angieogenesis therapy using targeted delivery of pacclitaxel to

Research ArticleTumor Angiogenesis Therapy Using Targeted Delivery ofPaclitaxel to the Vasculature of Breast Cancer Metastases

Shijun Zhu1 Walter Kisiel2 Yang J Lu1 Lars C Petersen3

John M Ndungu4 Terry W Moore5 Ernest T Parker1 Aiming Sun4 Dennis C Liotta4

Bassel F El-Rayes1 Daniel J Brat6 James P Snyder4 and Mamoru Shoji1

1Department of Hematology and Medical Oncology Winship Cancer Institute Emory University 1365 Clifton RoadNE Atlanta GA 30322 USA2Department of Pathology University of New Mexico Health Sciences Center Albuquerque NM 87131 USA3Biopharmaceuticals Research Unit Novo Nordisk AS 2760 Maloslashv Denmark4Department of Chemistry Emory University 1515 Dickey Drive Atlanta GA 30322 USA5Department of Medicinal Chemistry and Pharmacognosy and UIC Cancer Center University of Illinois at ChicagoChicago IL 60612 USA6Department of Pathology Winship Cancer Institute Emory University 1365 Clifton Road NE Atlanta GA 30322 USA

Correspondence should be addressed to Mamoru Shoji mshojiemoryedu

Received 14 August 2014 Revised 30 October 2014 Accepted 30 October 2014 Published 7 December 2014

Academic Editor Guru V Betageri

Copyright copy 2014 Shijun Zhu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Breast cancer aberrantly expresses tissue factor (TF) in cancer tissues and cancer vascular endothelial cells (VECs) TF plays a centralrole in cancer angiogenesis growth andmetastasis and as such is a target for therapy and drug delivery TF is the cognate receptorof factorVIIa (fVIIa)We have coupled PTX (paclitaxel also namedTaxol) with a tripeptide phenylalanine-phenylalanine-argininechloromethyl ketone (FFRck) and conjugated it with fVIIaThe key aim of the work is to evaluate the antiangiogenic effects of PTX-FFRck-fVIIa against a PTX-resistant breast cancer cell line Matrigel mixed with VEGF andMDA-231 was injected subcutaneouslyinto the flank of athymic nude mice Animals were treated by tail vein injection of the PTX-FFRck-fVIIa conjugate unconjugatedPTX or PBS The PTX-FFRck-fVIIa conjugate significantly reduces microvessel density in matrigel (119901 lt 001ndash005) compared toPBS and unconjugated PTXThe breast cancer lungmetastasis model in athymic nudemice was developed by intravenous injectionof MDA-231 cells expressing luciferase Animals were similarly treated intravenously with the PTX-FFRck-fVIIa conjugate or PBSThe conjugate significantly inhibits lungmetastasis as compared to the control highlighting its potential to antagonize angiogenesisin metastatic carcinoma In conclusion PTX conjugated to fVIIa is a promising therapeutic approach for improving selective drugdelivery and inhibiting angiogenesis

1 Introduction

ScopeTheobjective is to selectively deliver a highly toxic drugto tumor cells and the related tumor vascular endotheliumThe target is tissue factor (TF) a membrane bound proteinaberrantly expressed on cancer cells and their endotheliaThe drug carrier is factor VIIa (fVIIa) the natural ligandof TF fVIIa is transformed to its competitive inhibitor byconjugation with paclitaxel-FFRck resulting in paclitaxel-(PTX-) FFRck-fVIIa The conjugation prevents thromboem-bolic complications associated with fVII administration and

provides an effective antiangiogenic approach that targets TF-expressing endothelia and cancers

Tissue factor (TF) is a 47 kDa transmembrane glycopro-tein receptor of factor VIIVIIa (fVIIa) a critical regulatorof tissue hemostasis and one of the bodyrsquos most potentprocoagulants [1] Under normal conditions TF is expressedby stromal cells outer blood vessel layers (smoothmuscle andadventitia) but not by vascular endothelial cells (VECs) (theinner most layer) Injury of the vascular wall causes TF tobind to its highly specific activating ligand fVIIa from theplasma to initiate thrombosis with subsequent hemostasis by

Hindawi Publishing CorporationJournal of Drug DeliveryVolume 2014 Article ID 865732 12 pageshttpdxdoiorg1011552014865732

2 Journal of Drug Delivery

generating a thrombinfibrin deposit [2 3] In this contextangiogenesis is a crucial process for tumor progression andmetastasis [4] In a study of 328 patients with breast cancertumor angiogenesis was assessed by microvessel density(MVD) to show that the prognosis of patients with MVDgreater than 100microvesselsmm2 in a microscope fieldwas significantly worse (119901 lt 00001) than the prognosisof patients with lower MVD [5] Nawroth and colleaguesdemonstrated that transfection of the cDNA of full lengthTF (flTF) in a sense orientation accelerated angiogenesisand tumor growth whereas that in the antisense cDNAorientation decreased angiogenesis and tumor growth [6]Phosphorylation of the serine residues on the cytoplasmicdomain of flTF by protein kinase C (PKC) [7] leads to VEGFproduction Furthermore deletion of the flTF cytoplasmicdomain impairs VEGF production inmelanomas [8] Clearlythe aberrant production and activation of TF are a deleteriousfactor in the angiogenic process

A variety of cancers show increased expression of TF byneoplastic cells and a direct correlation between TF levelsand tumor grade has been noted for multiple tumor types[9ndash12] TF has also been shown to be aberrantly expressed bythe VECs of cancer tissue a highly pathologic finding thatpromotes thrombosis In breast cancers TF is abnormallyexpressed by the endothelium in cancer lesions which ishighly pathologic as it promotes thrombosis

On the other hand it also provides a means to selectivelytarget tumor tissue [9ndash12] The aberrant expression of TF ontumor VECs induced by VEGF and its central role in angio-genesis provides a solid rationale for drug delivery [9 13ndash16] including targeting TF for neovascular-targeted therapysuch as immunotherapy and photodynamic therapy [17 18]

Given the high affinity of fVIIa for TF an enzy-matically inactivated form was developed by conjugatingphenylalanine-phenylalanine-arginine chloromethyl ketone(FFRck) to the enzymatic site of fVIIa [19ndash21] The resultingFFRck-fVIIa is unable to initiate blood clotting yet it has a 5-fold greater binding affinity to TF than native fVIIa [22] Thepotential of this particular delivery method can be maxi-mized by inserting highly potent but also very toxic anti-cancer drugs such as paclitaxel (PTX) into tumor cells specif-ically to reduce toxic effects distinct from the target area

The present study evaluates the in vivo selective drugdelivery and antiangiogenic effects of PTX conjugated toFFRck-fVIIa against a PTX-resistant breast cancer cell lineThe results suggest that the drug-conjugate is directed atendothelial and neoplastic cellsThus PTX-FFRck-fVIIa canblock the vicious cycle of thrombosis necrosis hypoxiaVEGF secretion tumor angiogenesis and invasion

2 Materials and Methods

The human breast cancer cell line MDA-MB-231-luc-D3H1containing firefly luciferase gene was purchased fromCaliperLifesciencesThe human head and neck squamous carcinoma(SCC) cell line Tu212 was provided by Dr Dong M Shin(Emory University Atlanta GA) Eaglersquos MEM mediumwith Earlersquo salt containing 2mM L-glutamine (Cat number10-010-CV) DMEMF12 (1 1) (Cat number 12-719F) and

inactivated fetal bovine serum (FBS) (Cat number 26140-079) were purchased from Mediatech Inc (Herndon VA20171) Lonza Group Ltd (Basel Switzerland) and GIBCO(Grand Island NY) respectively Paclitaxel was purchasedfrom Natland International Corporation (Research TrianglePark NC) Recombinant factor VIIa and FFRck-fVIIa (XXV-22W 265mg proteinmL) were kindly provided by Dr Wal-ter Kisiel and Dr Lars C Petersen (University of NewMexicoandNovoNordisk resp) Factor VII-deficient plasma (Prod-uct number 0700) was purchased from George King Bio-Medical Inc (Overland Park KS 66210-4192) Reagent 1 STANeoplastine Cl (freeze-dried thromboplastin prepared fromrabbit cerebral tissue) (Cat number 00605) and Reagent 2solvent containing calcium and sodium azide (Cat num-ber 00666) were purchased from Diagnostica Stago Inc(Parsippany-Troy Hills NJ 07054) Matrigel (Cat number356231 deficient in growth factors) was purchased from BDBiosciences (San Jose CA) Recombinant human VEGF-165 (VEGF-A) (10 120583gvial Catalog number 293 VE) waspurchased fromRampDMinneapolisMNAntibodies to vWF(von Willebrand factor) (Abcam Catalog number ab9378)rabbit anti-mouse tissue factor antibody (American Diag-nostica Inc Catalog number 4515) and anti-paclitaxel anti-body (Novos Biologicals Catalog number NBP1-05003) werepurchased from Abcam (ab9378) American DiagnosticaInc (ab4515) and Novos Biologicals respectively Kits forimmunohistochemical staining were purchased from AbcamHRPDAB (ABC) Detection IHC kit (ab64264) Femaleathymic nude mice (nunu) were purchased from Harlan(Indianapolis IN)

21 Cell Culture MDA-MB-231-luc-D3H1 breast cancer cells(triple-negative for ER PR and Her2) and Tu212 humansquamous carcinoma cells (SCC) were maintained in EaglersquosMEMmedium with Earlersquo salt containing 2mM L-glutamineand DMEMF12 (1 1) respectively containing 10 heatinactivated FBS penicillin (100 unitsmL) streptomycin(100 120583gmL) 01mMnonessential amino acid at 37∘C and 5CO295 air in a humidified atmosphere

22 Synthesis of Paclitaxel- (PTX-) FFRck and Conjuga-tion of PTX-FFRck to fVIIa The tripeptide phenylalanine-phenylalanine-arginine chloromethyl ketone (FFRck) groupis bound in an irreversible reaction to histidine 193 in the cat-alytic domain of fVIIa inactivating its serine protease activ-ity The FFRck-fVIIa functions as a competitive inhibitor offVIIa to tissue factor (TF) and has no coagulant activity [22]PTX was chemically coupled to the first phenylalanine of thechloromethyl ketone (FFRck) The resulting PTX-FFRck wasthen incubated together with fVIIa under gentle stirring at4∘C overnight to form PTX-FFRck-fVIIa conjugate as previ-ously described [23]

The conjugation procedure is essentially the same as thatdescribed for conjugation of EF24 [24ndash27] Briefly C21015840-PTX-FFRck in 100DMSOwas added dropwise to the fVIIa (MW50000) solution in a molar ratio of 3 1 at room temperaturefor 1-2 h while stirring followed by gentle stirring at 4∘Covernight C71015840-PTX-FFRck is less active than C21015840-PTX-FFRck C21015840 and C71015840 indicate the location of hydroxyl groups

Journal of Drug Delivery 3

O O

O

O

O

(a) (b)

O O

O

ONH

O

O

O

ONH NH

OH

OHOH

OH

OHAcO

AcOAcO

AcO

C2998400OH

C7998400OH

ArgPhe Phe

OCOPh OCOPh

Scheme 1 Paclitaxel (PTX) (a) At left the locations of C21015840OH and C71015840OH are labeled (b) at right the C21015840OHmoiety has been coupled witha glutamic acid linker and the corresponding Phe-Phe-Arg tripeptide to give C21015840-PTX-FFRck

(OH) on PTX to which PTX-FFRck is conjugated and illus-trated in Scheme 1 [23]The unconjugated excess PTX-FFRck(FW 1500) was removed by exhaustive dialysis at 4∘C usinga dialysis membrane with a pore size of 13000ndash15000MWto exclude molecules below 13000MW in 4 L of 10mM Tris-HCl pH 75 with several changes of the buffer every 12 hfor several days to ensure all unbound PTX-FFRck is dialyzedout

23 Determination of Residual fVIIa Activity of the PTX-FFRck-fVIIa Conjugate The PTX-FFRck-fVIIa conjugatecontaining trace amounts of fVIIa was diluted to 10minus4 and10minus5 for the assay Residual fVIIa activity in the PTX-FFRck-fVIIa conjugate was then measured according to the man-ufacturerrsquos instruction (Diagnostica STAGO Parsippany NJ)using an STArt 4 instrument (Diagnostica STAGOAsnieres-sur-Seine France) A standard fVIIa curve was plotted as logUmL versus clotting time for linear regression and residualfVIIa was determined from the standard curve

24 Inhibition by the PTX-FFRck-fVIIa Conjugate of MDA-MB-231 Cells and VEGF-A-Induced Angiogenesis in MatrigelPlugs inMice Prior to testing the efficacy on lungmetastasisa matrigel experiment was performed in mice to assess theinhibition of angiogenesis by the PTX-FFRck-fVIIa conju-gate induced byMDA-MB-231 cells andVEGF-AMDA-MB-231 cells produce high levels of both VEGF and TF [9 11] Amatrigel level of 06mLmouse containing 600 ng of VEGF-A and 06 times 106 MDA-MB-231-luc-D3H1 cells was implantedsubcutaneously in the flanks of female athymic nude miceOn days 13 14 and 15 after matrigel implantation (a) 01mLof PTX-FFRck-fVIIa conjugate (containing 13 120583g PTXequivalentmouse = 65120583g PTXkg body weight) (b) 01mLof unconjugated PTX (13120583gmouse) or (c) 01mL of PBS(vehicle) was administered intravenously into the tail veinOn day 19 matrigel plugs were collected and paraffin embed-ded Five mice per each regimen were treated Microves-sel density (MVD) per whole matrigel was determinedusing hematoxylin and eosin (HampE) stained slides countedby a blinded examiner Figure 2(a) is a representative schema

of the treatment plan and Figure 2(b) shows microvesselnumbers in matrigel in each treatment

25 Neutral Red Dye Cell Viability Assays The efficacy ofPTX-FFRck-fVIIa against various cell lines was tested in vitrousing neutral red (NR) dye cell viability assays as previouslydescribed [28] Cell viability following 48 h drug treatmentwas assayed by using the NR dye The latter is taken up byviable cells Briefly at the termination of culture existingmedium was removed and 200120583L of fresh warm mediumcontaining 50 120583g of NRmL was added to each well in a 96-well plate Cells were incubated at 37∘C for 30min followedby two washes with 200120583L of PBS The NR taken up by cellswas dissolved by adding 200 120583L of 05 N HCl containing 35ethanol Then the amount of the dye in each well was read at570 nmwith an EL800 Universal Microplate Reader (Bio-TekInstruments Inc Tustin CA) Each experiment was per-formed in triplicate and analyzed by Studentrsquos 119905-test Dose-response curves of PTX and PTX-FFRck-fVIIa were deter-mined using MDA-MB-231 breast cancer cells and Tu212SCC

26 Immunohistochemical Staining of TF of VECs in LungMetastasis and of PTX in Breast Cancer Xenografts in the LungVECs were stained using anti-von Willebrand Factor (vWF)antibody in 1 100 dilution TF was stained using anti-mouseTF antibody in 1 100 dilution PTX in the lungs was stainedusing paclitaxel antibody The Mouse and Rabbit SpecificHRPDAB detection IHC kit (Abcam ab64264) was usedaccording to manufacturerrsquos instructions HampE staining wasused for histological diagnosis

27 Lung Metastasis (Colonization) Lung metastases ofbreast cancer were developed by injecting iv the MDA-MB-231-luc-D3H1 cells (2 times 106 cells01mLmouse) containingluciferase gene into the tail vein of athymic nudemice Tumorcells are stuck in the capillaries before the first pass to theheart and randomly distributed in the lungs but no otherorgans Animals were treated with PTX-FFRck-fVIIa con-jugate (13 120583g PTX in PTX-FFRck-fVIIa01mL 20 g body


05

101520253035404550

001 01 1 10

(s)

(UmL)

fVIIa standard curve (FACT)

Figure 1 Standard curve of the fVIIa activity A standard curveof the fVIIa activity was plotted as log UmL versus clot time (inseconds) for linear regression UnitsmL for the unknowns weredetermined from the standard curve

Table 1 PTX-FFRck-fVIIa as a competitive inhibitor of factor VIIa

Factor VIIa activity Clotting time (insec)

inhibition of fVIIaactivity

fVIIa 143 0PTX-FFRck-fVIIa 375 932

weight) or PBS (01mL) iv on days 10 11 14ndash18 23ndash26 and28 following tumor cell inoculation (119899 = 10 each) Schematicrepresentation of the experiment is presented in Figure 4 Toassess the therapeutic response we followed the change ofthe luciferase activity on days 7 22 29 36 and 43 followinginoculation using the Xenogen box (Caliper LifesciencesInc) All protocols for animal studies were reviewed andapproved by the Institutional Animal Care and Use Com-mittee at Emory University To perform bioluminescenceimaging of tumors in the lungs the animals were anesthetizedby intraperitoneal injection of 01mLof amixture of 08mLofketamine from a vial containing 10mgmL 01mL of xylazinefrom a vial containing 100mgmL and 09mL of sterile water

28 Statistical Analysis Data obtained were analyzed usingone-way ANOVA Results are considered to be significantlydifferent when 119901 values are less than 005

3 Results

31 The PTX-FFRck-fVIIa Conjugate as an fVIIa InhibitorMeasurement of the fVIIa activity of the PTX-FFRck-fVIIapreparation revealed that the residual clot activity was lessthan 5 that of the unconjugated fVIIa control indicatingthat approximately 95 fVIIa was bound by PTX-FFRck andconverted to PTX-FFRck-fVIIa a competitive inhibitor offVIIa [23] (Table 1 and Figure 1)

32 Inhibition of MDA-MB-231 Cells and VEGF-A-InducedAngiogenesis in Matrigel Plugs in Mice by the PTX-FFRck-fVIIa Conjugate PTX-FFRck-fVIIa significantly reduced

MVD as compared with PBS or unconjugated PTX alonein matrigels containing MDA-MB-231 cells and VEGF-A(Figure 2) PBS and PTX alone (in a dose equivalent to theamount of PTX in PTX-FFRck-fVIIa) did not reduce MVDThe 119901 values of the MVD in matrigel treated with PBS PTXalone and PTX-FFRck-fVIIa (119899 = 5regimen) between PBSversus PTX PBS versus PTX-FFRck-fVIIa and PTX versusPTX-FFRck-fVIIa are 119901 lt 005 119901 lt 005 and 119901 lt 001respectively

33 Cytotoxic Activity of PTX-FFRck-fVIIa against MDA-MB-231 Breast Cancer Cells and Tu212 SCC Cells In VitroPTX and PTX-FFRck-fVIIa require over 1000 nM to killMDA-MB-231 cells but between 10 and 100 nM to kill Tu212cells 100 respectively Hence MDA-MB-231 cells are moreresistant to PTX and PTX-FFRck-fVIIa than Tu212 SCC(Figure 3) It is unclear why Tu212 cells are more sensitiveto PTX than MDA-MB-231 cells PTX is slightly better thanPTX-FFRck-fVIIa to MDA-MB-231 cells in vitro but PTX-FFRck-fVIIa is clearly more efficacious than PTX at thesame concentration because the PTX-FFRck-fVIIa conjugatebinds TF on VECs in vivo but PTX alone does not as shownin Figure 2

34 PTX-FFRck-fVIIa Inhibits TumorAngiogenesis andAtten-uates Growth of Drug-Resistant Breast Cancer Xenografts inthe Lung The experiments were performed to demonstratethat antitumor angiogenesis therapy may inhibit a drug-resistant tumor MDA-MB-231 cells are relatively resistantto PTX-FFRck-fVIIa (Figure 3) However the PTX-FFRck-fVIIa conjugate demonstrates antitumor angiogenesis activ-ity whereas PBS and PTX alone do not (Figure 2) Thereforewe compared the efficacy of PTX-FFRck-fVIIa and PBS invivo

Cells were intravenously injected into ten mice per eachregimen Three mice in the PBS-treated group died beforecompletion of the experiments possibly due to excessivetumor growth whereas none died in the PTX-FFRck-fVIIa-treated group In the PBS group tumor xenografts grew largerafter completion of treatment on day 28 in 6 of the 7 miceIn the PTX-FFRck-fVIIa group tumor growth in 7 of the 10mice was inhibited during the treatment period between day10 and day 28 andor thereafter (Figure 4)

35 Immunohistochemical (IHC) Staining of TF on VascularEndothelial Cells in Lung Metastasis TF expressed on theendothelium in the cancermilieu is the target of drug deliveryby its ligand fVIIa used as a drug carrier In Figure 5(a) TFis expressed on the tumor endothelium (single layer) whichcoincides with an endothelial marker vWF (Figure 5(b)) inthe lung xenografts of breast cancer

36 Paclitaxel Is Localized in Breast Cancer Tumor Xenograftsin the Lung To confirm the delivery of PTX by PTX-FFRck-fVIIa to themetastatic tumors localization of PTXwas deter-mined by IHC staining using anti-PTX antibody In Figure 6the HampE stain was included to demonstrate the presenceof tumor in this case predominantly around blood vessels


Experimental scheme

Day 191514130Matrigel Imaged Sacrificedinjection

darr darr darr

Tx PTX-FFRck-fVIIa iv (darr)

(a)

MDA-MB-231 and VEGFPBS PTX PTX-fVIIa

Mic

rove

ssel

num

bers

mat

rigel

0

5

10

15

20

25

30

35

(b)

Figure 2 PTX-FFRck-fVIIa inhibits angiogenesis in matrigels Matrigel containing MDA-MB-231 cells and VEGF-A was implanted scin the flanks of female athymic nude mice On days 13 14 and 15 after the matrigel implantation PBS PTX alone and PTX-FFRck-fVIIaconjugate were administered intravenously (iv) into the tail vein On day 19 matrigel plugs were collected and microvessel density (MVD)per wholematrigel stained with hematoxylin amp eosin was counted (a) Experimental scheme (b)Microvessel number inmatrigel treated withPBS PTX alone and PTX-FFRck-fVIIa (119899 = 5regimen) The 119901 values between PBS versus PTX PBS versus PTX-FFRck-fVIIa and PTXversus PTX-FFRck-fVIIa are 119901 lt 005 119901 lt 005 and 119901 lt 001 respectively

Effect of PTX and PTX-FFRck-fVIIa on MDA-MB-231 cells

0

20

40

60

80

100

120

1 10 100 1000 10000Concentration (nM)

Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(a)

Effect of PTX and PTX-FFRck-fVIIa on Tu212 SCC cells

10 100 10000

20

40

60

80

100

120

10Concentration (nM)

Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(b)

Figure 3 Cytotoxic activity of PTX-FFRck-fVIIa against MDA-MB-231 breast cancer cells and Tu212 SCC cells in vitro PTX and PTX-FFRck-fVIIa require over 1000 nM to kill MDA-MB-231 cells but between 10 and 100 nM to kill Tu212 cells 100 respectively

(Figures 6(a) and 6(b)) The HampE stain demonstrates thathuman breast cancer MDA-MB-231 cells are enlarged cellswith atypical nuclei and numerousmitoses around the vascu-lature of the lung (Figure 6(a)) There are also mononuclearinflammatory cells adjacent to blood vessel that are muchsmaller than breast cancer cells (Figure 6(b))

The cytoplasm of rare scattered tumor cells is faintlystained brown by anti-rabbit IgG immunoglobulin in con-trast to the cells stainedwith anti-PTX antibody which showsintense brown staining of the cytoplasmThe tumor grows asa solid mass within the lung with almost no viable bronchio-lar spaces present except for one region in which bronchiolarspaces are pushed to the periphery (Figure 6 panel magnified200x) We stained PTX in lung tissues harvested two weeksafter PTX-FFRck-fVIIa was discontinued

4 Discussion

In this paper we demonstrate that PTX-FFRck-fVIIa deliversPTX selectively to TF-expressing vascular endothelium andtumor metastases in the lungs Conjugating PTX to factorVIIa allows for delivery of the drug to TF-expressing VECsSince only tumor VECs express TF on the luminal surfacethis approach provides a selective delivery of PTX to tumorVECs The cytotoxic effects of PTX can inhibit VECs lead-ing to suppression of angiogenesis and tumor growth Inaddition the drug carrier FFRck-fVIIa is the competitiveinhibitor of factor VIIa and is expected to inhibit intravas-cular thrombosis caused by TF-expressing tumor vasculatureand circulating TF released from tumor Thus the drug-con-jugate not only is directed at endothelial and neoplastic cells


D7 D43D36D29D22 D7 D43D36D29D22 D7 D43D36D29D22





PBS treated (Tx) group (day 10 harr day 28 Tx) Three mice died out of ten during the treatment (see Figure 4(c))

Paclitaxel- (PTX-)FFRck-fVIIa treated (Tx) group (day 10 harr day 28 treated) None died (see Figure 4(c))

Mouse 752 Tx D10 harr D28 Mouse 755 Tx D10 harr D28 Mouse 760 Tx D10 harr D28




Mouse 763 Tx D10 harr D28 Mouse 775 Tx D10 harr D28 Mouse lost Tx D10 harr D28

Day0 7 10 22 29 36 444338

MDA231l ImagedImagedImaged

(a)

Imaged SacrificedImagedinoculation


darrdarrdarrdarrdarrdarrdarrdarrdarrdarrdarrdarr

11 14 15 16 17 18 23 24 25 26 28

(b)

Figure 4 Continued


Mouse 755

Mouse 760 Mouse 763

Mouse 775 Mouse 762

Mouse 752PBS treated group three mice died out of ten during the treatment L and R indicate left and right lungs of each mouse number

Day 7 Day 22 Day 29 Day 36 Day 44Day of imaging

752L752R


755L755R


760L760R


763L763R


(c)

775L775R


762L762R

000E + 00

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Mouse 765


765L765R

000E + 00

500E + 04

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350E + 05

Figure 4 Continued



751L751R


756L756R


771L771R


774L774R

PTX-FFRck-fVIIa-treated group none died out of ten mice during the treatmentMouse 751

000E + 00

200E + 04

400E + 04

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140E + 05Mouse 774

000E + 00

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772L772R


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758L758R

Day 7

(c)

Day 22 Day 29 Day 36 Day 44Day of imaging

770L770R

000E + 00

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Figure 4 Continued


Mouse 757 Mouse 754


757L757R


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000E + 00

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(c)

PTX-FFRck-fVIIa-treated group none died out of ten mice during the treatment

Figure 4 PTX-FFRck-fVIIa reduced breast cancer lung metastasis by inhibiting tumor angiogenesis On day 0 human breast cancer MDA-MB-231-luc-D3H1 cells containing luciferase gene were injected iv into the tail vein of female athymic nude mice Twelve treatments withPTX-FFRck-fVIIa and PBS were administered iv into the tail veins on days 10 11 14ndash18 23ndash26 and 28 following tumor cell inoculation(119899 = 10 miceregimen) Therapeutic response of the tumor xenografts was followed by imaging the luciferase activity (a) Experimentalscheme (b) Images of the luciferase activity of breast cancer xenografts in the lungs (c) Quantitation of the luciferase activity of (b)

(200x) (400x)

(a)

(b)

Figure 5 TF and vWF stains of endothelium in breast cancer lung metastases (a) TF staining using an anti-mouse TF antibody (arrowsindicate a single layer of endothelial cell stained darker brown) Photographs are 200x and 400x respectively (b) vWF staining using anti-vWF antibody (arrows indicate a single layer of endothelial cell stained darker brown) All photographs are 200x

but also blocks the vicious cycle of thrombosis necrosishypoxia VEGF secretion tumor angiogenesis and invasion

A major concern in patients with advanced cancer isthe increased risk of thrombosis Factor VIIa is a natural

procoagulant and an activator of the coagulation cascadeFFRck-fVIIa was initially developed as a potential anticoag-ulant given that it inhibits the binding of factor VIIa to tissuefactor Clinical experience has shown that use of FFRck-fVIIa


Anti-rabbit IgG only (400x)

Anti-PTX antibody (400x)

(a)

Rabbit anti-PTX antibody (200x)


(b)

Figure 6 Paclitaxel (PTX) staining in MDA-MB-231 lung metastasis The HampE stain demonstrates human breast cancer MDA-MB-231 cellssurrounding a blood vessel in the lung (a) and mononuclear cells adjacent to a blood vessel that are much smaller than breast cancer cells (b)The IHC stains demonstrate that the cytoplasm of rare scattered tumor cells is faintly stained brown by anti-rabbit IgG immunoglobulin incontrast to the cells stained with anti-PTX antibody which shows intense brown staining of the cytoplasm

is safe [29 30] The main complications observed in clinicaltrials were related to slight increased risk of bleeding whichis expected in anticoagulant therapy Therefore the use ofFFRck-fVIIa as an approach for drug delivery is feasiblein the clinical setting Since the drug-conjugate specificallydelivers drug to target cells where TF is concentrated thisapproach achieves a cytotoxic effect at a lower drug con-centration than required for an unconjugated drug In turnthis will limit the potential side effects of the cytotoxic agentIn this paper we demonstrate that the conjugated PTX hasgreater anti-angiogenic activity than an equivalent amountof PTX using the matrigel experiment This provides furthersupport that targeted deliverymay improve activity and lowertoxicity for standard cytotoxic agents

In many instances targeted therapy is used to target asingle signaling pathway This is not the case for cancer cellsand the endothelium in cancer Targeting a single pathway hasnot proved to be effective for extended treatment since tumorcells develop alternative signaling pathways to circumvent theinhibition by a single inhibitor or a combination of severalsingle signaling pathway inhibitors [31ndash33] Moreover thesesignaling-based therapies exhibit the same frequency andseverity of toxicities as traditional cytotoxic agents the maindifference being the nature of the side effects [34]

The targeting of PTX to TF-expressing VECs and neo-plastic cells is a highly specific approach TF is only expressedby pathologic blood vessels and is therefore an ideal targetThe use of the high affinity factor VII ligand for TF as


the targeting agent is employed to take advantage of one ofthe bodyrsquos most specific protein interactions It is anticipatedthat it will be generally useful for directing other therapies toTF-expressing cells

5 Conclusions

The efficacy of antiangiogenesis therapy was tested againstpaclitaxel-resistant MDA-MB-231-luc-D3H1 breast cancerxenografts in the lung Paclitaxel-FFRck-fVIIa was deliveredto both TF-expressing tumor vasculature and tumors Thetargeted delivery of the drug-conjugate was efficacious andsuppressed tumor growth of the drug-resistant breast cancerxenografts Treated animals did not demonstrate any bleedingtendency or signs of physical impairmentThis approachmaybe useful for controlling drug-resistant tumor metastasis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Anthea Hammond for critical readingand editing of the paper This research was supported by theUS Department of Defense Division of US Army DAMD17-00-1-0241 and W81XWH-08-1-0494 (Mamoru Shoji ShijunZhu Yang J Lu Terry W Moore John M Ndungu AimingSun and James P Snyder) the National Institutes of Health(NIH) Grant nos R21CA82995-01A1 1 R21 CA139035-01A1(Mamoru Shoji Daniel J Brat Shijun Zhu Terry W MooreJohn M Ndungu Aiming Sun and James P Snyder) andthe Emory Institute for Drug Development (TerryW MooreJohn M Ndungu Aiming Sun James P Snyder and DennisC Liotta)

References

[1] F R Rickles ldquoMechanisms of cancer-induced thrombosis incancerrdquo Pathophysiology of Haemostasis andThrombosis vol 35no 1-2 pp 103ndash110 2006

[2] E W Davie K Fujikawa and W Kisiel ldquoThe coagulationcascade initiation maintenance and regulationrdquo Biochemistryvol 30 no 43 pp 10363ndash10370 1991

[3] Y Nemerson ldquoTissue factor and hemostasisrdquo Blood vol 71 no1 pp 1ndash8 1988

[4] J Folkman ldquoAngiogenesis an organizing principle for drugdiscoveryrdquoNature ReviewsDrugDiscovery vol 6 no 4 pp 273ndash286 2007

[5] M Toi K Inada H Suzuki and T Tominaga ldquoTumor angio-genesis in breast cancer its importance as a prognostic indicatorand the association with vascular endothelial growth factorexpressionrdquo Breast Cancer Research and Treatment vol 36 no2 pp 193ndash204 1995

[6] Y Zhang Y Deng T Luther et al ldquoTissue factor controls thebalance of angiogenic and antiangiogenic properties of tumorcells in micerdquo Journal of Clinical Investigation vol 94 no 3 pp1320ndash1327 1994

[7] T F Zioncheck S Roy and G A Vehar ldquoThe cytoplasmicdomain of tissue factor is phosphorylated by a protein kinaseC-dependent mechanismrdquoThe Journal of Biological Chemistryvol 267 no 6 pp 3561ndash3564 1992

[8] K AbeM Shoji J Chen et al ldquoRegulation of vascular endothe-lial growth factor production and angiogenesis by the cytoplas-mic tail of tissue factorrdquo Proceedings of the National Academyof Sciences of the United States of America vol 96 no 15pp 8663ndash8668 1999

[9] M Shoji W W Hancock K Abe et al ldquoActivation of coagu-lation and angiogenesis in cancer immunohistochemical local-ization in situ of clotting proteins and vascular endothelialgrowth factorrdquoThe American Journal of Pathology vol 152 no2 pp 399ndash411 1998

[10] J Contrino G Hair D L Kreutzer and F R Rickles ldquoInsitu detection of tissue factor in vascular endothelial cellscorrelation with the malignant phenotype of human breastdiseaserdquo Nature Medicine vol 2 no 2 pp 209ndash215 1996

[11] J N Zhou S Ljungdahl M C Shoshan J Swedenborg andS Linder ldquoActivation of tissue-factor gene expression in breastcarcinoma cells by stimulation of the RAF-ERK signalingpathwayrdquoMolecular Carcinogenesis vol 21 pp 234ndash243 1998

[12] Y W van den Berg L G van den Hengel H R Myers et alldquoAlternatively spliced tissue factor induces angiogenesisthrough integrin ligationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 106 no 46 pp19497ndash19502 2009

[13] M Clauss M Gerlach H Gerlach et al ldquoVascular permeabilityfactor a tumor-derived polypeptide that induces endothelialcell and monocyte procoagulant activity and promotes mono-cyte migrationrdquo Journal of Experimental Medicine vol 172 no6 pp 1535ndash1545 1990

[14] S Zucker H Mirza C E Conner et al ldquoVascular endothelialgrowth factor induces tissue factor and matrix metallopro-teinase production in endothelial cells conversion of prothrom-bin to thrombin results in progelatinase A activation and cellproliferationrdquo International Journal of Cancer vol 75 pp 780ndash786 1998

[15] D Mechtcheriakova A Wlachos H Holzmuller B R Binderand E Hofer ldquoVascular endothelial cell growth factor-inducedtissue factor expression in endothelial cells is mediated by EGR-1rdquo Blood vol 93 no 11 pp 3811ndash3823 1999

[16] A L Armesilla E Lorenzo P G del Arco S Martınez-MartınezAAlfranca and JMRedondo ldquoVascular endothelialgrowth factor activates nuclear factor of activated T cells inhuman endothelial cells a role for tissue factor gene expressionrdquoMolecular and Cellular Biology vol 19 no 3 pp 2032ndash20431999

[17] Z Hu and A Garen ldquoTargeting tissue factor on tumor vascularendothelial cells and tumor cells for immunotherapy in mousemodels of prostatic cancerrdquoProceedings of theNational Academyof Sciences of the United States of America vol 98 no 21 pp12180ndash12185 2001

[18] F Lu ZHu J SinardAGaren andRAAdelman ldquoFactorVII-verteporfin for targeted photodynamic therapy in a rat modelof choroidal neovascularizationrdquo InvestigativeOphthalmologyampVisual Science vol 50 no 8 pp 3890ndash3896 2009

[19] G Schoellmann and E Shaw ldquoDirect evidence for the presenceof histidine in the active center of chymotrypsinrdquo Biochemistryvol 2 pp 252ndash255 1963


[20] C Kettner and E Shaw ldquo[63] Inactivation of trypsin-likeenzymes with peptides of arginine chloromethyl ketonerdquoMeth-ods in Enzymology vol 80 pp 826ndash842 1981

[21] E B Williams S Krishnaswamy and K G Mann ldquoZymogenenzyme discrimination using peptide chloromethyl ketonesrdquoJournal of Biological Chemistry vol 264 no 13 pp 7536ndash75451989

[22] B B Sorensen E Persson P O Freskgard et al ldquoIncorporationof an active site inhibitor in factor VIIa alters the affinity fortissue factorrdquo The Journal of Biological Chemistry vol 272 no18 pp 11863ndash11868 1997

[23] J M Ndungu Y J Lu S Zhu et al ldquoTargeted delivery ofpaclitaxel to tumor cells synthesis and in vitro evaluationrdquoJournal of Medicinal Chemistry vol 53 no 8 pp 3127ndash31322010

[24] A SunM Shoji Y J Lu D C Liotta and J P Snyder ldquoSynthesisof EF24-tripeptide chloromethyl ketone a novel curcumin-related anticancer drug delivery systemrdquo Journal of MedicinalChemistry vol 49 no 11 pp 3153ndash3158 2006

[25] M Shoji A Sun W Kisiel et al ldquoTargeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conju-gated to factor VIIardquo Journal of Drug Targeting vol 16 no 3pp 185ndash197 2008

[26] B K Adams E M Ferstl M C Davis et al ldquoSynthesis andbiological evaluation of novel curcumin analogs as anti-cancerand anti-angiogenesis agentsrdquo Bioorganic and Medicinal Chem-istry vol 12 no 14 pp 3871ndash3883 2004

[27] B K Adams J Cai J Armstrong et al ldquoEF24 a novel syntheticcurcumin analog induces apoptosis in cancer cells via a redox-dependent mechanismrdquo Anti-Cancer Drugs vol 16 no 3 pp263ndash275 2005

[28] S-Z Zhang M M Lipsky B F Trump and I-C Hsu ldquoNeutralred (NR) assay for cell viability and xenobiotic-induced cyto-toxicity in primary cultures of human and rat hepatocytesrdquo CellBiology and Toxicology vol 6 no 2 pp 219ndash234 1990

[29] E Erhardtsen P Nilsson M Johannessen and M S ThomsenldquoPharmacokinetics and safety of FFR-rFVIIa after single dosesin healthy subjectsrdquo Journal of Clinical Pharmacology vol 41no 8 pp 880ndash885 2001

[30] T Soderstrom U Hedner and B Arnljots ldquoActive site-inactivated factor VIIa prevents thrombosis without increasedsurgical bleeding topical and intravenous administration in arat model of deep arterial injuryrdquo Journal of Vascular Surgeryvol 33 no 5 pp 1072ndash1079 2001

[31] D Torti and L Trusolino ldquoOncogene addiction as a founda-tional rationale for targeted anti-cancer therapy promises andperilsrdquo EMBO Molecular Medicine vol 3 no 11 pp 623ndash6362011

[32] T M Brand M Iida and D L Wheeler ldquoMolecular mecha-nisms of resistance to the EGFR monoclonal antibody cetux-imabrdquoCancer BiologyampTherapy vol 11 no 9 pp 777ndash792 2011

[33] M De Palma and D Hanahan ldquoThe biology of personalizedcancer medicine facing individual complexities underlyinghallmark capabilitiesrdquoMolecular Oncology vol 6 no 2 pp 111ndash127 2012

[34] G K Dy and A A Adjei ldquoUnderstanding recognizing andmanaging toxicities of targeted anticancer therapiesrdquo A CancerJournal for Clinicians vol 63 no 4 pp 249ndash279 2013


generating a thrombinfibrin deposit [2 3] In this contextangiogenesis is a crucial process for tumor progression andmetastasis [4] In a study of 328 patients with breast cancertumor angiogenesis was assessed by microvessel density(MVD) to show that the prognosis of patients with MVDgreater than 100microvesselsmm2 in a microscope fieldwas significantly worse (119901 lt 00001) than the prognosisof patients with lower MVD [5] Nawroth and colleaguesdemonstrated that transfection of the cDNA of full lengthTF (flTF) in a sense orientation accelerated angiogenesisand tumor growth whereas that in the antisense cDNAorientation decreased angiogenesis and tumor growth [6]Phosphorylation of the serine residues on the cytoplasmicdomain of flTF by protein kinase C (PKC) [7] leads to VEGFproduction Furthermore deletion of the flTF cytoplasmicdomain impairs VEGF production inmelanomas [8] Clearlythe aberrant production and activation of TF are a deleteriousfactor in the angiogenic process

A variety of cancers show increased expression of TF byneoplastic cells and a direct correlation between TF levelsand tumor grade has been noted for multiple tumor types[9ndash12] TF has also been shown to be aberrantly expressed bythe VECs of cancer tissue a highly pathologic finding thatpromotes thrombosis In breast cancers TF is abnormallyexpressed by the endothelium in cancer lesions which ishighly pathologic as it promotes thrombosis

On the other hand it also provides a means to selectivelytarget tumor tissue [9ndash12] The aberrant expression of TF ontumor VECs induced by VEGF and its central role in angio-genesis provides a solid rationale for drug delivery [9 13ndash16] including targeting TF for neovascular-targeted therapysuch as immunotherapy and photodynamic therapy [17 18]

Given the high affinity of fVIIa for TF an enzy-matically inactivated form was developed by conjugatingphenylalanine-phenylalanine-arginine chloromethyl ketone(FFRck) to the enzymatic site of fVIIa [19ndash21] The resultingFFRck-fVIIa is unable to initiate blood clotting yet it has a 5-fold greater binding affinity to TF than native fVIIa [22] Thepotential of this particular delivery method can be maxi-mized by inserting highly potent but also very toxic anti-cancer drugs such as paclitaxel (PTX) into tumor cells specif-ically to reduce toxic effects distinct from the target area

The present study evaluates the in vivo selective drugdelivery and antiangiogenic effects of PTX conjugated toFFRck-fVIIa against a PTX-resistant breast cancer cell lineThe results suggest that the drug-conjugate is directed atendothelial and neoplastic cellsThus PTX-FFRck-fVIIa canblock the vicious cycle of thrombosis necrosis hypoxiaVEGF secretion tumor angiogenesis and invasion

2 Materials and Methods

The human breast cancer cell line MDA-MB-231-luc-D3H1containing firefly luciferase gene was purchased fromCaliperLifesciencesThe human head and neck squamous carcinoma(SCC) cell line Tu212 was provided by Dr Dong M Shin(Emory University Atlanta GA) Eaglersquos MEM mediumwith Earlersquo salt containing 2mM L-glutamine (Cat number10-010-CV) DMEMF12 (1 1) (Cat number 12-719F) and

inactivated fetal bovine serum (FBS) (Cat number 26140-079) were purchased from Mediatech Inc (Herndon VA20171) Lonza Group Ltd (Basel Switzerland) and GIBCO(Grand Island NY) respectively Paclitaxel was purchasedfrom Natland International Corporation (Research TrianglePark NC) Recombinant factor VIIa and FFRck-fVIIa (XXV-22W 265mg proteinmL) were kindly provided by Dr Wal-ter Kisiel and Dr Lars C Petersen (University of NewMexicoandNovoNordisk resp) Factor VII-deficient plasma (Prod-uct number 0700) was purchased from George King Bio-Medical Inc (Overland Park KS 66210-4192) Reagent 1 STANeoplastine Cl (freeze-dried thromboplastin prepared fromrabbit cerebral tissue) (Cat number 00605) and Reagent 2solvent containing calcium and sodium azide (Cat num-ber 00666) were purchased from Diagnostica Stago Inc(Parsippany-Troy Hills NJ 07054) Matrigel (Cat number356231 deficient in growth factors) was purchased from BDBiosciences (San Jose CA) Recombinant human VEGF-165 (VEGF-A) (10 120583gvial Catalog number 293 VE) waspurchased fromRampDMinneapolisMNAntibodies to vWF(von Willebrand factor) (Abcam Catalog number ab9378)rabbit anti-mouse tissue factor antibody (American Diag-nostica Inc Catalog number 4515) and anti-paclitaxel anti-body (Novos Biologicals Catalog number NBP1-05003) werepurchased from Abcam (ab9378) American DiagnosticaInc (ab4515) and Novos Biologicals respectively Kits forimmunohistochemical staining were purchased from AbcamHRPDAB (ABC) Detection IHC kit (ab64264) Femaleathymic nude mice (nunu) were purchased from Harlan(Indianapolis IN)

21 Cell Culture MDA-MB-231-luc-D3H1 breast cancer cells(triple-negative for ER PR and Her2) and Tu212 humansquamous carcinoma cells (SCC) were maintained in EaglersquosMEMmedium with Earlersquo salt containing 2mM L-glutamineand DMEMF12 (1 1) respectively containing 10 heatinactivated FBS penicillin (100 unitsmL) streptomycin(100 120583gmL) 01mMnonessential amino acid at 37∘C and 5CO295 air in a humidified atmosphere

22 Synthesis of Paclitaxel- (PTX-) FFRck and Conjuga-tion of PTX-FFRck to fVIIa The tripeptide phenylalanine-phenylalanine-arginine chloromethyl ketone (FFRck) groupis bound in an irreversible reaction to histidine 193 in the cat-alytic domain of fVIIa inactivating its serine protease activ-ity The FFRck-fVIIa functions as a competitive inhibitor offVIIa to tissue factor (TF) and has no coagulant activity [22]PTX was chemically coupled to the first phenylalanine of thechloromethyl ketone (FFRck) The resulting PTX-FFRck wasthen incubated together with fVIIa under gentle stirring at4∘C overnight to form PTX-FFRck-fVIIa conjugate as previ-ously described [23]

The conjugation procedure is essentially the same as thatdescribed for conjugation of EF24 [24ndash27] Briefly C21015840-PTX-FFRck in 100DMSOwas added dropwise to the fVIIa (MW50000) solution in a molar ratio of 3 1 at room temperaturefor 1-2 h while stirring followed by gentle stirring at 4∘Covernight C71015840-PTX-FFRck is less active than C21015840-PTX-FFRck C21015840 and C71015840 indicate the location of hydroxyl groups


O O

O

O

O

(a) (b)

O O

O

ONH

O

O

O

ONH NH

OH

OHOH

OH

OHAcO

AcOAcO

AcO

C2998400OH

C7998400OH

ArgPhe Phe

OCOPh OCOPh










05

101520253035404550

001 01 1 10

(s)

(UmL)









3 Results










Experimental scheme


darr darr darr


(a)


Mic

rove

ssel

num

bers

mat

rigel

0

5

10

15

20

25

30

35

(b)



0

20

40

60

80

100

120


Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(a)


10 100 10000

20

40

60

80

100

120


Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(b)




4 Discussion















Day0 7 10 22 29 36 444338


(a)




11 14 15 16 17 18 23 24 25 26 28

(b)

Figure 4 Continued


Mouse 755

Mouse 760 Mouse 763

Mouse 775 Mouse 762



752L752R


755L755R


760L760R


763L763R


(c)

775L775R


762L762R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

140E + 06

160E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

700E + 05

800E + 05

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

Mouse 765


765L765R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

Figure 4 Continued



751L751R


756L756R


771L771R


774L774R


000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

140E + 05Mouse 774

000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

Mouse 756 Mouse 771

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

900E + 04

Mouse 772 Mouse 773

Mouse 758 Mouse 770

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05


772L772R


773L773R


758L758R

Day 7

(c)


770L770R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

Figure 4 Continued


Mouse 757 Mouse 754


757L757R


754L754R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

500E + 02

100E + 03

150E + 03

200E + 03

250E + 03

300E + 03

(c)



(200x) (400x)

(a)

(b)








(a)



(b)







5 Conclusions




Acknowledgments


References





































O O

O

O

O

(a) (b)

O O

O

ONH

O

O

O

ONH NH

OH

OHOH

OH

OHAcO

AcOAcO

AcO

C2998400OH

C7998400OH

ArgPhe Phe

OCOPh OCOPh










05

101520253035404550

001 01 1 10

(s)

(UmL)









3 Results










Experimental scheme


darr darr darr


(a)


Mic

rove

ssel

num

bers

mat

rigel

0

5

10

15

20

25

30

35

(b)



0

20

40

60

80

100

120


Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(a)


10 100 10000

20

40

60

80

100

120


Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(b)




4 Discussion















Day0 7 10 22 29 36 444338


(a)




11 14 15 16 17 18 23 24 25 26 28

(b)

Figure 4 Continued


Mouse 755

Mouse 760 Mouse 763

Mouse 775 Mouse 762



752L752R


755L755R


760L760R


763L763R


(c)

775L775R


762L762R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

140E + 06

160E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

700E + 05

800E + 05

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

Mouse 765


765L765R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

Figure 4 Continued



751L751R


756L756R


771L771R


774L774R


000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

140E + 05Mouse 774

000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

Mouse 756 Mouse 771

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

900E + 04

Mouse 772 Mouse 773

Mouse 758 Mouse 770

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05


772L772R


773L773R


758L758R

Day 7

(c)


770L770R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

Figure 4 Continued


Mouse 757 Mouse 754


757L757R


754L754R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

500E + 02

100E + 03

150E + 03

200E + 03

250E + 03

300E + 03

(c)



(200x) (400x)

(a)

(b)








(a)



(b)







5 Conclusions




Acknowledgments


References





































05

101520253035404550

001 01 1 10

(s)

(UmL)









3 Results










Experimental scheme


darr darr darr


(a)


Mic

rove

ssel

num

bers

mat

rigel

0

5

10

15

20

25

30

35

(b)



0

20

40

60

80

100

120


Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(a)


10 100 10000

20

40

60

80

100

120


Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(b)




4 Discussion















Day0 7 10 22 29 36 444338


(a)




11 14 15 16 17 18 23 24 25 26 28

(b)

Figure 4 Continued


Mouse 755

Mouse 760 Mouse 763

Mouse 775 Mouse 762



752L752R


755L755R


760L760R


763L763R


(c)

775L775R


762L762R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

140E + 06

160E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

700E + 05

800E + 05

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

Mouse 765


765L765R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

Figure 4 Continued



751L751R


756L756R


771L771R


774L774R


000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

140E + 05Mouse 774

000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

Mouse 756 Mouse 771

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

900E + 04

Mouse 772 Mouse 773

Mouse 758 Mouse 770

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05


772L772R


773L773R


758L758R

Day 7

(c)


770L770R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

Figure 4 Continued


Mouse 757 Mouse 754


757L757R


754L754R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

500E + 02

100E + 03

150E + 03

200E + 03

250E + 03

300E + 03

(c)



(200x) (400x)

(a)

(b)








(a)



(b)







5 Conclusions




Acknowledgments


References





































Experimental scheme


darr darr darr


(a)


Mic

rove

ssel

num

bers

mat

rigel

0

5

10

15

20

25

30

35

(b)



0

20

40

60

80

100

120


Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(a)


10 100 10000

20

40

60

80

100

120


Cel

l via

bilit

y (

of s

urvi

val)

PTXPTX-FFRck-fVIIa

(b)




4 Discussion















Day0 7 10 22 29 36 444338


(a)




11 14 15 16 17 18 23 24 25 26 28

(b)

Figure 4 Continued


Mouse 755

Mouse 760 Mouse 763

Mouse 775 Mouse 762



752L752R


755L755R


760L760R


763L763R


(c)

775L775R


762L762R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

140E + 06

160E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

700E + 05

800E + 05

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

Mouse 765


765L765R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

Figure 4 Continued



751L751R


756L756R


771L771R


774L774R


000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

140E + 05Mouse 774

000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

Mouse 756 Mouse 771

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

900E + 04

Mouse 772 Mouse 773

Mouse 758 Mouse 770

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05


772L772R


773L773R


758L758R

Day 7

(c)


770L770R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

Figure 4 Continued


Mouse 757 Mouse 754


757L757R


754L754R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

500E + 02

100E + 03

150E + 03

200E + 03

250E + 03

300E + 03

(c)



(200x) (400x)

(a)

(b)








(a)



(b)







5 Conclusions




Acknowledgments


References

















































Day0 7 10 22 29 36 444338


(a)




11 14 15 16 17 18 23 24 25 26 28

(b)

Figure 4 Continued


Mouse 755

Mouse 760 Mouse 763

Mouse 775 Mouse 762



752L752R


755L755R


760L760R


763L763R


(c)

775L775R


762L762R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

140E + 06

160E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

700E + 05

800E + 05

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

Mouse 765


765L765R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

Figure 4 Continued



751L751R


756L756R


771L771R


774L774R


000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

140E + 05Mouse 774

000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

Mouse 756 Mouse 771

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

900E + 04

Mouse 772 Mouse 773

Mouse 758 Mouse 770

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05


772L772R


773L773R


758L758R

Day 7

(c)


770L770R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

Figure 4 Continued


Mouse 757 Mouse 754


757L757R


754L754R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

500E + 02

100E + 03

150E + 03

200E + 03

250E + 03

300E + 03

(c)



(200x) (400x)

(a)

(b)








(a)



(b)







5 Conclusions




Acknowledgments


References





































Mouse 755

Mouse 760 Mouse 763

Mouse 775 Mouse 762



752L752R


755L755R


760L760R


763L763R


(c)

775L775R


762L762R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

140E + 06

160E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

700E + 05

800E + 05

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

Mouse 765


765L765R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

Figure 4 Continued



751L751R


756L756R


771L771R


774L774R


000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

140E + 05Mouse 774

000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

Mouse 756 Mouse 771

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

900E + 04

Mouse 772 Mouse 773

Mouse 758 Mouse 770

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05


772L772R


773L773R


758L758R

Day 7

(c)


770L770R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

Figure 4 Continued


Mouse 757 Mouse 754


757L757R


754L754R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

500E + 02

100E + 03

150E + 03

200E + 03

250E + 03

300E + 03

(c)



(200x) (400x)

(a)

(b)








(a)



(b)







5 Conclusions




Acknowledgments


References






































751L751R


756L756R


771L771R


774L774R


000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

140E + 05Mouse 774

000E + 00

200E + 04

400E + 04

600E + 04

800E + 04

100E + 05

120E + 05

Mouse 756 Mouse 771

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

000E + 00

100E + 04

200E + 04

300E + 04

400E + 04

500E + 04

600E + 04

700E + 04

800E + 04

900E + 04

Mouse 772 Mouse 773

Mouse 758 Mouse 770

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05


772L772R


773L773R


758L758R

Day 7

(c)


770L770R

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

200E + 05

400E + 05

600E + 05

800E + 05

100E + 06

120E + 06

000E + 00

100E + 05

200E + 05

300E + 05

400E + 05

500E + 05

600E + 05

Figure 4 Continued


Mouse 757 Mouse 754


757L757R


754L754R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

500E + 02

100E + 03

150E + 03

200E + 03

250E + 03

300E + 03

(c)



(200x) (400x)

(a)

(b)








(a)



(b)







5 Conclusions




Acknowledgments


References





































Mouse 757 Mouse 754


757L757R


754L754R

000E + 00

500E + 04

100E + 05

150E + 05

200E + 05

250E + 05

300E + 05

350E + 05

400E + 05

450E + 05

500E + 05

000E + 00

500E + 02

100E + 03

150E + 03

200E + 03

250E + 03

300E + 03

(c)



(200x) (400x)

(a)

(b)








(a)



(b)







5 Conclusions




Acknowledgments


References







































(a)



(b)







5 Conclusions




Acknowledgments


References






































5 Conclusions




Acknowledgments


References




































angieogenesis therapy using targeted delivery of pacclitaxel to

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