research article patched targeting peptides for...

Research ArticlePatched Targeting Peptides for Imaging and Treatment ofHedgehog Positive Breast Tumors

Daniel Smith1 Fanlin Kong2 David Yang2 Richard Larson1

Jennifer Sims-Mourtada3 and Wendy A Woodward1

1 Department of Radiation Oncology The University of Texas MD Anderson Cancer Center Houston TX 77030 USA2Department of Cancer Systems Imaging The University of Texas MD Anderson Cancer Center Houston TX 77030 USA3 Center for Translational Cancer Research Helen F Graham Cancer Center and Research Institute Christiana Care Health System4701 Ogletown-Stanton Road Newark DE 19713 USA

Correspondence should be addressed to Jennifer Sims-Mourtada jsimsmourtadachristianacareorg

Received 28 May 2014 Revised 11 July 2014 Accepted 15 July 2014 Published 8 September 2014

Academic Editor Mei-Hsiu Liao

Copyright copy 2014 Daniel Smith 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

High tumor hedgehog expression is correlated with poor prognosis in invasive ductal carcinoma Peptides which bind the patchedreceptor have recently been reported to have a growth inhibitory effect in tumors with activated hedgehog signaling We soughtto examine growth inhibition with these peptides in breast cancer cells and use these peptides as molecular imaging probes tofollow changes in hedgehog expression after chemotherapy Significant growth inhibition was observed in breast cancer cell linestreated with PTCH-blocking peptides Significant in vitro uptake was observed with both FITC- and 99mTc-EC-peptide conjugatesIn vivo imaging studies displayed greater accumulation of 99mTc-labeled peptides within tumors as compared to adjacent muscletissue Patched receptor expression increased after treatment and this correlatedwith an increase in tumor radiotracer uptakeThesestudies suggest that peptides which bind the sonic hedgehog docking site in patched receptor correlate with patched expression andcan be used to image patched in vivo Further our data suggest that radiolabeled peptides may enable us to examine the activity ofthe hedgehog signaling pathway and to evaluate response to anti-cancer therapies

1 Introduction

The hedgehog (Hh) signaling pathway plays a critical role inembryonic development and wound healing and its aberrantactivity is associated with several malignancies Recent stud-ies implicateHh signaling in breast cancer growth andmetas-tasis and high tumor sonic hedgehog (SHh) expression iscorrelated with poor prognosis in invasive ductal carcinomaSHh binds to the suppressive receptor patched-1 (PTCH-1) and relieves the inhibition of the transmembrane proteinsmoothened (Smo) by PTCH-1 resulting in the translocationof Gli transcription factors to the nucleus and activation ofHh target genes In tumors with activated Hh signaling highlevels of PTCH-1 have been reported especially within thetumor stroma

Previously we demonstrated strong detection of tumorxenografts using an iodinated derivative of the PTCH-1

binding ligand sonic hedgehog [1] Although this agent wascapable of delineating tumor tissue its clinical utility islimited due to poor stability and pharmacokinetics Imagingwith radiolabeled peptides has been shown to improvepharmacokinetics and the targeting of other tumor-basedreceptors Therefore we sought to develop radiolabeled pep-tides which dock inside the PTCH receptor Nakamura et alpreviously reported the synthesis of several peptides targetingthe PTCH-1 receptor [2] These peptides were shown to bindto the PTCH-1 receptor on the surface of pancreatic tumorsand decrease tumor growth

Here we selected technetium-99m (99mTc) as the radi-oisotope because of its favorable physical characteristics fordiagnostic imaging studies and due to the ease of using itsbenchtop generator-based system for clinical applications Itemits 140 keV gamma ray with an 89 branching fraction

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 525680 9 pageshttpdxdoiorg1011552014525680

2 BioMed Research International

which can be detected by single photon emission computedtomography (SPECT) In addition the half-life of 99mTcis relatively long (602 h) compared to most nuclear imag-ing radioisotopes which facilitates serial imaging that mayimprove the differentiation of tumor from inflammation Tolabel the peptide with 99mTc the chelator NN1015840-ethylene-di-L-cysteine (EC) is selected and used as a linker EC isknown to chelate 99mTc stably owing to the efficient bindingof the oxotechnetium group to the two thiols and two aminenitrogen atoms of EC

Here we report the radiolabeling of these peptides todetect the PTCH receptor on breast cancer cells and breastcancer stem cell-enriched populations These molecularimaging probes have the potential to identify Hh-inducedchanges in PTCH-1 expression which is useful for theimaging of aberrant Hh signaling in malignancies

2 Methods

21 Peptides PTCH-binding peptides Amdashsequence FAPVL-DGAVSTLLGVmdash and Bmdashsequence DNTRYSPPPPYSSHSmdashwere commercially synthesized with or without an N-ter-minal FITC-Ahx modification (GenScript Piscataway NJ)Peptides were resuspended at a stock concentration of200120583M in 10 DMSO in deionized water

22 Synthesis and Radiolabeling of PTCH Ethylenedicysteine(EC) was selected as a chelator for PTCH conjugationSodium bicarbonate (1 N 1mL) was added to a stirredsolution of EC (5mg 0019mmol) To this colorless solutionsulfo-NHS (4mg 0019mmol) and EC (5mg 0019mmol)were added PTCH (03mg) was then added The mixturewas stirred at room temperature for 24 hours The mixturewas dialyzed for 48 hours with a cutoff at molecular weight10000Da After dialysis the product was freeze-dried withthe product in the salt form weighing 05mg99mTc-pertechnetate was obtained from Mallinckrodt

(Houston TX) Radiosynthesis of 99mTc-EC-PTCH wasachieved by adding the required amount of 99mTc-pertechnetate into EC-PTCH (01mg) and tin chloride(II) (SnCl

2 100mg) The mixture was loaded on a sephadex

gel column (PD-10 G-25) (Sigma Chemical Company StLouis MO) and eluted with phosphate-buffered saline (pH74) Onemilliliter of each fractionwas collectedThe productwas collected at fraction 3 with a 70 yield Radiochemicalpurity was assessed by Radio-TLC (BioScan WashingtonDC) using saline as an eluant

23 Cell Lines and Culture Conditions The human cell linesT47-D SKBR3 andMCF-7were obtained from theAmericanType Tissue Company (ATCC) and cultured in DMEMwith 10 fetal bovine serum (Atlanta Biologicals FloweryBranch GA) and 1 antibiotic-antimycotic (Invitrogen LifeTechnologies Grand Island NY Life Technologies GrandIsland NY) The human cell line SUM159 was obtained fromAsterand (Detroit MI) and cultured in DMEM contain-ing 1 120583gmL hydrocortisone (Invitrogen Life TechnologiesGrand Island NY Life Technologies Grand Island NY)

5 120583gmL insulin (Invitrogen Life Technologies Grand IslandNY) and 1 antibioticantimycoticThe rat breast cancer cellline 13762 was derived from a tumor induced in a Fischer-344rat by giving an oral dose of 712-dimethylbenz[a]anthracene[3] and the cells were cultured in RPMI-1640 mediumsupplemented with 10 (vol vol) fetal bovine serum and1 antibiotic-antimycotic For mammosphere assays cellswere cultured in MEM media supplemented with 1X B27(Invitrogen Life Technologies Grand Island NY) 20 ngmLepidermal growth factor (EGF Invitrogen Life TechnologiesGrand Island NY) and 20 ngmL basic fibroblast growthfactor (bFGF Invitrogen Life Technologies Grand IslandNY) and seeded into ultralow attachment plates (CorningLife Sciences Salt Lake City UT) Cells were grown for 7ndash10 days and spheres were obtained All cells were culturedat 37∘C in a humidified atmosphere containing 5 carbondioxide

24 Survival Assays Breast cancer cell lines were seededinto 96-well plates at a density of 5000ndash7000 cells perwell Cells were grown overnight and media were replacedwith culture media containing unlabeled peptides A andB at the indicated concentrations Cells were cultured foran additional 48 hours and survival was determined usingthe MTT-based Cell Proliferation Assay (Biotium) Data isexpressed as treateduntreated

25 Fluorescence Microscopy Breast cancer cell lines wereseeded onto chamber slides (Nunc Roskilde Denmark) andgrown overnight For sphere assays cells were seeded in 3Dmedia as described above in 96-well low attachment platesat a density of 100ndash1000 cells per well and cultured for 7ndash10 days until spheres were formed Spheres were filteredusing a cell strainer and replated into low attachment platesCells or spheres were treated with 100 nM peptide A or Band incubated for 2 hours Media were removed and cellsor spheres were washed two times with 1X PBS Followingwashes 1mL of 1X PBS was added to the slide or plate andcells or spheres were analyzed by fluorescence microscopyusing a Zeiss motorized AxioObserver Z1 microscope Forco-localization experiments cells were seeded at a density of7000 cells per well in a chamber slide and cultured overnightCells were incubated with 100nM peptide and incubated for2 hours Cells were fixed in methanol at -20 degrees Celsiusfor 5 min and blocked with PBS containing 10 goat serumCells were stained with anti-PTCH antibody (Santa CruzBiotechnology Dallas TX) overnight at 4 degrees Celsiuswashed 3 time in 1XPBS and incubated with an anti-rabbitAlexa-555 secondary antibody for 1 hour at RT Slides werewashed 3 times with PBS and stained with DAPI ProlongGold (InvitrogenLife TechnologiesGrand IslandNY) Slideswere analyzed using a Zeiss motorized AxioObserver Z1microscope

26 Uptake Studies To measure uptake of the FITC-taggedpeptide cells were plated at a density of 5000ndash10000 cellsper well in a 24-well plate and grown overnight For sphereassays cells were plated as described above Cells or spheres

BioMed Research International 3

0

20

40

60

80

100

120U

ntre

ated

()

Peptide A

lowastlowast

lowastU

ntre

ated

20

nM

50

nM

100

nM

500

nM

1uM

5uM

(a)

0

20

40

60

80

100

120

Unt

reat

ed (

)

Peptide B

lowastlowast

Unt

reat

ed

20

nM

50

nM

100

nM

500

nM

1uM

5uM

(b)

Figure 1 Patched-binding peptides decrease growth of the SKBR3 breast cancer cell line Using theMTT assay peptides A andB administeredto SKBR3 significantly decreased growth compared to untreated cells Error bars represent standard deviation Significance is represented byasterisk lowast119875 le 005

were treated with 100 nM peptide and incubated for 2 hoursCells or spheres were washed 3X with PBS trypsinized andresuspended in 500 120583L culture media Cells were countedusing a Countess automated cell counter (Invitrogen LifeTechnologies Grand Island NY Life Technologies GrandIsland NY) after staining with trypan blue (Invitrogen LifeTechnologies Grand Island NY Life Technologies GrandIslandNY)Next 100 120583L of cell suspensionwas transferred toblack polystyrene 96-well plates Fluorescence was measuredat 485 nm excitation and 535 nm emission Uptake is reportedas mean fluorescence intensity per 1000 cells

The rat breast carcinoma cell line 13762 and the humanbreast cancer cell lines SUM159 and MDA-IBC3 were usedfor the in vitro radiotracer uptake One day before the uptakeexperiment 2 times 105 cellswell of each cell line were seededin six-well plates and incubated at 37∘C in 5 CO

2under

humidified conditions The following day 300 kBq of 99mTc-EC-peptideA or 99mTc-ECwas addedwith 2mL of the appro-priate media to each of the wellsThe cells were incubated for30 minutes 1 hour 2 hours or 4 hours after which the mediawas aspirated cells were washed twice with PBS and thencells were suspended with trypsin Radioactivity of collectedcells was measured on a gamma counter (Packard) with anenergy window of 126ndash154 keV for 99mTc and percent uptakewas calculated by using an appropriate standard Percentuptake was then normalized to milligrams of protein in thesample where the protein concentration was measured usingthe Bradford method (Bio-Rad Laboratories Berkeley CA)Each sample was run in triplicate with error bars indicatingstandard deviation

27 Animal Model and Chemotherapy Treatment All animalwork was carried out in the Small Animal Imaging Facility(SAIF) at the University of Texas MD Anderson CancerCenter under an approved Institutional Animal Care andUse Committee (IACUC) protocol Female Fischer 344 rats(150 plusmn 25 g 119899 = 6) (Harlan Sprague-Dawley IndianapolisIN) were inoculated subcutaneously with 01mL of a 13762

breast carcinoma cell suspension (105 cellsrat of a breasttumor cell line specific to Fischer rats) into the hind legsusing 25-gauge needles Studies were performed 12ndash14 daysafter inoculation when tumors reached approximately 1 cmin diameter For treatment studies rats were injected with20mgkg paclitaxel and reimaged 7 days later After theposttreatment scan tumors were removed and formalin sec-tions were made Control tumors were taken from untreatedmice 13 days after inoculation Sections were stained with ananti-patched antibody (Santa Cruz) using a peroxide-basedimmunohistochemical detection kit (Dako) according to themanufacturerrsquos instructions

28 Planar Imaging Planar scintigraphic images wereobtained using M-CAM (Siemens Medical Solutions Hoff-man Estates IL) equipped with a low energy high resolution(LEHR) collimator Anesthetized breast tumor-bearingrats were injected intravenously with 99mTc-EC-peptideA (03mgrat 300 120583Cirat 119899 = 3) before and 7 days afterpaclitaxel treatment 99mTc-EC (015mgrat 300 120583Cirat119899 = 3) was used as a control The images were acquiredat 1 hr 2 hr and 4 hr after administration of radiotracersComputer outlined regions of interest (ROIs in counts perpixel) between tumor and muscle were used to calculatetumor-to-muscle (TM) ratios

29 Statistical Analysis Statistical analysis was performedusing Graph Pad Prism 6 software (Graph Pad La Jolla CA)using ANOVA or unpaired 119905-test For all tests 119875 values lessthan 005 were considered to be significant

3 Results

31 Growth Inhibitory Effect of Peptides A and B in Breast Can-cer Lines Inhibition of hedgehog signaling has been shownto decrease growth and survival of breast cancer cells [2 4]Antibodies that disrupt the binding of sonic hedgehog tothe PTCH receptor have also been reported to inhibit breast


Peptide A Peptide B

(a)

Peptide A

0

20

40

60

80

T47D

SkBr

3

1376

2

SUM

159

MCF

-7

IBC3

Mea

n Fl

100

0 ce

lls

Peptide B

0

20

40

60

80

100

T47D

SkBr

3

1376

2

SUM

159

MCF

-7

IBC3

Mea

n Fl

100

0 ce

lls

(b)

Peptide A Patched Overlay

(c)

Figure 2 Patched-binding peptides have significant uptake in breast cancer cell lines (a) Fluorescencemicroscopy of FITC-labeled peptidesAandB in SKBR3 breast cancer cells (b)Quantification of FITC-peptidesA andBuptake in breast cancer cell lines (c) Fluorescencemicroscopyof SKBR3 cells showing colocalization of peptide B (green) with the PTCH receptor (red) Colocalization appears as yellow staining in theimage overlay

cancer growth [5] The PTCH-binding peptides referred toin this paper as peptidesA andB have previously been shownto decrease hedgehog-dependent growth of pancreatic cancercell lines Therefore we sought to determine their effect onbreast cancer cell lines As shown in Figure 1 treatment ofSkBr3 breast cancer cell lines with peptides A and B resultedin significant growth inhibition at higher concentrationsMinimal effect was observed at lower concentrations

32 Peptide Uptake in Breast Cancer Cell Lines and Mammo-spheres To validate the PTCH-binding peptides A and B asligands to detect breast cancer cells we evaluated the cellularuptake of the peptides labeled with FITC Fluorescencemicroscopy of breast cancer cell lines revealed uptake ofthe FITC-tagged peptides As shown in Figure 2(a) cytosolicfluorescence was observed 24 hours after treatment of thebreast cancer cell line SKBR3 with peptide A or peptide B To


Peptide A Peptide B

(a)

0

20

40

60

80

100

120

140

160

180

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

Mea

n Fl

100

0 ce

lls

Peptide B

lowastlowast

lowastPeptide A

lowast

lowast

lowast

0

50

100

150

200

250

300

350

Mea

n Fl

100

0 ce

lls

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

(b)

Figure 3 Uptake of patched-binding peptides is increased in mammospheres from breast cancer cell lines (a) Fluorescence microscopy ofFITC-labeled peptides A and B in mammospheres from T47-D (b) Quantification of FITC-peptides A and B uptake in breast cancer celllines in monolayer and mammosphere cultures Uptake was significantly higher in cells cultured in mammosphere promoting conditions (3dimensional cultures) than in those grown in standard monolayer conditions Significance is represented by asterisk lowast119875 le 001

99mTc-EC-peptide 10000

8000

6000

4000

2000

0

Cou

nts

0 50 100 150 200

Position (mm)

99mTc-EC

Cou

nts

800

600

400

200

0

0 50 100 150 200

Position (mm)

Figure 4 Radiochemical purity of 99mTc-EC and 99mTc-EC-peptide A Radiochemical purity was determined by RadioTLC with saline asthe mobile phase


0

5

10

15

20

0 50 100 150 200 250 300

Upt

ake (

mg

prot

ein)

()

SUM159

lowast

Incubation time (min)

(a)

0

1

2

3

4

5

6

Upt

ake (

mg

prot

ein)

()

IBC3

lowast

0 50 100 150 200 250 300Incubation time (min)

(b)

lowast

0

1

2

3

4

5

6

7

Upt

ake (

mg

prot

ein)

()

13762

99mTc-EC

99mTc-EC-peptide


(c)

Figure 5 In vitro uptake of 99mTc-EC-peptide A Results of in vitro assays showing significant uptake of peptide A compared to 99mTc-ECcontrol in (a) SUM159 (b) MDA-IBC3 and (c) 13762 breast cancer cell lines Data is represented as uptake per mgprotein Error barsrepresent standard deviations Significance is represented by asterisk lowast119875 le 0001

quantify these findings uptake studies were performed in apanel of breast cancer cell lines and fluorescence intensity wasmeasured As shown in Figure 2(b) significant uptake of thefluorescent peptides was observed Furthermore fluorescentintensity corresponded to PTCH expression as previouslyreported [1] suggesting that binding is specific to the PTCHreceptor To further confirm colocalization of PTCH-bindingpeptides with PTCH receptor expression we performedfluorescence microscopy using anti-PTCH antibodies oncells treated with FITC-labeled PTCH-binding peptides Asshown in Figure 2(c) uptake of PTCH-binding peptides(green) colocalized with PTCH receptor expression (red)

Hedgehog pathway members PTCH Gli-1 and Gli-2have been reported to be more highly expressed in normalmammary stem cells and theirmalignant counterparts breastcancer stem cells compared to more differentiated breastcancer cells [6] High expression of the PTCH receptorhas been reported in breast cancer cells cultured in stemcell-enriching conditions (mammospheres 3 dimensionalcultures) Consistent with these findings an increase in

peptide uptake was observed in mammospheres comparedto 2 dimensional monolayer cultures (Figure 3) These dataindicate that PTCH-binding peptides may provide a methodof targeting breast cancer stem cells

33 Synthesis and Radiolabeling of EC-Peptide A To establishthe uptake of PTCH-binding peptides in vivo we synthesizedchelator-peptide conjugates that could be radiolabeled with99mTc for gamma scintigraphy A simple and efficient synthe-sis of 99mTc EC-PTCH was developed EC was conjugated tothe lysine residue of peptide A 99mTc EC-PTCH was foundto be radiochemically pure (100 Figure 4)

34 In Vitro Uptake Studies In vitro cellular uptake of the99mTc-conjugated peptide was performed in three breastcancer cell lines SUM159 MDA-IBC3 and 13762 As shownin Figure 5 cellular uptake of the peptide conjugate wassignificantly higher than that of the chelator alone in all linesSimilar to the data for the FITC-tagged peptide the SUM159


TM = 470 TM = 347 TM = 552

1hr

TM = 402 TM = 351 TM = 454

2hr

TM = 425 TM = 317 TM = 540

4hr

Figure 6 In vivo uptake of 99mTc-EC-peptide A in rats bearing13762 breast carcinoma xenografts Tumor-to-muscle (TM) ratiosare given for 3 separate rats at multiple timepoints after injection ofthe radiotracer Arrows indicate tumor location

line showed the highest radiotracer uptake with approxi-mately 18 uptake per mg protein at 4 hours (Figure 5(a))Significant uptake of the radiotracer was also observed in twoother lines increasing steadily in MDA-IBC3 (Figure 5(b))and reaching saturation after 1-2 hours in 13762 (Figure 5(c))

35 In Vivo Imaging To investigate the utility of peptideimaging of the PTCH receptor in breast cancer planarscintigraphy was performed in a rat model of breast cancerusing 99mTc-EC-peptide A Fisher rats were inoculated withthe mammary carcinoma cell line 13762 and after tumorsgrew for two weeks rats were injected with approximately300 120583Ci of the 99mTc-labeled peptide Planar scintigraphy wasconducted at 1 2 and 4 hours after injection of the radio-labeled peptide and tumor-to-muscle ratios were calculatedAn average tumor-to-muscle ratio of 45 plusmn 007 was obtainedat 1 hour Significant retention of the peptide was observed inthe tumor tissue up to 4 hours after injection (Figure 6)

Several studies have reported that hedgehog signalinginduces resistance to chemotherapy [4 7 8] Therefore

we expect that residual cells which remain after treatmentwith chemotherapy would have high expression of hedgehogpathway members We examined PTCH expression in tumorxenografts before and after treatment with paclitaxel Asshown in Figure 7(a) an increase in PTCHprotein expressionis observed in the residual tumor seven days after treatmentAlthough there was a decrease in tumor volume after treat-ment planar imaging with 99mTc-EC-peptide A revealed nosignificant decrease in tumor accumulation of the peptide(Figure 7(b)) These findings suggest that PTCH receptorimaging may provide a useful method to assess resistanttumor tissue after chemotherapy treatment

4 Discussion

Neoadjuvant chemotherapy is commonly prescribed fortreatment of invasive or large tumors to allow for breast-conserving surgery However there is currently no reliablemethod to noninvasively follow response to chemotherapy Itis unclear whether the current standard for clinical imaging18F-FDG PET is predictive of treatment response due tofalse positive results following treatment In vitro and in vivostudies have demonstrated high FDGuptake in inflammatorylesions [5] Increased FDG uptake in macrophages andneutrophils caused by treatment-induced inflammation hasalso been reported [9 10]

We show that in vivo imaging with 99mTc-PTCH pep-tides may offer an alternative method to follow treatmentresponse and allow for tumor-specific imaging prior to andimmediately after chemotherapy treatment Our data suggestthat peptides which bind to the ligand docking site of thehedgehog receptor PTCH are localized to breast cancers invivo Furthermore we show that PTCH receptor expressionis increased after paclitaxel treatment in a rat model ofbreast cancer These results indicate that PTCH-positivetreatment-resistant cellsmay be enriched after chemotherapyIn addition to tumor uptake significant uptake of the peptidewas observed in liver and kidney tissues This may be due toclearance of the peptide and the FITC tag Additionally liveruptake may be due to low level endogenous expression of thePTCH receptor by liver tissue Although our work and thatof others [2] suggest that PTCH docking peptides specificallytarget the PTCH receptor on cancer cells binding to other cellsurface receptors cannot be ruled out

The cancer stem cell hypothesis states that tumors consistof a heterogeneous population of cells including both rapidlydividing differentiated cells that can be effectively targetedby chemotherapy and relatively resistant stem-like cells [11]Previous studies have reported that breast cancer stem cellsexpress high levels of the PTCH receptor and that hedgehogsignaling is required for the growth of these cells [6] Our dataindicate that PTCH-binding peptides have higher uptake incells cultured under stem cell-promoting conditions (mam-mospheres) andmay serve as a ligand to detect and target thiscell population Additionally our findings suggest that PTCHreceptor-positive cells are resistant to chemotherapy and that99mTc-peptide A may be a useful agent for the detection of


Untreated Treated

(a)

lowast

0

02

04

06

08

1

2 4(hr)

ID (

)

Before treatmentAfter treatment

(b)

Figure 7 PTCH expression is increased in breast cancer xenografts after chemotherapy treatment (a) Immunohistochemical detection ofPTCH before and after treatment with paclitaxel (b) Tumor uptake of 99mTc-EC-peptide A is increased after treatment with chemotherapyData represents injected dose in the ROI of the tumor Error bars represent standard deviation Significance is represented by asterisklowast119875 le 005

treatment-resistant breast cancer cells with active hedgehogsignaling

PTCH-blocking peptides have been shown to decreasegrowth of pancreatic cancer cell lines [2] Similar to previousstudies we show that treatment of breast cancer cell lines withPTCH-binding peptides decreases growth of breast cancercell lines While our preliminary results suggest that PTCH-binding peptides may slow growth of breast cancer furtherstudy is needed to validate the therapeutic effect Our dataalso indicate that further evaluation of the effect of PTCH-binding peptides on tumor detection growth and survivalin orthotopic models of breast cancer is warranted These

peptides may serve as useful theranostics which may be usedto both image and treat breast cancer

Conflict of Interests

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

Acknowledgments

This project was supported by the National Institutes ofHealth grant U54-GM104941 the NIHNational Cancer


Institute Grant RO1 CA138230-01 and the Delaware INBREprogram with a grant from the National Institute of GeneralMedical SciencesmdashNIGMS (8 P20 GM103446-13)

References

[1] J Sims-Mourtada D Yang I Tworowska et al ldquoDetection ofcanonical hedgehog signaling in breast cancer by 131-iodine-labeled derivatives of the sonic hedgehog proteinrdquo Journal ofBiomedicine and Biotechnology vol 2012 Article ID 639562 8pages 2012

[2] M Nakamura H Tanaka Y Nagayoshi et al ldquoTargetingthe hedgehog signaling pathway with interacting peptides toPatched-1rdquo Journal of Gastroenterology vol 47 no 4 pp 452ndash460 2012

[3] D J Yang W E Fogler J L Bryant et al ldquoAssessmentof antiangiogenic effect using 99mTc-EC-endostatinrdquo CancerBiotherapy andRadiopharmaceuticals vol 17 no 2 pp 233ndash2452002

[4] F Chai J Zhou C Chen et al ldquoThe Hedgehog inhibitorcyclopamine antagonizes chemoresistance of breast cancercellsrdquo OncoTargets andTherapy vol 6 pp 1643ndash1647 2013

[5] S LawM Fok S Chow K-M Chu and JWong ldquoPreoperativechemotherapy versus surgical therapy alone for squamous cellcarcinoma of the esophagus a prospective randomized trialrdquoJournal of Thoracic and Cardiovascular Surgery vol 114 no 2pp 210ndash117 1997

[6] S Liu G Dontu I D Mantle et al ldquoHedgehog signaling andBmi-1 regulate self-renewal of normal and malignant humanmammary stem cellsrdquoCancer Research vol 66 no 12 pp 6063ndash6071 2006

[7] J Sims-Mourtada J G Izzo J Ajani and K S C Chao ldquoSonicHedgehog promotes multiple drug resistance by regulation ofdrug transportrdquo Oncogene vol 26 no 38 pp 5674ndash5679 2007

[8] J Sims-Mourtada J G Izzo S Apisarnthanarax et al ldquoHedge-hog an attribute to tumor regrowth after chemoradiotherapyand a target to improve radiation responserdquo Clinical CancerResearch vol 12 no 21 pp 6565ndash6572 2006

[9] D R Jones L A Parker Jr F C Detterbeck and T M EganldquoInadequacy of computed tomography in assessing patientswith esophageal carcinoma after induction chemoradiother-apyrdquo Cancer vol 85 pp 1026ndash1032 1999

[10] G Zuccaro Jr T W Rice J Goldblum et al ldquoEndoscopicultrasound cannot determine suitability for esophagectomyafter aggressive chemoradiotherapy for esophageal cancerrdquoTheAmerican Journal of Gastroenterology vol 94 no 4 pp 906ndash912 1999

[11] G J Lindeman and J E Visvader ldquoInsights into the cell oforigin in breast cancer and breast cancer stem cellsrdquoAsia-PacificJournal of Clinical Oncology vol 6 no 2 pp 89ndash97 2010

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which can be detected by single photon emission computedtomography (SPECT) In addition the half-life of 99mTcis relatively long (602 h) compared to most nuclear imag-ing radioisotopes which facilitates serial imaging that mayimprove the differentiation of tumor from inflammation Tolabel the peptide with 99mTc the chelator NN1015840-ethylene-di-L-cysteine (EC) is selected and used as a linker EC isknown to chelate 99mTc stably owing to the efficient bindingof the oxotechnetium group to the two thiols and two aminenitrogen atoms of EC

Here we report the radiolabeling of these peptides todetect the PTCH receptor on breast cancer cells and breastcancer stem cell-enriched populations These molecularimaging probes have the potential to identify Hh-inducedchanges in PTCH-1 expression which is useful for theimaging of aberrant Hh signaling in malignancies

2 Methods

21 Peptides PTCH-binding peptides Amdashsequence FAPVL-DGAVSTLLGVmdash and Bmdashsequence DNTRYSPPPPYSSHSmdashwere commercially synthesized with or without an N-ter-minal FITC-Ahx modification (GenScript Piscataway NJ)Peptides were resuspended at a stock concentration of200120583M in 10 DMSO in deionized water

22 Synthesis and Radiolabeling of PTCH Ethylenedicysteine(EC) was selected as a chelator for PTCH conjugationSodium bicarbonate (1 N 1mL) was added to a stirredsolution of EC (5mg 0019mmol) To this colorless solutionsulfo-NHS (4mg 0019mmol) and EC (5mg 0019mmol)were added PTCH (03mg) was then added The mixturewas stirred at room temperature for 24 hours The mixturewas dialyzed for 48 hours with a cutoff at molecular weight10000Da After dialysis the product was freeze-dried withthe product in the salt form weighing 05mg99mTc-pertechnetate was obtained from Mallinckrodt

(Houston TX) Radiosynthesis of 99mTc-EC-PTCH wasachieved by adding the required amount of 99mTc-pertechnetate into EC-PTCH (01mg) and tin chloride(II) (SnCl

2 100mg) The mixture was loaded on a sephadex

gel column (PD-10 G-25) (Sigma Chemical Company StLouis MO) and eluted with phosphate-buffered saline (pH74) Onemilliliter of each fractionwas collectedThe productwas collected at fraction 3 with a 70 yield Radiochemicalpurity was assessed by Radio-TLC (BioScan WashingtonDC) using saline as an eluant

23 Cell Lines and Culture Conditions The human cell linesT47-D SKBR3 andMCF-7were obtained from theAmericanType Tissue Company (ATCC) and cultured in DMEMwith 10 fetal bovine serum (Atlanta Biologicals FloweryBranch GA) and 1 antibiotic-antimycotic (Invitrogen LifeTechnologies Grand Island NY Life Technologies GrandIsland NY) The human cell line SUM159 was obtained fromAsterand (Detroit MI) and cultured in DMEM contain-ing 1 120583gmL hydrocortisone (Invitrogen Life TechnologiesGrand Island NY Life Technologies Grand Island NY)

5 120583gmL insulin (Invitrogen Life Technologies Grand IslandNY) and 1 antibioticantimycoticThe rat breast cancer cellline 13762 was derived from a tumor induced in a Fischer-344rat by giving an oral dose of 712-dimethylbenz[a]anthracene[3] and the cells were cultured in RPMI-1640 mediumsupplemented with 10 (vol vol) fetal bovine serum and1 antibiotic-antimycotic For mammosphere assays cellswere cultured in MEM media supplemented with 1X B27(Invitrogen Life Technologies Grand Island NY) 20 ngmLepidermal growth factor (EGF Invitrogen Life TechnologiesGrand Island NY) and 20 ngmL basic fibroblast growthfactor (bFGF Invitrogen Life Technologies Grand IslandNY) and seeded into ultralow attachment plates (CorningLife Sciences Salt Lake City UT) Cells were grown for 7ndash10 days and spheres were obtained All cells were culturedat 37∘C in a humidified atmosphere containing 5 carbondioxide

24 Survival Assays Breast cancer cell lines were seededinto 96-well plates at a density of 5000ndash7000 cells perwell Cells were grown overnight and media were replacedwith culture media containing unlabeled peptides A andB at the indicated concentrations Cells were cultured foran additional 48 hours and survival was determined usingthe MTT-based Cell Proliferation Assay (Biotium) Data isexpressed as treateduntreated

25 Fluorescence Microscopy Breast cancer cell lines wereseeded onto chamber slides (Nunc Roskilde Denmark) andgrown overnight For sphere assays cells were seeded in 3Dmedia as described above in 96-well low attachment platesat a density of 100ndash1000 cells per well and cultured for 7ndash10 days until spheres were formed Spheres were filteredusing a cell strainer and replated into low attachment platesCells or spheres were treated with 100 nM peptide A or Band incubated for 2 hours Media were removed and cellsor spheres were washed two times with 1X PBS Followingwashes 1mL of 1X PBS was added to the slide or plate andcells or spheres were analyzed by fluorescence microscopyusing a Zeiss motorized AxioObserver Z1 microscope Forco-localization experiments cells were seeded at a density of7000 cells per well in a chamber slide and cultured overnightCells were incubated with 100nM peptide and incubated for2 hours Cells were fixed in methanol at -20 degrees Celsiusfor 5 min and blocked with PBS containing 10 goat serumCells were stained with anti-PTCH antibody (Santa CruzBiotechnology Dallas TX) overnight at 4 degrees Celsiuswashed 3 time in 1XPBS and incubated with an anti-rabbitAlexa-555 secondary antibody for 1 hour at RT Slides werewashed 3 times with PBS and stained with DAPI ProlongGold (InvitrogenLife TechnologiesGrand IslandNY) Slideswere analyzed using a Zeiss motorized AxioObserver Z1microscope

26 Uptake Studies To measure uptake of the FITC-taggedpeptide cells were plated at a density of 5000ndash10000 cellsper well in a 24-well plate and grown overnight For sphereassays cells were plated as described above Cells or spheres


0

20

40

60

80

100

120U

ntre

ated

()

Peptide A

lowastlowast

lowastU

ntre

ated

20

nM

50

nM

100

nM

500

nM

1uM

5uM

(a)

0

20

40

60

80

100

120

Unt

reat

ed (

)

Peptide B

lowastlowast

Unt

reat

ed

20

nM

50

nM

100

nM

500

nM

1uM

5uM

(b)




2under






3 Results



Peptide A Peptide B

(a)

Peptide A

0

20

40

60

80

T47D

SkBr

3

1376

2

SUM

159

MCF

-7

IBC3

Mea

n Fl

100

0 ce

lls

Peptide B

0

20

40

60

80

100

T47D

SkBr

3

1376

2

SUM

159

MCF

-7

IBC3

Mea

n Fl

100

0 ce

lls

(b)


(c)





Peptide A Peptide B

(a)

0

20

40

60

80

100

120

140

160

180

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

Mea

n Fl

100

0 ce

lls

Peptide B

lowastlowast

lowastPeptide A

lowast

lowast

lowast

0

50

100

150

200

250

300

350

Mea

n Fl

100

0 ce

lls

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

(b)



8000

6000

4000

2000

0

Cou

nts

0 50 100 150 200

Position (mm)

99mTc-EC

Cou

nts

800

600

400

200

0

0 50 100 150 200

Position (mm)



0

5

10

15

20

0 50 100 150 200 250 300

Upt

ake (

mg

prot

ein)

()

SUM159

lowast


(a)

0

1

2

3

4

5

6

Upt

ake (

mg

prot

ein)

()

IBC3

lowast


(b)

lowast

0

1

2

3

4

5

6

7

Upt

ake (

mg

prot

ein)

()

13762

99mTc-EC

99mTc-EC-peptide


(c)








TM = 470 TM = 347 TM = 552

1hr

TM = 402 TM = 351 TM = 454

2hr

TM = 425 TM = 317 TM = 540

4hr






4 Discussion





Untreated Treated

(a)

lowast

0

02

04

06

08

1

2 4(hr)

ID (

)


(b)







Acknowledgments




References

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120U

ntre

ated

()

Peptide A

lowastlowast

lowastU

ntre

ated

20

nM

50

nM

100

nM

500

nM

1uM

5uM

(a)

0

20

40

60

80

100

120

Unt

reat

ed (

)

Peptide B

lowastlowast

Unt

reat

ed

20

nM

50

nM

100

nM

500

nM

1uM

5uM

(b)




2under






3 Results



Peptide A Peptide B

(a)

Peptide A

0

20

40

60

80

T47D

SkBr

3

1376

2

SUM

159

MCF

-7

IBC3

Mea

n Fl

100

0 ce

lls

Peptide B

0

20

40

60

80

100

T47D

SkBr

3

1376

2

SUM

159

MCF

-7

IBC3

Mea

n Fl

100

0 ce

lls

(b)


(c)





Peptide A Peptide B

(a)

0

20

40

60

80

100

120

140

160

180

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

Mea

n Fl

100

0 ce

lls

Peptide B

lowastlowast

lowastPeptide A

lowast

lowast

lowast

0

50

100

150

200

250

300

350

Mea

n Fl

100

0 ce

lls

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

(b)



8000

6000

4000

2000

0

Cou

nts

0 50 100 150 200

Position (mm)

99mTc-EC

Cou

nts

800

600

400

200

0

0 50 100 150 200

Position (mm)



0

5

10

15

20

0 50 100 150 200 250 300

Upt

ake (

mg

prot

ein)

()

SUM159

lowast


(a)

0

1

2

3

4

5

6

Upt

ake (

mg

prot

ein)

()

IBC3

lowast


(b)

lowast

0

1

2

3

4

5

6

7

Upt

ake (

mg

prot

ein)

()

13762

99mTc-EC

99mTc-EC-peptide


(c)








TM = 470 TM = 347 TM = 552

1hr

TM = 402 TM = 351 TM = 454

2hr

TM = 425 TM = 317 TM = 540

4hr






4 Discussion





Untreated Treated

(a)

lowast

0

02

04

06

08

1

2 4(hr)

ID (

)


(b)







Acknowledgments




References

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Peptide A Peptide B

(a)

Peptide A

0

20

40

60

80

T47D

SkBr

3

1376

2

SUM

159

MCF

-7

IBC3

Mea

n Fl

100

0 ce

lls

Peptide B

0

20

40

60

80

100

T47D

SkBr

3

1376

2

SUM

159

MCF

-7

IBC3

Mea

n Fl

100

0 ce

lls

(b)


(c)





Peptide A Peptide B

(a)

0

20

40

60

80

100

120

140

160

180

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

Mea

n Fl

100

0 ce

lls

Peptide B

lowastlowast

lowastPeptide A

lowast

lowast

lowast

0

50

100

150

200

250

300

350

Mea

n Fl

100

0 ce

lls

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

(b)



8000

6000

4000

2000

0

Cou

nts

0 50 100 150 200

Position (mm)

99mTc-EC

Cou

nts

800

600

400

200

0

0 50 100 150 200

Position (mm)



0

5

10

15

20

0 50 100 150 200 250 300

Upt

ake (

mg

prot

ein)

()

SUM159

lowast


(a)

0

1

2

3

4

5

6

Upt

ake (

mg

prot

ein)

()

IBC3

lowast


(b)

lowast

0

1

2

3

4

5

6

7

Upt

ake (

mg

prot

ein)

()

13762

99mTc-EC

99mTc-EC-peptide


(c)








TM = 470 TM = 347 TM = 552

1hr

TM = 402 TM = 351 TM = 454

2hr

TM = 425 TM = 317 TM = 540

4hr






4 Discussion





Untreated Treated

(a)

lowast

0

02

04

06

08

1

2 4(hr)

ID (

)


(b)







Acknowledgments




References

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Peptide A Peptide B

(a)

0

20

40

60

80

100

120

140

160

180

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

Mea

n Fl

100

0 ce

lls

Peptide B

lowastlowast

lowastPeptide A

lowast

lowast

lowast

0

50

100

150

200

250

300

350

Mea

n Fl

100

0 ce

lls

T47-

D

T47-

D 3

D

1376

2

1376

2-3D

IBC3

IBC3

-3D

(b)



8000

6000

4000

2000

0

Cou

nts

0 50 100 150 200

Position (mm)

99mTc-EC

Cou

nts

800

600

400

200

0

0 50 100 150 200

Position (mm)



0

5

10

15

20

0 50 100 150 200 250 300

Upt

ake (

mg

prot

ein)

()

SUM159

lowast


(a)

0

1

2

3

4

5

6

Upt

ake (

mg

prot

ein)

()

IBC3

lowast


(b)

lowast

0

1

2

3

4

5

6

7

Upt

ake (

mg

prot

ein)

()

13762

99mTc-EC

99mTc-EC-peptide


(c)








TM = 470 TM = 347 TM = 552

1hr

TM = 402 TM = 351 TM = 454

2hr

TM = 425 TM = 317 TM = 540

4hr






4 Discussion





Untreated Treated

(a)

lowast

0

02

04

06

08

1

2 4(hr)

ID (

)


(b)







Acknowledgments




References

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

20

0 50 100 150 200 250 300

Upt

ake (

mg

prot

ein)

()

SUM159

lowast


(a)

0

1

2

3

4

5

6

Upt

ake (

mg

prot

ein)

()

IBC3

lowast


(b)

lowast

0

1

2

3

4

5

6

7

Upt

ake (

mg

prot

ein)

()

13762

99mTc-EC

99mTc-EC-peptide


(c)








TM = 470 TM = 347 TM = 552

1hr

TM = 402 TM = 351 TM = 454

2hr

TM = 425 TM = 317 TM = 540

4hr






4 Discussion





Untreated Treated

(a)

lowast

0

02

04

06

08

1

2 4(hr)

ID (

)


(b)







Acknowledgments




References

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TM = 470 TM = 347 TM = 552

1hr

TM = 402 TM = 351 TM = 454

2hr

TM = 425 TM = 317 TM = 540

4hr






4 Discussion





Untreated Treated

(a)

lowast

0

02

04

06

08

1

2 4(hr)

ID (

)


(b)







Acknowledgments




References

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Untreated Treated

(a)

lowast

0

02

04

06

08

1

2 4(hr)

ID (

)


(b)







Acknowledgments




References

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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