Patent Application: US-201213544631-A

Abstract:
to gain a better understanding of brain tumor angiogenesis , new techniques for isolating brain endothelial cells and evaluating gene expression patterns were developed . when transcripts from brain ecs derived from normal and malignant colorectal tissues were compared with transcripts from non - endothelial cells , genes predominantly expressed in the endothelium were identified . comparison between normal - and tumor - derived endothelium revealed genes that were specifically elevated in tumor - associated brain endothelium . these results confirm that neoplastic and normal endothelium in human brains are distinct at the molecular level , and have significant implications for the development of anti - angiogenic therapies in the future .

Description:
using sage ( serial analysis of gene expression ) profiling , this study was able to identify previously unrecognized , angiogenesis - specific markers that discriminate between non - proliferative and pathologic endothelial cells . we identified 255 human genes that were expressed at significantly higher levels in brain tumor endothelium than in normal brain endothelium . see table 1 . we have named these markers gems ( glioma endothelial markers ). any of the gems disclosed in any of the tables can be used in the methods of the present invention , according to the discretion of the skilled artisan . ecs represent only a minor fraction of the total cells within normal or tumor tissues , and only those ec transcripts expressed at the highest levels would be expected to be represented in libraries constructed from unfractionated tissues . the genes described in the current study should therefore provide a valuable resource for basic and clinical studies of human brain angiogenesis in the future . genes which have been identified as expressed more in glioma endothelial cells than in normal brain endothelial cells ( gems ) include those which correspond to tags shown in seq id nos : 1 - 32 . the tags correspond to the segment of the cdna that is 3 ′ of the 3 ′ most restriction endonuclease site for the restriction enzyme nlaiii which was used as the anchoring enzyme . the tag shown is the same strand as the mrna . other such genes are listed in tables 1 and 2 . isolated and purified nucleic acids , according to the present invention are those which are not linked to those genes to which they are linked in the human genome . moreover , they are not present in a mixture such as a library containing a multitude of distinct sequences from distinct genes . they may be , however , linked to other genes such as vector sequences or sequences of other genes to which they are not naturally adjacent . tags disclosed herein , because of the way that they were made , represent sequences which are 3 ′ of the 3 ′ most restriction enzyme recognition site for the tagging enzyme used to generate the sage tags . in this case , the tags are 3 ′ of the most 3 ′ most nlaiii site in the cdna molecules corresponding to mrna . nucleic acids corresponding to tags may be rna , cdna , or genomic dna , for example . such corresponding nucleic acids can be determined by comparison to sequence databases to determine sequence identities . sequence comparisons can be done using any available technique , such as blast , available from the national library of medicine , national center for biotechnology information . tags can also be used as hybridization probes to libraries of genomic or cdna to identify the genes from which they derive . thus , using sequence comparisons or cloning , or combinations of these methods , one skilled in the art can obtain full - length nucleic acid sequences . genes corresponding to tags will contain the sequence of the tag at the 3 ′ end of the coding sequence or of the 3 ′ untranslated region ( utr ), 3 ′ of the 3 ′ most recognition site in the cdna for the restriction endonuclease which was used to make the tags . the nucleic acids may represent either the sense or the anti - sense strand . nucleic acids and proteins although disclosed herein with sequence particularity , may be derived from a single individual . allelic variants which occur in the population of humans are included within the scope of such nucleic acids and proteins . those of skill in the art are well able to identify allelic variants as being the same gene or protein . given a nucleic acid , one of ordinary skill in the art can readily determine an open reading frame present , and consequently the sequence of a polypeptide encoded by the open reading frame and , using techniques well known in the art , express such protein in a suitable host . proteins comprising such polypeptides can be the naturally occurring proteins , fusion proteins comprising exogenous sequences from other genes from humans or other species , epitope tagged polypeptides , etc . isolated and purified proteins are not in a cell , and are separated from the normal cellular constituents , such as nucleic acids , lipids , etc . typically the protein is purified to such an extent that it comprises the predominant species of protein in the composition , such as greater than 50 , 60 70 , 80 , 90 , or even 95 % of the proteins present . using the proteins according to the invention , one of ordinary skill in the art can readily generate antibodies which specifically bind to the proteins . such antibodies can be monoclonal or polyclonal . they can be chimeric , humanized , or totally human . any functional fragment or derivative of an antibody can be used including fab , fab ′, fab2 , fab ′ 2 , and single chain variable regions . so long as the fragment or derivative retains specificity of binding for the endothelial marker protein it can be used . antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions . if the antibody binds to the appropriate antigen at least 2 , 5 , 7 , and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific . techniques for making such partially to fully human antibodies are known in the art and any such techniques can be used . according to one particularly preferred embodiment , fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes . multiple strains of such transgenic mice have been made which can produce different classes of antibodies . b cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody . see for example , nina d . russel , jose r . f . corvalan , michael l . gallo , c . geoffrey davis , liise - anne pirofski . production of protective human antipneumococcal antibodies by transgenic mice with human immunoglobulin loci infection and immunity april 2000 , p . 1820 - 1826 ; michael l . gallo , vladimir e . ivanov , aya jakobovits , and c . geoffrey davis . the human immunoglobulin loci introduced into mice : v ( d ) and j gene segment usage similar to that of adult humans european journal of immunology 30 : 534 - 540 , 2000 ; larry l . green . antibody engineering via genetic engineering of the mouse : xenomouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies journal of immunological methods 231 11 - 23 , 1999 ; yang x - d , corvalan j r f , wang p , roy c m - n and davis c g . fully human anti - interleukin - 8 monoclonal antibodies : potential therapeutics for the treatment of inflammatory disease states . journal of leukocyte biology vol . 66 , pp 401 - 410 ( 1999 ); yang x - d , jia x - c , corvalan j r f , wang p , c g davis and jakobovits a . eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy . cancer research vol . 59 , number 6 , pp 1236 - 1243 ( 1999 ); jakobovits a . production and selection of antigen - specific fully human monoclonal antibodies from mice engineered with human ig loci . advanced drug delivery reviews vol . 31 , pp : 33 - 42 ( 1998 ); green l and jakobovits a . regulation of b cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes . j . exp . med . vol . 188 , number 3 , pp : 483 - 495 ( 1998 ); jakobovits a . the long - awaited magic bullets : therapeutic human monoclonal antibodies from transgenic mice . exp . opin . invest . drugs vol . 7 ( 4 ), pp : 607 - 614 ( 1998 ); tsuda h , maynard - currie k , reid l , yoshida t , edamura k , maeda n , smithies o , jakobovits a . inactivation of mouse hprt locus by a 203 - bp retrotransposon insertion and a 55 - kb gene - targeted deletion : establishment of new hprt - deficient mouse embryonic sgem cell lines . genomics vol . 42 , pp : 413 - 421 ( 1997 ); sherman - gold , r . monoclonal antibodies : the evolution from &# 39 ; 80s magic bullets to mature , mainstream applications as clinical therapeutics . genetic engineering news vol . 17 , number 14 ( august 1997 ); mendez m , green l , corvalan j , jia x - c , maynard - currie c , yang x - d , gallo m , louie d , lee d , erickson k , luna j , roy c , abderrahim h , kirschenbaum f , noguchi m , smith d , fukushima a , hales j , finer m , davis c , zsebo k , jakobovits a . functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice . nature genetics vol . 15 , pp : 146 - 156 ( 1997 ); jakobovits a . mice engineered with human immunoglobulin yacs : a new technology for production of fully human antibodies for autoimmunity therapy . weir &# 39 ; s handbook of experimental immunology , the integrated immune system vol . iv , pp : 194 . 1 - 194 . 7 ( 1996 ); jakobovits a . production of fully human antibodies by transgenic mice . current opinion in biotechnology vol . 6 , no . 5 , pp : 561 - 566 ( 1995 ); mendez m , abderrahim h , noguchi m , david n , hardy m , green l , tsuda h , yoast s , maynard - currie c , garza d , gemmill r , jakobovits a , klapholz s . analysis of the structural integrity of yacs comprising human immunoglobulin genes in yeast and in embryonic sgem cells . genomics vol . 26 , pp : 294 - 307 ( 1995 ); jakobovits a . yac vectors : humanizing the mouse genome . current biology vol . 4 , no . 8 , pp : 761 - 763 ( 1994 ); arbones m , ord d , ley k , ratech h , maynard - curry k , otten g , capon d , tedder t . lymphocyte homing and leukocyte rolling and migration are impaired in l - selectin - deficient mice . immunity vol . 1 , no . 4 , pp : 247 - 260 ( 1994 ); green l , hardy m , maynard - curry k , tsuda h , louie d , mendez m , abderrahim h , noguchi m , smith d , zeng y , et . al . antigen - specific human monoclonal antibodies from mice engineered with human ig heavy and light chain yacs . nature genetics vol . 7 , no . 1 , pp : 13 - 21 ( 1994 ); jakobovits a , moore a , green l , vergara g , maynard - curry k , austin h , klapholz s . germ - line transmission and expression of a human - derived yeast artificial chromosome . nature vol . 362 , no . 6417 , pp : 255 - 258 ( 1993 ); jakobovits a , vergara g , kennedy j , hales j , mcguinness r , casentini - borocz d , brenner d , otten g . analysis of homozygous mutant chimeric mice : deletion of the immunoglobulin heavy - chain joining region blocks b - cell development and antibody production . proceedings of the national academy of sciences usa vol . 90 , no . 6 , pp : 2551 - 2555 ( 1993 ); kucherlapati et al ., u . s . pat . no . 6 , 1075 , 181 . antibodies can also be made using phage display techniques . such techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics . single chain fv can also be used as is convenient . they can be made from vaccinated transgenic mice , if desired . antibodies can be produced in cell culture , in phage , or in various animals , including but not limited to cows , rabbits , goats , mice , rats , hamsters , guinea pigs , sheep , dogs , cats , monkeys , chimpanzees , apes . antibodies can be labeled with a detectable moiety such as a radioactive atom , a chromophore , a fluorophore , or the like . such labeled antibodies can be used for diagnostic techniques , either in vivo , or in an isolated test sample . antibodies can also be conjugated , for example , to a pharmaceutical agent , such as chemotherapeutic drug or a toxin . they can be linked to a cytokine , to a ligand , to another antibody . suitable agents for coupling to antibodies to achieve an anti - tumor effect include cytokines , such as interleukin 2 ( il - 2 ) and tumor necrosis factor ( tnf ); photosensitizers , for use in photodynamic therapy , including aluminum ( iii ) phthalocyanine tetrasulfonate , hematoporphyrin , and phthalocyanine ; radionuclides , such as iodine - 131 ( 131 i ), yttrium - 90 ( 90 y ), bismuth - 212 ( 212 bi ), bismuth - 213 ( 213 bi ), technetium - 99m ( 99m tc ), rhenium - 186 ( 186 re ), and rhenium - 188 ( 188 re ); antibiotics , such as doxorubicin , adriamycin , daunorubicin , methotrexate , daunomycin , neocarzinostatin , and carboplatin ; bacterial , plant , and other toxins , such as diphtheria toxin , pseudomonas exotoxin a , staphylococcal enterotoxin a , abrin - a toxin , ricin a ( deglycosylated ricin a and native ricin a ), tgf - alpha toxin , cytotoxin from chinese cobra ( naja naja atra ), and gelonin ( a plant toxin ); ribosome inactivating proteins from plants , bacteria and fungi , such as restrictocin ( a ribosome inactivating protein produced by aspergillus restrictus ), saporin ( a ribosome inactivating protein from saponaria officinalis ), and rnase ; tyrosine kinase inhibitors ; ly207702 ( a difluorinated purine nucleoside ); liposomes containing antitumor agents ( e . g ., antisense oligonucleotides , plasmids which encode for toxins , methotrexate , etc . ); and other antibodies or antibody fragments , such as f ( ab ). those of skill in the art will readily understand and be able to make such antibody derivatives , as they are well known in the art . the antibodies may be cytotoxic on their own , or they may be used to deliver cytotoxic agents to particular locations in the body . the antibodies can be administered to individuals in need thereof as a form of passive immunization . characterization of extracellular regions for the cell surface and secreted proteins from the protein sequence is based on the prediction of signal sequence , transmembrane domains and functional domains . antibodies are preferably specifically immunoreactive with membrane associated proteins , particularly to extracellular domains of such proteins or to secreted proteins . such targets are readily accessible to antibodies , which typically do not have access to the interior of cells or nuclei . however , in some applications , antibodies directed to intracellular proteins may be useful as well . moreover , for diagnostic purposes , an intracellular protein may be an equally good target since cell lysates may be used rather than a whole cell assay . computer programs can be used to identify extracellular domains of proteins whose sequences are known . such programs include smart software ( schultz et al ., proc . natl . acad . sci . usa 95 : 5857 - 5864 , 1998 ) and pfam software ( bageman et al ., nucleic acids res . 28 : 263 - 266 , 2000 ) as well as psortii . typically such programs identify transmembrane domains ; the extracellular domains are identified as immediately adjacent to the transmembrane domains . prediction of extracellular regions and the signal cleavage sites are only approximate . it may have a margin of error + or − 5 residues . signal sequence can be predicted using three different methods ( nielsen et al , protein engineering 10 : 1 - 6 , 1997 , jagla et . al , bioinformatics 16 : 245 - 250 , 2000 , nakai , k . and horton , p . trends in biochem . sci . 24 : 34 - 35 , 1999 ) for greater accuracy . similarly transmembrane ( tm ) domains can be identified by multiple prediction methods . ( pasquier , et . al , protein eng . 12 : 381 - 385 , 1999 , sonnhammer et al ., in proc . of sixth int . conf . on intelligent systems for molecular biology , p . 175 - 182 , ed j . glasgow , t . littlejohn , f . major , r . lathrop , d . sankoff , and c . sensen menlo park , calif . : aaai press , 1998 , klein , et . al , biochim . biophys . acta , 815 : 468 , 1985 , nakai and kanehisa genomics , 14 : 897 - 911 , 1992 ). in ambiguous cases , locations of functional domains in well characterized proteins are used as a guide to assign a cellular localization . putative functions or functional domains of novel proteins can be inferred from homologous regions in the database identified by blast searches ( altschul et . al . nucleic acid res . 25 : 3389 - 3402 , 1997 ) and / or from a conserved domain database such as pfam ( bageman et . al , nucleic acids res . 27 : 260 - 262 1999 ) blocks ( henikoff , et . al , nucl . acids res . 28 : 228 - 230 , 2000 ) and smart ( ponting , et . al , nucleic acid res . 27 , 229 - 232 , 1999 ). extracellular domains include regions adjacent to a transmembrane domain in a single transmembrane domain protein ( out - in or type i class ). for multiple transmembrane domains proteins , the extracellular domain also includes those regions between two adjacent transmembrane domains ( in - out and out - in ). for type ii transmembrane domain proteins , for which the n - terminal region is cytoplasmic , regions following the transmembrane domain is generally extracellular . secreted proteins on the other hand do not have a transmembrane domain and hence the whole protein is considered as extracellular . membrane associated proteins can be engineered to delete the transmembrane domains , thus leaving the extracellular portions which can bind to ligands . such soluble forms of transmembrane receptor proteins can be used to compete with natural forms for binding to ligand . thus such soluble forms act as inhibitors . and can be used therapeutically as anti - angiogenic agents , as diagnostic tools for the quantification of natural ligands , and in assays for the identification of small molecules which modulate or mimic the activity of a gem : ligand complex . alternatively , the endothelial markers themselves can be used as vaccines to raise an immune response in the vaccinated animal or human . for such uses , a protein , or immunogenic fragment of such protein , corresponding to the intracellular , extracellular or secreted gem of interest is administered to a subject . the immogenic agent may be provided as a purified preparation or in an appropriately expressing cell . the administration may be direct , by the delivery of the immunogenic agent to the subject , or indirect , through the delivery of a nucleic acid encoding the immunogenic agent under conditions resulting in the expression of the immunogenic agent of interest in the subject . the gem of interest may be delivered in an expressing cell , such as a purified population of glioma endothelial cells or a populations of fused glioma endothelial and dendritic cells . nucleic acids encoding the gem of interest may be delivered in a viral or non - viral delivery vector or vehicle . non - human sequences encoding the human gem of interest or other mammalian homolog can be used to induce the desired immunologic response in a human subject . for several of the gems of the present invention , mouse , rat or other ortholog sequences are described herein or can be obtained from the literature or using techniques well within the skill of the art . endothelial cells can be identified using the markers which are disclosed herein as being endothelial cell specific . these include the human markers identified by seq id nos : 1 - 510 . antibodies specific for such markers can be used to identify such cells , by contacting the antibodies with a population of cells containing some endothelial cells . the presence of cross - reactive material with the antibodies identifies particular cells as endothelial . similarly , lysates of cells can be tested for the presence of cross - reactive material . any known format or technique for detecting cross - reactive material can be used including , immunoblots , radioimmunoassay , elisa , immunoprecipitation , and immunohistochemistry . in addition , nucleic acid probes for these markers can also be used to identify endothelial cells . any hybridization technique known in the art including northern blotting , rt - pcr , microarray hybridization , and in situ hybridization can be used . one can identify glioma endothelial cells for diagnostic purposes , testing cells suspected of containing one or more gems . one can test both tissues and bodily fluids of a subject . for example , one can test a patient &# 39 ; s blood for evidence of intracellular and membrane associated gems , as well as for secreted gems . intracellular and / or membrane associated gems may be present in bodily fluids as the result of high levels of expression of these factors and / or through lysis of cells expressing the gems . populations of various types of endothelial cells can also be made using the antibodies to endothelial markers of the invention . the antibodies can be used to purify cell populations according to any technique known in the art , including but not limited to fluorescence activated cell sorting . such techniques permit the isolation of populations which are at least 50 , 60 , 70 , 80 , 90 , 92 , 94 , 95 , 96 , 97 , 98 , and even 99 % the type of endothelial cell desired , whether normal , tumor , or pan - endothelial . antibodies can be used to both positively select and negatively select such populations . preferably at least 1 , 5 , 10 , 15 , 20 , or 25 of the appropriate markers are expressed by the endothelial cell population . populations of endothelial cells made as described herein , can be used for screening drugs to identify those suitable for inhibiting the growth of tumors by virtue of inhibiting the growth of the tumor vasculature . populations of endothelial cells made as described herein , can be used for screening candidate drugs to identify those suitable for modulating angiogenesis , such as for inhibiting the growth of tumors by virtue of inhibiting the growth of endothelial cells , such as inhibiting the growth of the tumor or other undesired vasculature , or alternatively , to promote the growth of endothelial cells and thus stimulate the growth of new or additional large vessel or microvasculature . inhibiting the growth of endothelial cells means either regression of vasculature which is already present , or the slowing or the absence of the development of new vascularization in a treated system as compared with a control system . by stimulating the growth of endothelial cells , one can influence development of new ( neovascularization ) or additional vasculature development ( revascularization ). a variety of model screening systems are available in which to test the angiogenic and / or anti - angiogenic properties of a given candidate drug . typical tests involve assays measuring the endothelial cell response , such as proliferation , migration , differentiation and / or intracellular interaction of a given candidate drug . by such tests , one can study the signals and effects of the test stimuli . some common screens involve measurement of the inhibition of heparanase , endothelial tube formation on matrigel , scratch induced motility of endothelial cells , platelet - derived growth factor driven proliferation of vascular smooth muscle cells , and the rat aortic ring assay ( which provides an advantage of capillary formation rather than just one cell type ). drugs can be screened for the ability to mimic or modulate , inhibit or stimulate , growth of tumor endothelium cells and / or normal endothelial cells . drugs can be screened for the ability to inhibit tumor endothelium growth but not normal endothelium growth or survival . similarly , human cell populations , such as normal endothelium populations or glioma endothelial cell populations , can be contacted with test substances and the expression of glioma endothelial markers and / or normal endothelial markers determined . test substances which decrease the expression of glioma endothelial markers ( gems ) are candidates for inhibiting angiogenesis and the growth of tumors . in cases where the activity of a gem is known , agents can be screened for their ability to decrease or increase the activity . for those glioma endothelial markers identified as containing transmembrane regions , it is desirable to identify drug candidates capable of binding to the gem receptors found at the cell surface . for some applications , the identification of drug candidates capable of blocking the gem receptor from its native ligand will be desired . for some applications , the identification of a drug candidate capable of binding to the gem receptor may be used as a means to deliver a therapeutic or diagnostic agent . for other applications , the identification of drug candidates capable of mimicking the activity of the native ligand will be desired . thus , by manipulating the binding of a transmembrane gem receptor : ligand complex , one may be able to promote or inhibit further development of endothelial cells and hence , vascularization . for those glioma endothelial markers identified as being secreted proteins , it is desirable to identify drug candidates capable of binding to the secreted gem protein . for some applications , the identification of drug candidates capable of interfering with the binding of the secreted gem it is native receptor . for other applications , the identification of drug candidates capable of mimicking the activity of the native receptor will be desired . thus , by manipulating the binding of the secreted gem : receptor complex , one may be able to promote or inhibit further development of endothelial cells , and hence , vascularization . expression can be monitored according to any convenient method . protein or mrna can be monitored . any technique known in the art for monitoring specific genes &# 39 ; expression can be used , including but not limited to elisas , sage , microarray hybridization , western blots . changes in expression of a single marker may be used as a criterion for significant effect as a potential pro - angiogenic , anti - angiogenic or anti - tumor agent . however , it also may be desirable to screen for test substances which are able to modulate the expression of at least 5 , 10 , 15 , or 20 of the relevant markers , such as the tumor or normal endothelial markers . inhibition of gem protein activity can also be used as a drug screen . human and mouse gems can be used for this purpose . test substances for screening can come from any source . they can be libraries of natural products , combinatorial chemical libraries , biological products made by recombinant libraries , etc . the source of the test substances is not critical to the invention . the present invention provides means for screening compounds and compositions which may previously have been overlooked in other screening schemes . nucleic acids and the corresponding encoded proteins of the markers of the present invention can be used therapeutically in a variety of modes . gems can be used to stimulate the growth of vasculature , such as for wound healing or to circumvent a blocked vessel . the nucleic acids and encoded proteins can be administered by any means known in the art . such methods include , using liposomes , nanospheres , viral vectors , non - viral vectors comprising polycations , etc . suitable viral vectors include adenovirus , retroviruses , and sindbis virus . administration modes can be any known in the art , including parenteral , intravenous , intramuscular , intraperitoneal , topical , intranasal , intrarectal , intrabronchial , etc . specific biological antagonists of gems can also be used to therapeutic benefit . for example , antibodies , t cells specific for a gem , antisense to a gem , and ribozymes specific for a gem can be used to restrict , inhibit , reduce , and / or diminish tumor or other abnormal or undesirable vasculature growth . such antagonists can be administered as is known in the art for these classes of antagonists generally . anti - angiogenic drugs and agents can be used to inhibit tumor growth , as well as to treat diabetic retinopathy , rheumatoid arthritis , psoriasis , polycystic kidney disease ( pkd ), and other diseases requiring angiogenesis for their pathologies . mouse counterparts to human gems can be used in mouse cancer models or in cell lines or in vitro to evaluate potential anti - angiogenic or anti - tumor compounds or therapies . their expression can be monitored as an indication of effect . mouse gems can be used as antigens for raising antibodies which can be tested in mouse tumor models . mouse gems with transmembrane domains are particularly preferred for this purpose . mouse gems can also be used as vaccines to raise an immunological response in a human to the human ortholog . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . in this study we employ sage transcript profiling to derive the transcriptomes from normal and neoplastic brain tissue . moreover , we have employed a new version of sage , long sage , allowing for the derivation of 21 bp sage tags . these longer tags allow for the direct interrogation of genomic dna , identifying unique locations of cell - specific transcription . endothelial cells from normal brain and different stages of gliomas were expression profiled and compared to each other and to the colon endothelial cell data . distinct sets of genes define global tumor and normal endothelial cell markers as well as defining glioma - specific endothelial markers . this expanded tumor endothelial cell database will likely provide further insights into the complex regulatory mechanisms governing tumor angiogenesis . tissue procurement and endothelial cell isolation . five separate brain tissue samples ( table 1 ) were resected and immediately subjected to endothelial cell isolation with slight modifications to the protocol described previously . st croix , b ., rago , c ., velculescu , v ., traverso , g ., romans , k . e ., montgomery , e ., lal , a ., riggins , g . j ., lengauer , c ., vogelstein , b ., and kinzler , k . w . ( 2000 ). genes expressed in human tumor endothelium . science 289 , 1197 - 202 . briefly , samples were surgically excised and submerged in dmem . the samples were minced into 2 centimeter cubes and subjected to tissue digestion with a collagenase cocktail . samples were mixed at 37 ° c . until dissolved . cells were spun down and washed two times with pbs / bsa and filtered through successive nylon mesh filters of 250 , 100 and 40 microns . samples were resuspended in pbs / bsa and applied to a 30 % percoll gradient centrifuging for 15 minutes at 800 g . 5 ml off the top of the percoll gradient was diluted in 50 ml dmem and cells pelleted , washed with pbs and resuspended in 3 ml pbs / bsa . cells were filtered through falcon blue top filter tubes , spun down and resuspended in 1 ml pbs / bsa . 100 microliters of prewashed ant - cd45 magnetic beads ( dynal ) were added and the solution allowed to gently mix for ten minutes . bead - bound cells were discarded and the supernatant transferred to a fresh microcentrifuge tube . 10 microliters of p1h12 mab ( 1 : 100 ) ( brain n1 , t1 , and t2 samples ) or uea - i lectin ( brain n2 and t3 samples ) was added and the samples were mixed gently at 4 ° c . for 45 minutes . cells were pelleted and washed 3 times in pbs / bsa and resuspended in 500 microliters pbs / bsa . prewashed goat anti - mouse m450 dynabeads were added to each tube and allowed to mix for 15 minutes at 4 ° c . bead - bound cells were washed 8 times with pbs / bsa and resuspended in a final volume of 500 microliters pbs . cells were counted and frozen at − 70 ° c . prior to rna extraction . rna isolation and sage library generation . rna was isolated from the selected cells and initially subjected to rt - pcr analysis to determine the relative abundance of specific , known endothelial cell markers . the microsage protocol st croix , b ., rago , c ., velculescu , v ., traverso , g ., romans , k . e ., montgomery , e ., lal , a ., riggins , g . j ., lengauer , c ., vogelstein , b ., and kinzler , k . w . ( 2000 ). genes expressed in human tumor endothelium . science 289 , 1197 - 202 ( server www , domain name sagenet . org , directory sage protocol ) was used to generate high - quality longsage libraries employing the tagging enzyme mmei instead of bsmfi . 21 base tags were defined by capillary sequencing using a combination of an abi 3700 and abi 3100 . the sample descriptions and sequencing depth are shown in table 3 . data analysis . long sage tags derived from the brain endothelial samples were reduced to short tags to allow for the integration of colon endothelial sage data . aggregate short tags were derived from the long tags . any short tag counts that had more than one corresponding long tag representative were summed and the counts represented as one short tag . both sequencing errors and legitimate long tag derivatives contribute to the generation of multiple long tags . for transcript and genome mapping , differential long tags were employed . differential gene expression was evaluated as follows : for the two normal brain samples , either the maximum or minimum value was used for determining tumor / normal and normal / tumor ratios , respectively . for the three brain tumor samples , the median value was used for the tumor / normal whereas the maximum value was used for the normal / tumor ratios . a two parameter family of beta distributions was used to assess the probability of observing two fold differences in the observed sage tag abundances . chen , h ., centola , m ., altschul , s . f ., and metzger , h . ( 1998 ). characterization of gene expression in resting and activated mast cells . j exp med 188 , 1657 - 68 . the following provides a detailed protocol useful for isolating brain endothelial cells . all steps were done at 4 ° c . in cold room and in centrifuge except digestion . 1 ) take sample from operating room and submerge in known volume of dmem + in 50 ml conical tube to measure tumor volume by displacement . cut off 2 small pieces of tumor on dry ice and store at − 70 ° c . for mrna extraction / immunohistochemistry / in situ analysis . 2 ) take sample from conical and place in small amount of dmem + in 10 cm tissue culture dish in hood . mince specimen into 2 mm cubes with sterile scalpel . 3 ) transfer minced specimen to small autoclaved erlenmeyer flask and add 5 × volume of digestion cocktail . sample volumes & gt ; 5 ml should be split into multiple flasks . 4 ) mix in bacterial shaker or in 37 ° c . room on rotating shaker for 45 minutes or until sample is dissolved . titrate with 10 ml piper every 15 minutes . once a good cell suspension is obtained , remove and transfer to 50 ml conical . 5 ) spin down at 1500 rpm ( 600 × g ) at 4 ° c . for 5 minutes . 6 ) wash 2 × with pbs / bsa and spin down again . pool samples . 7 ) filter through nylon mesh ( 250 , 100 , 40 micron ). 8 ) spin down . 9 ) resuspend n pbs / bsa at ½ the original tumor volume . 10 ) apply sample in 500 ul aliquots to preformed 30 % percoll gradient ( gradients needed = volume of original sample ). 11 ) spin at 1750 rpm ( 800 g ) for 15 minutes . 12 ) remove top 5 ml percoll from each tube and dilute with dmem to 50 ml volume . 13 ) pellet cells in centrifuge at 1500 rpm . pool pelleted cells . 14 ) wash 2 × with pbs / bsa and resuspend in 3 ml pbs / bsa . 15 ) filter through falcon blue top filter tube . 16 ) spin down and resuspend in 1 ml pbs / bsa in a 1 . 5 ml microcentrigufe tube . 17 ) add 100 μl of prewashed anti - cd45 beads ( hematopoietic depletion ) to solution and rotate end over end in cold room for ten minutes . [ for brain tissue isolation , an additional negative selection with berep4 epithelial depletion is not needed ] 18 ) remove bead - bound cells and transfer supernatant to a fresh microcentrifuge tube . save bead - bound sample by freezing at − 70 ° c . repeat extraction to ensure complete removal of all beads . 19 ) add 10 ul of p1h12 mab ( 1 : 100 ) to cells and mix in cold room with end - over - end rotation for 45 minutes . [ as an alternative , selection using uea1 lectin also provides quality endothelial cell selection .] 20 ) pellet cells and wash 3 × with pbs / bsa . 21 ) resuspend cells in 500 ul of pbs / bsa . 22 ) divide sample into four 1 . 5 ml microcentrifuge tubes ( 125 ul per tube ) and bring volume up to 800 ul . add 50 ul of prewashed goat anti - mouse m450 dynabeads to each tube . 23 ) rotate tubes in cold room for 15 minutes . 24 ) separate with magnet and save supernatant as staining control , tumor / brain fraction . 25 ) rinse 8 × with pbs / bsa . 26 ) pool beads into single microcentrifuge tube . 27 ) resuspend final cells in 500 ul plain pbs . 28 ) take 5 ul of solution and combine with 5 ul of magic dapi and count on hemacytometer . 29 ) remove 10 k cells for staining for quality control based on hemacytometer results 30 ) separate beads again and freeze remainder at − 70 ° c . for mrna extraction . this example describes the preparation of sage tags from mrna extracted from brain endothelial cells . the preparation is described with reference to standard sage tag preparation procedures as are known in the art . all of the template was used in the pcr sage ditag step . usually we take only a small portion of our template , dilute it and perform ˜ 300 pcr reactions . for these libraries we used all of our material , diluted it and performed ˜ 1200 pcr reactions . during the post - amplified pcr product purification step we normally do a standard large volume phenol / chloroform extraction and remove the aqueous layer which contains the product of interest . for these libraries we used eppendorfs phase lock product which creates a physical barrier between the aqueous and organic layers thereby decreasing the amount of product you leave behind . this product was used for all p / c extractions in the second half of the protocol . digesting the amplified pcr products with nlaiii to release the ditag of interest is usually done in one reaction . for these libraries i divided the material into thirds and performed 3 nlaiii reactions in the hopes of yielding more released ditag . due to the low amount of material , upon entering the concatemer and digested pzero ligation reaction , i modified the recipe for this reaction to accommodate this . standard reaction calls for 6 ul of concatemers , 2 ul of 5 × ligase buffer , 1 ul digested pzero vector , and 1 ul of high concentrate ligase . i modified it to 6 ul of concatemers , 2 ul of 5 × ligase buffer , 0 . 3 - 0 . 5 ul of digested pzero vector , 1 ul high concentrate ligase and filled the missing volume with water . my intention was to favor the concatemer to pzero ligation reaction relative to the competing pzero to pzero ligation reaction . most gels during the procedure showed weak amounts of product for visualization and the concatemer gels showed no visible product via the naked eye ( we cut out certain fractions regardless ). microarray analysis . custom 50 nucleotide oligomer arrays were constructed containing 606 unique gene elements . the 606 genes were derived from tumor and normal induced genes from both colon and brain data ( 328 genes ), as well as 278 genes from both literature reviews and housekeeping genes . arrays were interrogated with cy3 and cy5 dye - swapped labelled arna samples comparing hmvecs grown on plastic , collagen , fibrin , or matrigel . in situ hybridizations and immunohistochemistry . in situ hybridizations for pv i , vegfr2 and vwf were carried out as described previously ( 10 ). co - staining of pv1 and cd31 was carried out as follows : four 500 nucleotide riboprobe fragments specific for pv1 were transcribed and used to probe formalin fixed 5 micron tissue sections . final detection of the bound riboprobes were delayed until after the cd31 ihc staining . after pv1 hybridization and washing , tissue sections were fixed for 20 minutes in 4 % formaldehyde . after a brief rinse in tbs , antigen retrieval was carried out using dako target retrieval solution ( dako , cat # s 1699 ) according to manufacturer &# 39 ; s instructions . after a five minute wash in tbs , slides were digested with proteinase k at 20 ng / ml in tbs for 20 minutes at 37t , then blocked for 20 minutes at room temperature in block ( 10 % goat serum / 0 . 5 % casein / 0 . 05 % tween - 20 / pbs ). slides were incubated with dako cd31 ( cat # m0823 ) at a final concentration of 1 microgram / slide in block solution , for 60 minutes at room temperature . after two 5 minute tbst ( dako , cat # s3306 ) washes at room temperature , pv1 riboprobe and cd31 antibody were detected with streptavidin - cy2 ( jackson immunoresearch , cat # 016 - 220 - 084 ) at 5 micrograms / slide for the pv1 riboprobe , and goat anti - mouse - cy3 ( jackson immunoresearch , cat # 115 - 165 - 146 ) at 2 . 5 micrograms / slide for cd31 , for 60 minutes at room temperature . after three ywashes in tbst , the slides were mounted with antifade medium containing dapi nuclear counter - stain , cover - slipped and stored at − 20 ′ c until viewing . single images of dapi , cy2 and cy3 images were acquired separately on a zeiss axioplan at 40 × with a hammamatsu camera , then merged together to form a composite image using universal imaging metamorph software , and stored at − 20c until viewing . capillary - like tubule formation assay . the formation of capillary - like tubular structures was assessed in matrigel - coated multiwell plates essentially as described previously ( 12 ). briefly , 300 microliters of matrigel ( bd , bedford , mass .) was added to each well of a 24 well plate and allowed to polymerize at 37 ″ c for 30 minutes . hmvecs ( biowhittaker ) were infected with adenovius harboring tem . 1 or gfp gene or empty vector ( ev ) for 67 hours at 300 moi ( multiplicity of infection ). cells were then seeded at a density of 30 × 10 3 cells / well in 500 microliters egm - 2 medium with supplements ( biowhittaker ) in matrigel - coated plates and incubated at 37 ′ c for 24 hours and viewed using a nikon eclipse te200 microscope under a phase contrast and photographed . images were analyzed using software scion image ( scion corporation , frederick , md .) under the mode of integrated density . cell proliferation assay . hmvec proliferation was assessed by the cell titer - glo luminescent cell viability assay ( promega , madison , wis .) in 96 - well cell culture plates . hmvecs were seeded at 2 , 000 cells per well in 100 microliters medium and plates were incubated at 37 ′ c for 48 hours . reagent was added to each well according to manufacture &# 39 ; s instruction , and fluorescence was measured using the millipore cytofluor2350 . five independent endothelial cell populations were purified from glioma tumor tissue and normal brain tissue . in this study , the tissue defined as normal is derived from patients with epilepsy who have undergone a temporal lobectomy . the samples are summarized in table 3 . samples n1 , t1 and t2 were ultimately p1h12 - selected and samples n2 and t3 were uea - i selected . prior to sage analysis , each sample was assessed for the relative mrna abundance for vwf , glial fibrillary acidic protein ( gfap ) and ef1 by rt - pcr . abundant levels of vwf and the control housekeeper ef1 , and low levels of the glial cell - specific gene gfap suggested the cell population was primarily endothelial ( data not shown ). sage analysis was performed to a depth of approximately 50 , 000 tags ( table 3 ). for data analysis , each sage project was normalized to exactly 50 , 000 tags . pairwise comparisons between expression data derived from tumor samples selected with p1h12 or uea - i showed correlation coefficients around 80 %, slightly higher than a comparison between two tumor samples both selected with p1h12 . this suggests that selecting endothelial cells with either p1h12 or uea - i results in highly similar cell populations . moreover , nearly half of the tumor specific markers revealed in this study are induced 4 fold in each of the normal samples used , suggesting the normal samples are similar populations as well . with this in mind , we felt that combining data for the two normal samples and for the three tumor samples was appropriate . genes specific for endothelial cells showed expression levels consistent with the previously examined colon endothelial sage data ( table 4 ). additionally , markers specific for epithelial , hematopoeitic or glial cells showed limited or no expression in the brain endothelial libraries suggesting little contamination from non - endothelial cell populations ( table 4 ). finally , the data generated here allow for the derivation of alt gene ec prediction class of which 6 have been previously described as ec - specific ( huminiecki , l ., and bicknell , r . ( 2000 ). in silico cloning of novel endothelial - specific genes . genome res 10 , 1796 - 806 .) ( data not shown ). this provides further evidence of pure ec populations used for this study . genes expressed preferentially in glioma derived endothelial cells as opposed to normal endothelial cells are potentially involved in regulating angiogenesis - dependent tumor growth . specific parameters for the sorting of sage data and the layering of additional statistical filters allowed for a conservative estimate of legitimate differentially expressed genes ( see methods ). excluding mitochondrial genes , 131 genes were observed to be induced in the glioma endothelial cells based on a four fold induction ratio . only 14 genes can be entertained as glioma - specific when additional statistical filters are applied ( table 5 ). in this case , a two fold parameter family of distributions was used to establish a 90 % probability of observing at least a 2 fold difference in values . only one of these twelve genes , apolipoprotein d , shows higher expression in the stage iii glioma than at least one of the stage iv tumors . this suggests that many of the highly induced glioma endothelial genes revealed in this analysis may be involved in later stages of angiogenesis where the initiation of vascular sprouting has already occurred or are glioma type specific showing representation in the astrocytoma and not oligodendroglioma - derived ecs . less highly induced genes , or genes primarily induced in the less aggressive tumor stage , may be more reflective of angiogenesis initiation . several genes regulating extracellular matrix architecture are revealed as highly induced in this study . hspg2 ( perlecan ), several type iv collagen transcript variants , and matrix metalloprotease 14 ( mmp14 ) have all been shown to play a role in remodeling the extracellular matrix . interestingly , other genes that play roles in either cellular signaling or cell - cell communication are also highly expressed exclusively in glioma - associated endothelial cells . melanoma associated antigen ( mg50 ), endothelin receptor , the g - protein coupled receptor rdc - 1 , and integrin αv are all cell surface proteins previously demonstrated to play a role in signaling cascades . although the endothelin receptor , rdc - 1 and integrin αv have previously been shown to regulate angiogenesis , mg50 does not have an association with angiogenesis . moreover , mg50 was previously shown to be selectively associated with several types of tumor cells with a function yet to be defined . it is noteworthy that the p53 - induced , brain - specific angiogenesis inhibitor ( bai - 1 ) was expressed to significant levels but restricted to the earlier stage tumor present in this study ( data not shown ). it is possible that the loss of expression of bai - 1 in the later tumor stages reflects the need to more aggressively advance vascular development . other than the detection of a differential heyl sage tag , no other colon endothelial markers were observed to be preferentially expressed in the grade iii tumor . in total , of the 14 tumor induced genes listed , 12 are either present on the cell surface or secreted . the localization of the remaining two gene products has yet to be determined as these genes remain uncharacterized . finally , it is noteworthy that only a select few genes show significant (& gt ; 2 tags ) expression in a fetal brain library where angiogenesis is expected to be robust . in contrast to the highly biased localization of glioma - induced endothelial cell gene expression defined above , genes that are induced in the normal endothelial cells relative to glioma endothelial cells show a radically different cellular distribution . twenty - one genes are induced 4 fold or greater in the normal endothelial cells . filtering for genes with a 50 % or greater chance of having greater than 2 fold difference in transcript abundance reduces this list to 14 genes ( table 6 ). protein products predicted for these 14 genes show a range of cellular localizations with 4 gene products being intracellular , 5 being integral membrane proteins , 3 extracellular , and one each either secreted , on the cell surface or a nuclear membrane receptor . several of these genes have functions consistent with either tumor suppressor or anti - angiogenic functions . these anti - proliferative functions have been ascribed to the early growth response gene 1 ( egr1 ), btg2 , fruppel - like factor 4 ( klf4 ), and the serine protease inhibitor spint2 although associations with angiogenesis are limited to spint2 . the down - regulation of these genes in each of the three glioma tumors suggests that these genes may function to encode proteins with anti - angiogenic properties . both spint2 and btg2 are secreted and may act via paracrine mechanisms . also noteworthy is the preferential expression of the secreted protein mt1a as this metalothionein may serve as an antioxidant potentially attenuating dna damage within adjacent cells . interestingly , egr1 and klf4 ′ encode transcription factors suggesting that some part of the anti - angiogenic pathway revealed here may be initiated by these gene products . with the exception of mt1a , none of the above genes show differential expression in colon tumor ecs and may therefore be glioma - specific ec markers . the specificity of gene expression for tumor ec subtypes is important to define and can be addressed with the glioma ec data integrated with data obtained previously for colon ec populations . a limited number of genes are preferentially expressed in both brain and colon normal ec populations . in contrast , 16 genes were induced at least 4 fold in both colon and brain tumor ec fractions . 12 of these genes also met the criteria of having a greater than 50 % chance of being at least 2 fold differential ( table 7 ). the majority of these genes ( 7 ) are collagen transcripts . however , tumor endothalial marker 1 ( tem1 ), thy1 , and rdc - 1 also show consistent induction in the different tumor ec cells . this limited conservation of tumor - induced ec expression suggests highly specific ec expression profiles dependent on the tissue source . tem1 expression has been validated on tissue arrays harboring tissue slices from astrocytomas ( data not shown ). defining the specificity of gene expression to particular cell types can assist in determining function and designing therapeutics . our non - endothelial cell sage database currently contains 76 libraries encoding 255 , 000 unique sage transcripts . the epithelial cell lines derive from lung , ovary , kidney , prostate , breast , colon , pancreas . additional non - epithelial sources include cardiomyocytes , melanocytes , glioblastoma and monocytes . genes which show induction in glioma ecs and demonstrate a restricted expression in non - ec cells may be ideal targets for anti - angiogenic therapies . allowing for 1 or fewer tags in any non - ec library and at least a four - fold induction in glioma ecs yielded only 5 genes ( table 8 ). some of these genes are likely not ec - specific due to the relatively limited number of cell types included within the non - ec database . however , both pv - 1 and plexin a2 ( plxna2 ) are interesting genes with potential functional relevance to angiogenesis regulation . the sage tag that defines plxna2 falls outside of the current mrna boundaries residing 3 ′ of the ultimate exon . rt - pcr results , however , have confirmed transcription of mrna containing this tag in the tumor samples used to derive the sage data . plexins share homology with the scatter factor / hepatocyte growth factor ( sf / hgf ) family of receptors encoded by the met gene family [ tamagnone , l ., artigiani , s ., chen , h ., he , z ., ming , g . i ., song , h ., chedotal , a ., winberg , m . l ., goodman , c . s ., poo , m ., tessier - lavigne , m ., and comoglio , p . m . ( 1999 ). plexins are a large family of receptors for transmembrane , secreted , and gpi - anchored semaphorins in vertebrates . cell 99 , 71 - 80 .] earlier results have demonstrated a link between sf / hgf expression and increase tumorigencity [ bowers , d . c ., fan , s ., walter , k . a ., abounader , r ., williams , j . a ., rosen , e . m ., and laterra , j . ( 2000 ). scatter factor / hepatocyte growth factor protects against cytotoxic death in human glioblastoma via phosphatidylinositol 3 - kinase - and akt - dependent pathways . cancer res 60 , 4277 - 83 .] moreover , sf / hgf promotes this increased tumorigencity with concordant stimulation in angiogenesis [ lamszus , k ., laterra , j ., westphal , m ., and rosen , e . m . ( 1999 ). scatter factor / hepatocyte growth factor ( sf / hgf ) content and function in human gliomas . int j dev neurosci 17 , 517 - 30 .] in vivo targeting of sf / hgf was demonstrated to inhibit glioma growth and angiogenesis [ abounader , r ., lal , b ., luddy , c ., koe , g ., davidson , b ., rosen , e . m ., and laterra , j . ( 2002 ). in vivo targeting of sf / hgf and c - met expression via u1snrna / ribozymes inhibits glioma growth and angiogenesis and promotes apoptosis . faseb j 16 , 108 - 10 .]. plexins are known to function as coreceptors with neuropilin 1 functioning as a receptor for semaphorin and , in turn , regulating neuronal guidance and cell association [ tamagnone , 1999 , supra ]. as neuropilin - 1 and plexin association can serve to receive signals from semaphorins to guide neuronal growth , it is conceivable that a plexin - neuropilin association may regulate angiogenic growth in a manner analogous to kdr - neuropilin complexes signaling vegf responses . plexin a2 shows very low level expression in colon ecs and is not differentially induced in colon tumor ecs . it is noteoworthy that another plexin , plexin b2 ( plxnb2 ), also showed a five fold increase in glioma ec expression but did not make the statistical threshold demanded for table 8 . plexin b2 was previously shown to be differentially induced in brain tumors [ shinoura , n ., shamraj , o . i ., hugenholz , h ., zhu , j . g ., mcblack , p ., warnick , r ., tew , j . j ., wani , m . a ., and menon , a . g . ( 1995 ). identification and partial sequence of a cdna that is differentially expressed in human brain tumors . cancer lett 89 , 215 - 21 .] the upregulation of plexins in glioma ecs allows for a hypothesis whereby sf / hgf directly stimulates ec migration and proliferation . the novel discovery of a consistently upregulated level of plexin a2 in gliomas requires further evidence for a functional link between tumor levels of plexin a2 and angiogenesis regulation , particularly in the brain . pv - 1 ( also called plvap for plasmallema vesicle associated protein ), is a recently discovered type ii integral membrane glycoprotein shown to colocalize with caveolin - 1 . stan , r . v ., arden , k . c ., and palade , g . e . ( 2001 ). cdna and protein sequence , genomic organization , and analysis of cis regulatory elements of mouse and human plvap genes . genomics 72 , 304 - 13 . interestingly , this protein was the first to be shown to localize to the stomatal diaphragms and transendothelial channels within caveolae . the specific function of pv - 1 remains unknown . pv - 1 is expressed at substantial levels in colon ecs but is not expressed differentially between normal and tumor colon ecs . the upregulation of this caveolae - associated protein in gliomas may provide a means for specifically targeting glioma - associated endothelial cells as well as potentially providing a therapeutic delivery mechanism to the underlying tumorigenic cells ( marx , j . ( 2001 ). caveolae : a once - elusive structure gets some respect . science 294 , 1862 - 5 .)) from this study there is also the potential to define brain ec specific genes irrespective of function or differential expression in normal or tumor tissue . applying the same criteria as that applied for defining ec restricted glioma induced genes , only two genes , tnfα - induced protein 3 and junb , show consistent expression in the brain ec samples but severely limited expression in non - ec databases . the blood brain barrier within brain capillary endothelial cells results in a restricted diffusion of both small and large molecules as compared to non - brain ec junction complexes . as a result of this , brain capillary ecs facilitate molecular exchange via a tightly regulated , or catalyzed transport system . any differential expression of catalyzed membrane transporters between normal and tumor tissue may provide a means to selectively deliver therapies to tumor cells . the insulin receptor ( ir ) has been known for some time to be a marker for brain capillary ecs and to facilitate delivery of drugs . one of the most highly induced , glioma - specific genes in this study is the ir ( table 8 ). the high induction of ir transcripts in gliomas was not previously recognized and may provide a selective delivery mechanism to cancer cells as these receptors are also proposed to reside within caveolae structures [ smith , r . m ., jarret , l . ( 1988 ). lab . invest . 58 , 613 - 629 .] overall , very few transporters showed a differential induction in glioma - associated ecs as compared to their normal counterpart ( table 9 ). this is counter to previous suggestions linking altered expression of transporters with histologic grade of cns tumors [ guerin , c ., wolff , j . e ., laterra , j ., drewes , l . r ., brem , h ., and goldstein , g . w . ( 1992 ). vascular differentiation and glucose transporter expression in rat gliomas : effects of steroids . ann neurol 31 , 481 - 7 .] only one other gene , slc1a5 solute carrier family 1 member 5 ( neutral amino acid transporter ), showed a greater than 4 fold induction in glioma - derived ecs . it should be stated , however , that the standard sage tag for integrin αv is shared with aquaporin . long tag derivations of these two genes revealed that both integrin αv and aquaporin are induced in glioma ecs . aquaporin may play a role in caveolae swelling that accompanies vegf stimulated ec growth [ roberts , w . g ., and palade , g . e . ( 1997 ). neovasculature induced by vascular endothelial growth factor is fenestrated . cancer res 57 , 765 - 72 .] only one membrane transporter , na +/ k + transporting atp1a2 atpase , was reciprocally repressed in glioma - derived ecs . it remains possible that certain transporters were missed in this analysis due to incorrect functional assignment . nonetheless , the low number of differentially regulated transport facilitators suggests a small number of these genes need to be transcriptionally activated to accommodate any necessary increase in protein abundance required for tumor growth . table 10 shows genes induced in glioma endothelial cells but not in colon tumor or breast tumor endothelial cells . table 11 shows genes which encode transporters which are repressed in glioma endothelial cells . table 12 shows genes which encode proteins which are localized to the nucleus of both brain and colon tumor endothelial cells . table 13 shows genes which encode proteins which are localized to the cytoplasm of both brain and colon tumor endothelial cells . table 14 shows genes which encode proteins which are extracellular from both brain and colon tumor endothelial cells . table 15 shows genes which encode proteins which are localized to the membrane of both brain and colon tumor endothelial cells . table 16 shows genes which encode proteins which are induced in both brain and colon tumor endothelial cells . table 18 shows tumor endothelial markers in the brain which are cytoplasmic . table 19 shows tumor endothelial markers in the brain which are nuclear . table 20 shows tumor endothelial markers in the brain which are membrane associated . table 21 shows tumor endothelial markers in the brain which are extracellular . table 22 shows tumor endothelial markers in the brain which are unsorted with respect to cellular localization . abounader , r ., lal , b ., luddy , c ., koe , g ., davidson , b ., rosen , e . m ., and laterra , j . 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