tumor-specific targeting of pancreatic cancer with...

Preclinical Development

Tumor-Specific Targeting of Pancreatic Cancer withShiga Toxin B-Subunit

Matthias Maak1, Ulrich Nitsche1, Larissa Keller1, Petra Wolf2, Marianne Sarr4,5, Marine Thiebaud4,5,Robert Rosenberg1, Rupert Langer3, J€org Kleeff1, Helmut Friess1, Ludger Johannes4,5, and Klaus-Peter Janssen1

AbstractPancreatic carcinoma is one of themost aggressive tumor entities, and standard chemotherapyprovides only

modest benefit. Therefore, specific targeting of pancreatic cancer for early diagnosis and therapeutic inter-

vention is of great interest. We have previously shown that the cellular receptor for Shiga toxin B (STxB), the

glycosphingolipid globotriaosylceramide (Gb3 or CD77) is strongly increased in colorectal adenocarcinoma

and their metastases. Here, we report an upregulation of Gb3 in pancreatic adenocarcinoma (21 of 27 cases) as

compared with matched normal tissue (n¼ 27). The mean expression was highly significantly increased from

30 � 16 ng Gb3/mg tissue in normal pancreas to 61 � 41 ng Gb3/mg tissue (mean � SD, P ¼ 0.0006), as

evidencedby thin layer chromatography.Upregulation ofGb3 levels did not dependon tumor stage or grading

and showed no correlation with clinical outcome. Tumor cells and endothelial cells were identified as the

source of increased Gb3 expression by immunocytochemistry. Pancreatic cancer cell lines showed rapid

intracellular uptake of STxB to the Golgi apparatus, following the retrograde pathway. The therapeutic

application of STxB was tested by specific delivery of covalently coupled SN38, an active metabolite of the

topoisomerase I inhibitor irinotecan. The cytotoxic effect of the STxB-SN38 compound in pancreatic cancer cell

lineswas increasedmore than 100-fold comparedwith irinotecan.Moreover, this effectwas effectively blocked

by competing incubationwithnonlabeledSTxB, showing the specificity of the targeting. Thus, STxB constitutes

a promising new tool for specific targeting of pancreatic cancer. Mol Cancer Ther; �2011 AACR.

Introduction

Pancreatic carcinoma is one of the most aggressivetumor entities with a median survival of 6 months anda 5-year survival rate of less than 5% (1). Curative resec-tion often is not possible due to late diagnosis of thetumor, and the current standard chemotherapy withgemcitabine has increased the overall survival only mod-estly (2, 3). The targeting of pancreatic cancer for earlydiagnosis (e.g., by imaging methods) and for therapeuticpurposes (e.g., by specific delivery of cytotoxic com-pounds to pancreatic tumor cells) is therefore of greatclinical interest. The nontoxic B-subunit of the bacterialShiga toxin (STxB) is a promising new option for diag-

nosis and therapy of gastrointestinal tumors. We andothers showed previously that the expression of thecellular receptor of Shiga toxin, the glycosphingolipidGb3 (CD77), is significantly increased in colorectal carci-noma and their metastases (4, 5). Studies on geneticmouse models for human digestive cancer showed thatSTxB is suitable for noninvasive tumor imaging (6). STxBis taken up rapidly by tumor cells that express thereceptor Gb3 on their surface membrane, and STxB thenfollows the so-called retrograde route (7). The retrograderoute bypasses the late endocytic pathway and avoidsthe degrading environment of the lysosomes. Instead,STxB is transported from the endosome to the trans-Golgi network and then to the Golgi apparatus andis transported from there to the endoplasmic reticulum(8–10).

It is feasible that STxB has naturally evolved to with-stand inactivation by intestinal fluids. Moreover, STxBhas only low immunogenicity in humans (11, 12). Func-tionalized STxB can be used efficiently to target gastro-intestinal tumors in animal models (6), as well as short-term primary cultures from human colon cancer and livermetastases (5). Moreover, a recent study confirmed thefindings on Gb3 overexpression in colon cancer andreported an upregulation of Gb3 expression in humanpancreatic cancer patients (13). In the present study, wehave quantified Gb3 expression in human pancreatic

Authors' Affiliations: 1Department of Surgery, 2Institut f€ur MedizinischeStatistik und Epidemiologie, Klinikum rechts der Isar, 3Institute of Pathol-ogy and Pathological Anatomy, Technische Universit€at M€unchen, Munich,Germany; 4Traffic, Signaling and Delivery Laboratory, Institut Curie; and5CNRS UMR144, Paris, France

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Klaus-Peter Janssen, Department of Surgery,Klinikum rechts der Isar, TU M€unchen, Ismaninger Str. 22, 81675 Munich,Germany. Phone: 49-89-4140-2066; Fax: 49-89-4140-6031; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-0006

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on May 24, 2018. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 25, 2011; DOI: 10.1158/1535-7163.MCT-11-0006

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adenocarcinoma by lipid extraction and thin layer chro-matography (TLC), as well as by immunofluorescencemicroscopy on tissue sections, and tested pancreaticcancer cells for the uptake kinetics of functionalizedSTxB. Moreover, human astrocytoma xenografts in nudemice are eliminated rapidly, completely, and with long-term efficacy using verotoxin, supporting the concept oftherapeutic use of Shiga toxin–like toxins (14). Recently,successful coupling of STxB to 7-ethyl-10-hydroxycamp-tothecin (SN38), the active metabolite of the topoisome-rase inhibitor irinotecan (CPT-11), has been reported (15).SN38 is amenable to biochemical modification, making ita useful candidate drug for a proof-of-principle approachfor STxB-mediated tumor targeting. Furthermore, SN38 isseveral orders of magnitude more active than irinotecan,but its hydrophobicity and insolubility in most physio-logically compatible and pharmaceutically acceptablesolvents limits its clinical application (16, 17). Irinotecanhas already been reported in several clinical trials assecond-line chemotherapy of advanced pancreatic cancereither as single agent or in combination with gemcitabineor other agents (2, 18–20). Anticancer cytotoxicity wasreported, even though no obvious survival benefit wasreported so far. The STxB-SN38 compound has beenshown previously to possess highly increased cytotoxi-city as compared with irinotecan when tested on a color-ectal cancer cell line (15). Moreover, the STxB-SN38compound showed high receptor specificity, as the cyto-toxic effect relied on an intracellular uptake that ismediated by the receptor Gb3. The cytotoxic effect wasinhibited when the glycosphingolipid Gb3 was blocked(15). Therefore, STxB-SN38 could be a highly useful toolfor targeted chemotherapy, with greatly enhanced cyto-toxicity and low side effects. The aim of this study wastherefore to analyze the potential diagnostic and thera-peutic uses of STxB on pancreatic carcinoma. Takentogether, we report a significantly increased expressionof Gb3 in pancreatic cancer, as compared with matchednormal tissue samples. In addition, we have successfullytested a new compound for targeted chemotherapy,STxB-SN38, on pancreatic cancer cell lines.

Materials and Methods

Patient collectiveSamples were obtained from 28 patients with pancrea-

tic carcinoma admitted to our Surgical Department in2003 to 2006 (16male and 12 female, median age: 68 years,range: 42–83). Informed, written consent about the use oftissue samples had been obtained previously, withapproval of the local ethics committee. Median follow-up after surgery was 16.9 months. During this period, 3patients died because of unrelated causes and 17 diedbecause of cancer progression or disease recurrence.Tumors were classified according to the InternationalUnion Against Cancer, as summarized in Table 1 (21).Histology-guided sample selection was conducted by apathologist to identify tumor samples and to exclude

cancer cell contamination in the control samples. Allsamples were snap frozen in liquid nitrogen immediatelyafter resection and stored at �80�C until use.

Reagents and antibodiesTo allow for chemical coupling of fluorophores or

cytotoxic drugs to a defined acceptor site in STxB, aCys residue was added to the C-terminus of the wild-type protein. The recombinant mutant STxB-Cys protein

Table 1. Patient collective and clinical data

CollectivePatients, n 28 100%Female 12 42.9%Male 16 57.1%Age, y, median (range) 68 (42–83)Follow-up, mo, median 16.91

Tissue, n 55 100%Pancreatic carcinoma 28 50.9%Normal pancreas 27 49.1%

Gb3 expressionPancreatic carcinoma,ng/mg, median (range)

54.9 (9.7–181.5)

Normal pancreas, ng/mg,median (range)

29.0 (5.7–77.0)

Postoperative dataUICC stage, n 28 100%Ib 1 3.6%IIa 10 35.7%IIb 17 60.7%

Grade, n 28 100%G2 12 42.9%G3 16 57.1%

Tumor invasion, n 28 100%pT2 3 10.7%pT3 25 89.3%

Lymph node status, n 28 100%pN0 11 39.3%pN þ 17 60.7%N total, median (range) 17.5 (6–63)N positive, median (range) 1 (0–12)

Metastasis, n 28 100%M0 28 100%

Lymphatic invasion, n 28 100%Absent 26 92.9%Present 2 7.1%

Angioinvasion, n 28 100%Absent 27 96.4%Present 1 3.6%

R status, n 28 100%R0 17 60.7%R1 6 21.4%R2 0 0%Rx 5 17.9%

Abbreviation: UICC, International Union Against Cancer.

Maak et al.

Mol Cancer Ther; 2011 Molecular Cancer TherapeuticsOF2




was produced in endotoxin-free form as described (6).Antibodies and reagents used were as follows: anti-gol-gin p97 (Molecular Probes); anti-STxB (22); anti-CD31(BD Biosciences Pharmingen); DAPI, Ki67, and stauros-porine (from Sigma-Aldrich); and calnexin and anti-vimentin (both from Santa Cruz Biotechnology). Second-ary antibodies coupled to fluorophores were purchasedfrom Jackson Immunoresearch and cell culture reagentsfrom Invitrogen. Irinotecan was obtained from the Phar-macy of the Klinikum rechts der Isar. SN38 was coupledto STxB as described (15).

Indirect immunofluorescenceImmunostaining was conducted as described before

(6). Briefly, after paraformaldehyde fixation, cell lines oncoverslips were permeabilized with 0.1% Triton X-100,blocked with PBS containing 2% bovine serum albumin,and primary antibodies in blocking buffer were addedbefore counterstaining with secondary antibodies. At thisconcentration, Triton X-100 did not alter theGb3 detectionon cryosections. STxB was purified from bacteria, aspreviously described (23). Covalent coupling of STxBto fluorochrome Cy3 (Amersham Biosciences) was car-ried out according to the supplier’s instructions. Forstaining of endogenous Gb3, tissue cryosections werefixed with 3% paraformaldehyde for 20 minutes andincubated with STxB-Cy3 for 30 minutes at a final con-centration of 10 mg/mL, in PBS containing 0.2% bovineserum albumin. For counterstaining against the nuclearantigenKi67, 0.1% Triton X-100was used on cryosections.For image acquisition, epifluorescence or confocal micro-scopes (Zeiss) were used. Images were processed usingAdobe Photoshop Software CS3 and colocalization wastested with ImageJ version 1.42q (NIH).

Quantification of Gb3 expression by TLCGb3 expression was quantified as previously described

(refs. 5, 24–26; additional details in Supplementary Mate-rial). Briefly, unfixed tissue of pancreatic tumors andadjacent tissue were collected immediately after surgeryby an experienced pathologist. Tissueswere weighed andmechanically homogenized in 1 mL of aqueous buffer.The established pancreatic and colorectal cancer cell lineswere collected from cell culture dishes, washed, andanalyzed accordingly. The material in aqueous bufferwas injected immediately into 3.75 mL of chloroform/methanol (1:2). After mixing, 1.25 mL of chloroform and1.25 mL of water were added. The hydroalcoholic phasewas washed once with 1.5 mL of chloroform. The com-bined chloroform phases were dried under nitrogen, andlipids were saponified at 56�C for 1 hour in 1 mL ofmethanol/KOH. The saponification reaction was onceagain extracted as described above, and the chloroformphase was washed once with methanol/water (1:1). Theisolated neutral glycolipids were spotted on high-perfor-mance TLC plates (Merck) and separated with chloro-form/methanol/water (65:25:4). Dried plates weresoaked in 0.1% polyisobutylmethacrylate in hexane,

floated for 1 hour in blocking solution, followed byincubation with STxB (20 nmol/L), primary polyclonalantiserum against STxB, and secondary horseradish per-oxidase–coupled antirabbit antibodies. Reactive bandswere revealedwith enhanced chemiluminescence (Amer-sham Pharmacia Biotech).

Cell culture and STxB uptake assaysPancreatic (DanG, MiaPaCa2, and BxPC3) and color-

ectal (DLD1 and HT29) cancer cell lines were kept inDulbecco’s Modified Eagle’s Media (DMEM) with fetalcalf serum (FCS; 10% for BxPC3, 7% for all other cells), 1%Pen/Strep, and 1% glutamine. STxB covalently labeledwith the fluorophore Cy3 was added at final concentra-tion of 2.5 mg/mL as published earlier (5). After varioustime points, cells were fixed with 3% paraformaldehydeand analyzed by immunofluorescence as specified (seeabove). Cell lines were authenticated by the German cellline repository (DSMZ).

Cytotoxicity assaysCell growth analysis was conducted using the Cell

Proliferation Kit II (XTT) from Roche Diagnostics, theamount of dye corresponding to the number of metabo-lically active cells. The cell lines were split into 6-wellplates (at 1 � 105 cells per well) and grown for 3 days.Treatment with irinotecan or STxB-SN38 was carried outfor 6 hours in cell medium. Cells were washed withDMEM and substituted with fresh medium for an addi-tional 48 hours. Cellswere then seeded onto 96-well plates(in triplicates at 0.5� 104 cells perwell and 1� 105 cells perwell) inmediumcontaining0.5%FCS.After 12 to 18hours,the XTT solutionwas added inmediumwith 3% FCS, andthe cells were incubated for 30 hours. The amount offormazan was quantified with a Mithras LB 940 micro-plate reader (Berthold Technologies) at 450 to 500 nm.Mean values of 5 independent analyses are shown. Acompetition assay was conducted with 5 mmol/L STxB-SN38 or 50 mmol/L unlabeled STxB or 50 mmol/L ofunlabeled STxB 5 minutes before treatment with5 mmol/L STxB-SN38.

Statistical analysisStatistical analyses were conducted using SPSS (ver-

sion 16.0), Graph Pad Prism 5, and GraphPad InStat3(Graph Pad Software). Data are presented as mean � SDor where specified as median (range). The comparison ofGb3 expression in pancreatic adenocarcinoma and corre-sponding nondiseased pancreas was done using pairedStudent’s t test. For this calculation, the 27 pairwisecomplete observations were used. For evaluating theinfluence of Gb3 expression on survival time, Cox regres-sion was calculated. The Mann–Whitney U tests wereused for evaluating the correlation of Gb3 expressionwithhistopathologic data. All statistical comparisons weredone at a 0.05 level of significance. The IC50 values werecalculated using GraphPad Prism 5, a general-purposecurve fitting and scientific graphics program.

Targeting of Pancreatic Carcinoma

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Results

Gb3 expression is increased in pancreatic cancerPreviously, we have shown that the glycosphingolipid

Gb3 is overexpressed in human colorectal cancer (5).Here, we have analyzed surgically resected pancreaticadenocarcinoma and corresponding nondiseased pan-creas by lipid extraction and TLC. Gb3 expression wasdetectable in all tumors tested (n ¼ 28; Table 1). Averagemedian expression was 54.9 ng Gb3/mg tissue as com-pared with 29.0 ng Gb3/mg tissue in normal pancreas (n¼ 27). The mean difference of 31.2 ng Gb3/mg tissue washighly significant (95% CI: 14.8–47.7, P ¼ 0.0006). In adirect comparison of matched tissue samples from indi-vidual patients, we could detect an increased Gb3 expres-sion in 78% (21 of 27) of the tumors as compared directlywith the surrounding normal pancreas (Fig. 1A, Supple-mentary Fig. S1). In 48% (13 of 27) of the samples, the Gb3level was higher than the calculatedmedian expression of54.9 ng Gb3/mg tissue. In addition, established pancrea-tic cancer cell lines were tested and all showed Gb3expression (Fig. 1B). However, Gb3 expression in celllines was lower than in primary patient samples. Thus,Gb3 was clearly upregulated in pancreatic adenocarci-noma, in good accordance with recent observations (14).HT29 colon cancer cells, which have been described tobind STxB (16), had 40.9 ng Gb3/mg wet weight (notshown), whereas DLD1 colon cancer cells completelylacked Gb3. Our statistical analysis showed no correla-tion between Gb3 expression and histopathologic orsurvival data. In fact, the HR for the level of Gb3expression and survival time was 1.005. The correlationwith histopathologic data involved the tumor size (pT,P¼ 0.78) and lymph nodemetastasis status (pN category,P ¼ 0.52) as well as grading (G, P ¼ 0.21), lymphangiosis(P ¼ 0.19), and angioinvasion (P ¼ 0.48; SupplementaryTable SI). For further testing on differential expression,the parameter Gb3-delta was devised, a calculationbased on the level of Gb3 in carcinomas minus Gb3

expression level in normal tissue for each of the 27matched pairs. In normal pancreatic tissue, Gb3 wasdetectable by lipid extraction and by STxB overlay oncryosections, albeit at lower levels than in carcinomasamples. However, comparison of Gb3 delta with his-topathology failed to show significant association (Sup-plementary Table SII). Thus, Gb3 seems to be broadlyexpressed in pancreatic neoplasm, irrespective of grad-ing or tumor stage.

Immunocytochemistry reveals tumor cells andendothelia as the source of increased Gb3

expressionAnalysis of tissue sections indicated that pancreatic

cancer cells, but not stroma components, were the majorsource of upregulated intratumoral Gb3 levels. Previouswork showed that endothelial, immune, and enteroendo-crine cells express Gb3 (5, 6, 27). Moreover, tumor cells ofepithelial origin, as well as tumor-associated blood ves-sels and stroma are the source of increased Gb3 expres-sion in colorectal adenocarcinoma (5). In the presentstudy, tissue from patients with high (n ¼ 3) or lowGb3 expression (n ¼ 3) was analyzed by immunohisto-chemistry (Fig. 2), and in parallel, by TLC. Despite dif-ferent sensitivities, both detection methods were in goodaccordance. In normal pancreas, Gb3 expression wasmainly detected in endothelia (Fig. 2A–C). In tumorswith low Gb3 expression, stromal components like bloodvessels were the major source of Gb3 expression (Fig. 2D–F). However, in tumors with high Gb3 expression, duct-like cancer cells were strongly stained with fluorescentlylabeled STxB (Fig. 2G–L). Tumor-associated endothelialcells were onlyweakly stained for STxB (Fig. 2F and J). Noassociation of tumor cell growth with Gb3 expression wasdetected in carcinoma with Gb3 overexpression (Supple-mentary Table SIII). However, in carcinomawith lowGb3expression, the few cells that were positive for Gb3 werenegative for the proliferation marker Ki67 (Supplemen-tary Table SII).

0 0

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MiaP

aCa2

BxPC3

DLD1

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15

20

Normal pancreas Pancreatic carcinoma

50

100

ng G

b 3/m

g tis

sue

Gb 3

(ng/

mg

cells

)150

200

A B

Figure 1. Gb3 is overexpressed inpancreatic carcinoma ascompared with nondiseasedpancreas. A, Gb3 was quantifiedby TLC from tissue extracts ofpancreatic carcinoma (n¼ 28) andmatched normal tissue (n ¼ 27).The difference in Gb3 expressionwas highly significant(P ¼ 0.0006). B, Gb3 is expressedin pancreatic cancer cell lines(DanG, MiaPaCa2, and BxPC3)but is undetectable in the controlcell line DLD1 (colorectal cancer).

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Intracellular uptake of STxB in pancreatic cancercellsNext, we investigated whether pancreatic cancer cells

actively take up STxB along the retrograde route. Incuba-tion of pancreatic cancer cell lines with fluorescentlylabeled STxB-Cy3 at 4�C allowed binding to the receptorin the absence of cellular uptake and resulted in STxBstaining at the plasma membrane (Fig. 3). Upon incuba-tion at 37�C, STxB-Cy3 was rapidly internalized andcolocalized with the Golgi marker Golgin p97 or withthe endoplasmic reticulum marker calnexin (Fig. 3, Sup-plementary Figs. S2 and 3), indicating that STxB followedthe retrograde route to the Golgi apparatus. Two daysafter the initial uptake, STxB was still detectable in punc-

tate structures, colocalizing with the Golgi in DanG andMiaPaCa2 cells. However, no obvious colocalization ofSTxB could be detected with Golgi structures or endo-plasmic reticulum in BxPC3 cells after 48 hours (Supple-mentary Fig. S2). In accordance with our findings oncolorectal cancer cells, we did not observe an inductionof apoptosis by STxB in any of the cell lines tested, asevidenced by staining for cleaved caspase-3 (data notshown). The cell line DLD1, which lacks Gb3, did notshow STxB binding and uptake (Fig. 3). The percentageSTxB-positive cells was quantified after 60 minutes: 19%of BxPC3, 20% of DanG, 35% of MiaPaCa2, comparedwith 56% for HT29 colon cancer cells (positive control),and 0% of DLD1 cells (negative control; not shown).

A B C

D E F

G H I

J K L

Figure 2. Immunocytochemical analysis of Gb3 expression. Hematoxylin and eosin-stained sections show nondiseased pancreas (A) and pancreaticcarcinoma (D and G). Fluorescently labeled STxB-Cy3 (red) was used on consecutive sections (B, E, and H); nuclear staining is shown in blue (DAPI). A–C,nondiseased tissue. Note the colocalization of STxB-Cy3 and CD31-positive blood vessels (green), high-power view in C. D–F, pancreatic carcinoma with lowGb3 expression. Costaining for blood vessels with anti-CD31 (green) reveals minor expression of Gb3 in blood vessels, and duct-like pancreatic cancer cellsare weakly positive for Gb3 staining (high-power view in F). G–J, pancreatic carcinoma with high Gb3 expression. Costaining for blood vessels reveals minorexpression of Gb3 in blood vessels, whereas duct-like pancreatic cancer cells are strongly positive for Gb3 staining (high-power views in I and J). K, associationof Gb3 expression with cell proliferation: double-positive cells are stained both for STxB as well as for Ki67 (arrow), whereas other proliferating cells arenegative for Gb3 expression (arrowhead). L, Gb3 expression is confined to the bona fide pancreatic cancer cells (top left). The mesenchymal marker vimentin(green) shows no colocalization with STxB. A, B, D, E, G, H, and L: magnification, �200 (size bar, 100 mm). All other: magnification, �400 (size bar, 20 mm).DAPI, 40,6-diamidino-2-phenylindole.






Supplementary Fig. S3 shows the distribution of STxBuptake in analyzed cell lines at the tested time points,distinguishing between plasma membrane, Golgi, andvesicular staining.

STxB specifically delivers a topoisomerase type Iinhibitor to pancreatic cancer cells

Because STxB was taken up efficiently by pancreaticcancer cells, we investigated the feasibility of STxB-mediated chemotherapy with a topoisomerase I inhibitorcoupled covalently to STxB (15). SN38 is linked to STxB viaa cleavable linker arm that allows for SN38 release from

STxB in membrane of the endoplasmic reticulum or Golgiapparatus. On pancreatic cancer cells, the cytotoxic effectsof this compound were compared with the effects of thestandard drug irinotecan (Fig. 4). The left column indicatescell growth in response to increasing concentrations ofirinotecan, the right column shows cell growth after treat-ment with STxB-SN38. All pancreatic cancer cell linesshowed significant growth inhibition upon treatment withirinotecan, and cell growthwasmore strongly inhibited bySTxB-SN38 (right column), with IC50 values in the sub-micromolar range (Supplementary Table SII and Supple-mentary Fig. S4). The inhibitory concentration 50% (IC50) is

Figure 3. Analysis of intracellular uptake kinetics of STxB in pancreatic cancer cells (DanG, MiaPaCa2, and BxPC3) and a colon cancer cell line (DLD1). Thewhite bars indicate 20 mm. STxB-Cy3 (red), Golgi staining with anti-golgin p97 (green), and nuclear staining (blue; overlay, nuclear staining combined withSTxB-Cy3 staining). Incubation with STxB was carried out on ice for 15 minutes, leading to distinct plasma membrane staining for STxB. After incubation withSTxB at 37�C for 15 minutes, colocalization with the Golgi apparatus is visible (arrow). After incubation with STxB at 37�C for 60 minutes, prominentcolocalization with Golgi structures occurs. After 48 hours in culture, STxB is still detectable within tumor cells. However, part of the STxB is found inintracellular vesicles that are clearly distinct from the Golgi apparatus (arrowhead). Note that Gb3-negative DLD1 cells show no uptake.

Maak et al.





the concentration that exerts half the specific cytotoxicity.Moreover, even when cells were treated with the che-motherapeutic agent for only 15 minutes, STxB-SN38showed a 20-fold greater efficacy than irinotecan (Sup-plementary Fig. S5). Irinotecan had a cytotoxic effecton DLD1 cells that lack the receptor Gb3, whereasSTxB-SN38 did not significantly inhibit cell growth(Fig. 4). At highest concentrations of STxB-SN38, areduction of cell growth was observable for DLD1cells, which may be due to nonspecific pinocytosis.

Figure 5 shows the results of a competition assay forMiaPaCa2 cells. Treatment of cells with unlabeled STxBinduced only a slight change in cell growthwhereas STxB-SN38 had a pronounced cytotoxic effect. After prior treat-ment with a 10-fold molar excess of unlabeled STxB, theeffect of STxB-SN38 was significantly diminished. Eventhough the competition with unlabeled STxB did notcompletely block the cytotoxic effects of STxB-SN38,this indicates that the effect of STxB-SN38 is mediatedspecifically by the Gb3 receptor.

Figure 4. STxB-SN38 inhibits cellgrowth in pancreatic cancer cells.Results of cell proliferationanalysis (n ¼ 5 independent testsfor each cell line and condition).Left, relative cell growthdetermined at fixed time pointsafter treatment with increasingconcentrations of irinotecan.Right, cell proliferation aftertreatment with increasingconcentrations of STxB-SN38.






Discussion

In the present work, we show the use of the nontoxicSTxB for specific targeting of human pancreatic cancercells. Pancreatic cancer is one of the most aggressivetumor entities and represents the fourth leading causeof cancer-related death in Western countries. So far, theonly curative therapy is surgery, but because of latediagnosis, most patients do not qualify for this treat-ment. The standard chemotherapy with gemcitabineand chemotherapeutic combinations improves cancer-related symptoms but enhances survival only modestly(2, 20). Therefore, new options for diagnostic andtherapeutic approaches are urgently needed. Pre-viously, we were able to show that STxB allows effi-cient targeting of colorectal carcinoma due to anincreased expression of its specific receptor, the glyco-sphingolipid Gb3 (or CD77; refs. 5, 6). The bindingkinetics of STxB to the receptor Gb3 are complex.The pentameric STxB has 3 binding sites per monomer.Thus, up to 15 Gb3 molecules per pentamer are pre-dicted to bind to STxB (7, 28). When bound to Gb3, STxBis internalized and transported to the Golgi apparatusand the endoplasmic reticulum via the retrograde path-way, avoiding degradation in the lysosomes (8–10).The protein is detectable in cancer cells for as longas 5 days (5, 7, 22, 23).

Consistent with recent data, we detected increasedlevels of Gb3 in 78% of pancreatic carcinoma comparedwith nondiseased pancreas (n ¼ 21 of 27 cases; ref. 13).Distler and colleagues reported overexpression of Gb3 in62% of pancreatic carcinoma (n ¼ 21) based on a newlydeveloped mass spectrometry method (13). We used aTLC approach, which yielded an absolute quantificationof Gb3 levels, based on wet weight of the tissue samplesanalyzed (5). The median expression of Gb3 for nondi-seased pancreas was 29 ng/mg of tissue, as opposed to55 ng Gb3/mg tumor tissue. However, the data reported

here show differences to previous reports, which may beexplained by the relatively small sizes of the patientcollectives. The difference in expression between normaland carcinoma tissue was statistically highly significantin our case (P ¼ 0.0006) but not in a previous study (13).Gb3 expression has been reported to lack correlation withany clinicopathologic parameter, except with tumor dif-ferentiation (13). This indicated high levels of Gb3 in lessdifferentiated tissue. However, we could not confirm acorrelation between tumor grading and Gb3 expressionon our collective data (P ¼ 0.21). Furthermore, our ana-lysis provided no evidence for a correlation of Gb3expression with clinical or pathologic parameters. Survi-val analysis showed no significant difference concerningGb3 levels. This is in accordance with our earlier findingsobtained on colon cancer (6) and indicates that elevatedGb3 expression is a frequent phenomenon in malignantgastrointestinal tumors, irrespective of stage and differ-entiation. The pathophysiologic role of upregulated Gb3expression in tumors is still unclear (29, 30). Recentlypublished data show that cisplatin induces Gb3 expres-sion in cancer cells and that Gb3 expression is linked toacquired cisplatin resistance (31). Moreover, increasedGb3 levels were correlated to increased expression ofmultidrug resistant 1/permeability glycoprotein(MDR1/PgP), a glycoprotein involved in the biosynthesisof glycolipids such as Gb3 (29). Gb3 and MDR1/PgPpartially colocalize, with Gb3-containing lipid rafts beingcrucial for intracellular MDR1/PgP surface trafficking.Furthermore, elevated levels of Gb3 were described indrug-resistant cancers (32, 33) and a functional interplaybetween MDR1/PgP and membrane Gb3 was suggested.

Importantly, carcinoma cells were the major Gb3source within the tumors. In contrast, the stroma wasessentially negative for Gb3. Therefore, the large stromacontent that is frequently found in pancreatic cancermay lead to an underestimation of the actual Gb3 levels.Interestingly, not all tumor cells within a given samplewere positive for Gb3. This heterogeneous expressionpattern is in accordance with earlier findings, reportingan association of the invasive status of colorectal cancercells with high Gb3 expression (4). Even though Gb3expression may be restricted to a subset of pancreaticcancer cells, a therapeutic intervention based on vector-ized STxB could still yield a clinical benefit, for exam-ple, by eliciting an antitumoral immune responsecaused by dying tumor cells. In the present study, anassociation of the proliferative status of cancer cellswith Gb3 levels was tested. However, there was noclear correlation of Gb3 content with the proliferationmarker Ki67. The Gb3 levels found in normal tissueare most likely due to endothelial cells, immune cells,and myofibroblasts, which have been reported toexpress Gb3 (24, 34, 35). We validated the endothelialexpression via CD31 staining of tissue sections.Endothelial cells in normal tissue were positive forGb3 and intratumoral blood vessels were not stronglymarked. However, we and others described a strong

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5 min 50 µmol/L STxB-WT + 6 h 5 µmol/L STxB-SN38

6 h 5 µmol/L STxB-SN38****

**

Figure 5. The cytotoxic effect of STxB-SN38 is mediated by the receptorGb3. XTT analysis of MiaPaCa2 cells after competing incubation withSTxB-SN38 and unlabeled STxB (n ¼ 3 assays). Mock treatment withunlabeled recombinant STxB (STxB-WT at 50 mmol/L) had only minoreffects, whereas treatment with STxB-SN38 (5 mmol/L) leads to significantgrowth inhibition. Five minutes of preincubation with 50 mmol/L STxB-WT,before treatment with STxB-SN38 (5 mmol/L), significantly reduces thecytotoxic effects of STxB-SN38 (*, significance, P < 0.05; **, highsignificance, P < 0.001).

Maak et al.





Gb3 expression in tumor-associated neovascularizationin human bladder cancer and colorectal carcinoma(35–38). Our findings indicate a less pronouncedexpression of Gb3 in intratumoral blood vessels inpancreatic cancer, which may result from organ-speci-fic variances concerning tumor vascularization. How-ever, it cannot be excluded that Gb3 may also beexpressed by further cells at levels that are below thedetection threshold in immunocytochemistry.Here, we could validate an intracellular uptake along

the retrograde pathway in pancreatic cancer cells,whereas no uptake of STxB was observed in DLD1 cellslacking the Gb3 receptor. Colocalization of fluorescentlylabeled STxB and the Golgi marker Golgin p97 occurredwithin 60 minutes, and intracellular STxB was stilldetectable after 2 days, even though cell-specific differ-ences were observable at late time points. Therefore,targeted chemotherapy mediated by STxB may be fea-sible on pancreatic cancer. As proof of principle, weused a previously established compound that consistsof a topoisomerase type I inhibitor, coupled covalentlyto STxB (16). SN38 is the active metabolite of irinotecan,which inhibits the DNA topoisomerase I (15, 17, 39).Irinotecan is activated by hydrolysis to SN38, but only2% to 8% of irinotecan is converted to SN38 in the liverand the tumor cells, which requires higher dosage toobtain therapeutic efficacy (40, 41). SN38 is far moreefficient than irinotecan, but due to its hydrophobicityand low stability in serum, its clinical use is limited (40,41). In vitro, the cytotoxicity was successfully shown forlung, ovarian, colorectal, and gastric cancer, and severaltrials investigate the clinical use of the agent (16, 17, 39,32–43). Covalent coupling to STxB could be an effectivemeans to stabilize SN38 (15) and has the advantage ofgreatly enhanced specificity. Only cells that express theGb3 receptor are targeted, and consequently, the clinicalside effects could be expected to be greatly diminished.Endothelial cells or kidney epithelia express Gb3 andmight therefore be negatively affected by a cytotoxicSTxB-based compound (44, 45). However, although Gb3levels and Shiga toxin (STx) binding does not changedepending on patients age, the effects, in particular thehemolytic uremic syndrome, appear age dependent.Therefore, the renal damage caused by STx and con-clusively in the case of therapeutic usage of STxB, thepossible renal side effects cannot be estimated yet andshould be evaluated (46).The standard therapy for pancreatic cancer is gemci-

tabine (2, 3, 47, 48). However, irinotecan is currentlyanalyzed as first-, second-, and third-line therapy for

advanced and metastatic pancreatic cancer in gemcita-bine refractory cases and showed slight but objectiveresponses as single agent or in combination (FOLFIRI;refs. 21, 49, 50). We therefore decided to test STxB-SN38as a novel therapeutic compound on pancreatic cancercells. As positive control, STxB-SN38 was tested on theGb3-expressing colon cancer cell line HT29 (15). Prolif-eration assays showed cytotoxicity of STxB-SN38 onHT29 cells (not shown). To validate the receptor speci-ficity, tests were conducted on DLD1, a Gb3-negativecolon cancer cell line. Reduced cytotoxicity wasobserved for STxB-SN38, whereas the cells respondedto irinotecan. All pancreatic cancer cell lines tested weresensitive to treatment with irinotecan or STxB-SN38.Importantly, STxB-SN38 had a significantly strongercytotoxic effect than irinotecan; the IC50 values forSTxB-SN38 were enhanced over 100-fold as comparedwith irinotecan, in accordance with earlier findings oncolorectal cancer cells (ref. 15; Fig. 4). However, therewas no positive correlation between the Gb3 expressionlevels in the cell lines and sensitivity toward STxB-SN38. This may indicate that additional parameters,such as regulation of apoptotic pathways, may deter-mine the cellular sensitivity to the cytotoxic compound.Moreover, a competition assay with a 10-fold molarexcess of unlabeled STxB in competition with STxB-SN38 showed that the cytotoxicity is mainly mediatedby Gb3-triggered uptake. Taken together, STxB-mediated tumor targeting offers a promising newapproach to diagnose or treat pancreatic cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Alexandra Gnann and Widya Johannes forexcellent technical assistance and Prof. Bernhard Holzmann for criticaldiscussion.

Grant Support

M.Maak received funding from theKommission f€urKlinischeForschung (KKF/MRI). L. Johannes was supported by Fondation InNaBioSant�e (n�2009CS026) andFondation Pierre-Gilles de Gennes pour la Recherche. L. Keller and K.-P. Janssenwere funded by the Wilhelm Sander-Stiftung.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received January 4, 2011; revised June 17, 2011; accepted July 15, 2011;published OnlineFirst July 25, 2011.

References1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics,

2009. CA Cancer J Clin 2009;59:225–49.2. Brus C, Saif MW. Second line therapy for advanced pancreatic

adenocarcinoma: where are we and where are we going? Highlightsfrom the "2010 ASCO Annual Meeting". Chicago, IL, USA. June 4-8,2010. JOP 2010;11:321–3.

3. Herrmann R, Bodoky G, Ruhstaller T, Glimelius B, Bajetta E, Sch€ullerJ, et al. Gemcitabine plus capecitabine compared with gemcitabinealone in advanced pancreatic cancer: a randomized, multicenter,phase III trial of the Swiss Group for Clinical Cancer Research andthe Central European Cooperative Oncology Group. J Clin Oncol2007;25:2212–7.






4. Kovbasnjuk O, Mourtazina R, Baibakov B, Wang T, Elowsky C, ChotiMA, et al. The glycosphingolipid globotriaosylceramide in the meta-static transformation of colon cancer. Proc Natl Acad Sci U S A2005;102:19087–92.

5. Falguieres T, Maak M, von Weyhern C, Sarr M, Sastre X, Poupon MF,et al. Human colorectal tumors and metastases express Gb3 and canbe targeted by an intestinal pathogen-based delivery tool. Mol CancerTher 2008;7:2498–508.

6. Janssen KP, Vignjevic D, Boisgard R, Falgui�eres T, Bousquet G,Decaudin D, et al. In vivo tumor targeting using a novel intestinalpathogen-based delivery approach. Cancer Res 2006;66:7230–6.

7. Johannes L, Romer W. Shiga toxins–from cell biology to biomedicalapplications. Nat Rev Microbiol 2010;8:105–16.

8. Lingwood CA, Binnington B, Manis A, Branch DR. Globotriaosylceramide receptor function - where membrane structure and pathol-ogy intersect. FEBS Lett 2010;584:1879–86.

9. Muthing J, Schweppe CH, Karch H, Friedrich AW. Shiga toxins,glycosphingolipid diversity, and endothelial cell injury. Thromb Hae-most 2009;101:252–64.

10. Sandvig K, Torgersen ML, Engedal N, Skotland T, Iversen TG. Proteintoxins from plants and bacteria: probes for intracellular transport andtools in medicine. FEBS Lett 2010;584:2626–34.

11. Bast DJ, Brunton JL, Karmali MA, Richardson SE. Toxicity andimmunogenicity of a verotoxin 1 mutant with reduced globotriaosyl-ceramide receptor binding in rabbits. Infect Immun 1997;65:2019–28.

12. Chart H, Law D, Rowe B, Acheson DW. Patients with haemolyticuraemic syndrome caused by Escherichia coli O157: absence ofantibodies to Vero cytotoxin 1 (VT1) or VT2. J Clin Pathol 1993;46:1053–4.

13. Distler U, Souady J, Hulsewig M, Drmi�c-Hofman I, Haier J, FriedrichAW, et al. Shiga toxin receptor Gb3Cer/CD77: tumor-association andpromising therapeutic target in pancreas and colon cancer. PLoS One2009;4:e6813.

14. Arab S, Rutka J, Lingwood C. Verotoxin induces apoptosis and thecomplete, rapid, long-term elimination of human astrocytoma xeno-grafts in nude mice. Oncol Res 1999;11:33–9.

15. El Alaoui A, Schmidt F, Amessou M, Sarr M, Decaudin D, Florent JC,et al. Shiga toxin-mediated retrograde delivery of a topoisomerase Iinhibitor prodrug. Angew Chem Int Ed Engl 2007;46:6469–72.

16. Ebrahimnejad P, Dinarvand R, Sajadi A, Jaafari MR, Nomani AR, AziziE, et al. Preparation and in vitro evaluation of actively targetablenanoparticles for SN-38 delivery against HT-29 cell lines. Nanome-dicine 2010;6:478–85.

17. Sapra P, Zhao H, Mehlig M, Malaby J, Kraft P, Longley C, et al. Noveldelivery of SN38 markedly inhibits tumor growth in xenografts, includ-ing a camptothecin-11-refractory model. Clin Cancer Res 2008;14:1888–96.

18. Burtness B, Thomas L, Sipples R,McGurkM, Salikooti S, ChristoforouM, et al. Phase II trial of weekly docetaxel/irinotecan combination inadvanced pancreatic cancer. Cancer J 2007;13:257–62.

19. de la Fouchardiere C, Negrier S, Labrosse H,Martel Lafay I, DesseigneF, M�eeus P, et al. Phase I study of daily irinotecan as a radiationsensitizer for locally advanced pancreatic cancer. Int J Radiat OncolBiol Phys 2010;77:409–13.

20. Gebbia V, Maiello E, Giuliani F, Borsellino N, Arcara C, Colucci G.Irinotecan plus bolus/infusional 5-fluorouracil and leucovorin inpatients with pretreated advanced pancreatic carcinoma: a multi-center experience of the Gruppo Oncologico Italia Meridionale. AmJ Clin Oncol 2010;33:461–4.

21. Sobin LH. TNM, sixth edition: new developments in general conceptsand rules. Semin Surg Oncol 2003;21:19–22.

22. Johannes L, Tenza D, Antony C, Goud B. Retrograde transport ofKDEL-bearing B-fragment of Shiga toxin. J Biol Chem 1997;272:19554–61.

23. Mallard F, Johannes L. Shiga toxin B-subunit as a tool to studyretrograde transport. Methods Mol Med 2003;73:209–20.

24. Falguieres T, Mallard F, Baron C, Hanau D, Lingwood C, Goud B, et al.Targeting of Shiga toxin B-subunit to retrograde transport route inassociation with detergent-resistant membranes. Mol Biol Cell2001;12:2453–68.

25. Pellizzari A, Pang H, Lingwood CA. Binding of verocytotoxin 1 to itsreceptor is influenced by differences in receptor fatty acid content.Biochemistry 1992;31:1363–70.

26. Falguieres T, Roemer W, Amessou M, Afonso C, Wolf C, Tabet JC,et al. Functionally different pools of Shiga toxin receptor, globotriaosylceramide, in HeLa cells. FEBS J 2006;273:5205–18.

27. Schuller S, Heuschkel R, Torrente F, Kaper JB, Phillips AD. Shiga toxinbinding in normal and inflamed human intestinal mucosa. MicrobesInfect 2007;9:35–9.

28. Bast DJ, Sandhu J, Hozumi N, Barber B, Brunton J. Murine antibodyresponses to the verotoxin 1 B subunit: demonstration of majorhistocompatibility complex dependence and an immunodominantepitope involving phenylalanine 30. Infect Immun 1997;65:2978–82.

29. Hakomori SI. Cell adhesion/recognition and signal transductionthroughglycosphingolipidmicrodomain.Glycoconj J 2000;17:143–51.

30. Ono M, Hakomori S. Glycosylation defining cancer cell motility andinvasiveness. Glycoconj J 2004;20:71–8.

31. Johansson D, Andersson C, Moharer J, Johansson A, Behnam-Motlagh P. Cisplatin-induced expression of Gb3 enables verotoxin-1 treatment of cisplatin resistance in malignant pleural mesotheliomacells. Br J Cancer 2010;102:383–91.

32. De Rosa MF, Ackerley C, Wang B, Ito S, Clarke DM, Lingwood C.Inhibition of multidrug resistance by adamantylgb3, a globotriaosyl-ceramide analog. J Biol Chem 2008;283:4501–11.

33. Lingwood CA, Khine AA, Arab S. Globotriaosyl ceramide (Gb3)expression in human tumour cells: intracellular trafficking defines anew retrograde transport pathway from the cell surface to the nucleus,which correlates with sensitivity to verotoxin. Acta Biochim Pol1998;45:351–9.

34. Miyamoto Y, Iimura M, Kaper JB, Torres AG, Kagnoff MF. Role ofShiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia colisignalling of human colon epithelium in vivo. Cell Microbiol 2006;8:869–79.

35. Obrig TG, Louise CB, LingwoodCA, Boyd B, Barley-Maloney L, DanielTO. Endothelial heterogeneity in Shiga toxin receptors and responses.J Biol Chem 1993;268:15484–8.

36. Heath-Engel HM, Lingwood CA. Verotoxin sensitivity of ECV304 cellsin vitro and in vivo in a xenograft tumour model: VT1 as a tumourneovascular marker. Angiogenesis 2003;6:129–41.

37. Lingwood CA. Verotoxin/globotriaosyl ceramide recognition: angio-pathy, angiogenesis and antineoplasia. Biosci Rep 1999;19:345–54.

38. Viel T, Dransart E, Nemati F, Henry E, Theze B, Decaudin D, et al. Invivo tumor targeting by the B-subunit of shiga toxin. Mol Imaging2008;7:239–47.

39. Ebrahimnejad P, Dinarvand R, Sajadi SA, Atyabi F, Ramezani F,Jaafari MR. Preparation and characterization of poly lactide-co-gly-colide nanoparticles of SN-38. PDA J Pharm Sci Technol 2009;63:512–20.

40. Kunii R, Onishi H, Machida Y. Preparation and antitumor character-istics of PLA/(PEG-PPG-PEG) nanoparticles loaded with camptothe-cin. Eur J Pharm Biopharm 2007;67:9–17.

41. Zhang JA, Xuan T, Parmar M, Ma L, Ugwu S, Ali S, et al. Developmentand characterization of a novel liposome-based formulation of SN-38.Int J Pharm 2004;270:93–107.

42. Koizumi F, Kitagawa M, Negishi T, Onda T, Matsumoto S, HamaguchiT, et al. Novel SN-38-incorporating polymeric micelles, NK012, era-dicate vascular endothelial growth factor-secreting bulky tumors.Cancer Res 2006;66:10048–56.

43. Nakajima TE, Yasunaga M, Kano Y, Koizumi F, Kato K, Hamaguchi T,et al. Synergistic antitumor activity of the novel SN-38-incorporatingpolymeric micelles, NK012, combined with 5-fluorouracil in a mousemodel of colorectal cancer, as comparedwith that of irinotecan plus 5-fluorouracil. Int J Cancer 2008;122:2148–53.

44. Kaye SA, Louise CB, Boyd B, Lingwood CA, Obrig TG. Shiga toxin-associated hemolytic uremic syndrome: interleukin-1 beta enhance-ment of Shiga toxin cytotoxicity toward human vascular endothelialcells in vitro. Infect Immun 1993;61:3886–91.

45. Taguchi T, Uchida H, Kiyokawa N, Mori T, Sato N, Horie H, et al.Verotoxins induce apoptosis in human renal tubular epitheliumderived cells. Kidney Int 1998;53:1681–8.

Maak et al.





46. Ergonul Z, Clayton F, Fogo AB, Kohan DE. Shigatoxin-1 binding andreceptor expression in human kidneys do not changewith age. PediatrNephrol 2003;18:246–53.

47. Burris H, Storniolo AM. Assessing clinical benefit in the treatment ofpancreas cancer: gemcitabine compared to 5-fluorouracil. Eur JCancer 1997;33:S18–22.

48. Heinemann V, Labianca R, Hinke A, Louvet C. Increased survival usingplatinum analog combined with gemcitabine as compared to single-agent gemcitabine in advanced pancreatic cancer: pooled analysis of

two randomized trials, the GERCOR/GISCAD intergroup study and aGerman multicenter study. Ann Oncol 2007;18:1652–9.

49. Klapdor R, Fenner C. Irinotecan(Campto R): efficacy as third/forth linetherapy in advanced pancreatic cancer. Anticancer Res 2000;20:5209–12.

50. Yoo C, Hwang JY, Kim JE, Kim TW, Lee JS, Park DH, et al. Arandomised phase II study of modified FOLFIRI.3 vs modified FOL-FOX as second-line therapy in patients with gemcitabine-refractoryadvanced pancreatic cancer. Br J Cancer 2009;101:1658–63.






Published OnlineFirst July 25, 2011.Mol Cancer Ther Matthias Maak, Ulrich Nitsche, Larissa Keller, et al. Toxin B-SubunitTumor-Specific Targeting of Pancreatic Cancer with Shiga

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