Assessment of Antiangiogenic Effect Using 99mTc-EC-Endostatin

CANCER BIOTHERAPY amp RADIOPHARMACEUTICALSVolume 17 Number 2 2002copy Mary Ann Liebert Inc

Assessment of Antiangiogenic Effect Using 99mTc-EC-Endostatin

David J Yang12 Kil-Dong Kim3 Naomi R Schechter4 Dong-Fang Yu2 Peng Wu2

Ali Azhdarinia2 Jennifer S Roach3 Saady K Kalimi2 Kaoru Ozaki2 William E Fogler5

Jerry L Bryant2 Roy Herbst3 James Abbruzzes3 E Edmund Kim2 Donald A Podoloff2

Departments of Nuclear Medicine2 and ThoracicHead amp Neck Medical Oncology3 and RadiationOncology4 The University of Texas M D Anderson Cancer Center1ndash4 Houston Texas andEntreMed Inc5 Rockville Maryland

Purpose Tumor vascular density may provide a prognostic indicator of metastatic potential or survivalThe purpose of this study was to develop 99mTc-ethylenedicysteine-endostatin (99mTc-EC-endostatin) forthe evaluation of anti-angiogenesis therapy Method 99mTc-EC-endostatin was prepared by conjugatingethylenedicysteine (EC) to endostatin followed by adding pertechnetate and tin chloride Radiochemicalpurity was 95 In vitro cell viability affinity and TUNEL assays were performed Tissue distributionand planar imaging of radiolabeled endostatin were determined in tumor-bearing rats To assess anti-an-giogenic treatment response rats were treated with endostatin paclitaxel and saline followed by imag-ing with 99mTc-EC-endostatin Tumor response to endostatin therapy in tumor-bearing animal models wasassessed by correlating tumor uptake dose with microvessel density VEGF bFGF and IL-8 expressionduring endostatin therapy Results In vitro cell viability and TUNEL assays indicated no marked differ-ence between EC-endostatin and endostatin Cellular uptake assay suggests that endostatin binds to en-dostatin receptor Biodistribution of 99mTc-EC-endostatin in tumor-bearing rats showed increased tumor-to-tissue count density ratios as a function of time Tumor uptake (IDg) of 99mTc-EC-endostatin was02ndash05 Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-endostatinThe optimal time for imaging using radiolabeled endostatin was 2 hrs 99mTc-EC-endostatin could assesstreatment response There was a correlation between tumor uptake and cellular targets expression Con-clusion The results indicate that it is feasible to use 99mTc-EC-endostatin to assess efficiency of anti-an-giogenesis therapy

Key Words 99mTc-EC-endostatin antiangiogenesis biodistribution imaging

233

INTRODUCTION

Angiogenesis the proliferation of endothelial andsmooth muscle cells to form new blood vesselsis an essential component of the metastatic path-way These vessels provide the principal route by

which tumor cells exit the primary tumor site andenter the circulation For many tumors the vas-cular density can provide a prognostic indicatorof metastatic potential or survival with highlyvascularized tumors having a higher incidence ofmetastasis than poorly vascularized tumors1ndash3

It may be feasible to block angiogenesis andtumor progression by using anti-angiogenicagents At present antiangiogenic agents underclinical testing include naturally occurring in-hibitors of angiogenesis (eg angiostatin endo-statin platelet factor-4)4ndash7 specific inhibitors of

Address reprint requests to Dr David J Yang Universityof Texas M D Anderson Cancer Center Department ofNuclear Medicine Box 59 1515 Holcombe BoulevardHouston Texas 77030 Tel (713) 794-1053 Fax (713) 794-5456 E-mail dyangdimdacctmcedu

endothelial cell growth (eg TNP-470 thalido-mide interleukin-12)8ndash10 agents neutralizing an-giogenic peptides (eg antibodies to fibroblastgrowth factor or vascular endothelial growth fac-tor suramin and analogues tecogalan)11ndash12 ortheir receptors13 agents that interfere with vas-cular basement membrane and extracellular ma-trix (eg metalloprotease inhibitors angiostaticsteroids)14ndash16 anti-adhesion molecules17 anti-bodies such as anti-integrin avb318 and miscel-laneous drugs that modulate angiogenesis by di-verse mechanisms of action19

Malignant tumors are angiogenesis-dependentSeveral experimental studies suggest that primarytumor growth invasiveness and metastasis re-quire neovascularization20ndash22 Tumor-associatedangiogenesis is a complex multistep process un-der the control of positive and negative solublefactors Acquisition of the angiogenic phenotypeis a common pathway for tumor progression andactive angiogenesis is associated with molecularmechanisms leading to tumor progression23 Forinstance vascular endothelial growth factor(VEGF) is a mitogen morphogen and chemoat-tractant for endothelial cells and in vivo is apowerful mediator of vessel permeability24 In-terleukin-8 (IL-8) is a chemo-attractant for neu-trophils and is a potent angiogenic factor25 Ba-sic fibroblast growth factor (bFGF) has beenassociated with tumorigenesis and metastasis inseveral human cancers26 The prognostic value ofangiogenesis factor expression (eg VEGFbFGF microvessel density IL-8 MMP-2 andMMP-9) has been determined for cancer patientstreated with chemotherapy2728 These factorsregulate metastasis and angiogenesis and maypredict the metastatic potential in individual can-cer patients29

The use of anti-angiogenic therapy agents suchas endostatin represents one of the more promis-ing new approaches to anti-cancer therapy En-dostatin a fragment of the COOH-terminal do-main of mouse collagen XVIII is an endogenousinhibitor of tumor angiogenesis and endothelialcell growth Treatment of cow pulmonary arteryendothelial cells with endostatin has been shownto cause apoptosis Furthermore addition of en-dostatin led to a marked reduction of the Bcl-2and Bcl-XL anti-apoptotic proteins whereas Bax(pro-apoptotic) protein levels were unaffected5

These effects were not seen in several non-endo-thelial cells30ndash34 Though angiogenic factors re-flect angiogenesis status these agents may notadequately reflect the therapeutic response of tu-

mors Currently methods of assessing angiogen-esis in tumors rely on counting microvessel den-sity in the areas of neovascularization After tis-sue biopsy immunohistochemistry of tissuespecimen is performed The technique is invasiveand can not be repeatedly performed Thus thedevelopment of noninvasive imaging techniquesto measure anti-angiogenic effects would be awelcome contribution to the clinical arsenal

Radionuclide imaging modalities (positronemission tomography PET single photon emis-sion computed tomography SPECT) are diag-nostic cross-sectional imaging techniques thatmap the location and concentration of radionu-clide-labeled radiotracers Although computedtomography (CT) and magnetic resonance imag-ing (MRI) provide considerable anatomic infor-mation about the location and the extent of tumors these imaging modalities cannot ade-quately differentiate invasive lesions fromedema radiation necrosis grading or gliosisPET and SPECT have been used to localize and characterize tumors by measuring functionaland metabolic activities They have been also uti-lized in differentiating malignant and benign lesions evaluating and predicting therapeutic responses35ndash38

Due to favorable physical characteristics easyavailability and low price ($021mCi vs$50mCi of 18F) 99mTc is an agent of choice forlabeling radiopharmaceuticals 99mTc-ethylenedi-cysteine (99mTc-EC) is a successful example of astable N2S2 chelate39 Using a standard couplingtechnique 99mTc-EC-drug conjugates have beendeveloped to characterize tumors39ndash42 Thismethod may become a universal technique toevaluate various molecular targets for cancer di-agnosis

Athough endostatin causes endothelial apopto-sis5 the exact mechanism of endostatin bindinghas not been elucidated Our hypothesis statesthat endostatin may bind to endostatin receptorsand that this binding reflects therapeutic re-sponse If the uptake of tumor endothelial cellscan be assessed by 99mTc-EC-endostatin usingplanar scintigraphy this agent could be used toevaluate the efficiency of endostatin therapy forcancer Assessing angiogenesis in tumors with99mTc-EC-endostatin may prove to be a rationalmeans of selecting patients for treatment withanti-angiogenic agents In this report in vitro cel-lular uptake assay biodistribution imaging stud-ies and anti-angiogenic therapy studies using99mTc-EC-endostatin in mammary tumor-bearing

234

rats were evaluated Further tumor uptake andexpression of various angiogenic factors (tumoralVEGF bFGF IL-8 and microvessel density)were correlated

MATERIALS AND METHODS

Synthesis of LL-Ethylenedicysteine (EC)

EC was prepared in a two-step synthesis accord-ing to previously described methods25 Brieflythe precursor L-thiazolidine-4-carboxylic acid(mp 195deg reported 196-197deg) was synthesizedby reacting cysteine with formaldehyde EC (mp243degC reported 251degC) was then prepared via re-duction of L-thiazolidine-4-carboxylic acid inliquid ammonia and sodium

Synthesis of 99mTc-EC-endostatin

Sodium bicarbonate (1N 1 mL) was added to astirred solution of EC (391 mg 146 mmol) Tothis colorless solution sulfo-N-hydroxysulfosuc-cinimide (Pierce Chemical Co Radford IL) (313mg 146 mmol) and 1-ethyl-3-(3-dimethyl-aminopropyl) carbo-diimide (EDC 313 mg 19mmol) (Aldrich Chemical Co Milwaukee WI)were added Endostatin (Entremed IncRockville MD) (15 mg 075 mmol) was thenadded The mixture was stirred at room temper-ature for 24 hrs The mixture was dialyzed for 48hrs (MW cut-off of 10000) EC-endostatinweighed 17 mg (yield 100) after freeze dry-ing 99mTc-pertechnetate (55 mCi) (Syncor Phar-maceutical Inc Houston TX) was added to a vialcontaining the lyophilized residue of EC-endo-statin (10 mg 05 nmol) and tin chloride (II)(SnCl2 100 mg 053 mmol) in 02 mL water Theproduct was purified by using a sephadex G-25column (bedvolume 10 mL) (Sigma ChemicalCompany St Louis MO) and eluted with PBS(5 mL) One mL of eluent was collected in eachtest tube The product was isolated in Tubes 3and 4 yielded 39 mCi (70) Radiochemical purity was assessed by Radio-TLC scanner(Bioscan Washington DC) using 1M ammo-nium acetate methanol (41) as an eluant Highperformance liquid chromatograph (HPLC)equipped with a GPC column (Biosep SEC-S3000 78 3 300 mm Phenomenex TorranceCA) and two detectors (NaI and UV) was usedto analyze the purity of the product The eluantwas 01 LiBr in PBS (10 mM) and the flow ratewas 10 mLmin

The stability of labeled endostatin was testedin serum samples Briefly labeled endostatin(100 mCi) was incubated in dog serum (200 mL)at 37degC at 05ndash24 hrs The serum samples werediluted with 50 methanol in water and radio-TLC was used to analyze the product

In Vitro Cell Viability Studies

To determine whether EC-endostatin acts like en-dostatin in vitro potency was evaluated in RBACRL-1747 rat breast cancer cell line (AmericanType Culture Collection Rockville MD) Thiscell line was derived from a tumor induced in aFischer-344 rat by giving an oral dose of 712-dimethyl-benz[a]anthracene This cell line isknown to respond to endostatin treatment43 Cells(5000well 96-well plate) were treated with var-ious doses (01ndash100 mM) of EC-endostatin andendostatin The cells were incubated for 3 daysat 37degC Cell viability was determined by anMTT assay42

Immunofluorescence double staining deoxynu-cleotidyl transferase-mediated 29-deoxyuridine59-triphosphate-biotin nick end labeling (TUNEL)cell assay was used to evaluate EC-endostatin in-duced apoptosis Briefly sparse cultures of hu-man dermal microvascular endothelial cells-HD-MEC-nd (Cascade Biologics Portland OR) wereplated in 2-well chamber slides and allowed toattach for 24 hrs They were incubated over 18hrs at 37degC with 2 mgmL endostatin EC-endo-statin or EC alone TUNEL was performed usinga commercially available apoptosis detection kit(Promega Corporation Madison WI) A mount-ing medium-containing propidium iodide (Vec-tor Laboratories Burlingame CA) was used tocoverslip the samples Immunofluorescence mi-croscopy was performed using a 203-objective(Zeiss Plan-Neopluar) on an epifluorescence mi-croscope equipped with narrow bandpass excita-tion filters mounted in a filter wheel (Ludl Elec-tronic Products Hawthorne NY) to individuallyselect for green and red fluorescence Imageswere captured using a cooled CCD camera (Pho-tometrics Tucson AZ) and SmartCapture soft-ware (Digital Scientific Cambridge England) ona Macintosh computer Images were subse-quently processed using Adobe Photoshop soft-ware (Adobe Systems Mountain View CA) En-dothelial cells were identified by red fluorescenceand DNA fragmentation was detected by local-ized yellow and green fluorescence within the nu-cleus of apoptotic endothelial cells Quantifica-

235

tion of apoptotic endothelial cells was expressedas an average of apoptotic cells to the total num-ber of endothelial cells in 10 random 024 mm2

fields at 2003 magnification Triplicate assayswere performed and total TUNEL expression wasgiven as the average

In Vitro Cell Affinity Studies

Cellular binding was evaluated in the RBA CRL-1747 rat breast cancer cell line to determinewhether 99mTc-EC-endostatin binds to endostatinreceptors Cells (33000well) were treated withvarious doses (0ndash16 mMwell) of endostatin and99mTc-EC-endostatin (031 mMwell 01 mCiwell) After 2 hrs incubation the cells werewashed twice with ice cold PBS (1 mL) andtrypsin EDTA (01 mL) was added After 2 minPBS (04 mL) was added and the total volumecontaining cells was transferred to a test tube tocount the activity Cell viability was determinedby an MTT assay as described previously Eachdata represents an average of three measurementsand that were calculated as percentage of uptakeper number of viable cells

Tissue Distribution Studies

Eighteen female Fischer 344 rats (150 6 25 g)(Harlan Sprague-Dawley Indianapolis IN) (n 53 ratstime point) were inoculated (im) withmammary tumor cells derived from the RBACRL-1747 cell line The cells were cultured inEaglersquos MEM with Earlersquos BSS (90) and fetalbovine serum (10) Tumor cells (106 cellsrat)were injected (im) into the hind legs Studieswere performed 14 to 17 days after implantationwhen tumors were approximately 1 cm in diam-eter

In tissue distribution studies each animal wasinjected (iv 10 mCirat 10 mgrat) with 99mTc-EC-endostatin or 99mTc-EC Rats were sacrificedat 05ndash4 hrs The selected tissues were excisedweighed and counted for radioactivity by using agamma counter (Packard Instruments DownersGrove IL) The biodistribution of tracer in eachsample was calculated as percentage of the in-jected dose per gram of tissue wet weight(IDg)

Scintigraphic Imaging Studies

Scintigraphic images using a gamma camera(Siemens Medical Systems Inc Hoffman Es-tates IL) equipped with low-energy parallel-hole

collimator were obtained at 05ndash40 hrs after ivinjection of each radiotracer respectively To as-certain whether the tumor uptake by with 99mTc-EC-endostatin was related to endostatin receptorwe performed a blocking study Each rat was pre-treated with endostatin (n 5 3 50 mgkg iv) 1 hrprior to receiving 99mTc-EC-endostatin (300mCirat iv) and imaged at 05ndash40 hrs In a separate study rats (n 5 3) received 99mTc-EC-K1XaK2tPA (Entremed Rockville MD)K1XaK2tPA is a human plasminogen peptide withmolecular size of 20 kDa which is similar to en-dostatin44 This peptide was used as a non-specificcontrol Computer outlined regions of interest(ROI) (counts per pixel) were used to determinetumor-to-background count density ratios

Therapeutic Response Assessment

Mammary tumor-bearing rats (n 5 5 ratsagent)received (iv) endostatin (50 mgkg) paclitaxel(20 mgkg) or saline After one week tumor-bearing rats were imaged with 99mTc-EC-endo-statin at 05ndash40 hrs Computer outlined regionsof interest (ROI) (counts per pixel) of tumor le-sion site and symmetric normal muscle site wasused to determine tumor-to-background countdensity ratios The ratios were used to comparedynamic tumor uptake pre- and post-treatmentROI changes have been used as a standard to cor-respond to anatomically relevant features4546

In-Situ Hybridization (ISH) Assays

To determine whether tumor uptake of 99mTc-EC-endostatin correlates with mRNA expressionin the tumor specimen of rats ISH was per-formed47ndash50 ISH was carried out using the Mi-croprobe manual staining system (Fisher Scien-tific Pittsburgh PA) which detects hybridizationof a biotinylated DNA probe with mRNA Tis-sue sections (4 mm) of formalin-fixed paraffin-embedded specimens were mounted on silane-coated slides The slides were placed in theMicroprobe slide holder dewaxed and dehy-drated with alcohol followed by enzymatic di-gestion with pepsin51 Hybridization of the bio-tinylated DNA probe was carried out for 60 minat 45degC The samples were incubated for 30 minin alkaline phosphatase-labeled avidin at 45degCbriefly rinsed in 50 mM Tris buffer (pH 76)rinsed for 1 min with alkaline phosphatase en-hancer (Biomeda Corp Foster City CA) and in-cubated for 30 min with the chromogen substrateFastRed at 45degC A positive reaction in this as-

236

say stained red Control for endogenous alkalinephosphatase included treatment of the samples inthe absence of the biotinylated probe and use ofchromogen in the absence of any oligonucleotideprobes To check the specificity of the hy-bridization signal the following controls wereused (1) Rnase pretreatment of tissue sections(2) a biotin-labeled sense probe and (3) a com-petition assay with unlabeled antisense probe

To assess microvessel density changes52 anti-CD-31 immunohistochemical analysis was per-formed Briefly fresh frozen tumor specimenswere prepared and stained with anti-CD-31 anti-bodies The vessels were imaged with a fluores-cence microscope Vessel density was analyzedby quantifying PE-positive pixels per recordedfield Histopathology of VEGF microvessel den-sity IL-8 and bFGF from tumor specimen ob-tained on days 1 2 4 and 7 post-treatment of en-dostatin were performed

RESULTS

Chemistry

EC a dicarboxylic acid chelator may be conju-gated to amino or lysine residues of endostatinFrom the reaction yield (70ndash75) ten ECresidues was estimated to conjugate to endostatin(MW 20 kDa) 99mTc-EC-endostatin was foundto be radiochemically pure (97) (Figure 1)The retention time of 99mTc-EC-endostatin was107 min The amount of 99mTc-EC-endostatin in-jected to HPLC was 1 mCi per 225 ng The spe-cific activity was calculated to be 1 Cimmol Un-der the same condition the retention time of99mTc-EC and endostatin was 2 min and 103min Using radio-TLC analysis of 99mTc-EC-en-dostatin there was no difference in retardationfactor (Rf 5 04) at various incubation timessuggesting its in vivo stability (results not shown) Together these results suggest that this

237

Figure 1 HPLC analysis of 99mTc-EC-endostatin(Top Radioactive Bottom UV 254-nm) showed thathigh specific activity (1 Cimmol) 99mTc-EC-endo-statin could be prepared

methodology results in a simple and efficient syn-thesis of 99mTc-EC-endostatin

In Vitro Cell Viability and Cell Affinity Studies

Cell viability assays showed no marked differ-ence between EC-endostatin and endostatin (Fig-ure 2) At higher dose (100 mM) significantcell death was observed in both groups Therewas an unexpected drop in inhibition curvewhich might be due to experimental errorTUNEL assays showed that EC-endostatin andendostatin caused 1987 6 036 and 1943 6067 cell apoptosis respectively There was nomarked difference between EC-endostatin andendostatin groups (Figure 3)

In in vitro cell affinity assays there was amarkedly decreased uptake (20ndash50) of 99mTc-EC-endostatin after adding excess unlabeled en-dostatin (Table 1) We hypothesize that this de-creased uptake might be due to the specificbinding of endostatin to endostatin receptors

In Vivo Biodistribution Studies

Tumor-to-tissue uptake ratios for 99mTc-EC-endo-statin groups increased as a function of time whencompared to 99mTc-EC (Tables 2 and 3) The timefor the optimal uptake of endostatin in tumors was2ndash4 hrs postinjection of 99mTc-EC-endostatin Tu-mor uptake (IDG) in 99mTc-EC-endostatingroups was higher than 99mTc-EC (Table 3) Liveruptake in 99mTc-EC-endostatin groups was higherthan 99mTc-EC groups The difference in liver up-take may be due to different molecular weight(MW 20 kDa of endostatin vs MW 268 of EC)High kidney uptake was observed for both 99mTc-EC and 99mTc-EC-endostatin groups (92 6 005and 257 6 083IDg at 2 hrs respectively) pre-sumably because these agents are excreted throughthe kidney


Scintigraphic images of rats given 99mTc-EC-en-dostatin demonstrated that tumors could be well

238

Figure 2 MTT assays of viable cells indicated no markeddifference between endostatin (top) and EC-endostatin (bot-tom) at the drug concentrations tested Both agents showedsimilar percentage of cell inhibition at the concentrations(01ndash100 mM) tested

visualized at 05ndash40 hrs 14 days after inocula-tion of tumor cells (Figure 4) In vivo blockingstudies showed that there was 30ndash40 decreaseduptake after pretreament with unlabeled endo-statin in molar excess (data not shown) Also tu-mor had poor uptake (2ndash4 hrs) of 99mTc-EC-K1XaK2tPA a non-specific peptide (Figure 5)The findings suggest that tumor uptake of 99mTc-

EC-endostatin may be via an endostatin receptor-mediated process


A curve showing anti-rat mammary tumor growthactivity caused by paclitaxel and endostatin ispresented in Figure 6 Scintigraphic images of

239

Figure 3 Immunofluorescence double staining of apoptotic human dermal microvascular endothelial cells (TUNEL assay) in-dicated no marked difference between EC-endostatin and endostatin at the drug concentration (2 mgmL) tested EC-endostatinand endostatin induced apoptosis in 199 6 036 and 194 6 067 of the cells respectively

99mTc-EC-endostatin demonstrated that the tu-mor could be well visualized on day 7 post-treat-ment with endostatin and paclitaxel A selectedimage at 1 hour post-99mTc-EC-endostatin isshown in Figure 6 Tumor appeared to havehigher uptake of 99mTc-EC-endostatin in the con-trol (saline) group than in either of the groupstreated with endostatin or paclitaxel Using com-puter outlined ROI of the tumor lesion site andsymmetric non-tumor muscle site tumor to non-

tumor count density ratios for endostatin pacli-taxel and saline on day 7 post-treatment were calculated to be 240 6 003 238 6 003 and377 6 004 respectively The weights of tumorsat sacrifice were 55 6 033 g 31 6 011 g and79 6 042 g respectively There was 36 lessuptake of 99mTc-EC-endostatin by tumor in theendostatin-treated group vs the saline group(Figure 7) There was a correlation between thisdecreased uptake and decreased tumor volume

240


Immunohistochemical staining of tumor cross-sec-tion indicated reduced expression (by visualization)of tumor VEGF bFGF IL-8 and microvessel den-sity on day 2 post-endostatin treatment as comparedto pre-treatment (Figure 8)

DISCUSSION

Studies have suggested neovascularization as arequirement for primary tumor growth invasion

and metastasis Reports also give evidence thatacquisition of the angiogenic phenotype is a com-mon pathway for tumor progression and that active angiogenesis is associated with other mo-lecular mechanisms leading to tumor progres-sion20ndash23 Antiangiogenic therapy agents such asendostatin represent some of the more promis-ing new approaches to anticancer therapy Endo-statin causes apoptosis in endothelial cells hin-dering tumor progress Though measuringangiogenesis (blood vessel density) andor its

241

Figure 4 Scintigraphic images of mammary-tumor bearing rats following administration of 99mTc-EC-endostatin and 99mTc-EC (100 mCi10 mgrat iv) at 05ndash40 hrs on day 14 after inoculation of tumor cells Tumor located in right hind leg was wellvisualized with 99mTc-EC-endostatin

Figure 5 Scintigraphic images of mammary-tumor bearing rats following administration of99mTc-EC-K1XaK2tPA (100 mCi10 mgrat iv)at 05ndash40 hrs on day 14 after inoculation of tu-mor cells Tumor located in right hind leg wasvisualized at 05 hr but not at 2ndash4 hrs

main regulators such as IL-8 VEGF and bFGFin solid tumors may provide new and sensitivemarkers for tumor progression metastasis andprognosis the therapeutic response of tumors

may not be adequately reflected by these mea-surements These invasive methods also havelimitations in repeatedly obtaining specimens99mTc-EC-endostatin was synthesized and stud-

242

Figure 7 Scintigraphic images ofmammary-tumor bearing rats fol-lowing administration of 99mTc-EC-endostatin (10 mCi10 mgrat iv)at 1 hr on day 7 after treatment withendostatin (50 mgkg iv) pacli-taxel (20 mgkg) or saline (n 5 3per group)

Figure 6 Mammary tumor-bearing rats were dosed a single injection of endostatin (50 mgkg) or paclitaxel (20 and 40 mgkg)intravenously The weights of tumors at sacrifice on day 7 post-treatment were 55 6 033 g 31 6 011 g and 79 6 042 g re-spectively

ied for its potential as a noninvasive imagingtechnique for the evaluation and measurement oftumor response to antiangiogenic therapy

Despite stability of 99mTc-EC-endostatin inserum in vivo biodistribution data indicated highkidney uptake and bladder activity The findingssuggest that in vivo enzymatic hydrolysis of99mTc-EC-endostatin might have occurred Mo-lecular weight of the fragments of 99mTc-EC-en-dostatin is higher than 99mTc-EC Higher molec-ular weight may retain in kidney with a longertime thus 99mTc-EC-endostatin has higher kid-ney uptake than 99mTc-EC Liver uptake in99mTc-EC-endostatin groups was higher than99mTc-EC groups The difference in liver uptakemay be due to different molecular weight (MW20 kDa of endostatin vs MW 268 of EC) Highbackground in liver and kidneys for 99mTc-EC-endostatin may limit its clinical application inimaging tumors not in peripheral limbs

In vitro and in vivo cellular uptake studies sug-gest that endostatin may have its own receptoruptake mechanism The role of 99mTc-EC-endo-statin in imaging other tumors that overexpressendothelium activity needs to be evaluated Invivo studies revealed that endostatin was less po-tent than paclitaxel at the doses tested in thismodel The findings suggest that the combinationuse of endostatin with cytotoxic chemotherapeu-tic agents or radiation therapy may enhance theefficiency of endostatin therapy However radi-olabeled endostatin was found to be useful as abiological response marker in assessing endo-statin therapy Scintigraphic images showed goodvisualization of tumor as well as a correlationbetween tumor uptake and treatment effects De-creased tumor-to-nontumor uptake of radiola-beled endostatin following endostatin or pacli-taxel treatment correlated to decreased tumorvolume as well as decreased expression of an-

243

Figure 8 Immunohistochemical staining of tumor sectionsfrom mammary tumor-bearing rats treated with a single injec-tion of endostatin (50 mgkg) intravenously Tumor specimenswere excised at pre- and post-treatment (48 hrs later)

giogenic factors In other words the decrease intumor uptake of 99mTc-EC-endostatin signifiedeffective anti-tumor activity To ascertain whichpatients should be under endostatin therapy athreshold tumor uptake of 99mTc-EC-endostatinand therapeutic response of endostatin need to beevaluated and that may assist in prediction of ef-ficiency for endostatin therapy

In summary a convenient and simple methodfor radiolabeling endostatin to a high specific ac-tivity was achieved In vitro and in vivo cellularuptake and planar imaging studies demonstratedthe pharmacokinetic distribution and feasibilityof using this radiolabeled endostatin to image tu-mors Therapeutic response of endostatin and an-tiangiogenic (eg paclitaxel) therapy was as-sessed by using 99mTc-EC-endostatin There wasa positive correlation between tumor uptake andangiogenic factor expression The findings sug-gest that 99mTc-EC-endostatin a specific markerfor endothelial cells may be useful in assessingthe outcome of antiangiogenic treatment

ACKNOWLEDGMENTS

The authors wish to thank Eloise Daigle for hersecretarial support This work was supported inpart by the John S Dunn Foundation and En-treMed Research Fund (SR 00-248) The animalresearch is supported by M D Anderson CancerCenter (CORE) Grant NIH CA-16672

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2 Cao Y Therapeutic potentials of angiostatin in the treat-ment of cancer Haematologica 199984643ndash50

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5 Dhanabal M Ramchandran R Waterman MJ Lu HKnebelmann B Segal M and Sukhatme VP Endostatininduces endothelial cell apoptosis J Biol Chem199927411721ndash6

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7 Jouan V Canron X Alemany M Caen JP Quentin GPlouet J and Bikfalvi A Inhibition of in vitro angio-genesis by platelet factor-4-derived peptides and mech-anism of action Blood 199994984ndash93

8 Logothetis CJ Wu KK Finn LD Daliani D Figg WGhaddar H and Gutterman JU Phase I trial of the an-giogenesis inhibitor TNP-470 for progressive androgen-independent prostate cancer Clin Cancer Res 200171198ndash203

9 Moreira AL Friedlander DR Shif B Kaplan G and Za-gzag D Thalidomide and a thalidomide analogue in-hibit endothelial cell proliferation in vitro J Neuroon-col 199943109ndash14

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11 Bocci G Danesi R Benelli U Innocenti F Di Paolo AFogli S and Del Tacca M Inhibitory effect of suraminin rat models of angiogenesis in vitro and in vivo Can-cer Chemother Pharmacol 199943205ndash12

12 Sakamoto T Ishibashi T Kimura H Yoshikawa HSpee C Harris MS Hinton DR and Ryan SJ Effect oftecogalan sodium on angiogenesis in vitro by choroidalendothelial cells Invest Ophthalmol Vis Sci 1995361076ndash83

13 Pedram A Razandi M and Levin ER Natriuretic pep-tides suppress vascular endothelial cell growth factorsignaling to angiogenesis Endocrinology 20011421578ndash86

14 Lozonschi L Sunamura M Kobari M Egawa S DingL and Matsuno S Controlling tumor angiogenesis andmetastasis of C26 murine colon adenocarcinoma by anew matrix metalloproteinase inhibitor KB-R7785 intwo tumor models Cancer Res 1999591252ndash8

15 Maekawa R Maki H Yoshida H Hojo K Tanaka HWada T Uchida N Takeda Y Kasai H Okamoto HTsuzuki H Kambayashi Y Watanabe F Kawada KToda K Ohtani M Sugita K and Yoshioka T Correla-tion of antiangiogenic and antitumor efficacy of N-biphenyl sulfonyl-phenylalanine hydroxiamic acid(BPHA) an orally-active selective matrix metallopro-teinase inhibitor Cancer Res 1999591231ndash5

16 Banerjeei SK Zoubine MN Sarkar DK Weston APShah JH and Campbell DR 2-Methoxyestradiol blocksestrogen-induced rat pituitary tumor growth and tumorangiogenesis possible role of vascular endothelialgrowth factor Anticancer Res 2000202641ndash5

17 Liao F Li Y OrsquoConnor W Zanetta L Bassi R Santi-ago A Overholser J Hooper A Mignatti P Dejana EHicklin DJ and Bohlen P Monoclonal antibody to vas-cular endothelial-cadherin is a potent inhibitor of an-

244

giogenesis tumor growth and metastasis Cancer Res2000606805ndash10

18 Yeh CH Peng HC Yang RS and Huang TF Rhodos-tomin a snake venom disintegrin inhibits angiogene-sis elicited by basic fibroblast growth factor and sup-presses tumor growth by a selective alpha(v)beta(3)blockade of endothelial cells Mol Pharmacol 2001591333ndash42

19 Gasparini G The rationale and future potential of an-giogenesis inhibitors in neoplasia Drugs 19995817ndash38

20 Sion-Vardy N Fliss DM Prinsloo I Shoham-Vardi Iand Benharroch D Neoangiogenesis in squamous cellcarcinoma of the larynxmdashbiological and prognostic as-sociations Pathol Res Pract 20011971ndash5

21 Guang-Wu H Sunagawa M Jie-En L Shimada S GangZ Tokeshi Y and Kosugi T The relationship betweenmicrovessel density the expression of vascular endo-thelial growth factor (VEGF) and the extension of na-sopharyngeal carcinoma Laryngoscope 20001102066ndash9

22 Xiangming C Hokita S Natsugoe S Tanabe G BabaM Takao S Kuroshima K and Aikou T Angiogenesisas an unfavorable factor related to lymph node metas-tasis in early gastric cancer Ann Surg Oncol 19985585ndash9

23 Ugurel S Rappl G Tilgen W and Reinhold U Increasedserum concentration of angiogenic factors in malignantmelanoma patients correlates with tumor progressionand survival J Clin Oncol 200119577ndash83

24 Lamszus K Lengler U Schmidt NO Stavrou D ErgunS and Westphal M Vascular endothelial growth factorhepatocyte growth factorscatter factor basic fibroblastgrowth factor and placenta growth factor in humanmeningiomas and their relation to angiogenesis and ma-lignancy Neurosurgery 200046938ndash48

25 Petzelbauer P Watson CA Pfau SE and Pober JS IL-8 and angiogenesis evidence that human endothelialcells lack receptors and do not respond to IL-8 in vitroCytokine 19957267ndash72

26 Smith K Fox SB Whitehouse R Taylor M GreenallM Clarke J and Harris AL Upregulation of basic fi-broblast growth factor in breast carcinoma and its rela-tionship to vascular density oestrogen receptor epi-dermal growth factor receptor and survival Ann Oncol199910707ndash13

27 Inoue K Slaton JW Karashima T Yoshikawa C ShuinT Sweeney P Millikan R and Dinney CP The prog-nostic value of angiogenesis factor expression for pre-dicting recurrence and metastasis of bladder cancer af-ter neoadjuvant chemotherapy and radical cystectomyClin Cancer Res 200064866ndash73

28 Burian M Quint C and Neuchrist C Angiogenic fac-tors in laryngeal carcinomas do they have prognosticrelevance Acta Otolaryngol 1999119289ndash92

29 Slaton JW Inoue K Perrotte P El-Naggar AK Swan-son DA Fidler IJ and Dinney CP Expression levels ofgenes that regulate metastasis and angiogenesis corre-

late with advanced pathological stage of renal cell car-cinoma Am J Pathol 2001158735ndash43

30 Ramchandran R Dhanabal M Volk R Waterman MJSegal M Lu H Knebelmann B and Sukhatme VP An-tiangiogenic activity of restin NC10 domain of humancollagen XV comparison to endostatin Biochem Bio-phys Res Commun 1999255735ndash9

31 Szabo S and Sandor Z The diagnostic and prognosticvalue of tumor angiogenesis Eur J Surg Suppl 199858299ndash103

32 OrsquoReilly MS Boehm T Shing TY Fukai N Vasios GLane WS Flynn E Birkhead JR Olsen BR and Folk-man J Endostatin an endogenous inhibitor of angio-genesis and tumor growth Cell 199788277ndash85

33 Gibaldi M Regulating angiogenesis a new therapeuticstrategy J Clin Pharmacol 199838898ndash903

34 Zetter BR Angiogenesis and tumor metastasis AnnuRev Med 199849407ndash24

35 De Schepper AM De Beuckeleer L Vandevenne J andSomville J Magnetic resonance imaging of soft tissuetumors Eur Radiol 200010213ndash23

36 Nowak B Di Martino E Janicke S Cremerius U AdamG Zimny M Reinartz P and Bull U Diagnostic eval-uation of malignant head and neck cancer by F-18-FDGPET compared to CTMRI Nuklearmedizin 199938312ndash18

37 Kihlstrom L and Karlsson B Imaging changes after ra-diosurgery for vascular malformations functional tar-gets and tumors Neurosurg Clin N Am 199910167ndash80

38 Barker FG Chang SM Valk PE Pounds TR and Pra-dos MD18-Fluorodeoxyglucose uptake and survival ofpatients with suspected recurrent malignant gliomaCancer 199779115ndash26

39 Yang DJ Ilgan S Higuchi T Zareneyrizi F Oh CS LiuCW Kim EE and Podoloff DA Noninvasive assess-ment of tumor hypoxia with 99mTc-labeled metronida-zole Pharm Res 199916743ndash50

40 Ilgan S Yang DJ Higuchi T Zareneyrizi F Bayhan HYu DF Kim EE and Podoloff DA 99m Tc-Ethylenedi-cysteine-Folate A new tumor imaging agent Synthe-sis labeling and evaluation in animals Cancer Bio-therapy and Radiopharm 199813427ndash35

41 Zareneyrizi F Yang DJ Oh CS Ilgan S Yu DF TanseyW Liu CW Kim EE and Podoloff DA Synthesis of99mTc-ethylenedicysteine-colchicine for evaluation ofantiangiogenic efffect Anti-Cancer Drugs 199910685ndash92

42 Yang DJ Azhdarinia A Wu P Yu DF Tansey W Ko-hanim S Kim EE and Podoloff DA In vivo and in vitromeasurement of apoptosis in breast cancer cells using99mTc-EC- annexin V Cancer Biotherapy and Radio-pharmaceuticals 20011673ndash84

43 Perletti G Concari P Giardini R Marras E PiccininiF Folkman J and Chen L Antitumor activity of endo-statin against carcinogen-induced rat primary mammarytumors Cancer Res 2000601793ndash96

44 Sim BKL Fogler WE Zhou XH Liang H Madsen JW

245

Luu K OrsquoReilly MS Tomaszewski JE Fortier AHZinc ligand-disrupted recombinant human endostatinPotent inhibition of tumor growth safety and pharma-cokinetic profile Angiogenesis 1999341ndash51

45 Stodilka RZ Kemp BJ Prato FS Kertesz A Kuhl Dand Nicholson RL Scatter and attenuation correctionfor brain SPECT using attenuation distributions inferredfrom a head atlas J Nucl Med 2000411569ndash78

46 Kao CH ChangLai SP Chieng PU and Yen TC Tech-netium-99m methoxyisobutylisonitrile chest imaging ofsmall cell lung carcinoma relation to patient prognosisand chemotherapy responsemdasha preliminary reportCancer 19988364ndash8

47 Fasciani A DrsquoAmbrogio G Bocci G Monti M Genaz-zani AR and Artini PG High concentrations of the vas-cular endothelial growth factor and interleukin-8 inovarian endometriomata Mol Hum Reprod 2000650ndash4

48 Pataky DM Borisoff JF Fernandes KJ Tetzlaff W andSteeves JD Fibroblast growth factor treatment producesdifferential effects on survival and neurite outgrowth

from identified bulbospinal neurons in vitro Exp Neu-rol 2000163357ndash72

49 Radinsky R Bucana CD Ellis LM et al A rapid col-orimetric in situ messenger RNA hybridization tech-nique for analysis of epidermal growth factor receptorin paraffin-embedded surgical specimens of humancolon carcinomas Cancer Research 199353937ndash43

50 Bucana CD Fabra A Sanchez R et al Different pat-terns of macrophage infiltration into allogeneic-murineand xenogeneic-human neoplasms growing in nudemice American Journal of Pathology 19921411225ndash36

51 Pearson WR Lipman DJ Improved tools for biologi-cal sequence comparison Proceedings of the NationalAcademy of Sciences of the United States of America1988852444ndash48

52 Wild R Ramakrishnan S Sedgewick J and GriffioenAW Quantitative assessment of angiogenesis and tu-mor vessel architecture by computer-assisted digital im-age analysis effects of VEGF-toxin conjugate on tumormicrovessel density Microvasc Res 200059368ndash76

246

endothelial cell growth (eg TNP-470 thalido-mide interleukin-12)8ndash10 agents neutralizing an-giogenic peptides (eg antibodies to fibroblastgrowth factor or vascular endothelial growth fac-tor suramin and analogues tecogalan)11ndash12 ortheir receptors13 agents that interfere with vas-cular basement membrane and extracellular ma-trix (eg metalloprotease inhibitors angiostaticsteroids)14ndash16 anti-adhesion molecules17 anti-bodies such as anti-integrin avb318 and miscel-laneous drugs that modulate angiogenesis by di-verse mechanisms of action19

Malignant tumors are angiogenesis-dependentSeveral experimental studies suggest that primarytumor growth invasiveness and metastasis re-quire neovascularization20ndash22 Tumor-associatedangiogenesis is a complex multistep process un-der the control of positive and negative solublefactors Acquisition of the angiogenic phenotypeis a common pathway for tumor progression andactive angiogenesis is associated with molecularmechanisms leading to tumor progression23 Forinstance vascular endothelial growth factor(VEGF) is a mitogen morphogen and chemoat-tractant for endothelial cells and in vivo is apowerful mediator of vessel permeability24 In-terleukin-8 (IL-8) is a chemo-attractant for neu-trophils and is a potent angiogenic factor25 Ba-sic fibroblast growth factor (bFGF) has beenassociated with tumorigenesis and metastasis inseveral human cancers26 The prognostic value ofangiogenesis factor expression (eg VEGFbFGF microvessel density IL-8 MMP-2 andMMP-9) has been determined for cancer patientstreated with chemotherapy2728 These factorsregulate metastasis and angiogenesis and maypredict the metastatic potential in individual can-cer patients29

The use of anti-angiogenic therapy agents suchas endostatin represents one of the more promis-ing new approaches to anti-cancer therapy En-dostatin a fragment of the COOH-terminal do-main of mouse collagen XVIII is an endogenousinhibitor of tumor angiogenesis and endothelialcell growth Treatment of cow pulmonary arteryendothelial cells with endostatin has been shownto cause apoptosis Furthermore addition of en-dostatin led to a marked reduction of the Bcl-2and Bcl-XL anti-apoptotic proteins whereas Bax(pro-apoptotic) protein levels were unaffected5

These effects were not seen in several non-endo-thelial cells30ndash34 Though angiogenic factors re-flect angiogenesis status these agents may notadequately reflect the therapeutic response of tu-

mors Currently methods of assessing angiogen-esis in tumors rely on counting microvessel den-sity in the areas of neovascularization After tis-sue biopsy immunohistochemistry of tissuespecimen is performed The technique is invasiveand can not be repeatedly performed Thus thedevelopment of noninvasive imaging techniquesto measure anti-angiogenic effects would be awelcome contribution to the clinical arsenal

Radionuclide imaging modalities (positronemission tomography PET single photon emis-sion computed tomography SPECT) are diag-nostic cross-sectional imaging techniques thatmap the location and concentration of radionu-clide-labeled radiotracers Although computedtomography (CT) and magnetic resonance imag-ing (MRI) provide considerable anatomic infor-mation about the location and the extent of tumors these imaging modalities cannot ade-quately differentiate invasive lesions fromedema radiation necrosis grading or gliosisPET and SPECT have been used to localize and characterize tumors by measuring functionaland metabolic activities They have been also uti-lized in differentiating malignant and benign lesions evaluating and predicting therapeutic responses35ndash38

Due to favorable physical characteristics easyavailability and low price ($021mCi vs$50mCi of 18F) 99mTc is an agent of choice forlabeling radiopharmaceuticals 99mTc-ethylenedi-cysteine (99mTc-EC) is a successful example of astable N2S2 chelate39 Using a standard couplingtechnique 99mTc-EC-drug conjugates have beendeveloped to characterize tumors39ndash42 Thismethod may become a universal technique toevaluate various molecular targets for cancer di-agnosis

Athough endostatin causes endothelial apopto-sis5 the exact mechanism of endostatin bindinghas not been elucidated Our hypothesis statesthat endostatin may bind to endostatin receptorsand that this binding reflects therapeutic re-sponse If the uptake of tumor endothelial cellscan be assessed by 99mTc-EC-endostatin usingplanar scintigraphy this agent could be used toevaluate the efficiency of endostatin therapy forcancer Assessing angiogenesis in tumors with99mTc-EC-endostatin may prove to be a rationalmeans of selecting patients for treatment withanti-angiogenic agents In this report in vitro cel-lular uptake assay biodistribution imaging stud-ies and anti-angiogenic therapy studies using99mTc-EC-endostatin in mammary tumor-bearing

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Documents

Assessment of Antiangiogenic Effect Using 99mTc-EC-Endostatin