3D [];=0A= if (arguments.length >=3D 3) {=0A= this._async =3D async;=0A= }=0A= if (arguments.length > 3) {=0A= opera.postError('XMLHttpRequest.open() - user/password not = supported');=0A= }=0A= this.readyState =3D 1;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= };=0A= this.send =3D function(data) {=0A= if (!navigator.javaEnabled()) {=0A= alert("XMLHttpRequest.send() - Java must be installed and = enabled.");=0A= return;=0A= }=0A= if (this._async) {=0A= setTimeout(this._sendasync, 0, this, data);=0A= // this is not really asynchronous and won't execute until the = current=0A= // execution context ends=0A= } else {=0A= this._sendsync(data);=0A= }=0A= }=0A= this._sendasync =3D function(req, data) {=0A= if (!req._aborted) {=0A= req._sendsync(data);=0A= }=0A= };=0A= this._sendsync =3D function(data) {=0A= this.readyState =3D 2;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= // open connection=0A= var url =3D new java.net.URL(new = java.net.URL(window.location.href), this.url);=0A= var conn =3D url.openConnection();=0A= for (var i =3D 0; i < this._headers.length; i++) {=0A= conn.setRequestProperty(this._headers[i].h, this._headers[i].v);=0A= }=0A= this._headers =3D [];=0A= if (this.method =3D=3D 'POST') {=0A= // POST data=0A= conn.setDoOutput(true);=0A= var wr =3D new = java.io.OutputStreamWriter(conn.getOutputStream(), this._getCharset());=0A= wr.write(data);=0A= wr.flush();=0A= wr.close();=0A= }=0A= // read response headers=0A= // NOTE: the getHeaderField() methods always return nulls for me :(=0A= var gotContentEncoding =3D false;=0A= var gotContentLength =3D false;=0A= var gotContentType =3D false;=0A= var gotDate =3D false;=0A= var gotExpiration =3D false;=0A= var gotLastModified =3D false;=0A= for (var i =3D 0; ; i++) {=0A= var hdrName =3D conn.getHeaderFieldKey(i);=0A= var hdrValue =3D conn.getHeaderField(i);=0A= if (hdrName =3D=3D null && hdrValue =3D=3D null) {=0A= break;=0A= }=0A= if (hdrName !=3D null) {=0A= this._headers[this._headers.length] =3D {h:hdrName, = v:hdrValue};=0A= switch (hdrName.toLowerCase()) {=0A= case 'content-encoding': gotContentEncoding =3D true; break;=0A= case 'content-length' : gotContentLength =3D true; break;=0A= case 'content-type' : gotContentType =3D true; break;=0A= case 'date' : gotDate =3D true; break;=0A= case 'expires' : gotExpiration =3D true; break;=0A= case 'last-modified' : gotLastModified =3D true; break;=0A= }=0A= }=0A= }=0A= // try to fill in any missing header information=0A= var val;=0A= val =3D conn.getContentEncoding();=0A= if (val !=3D null && !gotContentEncoding) = this._headers[this._headers.length] =3D {h:'Content-encoding', v:val};=0A= val =3D conn.getContentLength();=0A= if (val !=3D -1 && !gotContentLength) = this._headers[this._headers.length] =3D {h:'Content-length', v:val};=0A= val =3D conn.getContentType();=0A= if (val !=3D null && !gotContentType) = this._headers[this._headers.length] =3D {h:'Content-type', v:val};=0A= val =3D conn.getDate();=0A= if (val !=3D 0 && !gotDate) this._headers[this._headers.length] = =3D {h:'Date', v:(new Date(val)).toUTCString()};=0A= val =3D conn.getExpiration();=0A= if (val !=3D 0 && !gotExpiration) = this._headers[this._headers.length] =3D {h:'Expires', v:(new = Date(val)).toUTCString()};=0A= val =3D conn.getLastModified();=0A= if (val !=3D 0 && !gotLastModified) = this._headers[this._headers.length] =3D {h:'Last-modified', v:(new = Date(val)).toUTCString()};=0A= // read response data=0A= var reqdata =3D '';=0A= var stream =3D conn.getInputStream();=0A= if (stream) {=0A= var reader =3D new java.io.BufferedReader(new = java.io.InputStreamReader(stream, this._getCharset()));=0A= var line;=0A= while ((line =3D reader.readLine()) !=3D null) {=0A= if (this.readyState =3D=3D 2) {=0A= this.readyState =3D 3;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= }=0A= reqdata +=3D line + '\n';=0A= }=0A= reader.close();=0A= this.status =3D 200;=0A= this.statusText =3D 'OK';=0A= this.responseText =3D reqdata;=0A= this.readyState =3D 4;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= if (this.onload) {=0A= this.onload();=0A= }=0A= } else {=0A= // error=0A= this.status =3D 404;=0A= this.statusText =3D 'Not Found';=0A= this.responseText =3D '';=0A= this.readyState =3D 4;=0A= if (this.onreadystatechange) {=0A= this.onreadystatechange();=0A= }=0A= if (this.onerror) {=0A= this.onerror();=0A= }=0A= }=0A= };=0A= };=0A= }=0A= // ActiveXObject emulation=0A= if (!window.ActiveXObject && window.XMLHttpRequest) {=0A= window.ActiveXObject =3D function(type) {=0A= switch (type.toLowerCase()) {=0A= case 'microsoft.xmlhttp':=0A= case 'msxml2.xmlhttp':=0A= case 'msxml2.xmlhttp.3.0':=0A= case 'msxml2.xmlhttp.4.0':=0A= case 'msxml2.xmlhttp.5.0':=0A= return new XMLHttpRequest();=0A= }=0A= return null;=0A= };=0A= }=0A= ------=_NextPart_000_0000_01C85870.B701F780 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://cancerres.aacrjournals.org/javascript/ajax/utility.js /************************************************************************= *****=0A= * javascript/ajax/utility.js=0A= *=0A= * Utility functions for working with XMLHttpRequest data.=0A= *=0A= * Copyright 2006 Board of Trustees of the Leland Stanford Junior = University.=0A= = *************************************************************************= ***/=0A= =0A= /*=0A= * Copy XML nodes into an HTMLElement. This effectively=0A= * clones XML markup which uses XHTML naming conventions=0A= * into an HTML DOM.=0A= */=0A= function copy_xml_to_html(src, dst) {=0A= if (src.nodeType =3D=3D 1) { /* Node.ELEMENT_NODE */=0A= var e =3D document.createElement(src.nodeName);=0A= for (var i =3D 0; i < src.childNodes.length; i++) {=0A= copy_xml_to_html(src.childNodes[i], e);=0A= }=0A= for (var i =3D 0; i < src.attributes.length; i++) {=0A= var n =3D src.attributes[i].name;=0A= var v =3D unescape_xml_string(src.attributes[i].value); =0A= e.setAttribute(n, v);=0A= if (n =3D=3D "class") {=0A= e.className =3D v;=0A= }=0A= else if (n =3D=3D "style") {=0A= set_css_style(v, e, "");=0A= }=0A= }=0A= dst.appendChild(e);=0A= }=0A= else if (src.nodeType =3D=3D 3) { /* Node.TEXT_NODE */=0A= dst.appendChild(document.createTextNode(src.nodeValue));=0A= }=0A= }=0A= =0A= /* =0A= * It is unclear that this is the right thing to be calling=0A= * from copy_xml_to_html, but it appears that Safari decides=0A= * to convert & to the NCR #, and then encodes that=0A= * NCR to &%26%2338;. So, I'm going to treat the DOM Attr=0A= * value as a plain string, and run our XML string input=0A= * through the decoding routine below.=0A= */=0A= function unescape_xml_string(s) {=0A= return s.replace(/'/g, "'")=0A= .replace(/'/g, "'")=0A= .replace(/"/g, "\"")=0A= .replace(/"/g, "\"")=0A= .replace(/>/g, ">")=0A= .replace(/>/g, ">")=0A= .replace(/</g, "<")=0A= .replace(/</g, "<")=0A= .replace(/&/g, "&")=0A= .replace(/&/g, "&");=0A= }=0A= =0A= /*=0A= * Parse set of CSS rules and apply them to an element.=0A= * This is quite horrifying, but I'm unable to determine=0A= * how else to handle this with IE 6. FireFox and other=0A= * sane browsers let you simply set the style attribute=0A= * or use e.style.setProperty(rule, value, priority),=0A= * IE 6 appears to have neither of these capabilities..=0A= */=0A= function set_css_style(css, e, priority) {=0A= var rules =3D css.split(";");=0A= for (var i =3D 0; i < rules.length; i++) {=0A= var nvpair =3D rules[i].split(":");=0A= if (nvpair.length =3D=3D 2) {=0A= try {=0A= var name =3D nvpair[0]; /* style attribute */=0A= var value =3D nvpair[1]; /* attribute value */=0A= =0A= /*=0A= * For each possible style attribute, set the=0A= * appropriate style property in the element.=0A= */=0A= if (name =3D=3D "background") {=0A= e.style.background =3D value;=0A= }=0A= else if (name =3D=3D "background-attachment") {=0A= e.style.backgroundAttachment =3D value;=0A= }=0A= else if (name =3D=3D "background-color") {=0A= e.style.backgroundColor =3D value;=0A= }=0A= else if (name =3D=3D "background-image") {=0A= e.style.backgroundImage =3D value;=0A= }=0A= else if (name =3D=3D "background-position") {=0A= e.style.backgroundPosition =3D value;=0A= }=0A= else if (name =3D=3D "background-position-x") {=0A= e.style.backgroundPositionX =3D value;=0A= }=0A= else if (name =3D=3D "background-position-y") {=0A= e.style.backgroundPositionY =3D value;=0A= }=0A= else if (name =3D=3D "background-repeat") {=0A= e.style.backgroundRepeat =3D value;=0A= }=0A= else if (name =3D=3D "behavior") {=0A= e.style.behavior =3D value;=0A= }=0A= else if (name =3D=3D "border") {=0A= e.style.border =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom") {=0A= e.style.borderBottom =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-color") {=0A= e.style.borderBottomColor =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-style") {=0A= e.style.borderBottomStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-bottom-width") {=0A= e.style.borderBottomWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-collapse") {=0A= e.style.borderCollapse =3D value;=0A= }=0A= else if (name =3D=3D "border-color") {=0A= e.style.borderColor =3D value;=0A= }=0A= else if (name =3D=3D "border-left") {=0A= e.style.borderLeft =3D value;=0A= }=0A= else if (name =3D=3D "border-left-color") {=0A= e.style.borderLeftColor =3D value;=0A= }=0A= else if (name =3D=3D "border-left-style") {=0A= e.style.borderLeftStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-left-width") {=0A= e.style.borderLeftWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-right") {=0A= e.style.borderRight =3D value;=0A= }=0A= else if (name =3D=3D "border-right-color") {=0A= e.style.borderRightColor =3D value;=0A= }=0A= else if (name =3D=3D "border-right-style") {=0A= e.style.borderRightStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-right-width") {=0A= e.style.borderRightWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-style") {=0A= e.style.borderStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-top") {=0A= e.style.borderTop =3D value;=0A= }=0A= else if (name =3D=3D "border-top-color") {=0A= e.style.borderTopColor =3D value;=0A= }=0A= else if (name =3D=3D "border-top-style") {=0A= e.style.borderTopStyle =3D value;=0A= }=0A= else if (name =3D=3D "border-top-width") {=0A= e.style.borderTopWidth =3D value;=0A= }=0A= else if (name =3D=3D "border-width") {=0A= e.style.borderWidth =3D value;=0A= }=0A= else if (name =3D=3D "bottom") {=0A= e.style.bottom =3D value;=0A= }=0A= else if (name =3D=3D "clear") {=0A= e.style.clear =3D value;=0A= }=0A= else if (name =3D=3D "clip") {=0A= e.style.clip =3D value;=0A= }=0A= else if (name =3D=3D "color") {=0A= e.style.color =3D value;=0A= }=0A= else if (name =3D=3D "cssText") {=0A= e.style.Sets =3D value;=0A= }=0A= else if (name =3D=3D "cursor") {=0A= e.style.cursor =3D value;=0A= }=0A= else if (name =3D=3D "direction") {=0A= e.style.direction =3D value;=0A= }=0A= else if (name =3D=3D "display") {=0A= e.style.display =3D value;=0A= }=0A= else if (name =3D=3D "font") {=0A= e.style.font =3D value;=0A= }=0A= else if (name =3D=3D "font-family") {=0A= e.style.fontFamily =3D value;=0A= }=0A= else if (name =3D=3D "font-size") {=0A= e.style.fontSize =3D value;=0A= }=0A= else if (name =3D=3D "font-style") {=0A= e.style.fontStyle =3D value;=0A= }=0A= else if (name =3D=3D "font-variant") {=0A= e.style.fontVariant =3D value;=0A= }=0A= else if (name =3D=3D "font-weight") {=0A= e.style.fontWeight =3D value;=0A= }=0A= else if (name =3D=3D "height") {=0A= e.style.height =3D value;=0A= }=0A= else if (name =3D=3D "ime-mode") {=0A= e.style.imeMode =3D value;=0A= }=0A= else if (name =3D=3D "layout-flow") {=0A= e.style.layoutFlow =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid") {=0A= e.style.layoutGrid =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-char") {=0A= e.style.layoutGridChar =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-line") {=0A= e.style.layoutGridLine =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-mode") {=0A= e.style.layoutGridMode =3D value;=0A= }=0A= else if (name =3D=3D "layout-grid-type") {=0A= e.style.layoutGridType =3D value;=0A= }=0A= else if (name =3D=3D "left") {=0A= e.style.left =3D value;=0A= }=0A= else if (name =3D=3D "letter-spacing") {=0A= e.style.letterSpacing =3D value;=0A= }=0A= else if (name =3D=3D "line-break") {=0A= e.style.lineBreak =3D value;=0A= }=0A= else if (name =3D=3D "line-height") {=0A= e.style.lineHeight =3D value;=0A= }=0A= else if (name =3D=3D "list-style") {=0A= e.style.listStyle =3D value;=0A= }=0A= else if (name =3D=3D "list-style-image") {=0A= e.style.listStyleImage =3D value;=0A= }=0A= else if (name =3D=3D "list-style-position") {=0A= e.style.listStylePosition =3D value;=0A= }=0A= else if (name =3D=3D "list-style-type") {=0A= e.style.listStyleType =3D value;=0A= }=0A= else if (name =3D=3D "margin") {=0A= e.style.margin =3D value;=0A= }=0A= else if (name =3D=3D "margin-bottom") {=0A= e.style.marginBottom =3D value;=0A= }=0A= else if (name =3D=3D "margin-left") {=0A= e.style.marginLeft =3D value;=0A= }=0A= else if (name =3D=3D "margin-right") {=0A= e.style.marginRight =3D value;=0A= }=0A= else if (name =3D=3D "margin-top") {=0A= e.style.marginTop =3D value;=0A= }=0A= else if (name =3D=3D "min-height") {=0A= e.style.minHeight =3D value;=0A= }=0A= else if (name =3D=3D "overflow") {=0A= e.style.overflow =3D value;=0A= }=0A= else if (name =3D=3D "overflow-x") {=0A= e.style.overflowX =3D value;=0A= }=0A= else if (name =3D=3D "overflow-y") {=0A= e.style.overflowY =3D value;=0A= }=0A= else if (name =3D=3D "padding") {=0A= e.style.padding =3D value;=0A= }=0A= else if (name =3D=3D "padding-bottom") {=0A= e.style.paddingBottom =3D value;=0A= }=0A= else if (name =3D=3D "padding-left") {=0A= e.style.paddingLeft =3D value;=0A= }=0A= else if (name =3D=3D "padding-right") {=0A= e.style.paddingRight =3D value;=0A= }=0A= else if (name =3D=3D "padding-top") {=0A= e.style.paddingTop =3D value;=0A= }=0A= else if (name =3D=3D "page-break-after") {=0A= e.style.pageBreakAfter =3D value;=0A= }=0A= else if (name =3D=3D "page-break-before") {=0A= e.style.pageBreakBefore =3D value;=0A= }=0A= else if (name =3D=3D "pixelBottom") {=0A= e.style.pixelBottom =3D value;=0A= }=0A= else if (name =3D=3D "pixelHeight") {=0A= e.style.pixelHeight =3D value;=0A= }=0A= else if (name =3D=3D "pixelLeft") {=0A= e.style.pixelLeft =3D value;=0A= }=0A= else if (name =3D=3D "pixelRight") {=0A= e.style.pixelRight =3D value;=0A= }=0A= else if (name =3D=3D "pixelTop") {=0A= e.style.pixelTop =3D value;=0A= }=0A= else if (name =3D=3D "pixelWidth") {=0A= e.style.pixelWidth =3D value;=0A= }=0A= else if (name =3D=3D "posBottom") {=0A= e.style.posBottom =3D value;=0A= }=0A= else if (name =3D=3D "posHeight") {=0A= e.style.posHeight =3D value;=0A= }=0A= else if (name =3D=3D "position") {=0A= e.style.position =3D value;=0A= }=0A= else if (name =3D=3D "posLeft") {=0A= e.style.posLeft =3D value;=0A= }=0A= else if (name =3D=3D "posRight") {=0A= e.style.posRight =3D value;=0A= }=0A= else if (name =3D=3D "posTop") {=0A= e.style.posTop =3D value;=0A= }=0A= else if (name =3D=3D "posWidth") {=0A= e.style.posWidth =3D value;=0A= }=0A= else if (name =3D=3D "right") {=0A= e.style.right =3D value;=0A= }=0A= else if (name =3D=3D "ruby-align") {=0A= e.style.rubyAlign =3D value;=0A= }=0A= else if (name =3D=3D "ruby-overhang") {=0A= e.style.rubyOverhang =3D value;=0A= }=0A= else if (name =3D=3D "ruby-position") {=0A= e.style.rubyPosition =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-3dlight-color") {=0A= e.style.scrollbar3dLightColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-arrow-color") {=0A= e.style.scrollbarArrowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-base-color") {=0A= e.style.scrollbarBaseColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-darkshadow-color") {=0A= e.style.scrollbarDarkShadowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-face-color") {=0A= e.style.scrollbarFaceColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-highlight-color") {=0A= e.style.scrollbarHighlightColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-shadow-color") {=0A= e.style.scrollbarShadowColor =3D value;=0A= }=0A= else if (name =3D=3D "scrollbar-track-color") {=0A= e.style.scrollbarTrackColor =3D value;=0A= }=0A= else if (name =3D=3D "float") {=0A= e.style.styleFloat =3D value;=0A= }=0A= else if (name =3D=3D "table-layout") {=0A= e.style.tableLayout =3D value;=0A= }=0A= else if (name =3D=3D "text-align") {=0A= e.style.textAlign =3D value;=0A= }=0A= else if (name =3D=3D "text-align-last") {=0A= e.style.textAlignLast =3D value;=0A= }=0A= else if (name =3D=3D "text-autospace") {=0A= e.style.textAutospace =3D value;=0A= }=0A= else if (name =3D=3D "text-decoration") {=0A= e.style.textDecoration =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationBlink") {=0A= e.style.textDecorationBlink =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationLineThrough") {=0A= e.style.textDecorationLineThrough =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationNone") {=0A= e.style.textDecorationNone =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationOverline") {=0A= e.style.textDecorationOverline =3D value;=0A= }=0A= else if (name =3D=3D "textDecorationUnderline") {=0A= e.style.textDecorationUnderline =3D value;=0A= }=0A= else if (name =3D=3D "text-indent") {=0A= e.style.textIndent =3D value;=0A= }=0A= else if (name =3D=3D "text-justify") {=0A= e.style.textJustify =3D value;=0A= }=0A= else if (name =3D=3D "text-kashida-space") {=0A= e.style.textKashidaSpace =3D value;=0A= }=0A= else if (name =3D=3D "text-overflow") {=0A= e.style.textOverflow =3D value;=0A= }=0A= else if (name =3D=3D "text-transform") {=0A= e.style.textTransform =3D value;=0A= }=0A= else if (name =3D=3D "text-underline-position") {=0A= e.style.textUnderlinePosition =3D value;=0A= }=0A= else if (name =3D=3D "top") {=0A= e.style.top =3D value;=0A= }=0A= else if (name =3D=3D "unicode-bidi") {=0A= e.style.unicodeBidi =3D value;=0A= }=0A= else if (name =3D=3D "vertical-align") {=0A= e.style.verticalAlign =3D value;=0A= }=0A= else if (name =3D=3D "visibility") {=0A= e.style.visibility =3D value;=0A= }=0A= else if (name =3D=3D "white-space") {=0A= e.style.whiteSpace =3D value;=0A= }=0A= else if (name =3D=3D "width") {=0A= e.style.width =3D value;=0A= }=0A= else if (name =3D=3D "word-break") {=0A= e.style.wordBreak =3D value;=0A= }=0A= else if (name =3D=3D "word-spacing") {=0A= e.style.wordSpacing =3D value;=0A= }=0A= else if (name =3D=3D "word-wrap") {=0A= e.style.wordWrap =3D value;=0A= }=0A= else if (name =3D=3D "writing-mode") {=0A= e.style.writingMode =3D value;=0A= }=0A= else if (name =3D=3D "z-index") {=0A= e.style.zIndex =3D value;=0A= }=0A= else if (name =3D=3D "zoom") {=0A= e.style.zoom =3D value;=0A= }=0A= }=0A= catch (e) {=0A= /* ignore error on attempt to set e.style.[property] */=0A= }=0A= }=0A= }=0A= }=0A= ------=_NextPart_000_0000_01C85870.B701F780 Content-Type: application/octet-stream Content-Transfer-Encoding: quoted-printable Content-Location: http://cancerres.aacrjournals.org/javascript/entrez/callback.js /************************************************************************= *****=0A= * javascript/entrez/callback.js=0A= *=0A= * Entrez Linking callback to populate content box.=0A= *=0A= * Copyright 2006 Board of Trustees of the Leland Stanford Junior = University.=0A= = *************************************************************************= ***/=0A= =0A= /*=0A= * Execute callback to fill content box with Entrez Linking information.=0A= */=0A= function entrez_callback(pmid, callback_url) {=0A= /*=0A= * MSIE 5.5 and below have issues with the JavaScript=0A= * used for Entrez Linking. 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