proteoglycans as target for an innovative therapeutic...

Small Molecule Therapeutics

Proteoglycans as Target for an InnovativeTherapeutic Approach in Chondrosarcoma:Preclinical Proof of ConceptCaroline Peyrode1,2, Val�erie Weber1,2, Aur�elien Voissi�ere1,2, Aur�elie Maisonial-Besset1,2,Aur�elien Vidal3, Philippe Auzeloux1,2, Vincent Gaumet1,2, Mich�ele Borel1,2,Marie-M�elanie Dauplat4, Mercedes Quintana1,2, Francoise Degoul1,2,Francoise R�edini5, Jean-Michel Chezal1,2, and Elisabeth Miot-Noirault1,2

Abstract

To date, surgery remains the only option for the treatment ofchondrosarcoma, which is radio- and chemoresistant due inpart to its large extracellular matrix (ECM) and poor vascularity.In case of unresectable locally advanced or metastatic diseaseswith a poor prognosis, improving the management of chon-drosarcoma still remains a challenge. Our team developed anattractive approach of improvement of the therapeutic index ofchemotherapy by targeting proteoglycan (PG)-rich tissues usinga quaternary ammonium (QA) function conjugated to mel-phalan (Mel). First of all, we demonstrated the crucial role ofthe QA carrier for binding to aggrecan by surface plasmonresonance. In the orthotopic model of Swarm rat chondrosar-coma, an in vivo biodistribution study of Mel and its QAderivative (Mel-QA), radiolabeled with tritium, showed rapid

radioactivity accumulation in healthy cartilaginous tissues andtumor after [3H]-Mel-QA injection. The higher T/M ratio of theQA derivative suggests some advantage of QA-active targetingof chondrosarcoma. The antitumoral effects were characterizedby tumor volume assessment, in vivo 99mTc-NTP 15-5 scinti-graphic imaging of PGs, 1H-HRMAS NMR spectroscopy, andhistology. The conjugation of a QA function to Mel did nothamper its in vivo efficiency and strongly improved the toler-ability of Mel leading to a significant decrease of side effects(hematologic analyses and body weight monitoring). Thus, QAconjugation leads to a significant improvement of the thera-peutic index, which is essential in oncology and enable repeat-ed cycles of chemotherapy in patients with chondrosarcoma.Mol Cancer Ther; 15(11); 2575–85. �2016 AACR.

IntroductionOne of the key problems in chondrosarcoma, the second most

common type of skeletal malignancy after osteosarcoma, is thelack of response to both chemotherapy and radiotherapy, givingrise to poor patient outcomes and leaving surgery as the onlyeffective curative treatment. The type of required surgical proce-dure may vary according to the grade, the initial location, and theextent of the disease. Large en bloc dramatic resection or amputa-tion, often necessary to allow the local control of the tumor and toavoid metastases, are at the origin of strong disability and mor-bidity (1). Nevertheless, wide resection is not always possible inlarge tumors and critical anatomic locations such as pelvis, axial

skeleton, and also in many cases of local recurrence. The clinicalbehavior and the prognosis for patients with chondrosarcomavary widely with a 10-year survival rate ranging from 29% to 83%depending on the tumor grades (2, 3).

For chondrosarcoma, the current clinical challenge is to preventrecurrences and to find better treatment options for patients withinoperable primary or recurrent disease, and metastases. Pub-lished data regarding the sensitivity of chondrosarcoma to cyto-toxic agents are scarce in both preclinical and clinical reports. Arecent study has shown that some chondrosarcoma cell lines maydisplay moderate sensitivity to doxorubicin and cisplatin, themost commonly used chemotherapeutic agents in sarcomas (4).In 2012, in a large retrospective and multicenter series, chemo-therapy using anthracycline was associated with a median pro-gression-free survival of 4.7 months in the first-line (2, 3). Thestudy of VanMaldegen and colleagues also demonstrated that thesurvival rate in patients with unresectable chondrosarcoma wasalso significantly higher in the case of systemic treatments (doxo-rubicin-based chemotherapy, and nonchemotherapy-basedagents such as imatinib and sirolimus) compared with patientsmanaged with best supportive care only (overall survival at 3years: 26% vs. 8% respectively, P < 0.05; ref. 5). According to suchpoor outcome and no global improvement in the survival ofpatients with chondrosarcoma over the past 30 years, personal-ized medicine including a combination of targeted therapies willmost likely be the best approach to tackle the resistance ofhuman chondrosarcoma.

1Clermont Universit�e, Universit�e d'Auvergne, Imagerie Mol�eculaire etTh�erapie Vectoris�ee, Clermont-Ferrand, France. 2INSERM, UMR990,Clermont-Ferrand, France. 3Arronax, Saint-Herblain, France. 4CLCCJean-Perrin, Clermont-Ferrand, France. 5INSERM, UMR S957, NantesAtlantique Universit�e, Nantes, France.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

C. Peyrode and V. Weber contributed equally to this article.

Corresponding Author: Caroline Peyrode, UMR 990 Inserm/UdA, 58 rue Mon-talembert, BP 184, 63005 Clermont-Ferrand Cedex, France. Phone: 334-7315-0825; Fax: 347-315-0801; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0003

�2016 American Association for Cancer Research.

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Treatments targeting tumor microenvironment have thereforebeen suggested as complements to surgical excision (6, 7). Nev-ertheless, due to the large amount of extracellular matrix (ECM)and the poor vascularity of chondrosarcoma, the anticanceragents have to diffuse over a relatively long distance to reachtumor cells and be effective (5).

The best approach for a personalized medicine of chondro-sarcoma might be to take advantage of such phenotypic micro-environmental features, by developing an attractive approachof targeting the proteoglycan (PG)-rich cartilaginous tissuesusing a quaternary ammonium (QA) function as a carrier fordrugs or radionuclides. The positively charged QA interactswith the sulfate and carboxylate groups present in PGs highlyexpressed in the chondrogenic ECM (8). With this strategy, wesuccessfully delivered imaging agents as well as therapeuticdrugs (such as anti-inflammatory drugs, metalloproteinaseinhibitors, cytotoxics, and more recently radiosensitizing nano-particles; refs. 8–13) selectively to PG-rich tissues such ascartilage and chondrosarcoma. In particular, the suitability andhigh sensitivity of the radiotracer N-[triethylammonium]-3-propyl-1,4,7,10,13-pentaazacyclopentadecane radiolabeledwith 99mTc (99mTc-NTP 15-5) was validated for in vivo diagnosisand follow up of chondrosarcoma via scintigraphic imaging(14–16). This radiotracer is currently transferred into clinicfor a first into human study. In parallel, we developed a thera-peutic approach with a QA derivative of melphalan (Mel-QA),an alkylating agent, that showed promising results from in vitroand in vivo preclinical experiments (13). The aim of the currentstudy was to characterize this therapeutic strategy.

First of all, the binding ability ofMel-QA to aggrecan (major PGin chondrosarcoma) was demonstrated by surface plasmon res-onance (SPR) respectively to its nontargeted equivalent (Mel).Quantitative whole-body autoradiography showed rapid accu-mulation of radioactivity in cartilaginous tissue after injection ofthe tritium-radiolabeled Mel-QA, as compared with Mel. Themain interest of the QA vector was to strongly attenuate sideeffects, monitored by hematologic analyses and body weight,while maintaining a tumor growth inhibition assessed byin vivo99mTc-NTP 15-5 scintigraphic imaging, 1H-HRMAS NMRspectroscopy, and histologic analyses of biopsies.

The work reported here clearly validates the relevance ofPGs as target for an innovative therapeutic approach inchondrosarcoma.

Materials and MethodsChemical synthesis

Mel was used as its hydrochloride form after treatment of Mel(Interchim) with a solution of 2 N hydrochloric acid in diethylether. The QA conjugate of Mel (Mel-QA) was prepared asalready published (13, 17) and fully characterized (Supple-mentary Fig. S1).

The NTP 15-5 and its nontargeted equivalent (i.e., 15-5)were synthesized and radiolabeled with 99mTc as describedpreviously (18).

SPR binding assaysTo demonstrate the binding affinity between the QA entity and

aggrecan, we used the SPR method with aggrecan immobilizedonto the surface of the sensor chip. When studied compoundsinteract with aggrecan, they form a complex that modifies the

refractive index. This change is measured in real-time and thesignal obtained is plotted in resonance unit (RU) versus time(19, 20). SPR assayswere carriedout onaBiacore T200 instrumentwith CM4 sensor chips (GE Healthcare). Sensor chip CM4 has adextran matrix with a low degree of carboxylation which may beof value for reducing nonspecific binding with highly positivecharged analytes and may be useful for kinetic analysis. Toimmobilize the ligand, sensor chip was firstly activated using a1:1, v:v, mixture of a 0.2 mol/L aqueous 1-ethyl-3-(3-dimethyla-minopropyl) carbodiimide hydrochloride salt solution and a0.5 mol/L aqueous N-hydroxysuccinimide (Amine CouplingKit, GE Healthcare) solution at a flow rate of 5 mL/minute for10 minutes. After that, aggrecan from bovine articular cartilage(Sigma Aldrich) was coated to the sensor chips at 400 mg/mL inrunning buffer HBS-Pþ 1� (0.01mol/L HEPES, 0.15mol/L NaCl,0.05%, v/v, surfactant p20, pH 7.4) containing 6 mmol/L ofhexadecyltrimethylammoniumbromide (CTAB) with a flow rateof 5 mL/minute to a level of approximately 500 RU. Unoccupiedbinding sites were blocked using an aqueous 1 mol/L ethanol-amine solution (pH 8.5; Amine Coupling Kit, GE Healthcare)with a flow rate of 5 mL/minute for 10 minutes. Mel and Mel-QAwere injected with a flow rate of 30 mL/minute in increasingconcentrations (0–2 mmol/L) using five 600-second injectionsand a 200-second dissociation time. After each experiment, flowcells were regenerated twice with NaCl 2 mol/L for 150 seconds.The interactions were recorded as the difference in responsebetween the immobilized aggrecan flow cell and a correspondingcontrol flow cell (activated and blocked, but without immobi-lized aggrecan). Kinetic and statistic parameters (Kd and c2) weredetermined by Biacore T200 Evaluation software by fitting thesteady-state values at equilibrium (Req). Experiments were per-formed in triplicate.

Orthotopic model of Swarm rat chondrosarcomaThe Swarm rat chondrosarcoma (SRC) line (Dr. Patrick

A. Guerne, Geneva, Switzerland) was delivered as tissue frag-ments which were frozen until use. Protocols were led underthe authorization of the French Directorate of Veterinary Ser-vices and in accordance with the 2010/63/UE Directive andwere conducted as described previously (13) using male Spra-gue–Dawley rats (Charles River Laboratories). All experimentswere conducted after ethics committee approval (C2E2A, n�CE82-12). Allograft transplantation of a 10-mm3 SRC fragmentwas performed on the right paw, the other paw being used asthe contralateral control. These SRC fragments were collectedfrom well-developed tumors on the paratibial area of donorSprague–Dawley rats.

Radiosyntheses of [3H]-Mel and [3H]-Mel-QAAn excess of N-t-Boc-4-amino-L-phenylalanine ethyl ester 1

was alkylated by reaction with [3H]-2-bromoethanol ([3H]-3),previously obtained in situ from ethyl bromoacetate 2 (2 mmol)and [3H]-NaBH4 (3.7 GBq, diluted with 2.2 mmol of unlabeledNaBH4). Then the crude reactionmixture containing amine 1 andN-t-Boc-4-(2-hydroxy-2-[3H]ethyl)amino-L-phenylalanine ethylester ([3H]-4) reacted with ethylene oxide (approximately 3 mL)in acetic acid to provide the corresponding 4-[bis(2-hydroxy-2-[3H]ethyl)]amino-phenyl derivative ([3H]-5) in 50% radiochem-ical yield (2.15 GBq, 2 mmol). Appel chlorination with triphe-nylphosphine (5.9 mmol) and carbon tetrachloride (2 mL) indichloromethane (20 mL) gave, after silica gel chromatographic

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purification, the corresponding tritiated dichloro derivative [3H]-6 (0.96 mmol, 962 MBq) in 48% chemical yield. Then, the esterand the t-Boc protecting group of compound [3H]-6 were hydro-lyzed with an aqueous 6 N hydrochloric acid solution (5 mL) toprovide the hydrochloride salt of tritiated Mel ([3H]-Mel, 0.96mmol, 740 MBq) in quantitative chemical yield. On the basis ofthis radiolabeling sequence, [3H]-Mel was isolated with a specificactivity of 777 MBq/mmol and a radiochemical purity > 98%, asdetermined by HPLC analyses (Supplementary Fig. S2). Theoverall yield of this radiochemical procedure was 20%.

For the preparation of tritiated Mel-QA, a five-step radiosynth-esis starting from hydrochloride salt of [3H]-Mel was developed.Thus, after Boc protection of the amino group of [3H]-Mel (0.69mmol, 573 MBq), N-protected Mel was immediately coupledwith 3-(dimethylamino)propylamine (0.83 mmol) in the pres-ence of dicyclohexylcarbodiimide (DCC, 1.03 mmol) and1-hydroxybenzotriazole (HOBt, 0.74 mmol) to give the corre-sponding amide intermediate [3H]-7 in 75% chemical yield afterpurification by column chromatography. Treatment with methyliodide (2.3 mmol) in ethanol gave the QA compound [3H]-8in quantitative yield. Finally, the t-Boc–protecting group wasremoved using a 2 N ethanolic hydrochloric acid solution(12 mL). A chromatography over a chloride ion-exchange resin(Dowex 1 � 8–200 mesh, Aldrich) provided the desired QAderivative of Mel ([3H]-Mel-QA, 0.46 mmol, 407 MBq) in 75%overall chemical yield from [3H]-Mel.

Finally, [3H]-Mel-QA was isolated with a specific activity of814 MBq/mmol and a radiochemical purity > 97%, as deter-mined by HPLC analyses (Supplementary Fig. S2). The overallyield of this radiosynthesis was 15% based on [3H]-NaBH4.

Tissue distribution study of [3H]-Mel and [3H]-Mel-QAconjugate by whole-body autoradiography and urinaryexcretion

This study was performed on thirty SRC-bearing rats at stageday 20 postimplantation. Animals were randomized into twogroups (n ¼ 15 per group) and received intravenous injectionof 2.5 MBq (in 200 mL of physiologic saline) of [3H]-Mel or[3H]-Mel-QA.

At several time points after injection (5 and 15 minutes, then1, 6, and 24 hours), intracardiac blood collection were per-formed (3 rats from each group per time point) under anes-thesia by inhalation of isoflurane (CSP) in air (1.5%, 1 L/minute). Animals were then immediately sacrificed by carbondioxide inhalation, frozen in liquid nitrogen, and embedded inblocks of carboxymethyl cellulose 2%, that were sagittallysectioned into 40-mm slices at �22�C with a cryomicro-tome(Reichert-Jung Cryopolycut). Each slice was dehydrated for 48hours in a cryochamber. Whole-body slices (25 slices peranimal and per time point) were then exposed for 1,000minutes in a digital autoradiographic analyzer (bImager,Biospace Measures). Surfacic activity (counts in cpm/mm2)was quantified in regions of interest (ROI) delineated overorgans of interest including tumors, corrected for radioactivedecay, and expressed as % of injected dose (ID)/g of tissue,after calibration with tritium microscale standards[Autoradiographic [3H] Microscale, Amersham Biosciences].Tumor-to-muscle ratio was calculated as follows: T/M ¼(counts per minute/mm2 in tumor)/(counts per minute/mm2

in muscle).

In a parallel experiment, groups of 6 rats received [3H]-Mel or[3H]-Mel-QA, and were housed in metabolic cages for urinecollection from 6 to 24 hours after injection.

Radioactivity of urine or blood samples was directly measuredafter addition of Packard Ultima Gold cocktail (PerkinElmer) in aWallac Winspectral 1414 Liquid Scintillation Spectrometer(PerkinElmer).

Tumor growth inhibition assessmentExperiments were conducted on SRC-bearing rats being

randomized into three groups (control, Mel-QA, and Mel),with a number of 6–8 animals/group. Treated rats receivedthree doses of Mel or Mel-QA (16 mmol/kg per injection) byintravenous route at 4-day intervals (q4d � 3 schedule), begin-ning on day 8 postimplantation. Controls received the excip-ient by intravenous route according to the same schedule.Animal weight and tumor volume were recorded twice a week.The tumor volume (TV) of each tumor was estimated using theformula: TV (mm3) ¼ (L � W2)/2 where L is the length in mm,and W the width in mm.

At the end of the study, tumors were removed, fixed in 10%buffered formalin, and then embedded in paraffin. Slices (5 mm)were then stained with hematoxylin–eosin.

Antitumor efficacy assessed by in vivo 99mTc-NTP 15-5scintigraphic imaging targeting PGs

Antitumor efficacy was monitored by scintigraphic imaging,using the radiotracer 99m Tc-NTP 15-5 developed by our groupat day 7 (one day before treatment) and day 30 (2 weeks aftertreatment; ref. 15). Acquisitions were performed 30 minutesafter intravenous tracer administration (25 MBq/animal), witha 10-minute planar acquisition for each posterior paw posi-tioned over the parallel-hole collimator of a small-animal gcamera (GammaImager, Biospace) and with a 15% windowcentered on the 140-keV photopeak of 99mTc. All the scans wereevaluated by the same experienced investigator, using fixed-size ROIs delineated over tumor and muscle patterns. For eachROI, total activity, count in cpm (counts per minutes) per mm2

were obtained. Tumor-to-muscle ratio was calculated: T/M ¼(counts per minute/mm2 in tumor)/(counts per minute/mm2

in muscle).To assess the in vivo specificity of 99mTc-NTP 15-5 imaging,

additional SRC animals (n ¼ 10) were injected with 99mTc-NTP15-5 or its nontargeted equivalent (99mTc-15-5) at day 20 post-implantation when tumors exhibited a mean volume of 949 �223 mm3. Dynamic planar imaging (DPI) was performed todetermine the kinetics of the tracer distribution in tumor. SRCanimals were intravenously injected via the tail vein with 25MBqof 99mTc-NTP15-5or 99mTc-15-5 simultaneously to the starting ofa 120-minute duration list mode acquisition using a 5-minutesampling time. Time–activity curves were obtained from ROIsdrawn around tumor and muscle with all measured activitiescorrected for radioactive decay and T/M ratio calculated at eachtime point.

Antitumor efficacy analysis by 1H HRMAS NMR spectroscopyprofile

1H HRMAS spectroscopy profile analyses were performed in aparallel study following the same protocol with additional ani-mals (4 animals per group). Tumors were excised one week afterthe final injection, rapidly frozen in liquid nitrogen and stored at�80�C until analysis.

Proteoglycans as Target for Therapy of Chondrosarcoma

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All experiments were performed on frozen tumor samplesflushed for a few seconds in cooled D2O. 1H NMR spectroscopywas performed on a small-bore Bruker DRX 500magnet (Bruker)equipped with an HRMAS probe. Samples were then set into4-mm diameter, 50-mL free volume ZrO2 rotor tubes withoutupper spacer. A total of 3 mL of D2O containing 0.75% 3-(tri-methylsilyl)propionic-2,2,3,3-d4 acid (TSP; Sigma), used forpeak referencing, was added to the rotor tubes to lock thespectrometer. Rotors were spun at 4 kHz to keep the rotationsidebands out of the acquisition window. A Bruker cooling unitwas used to maintain the sample temperature at 277 K to min-imize tissue degradation. One-dimensional 1H NMR sequencewas a saturation recovery sequence. The resonances were assignedon the basis of the known chemical shifts of the major structuralgroups using tissue data (21). The relative content of metaboliteswas estimated by peak area integration. To compare themetabolicprofiles between experimental groups, each peak was normalizedwith the respect of the total integral of the spectrum in the 0.5 to4.7 ppm region (21–23). Sample stability inside the rotor wasassessed at a temperature of 277 K over several hours by a series ofanalyses. During the 48-minute overall acquisition period, nosignificant changes in NMR signals of biological material wereobserved. Each experiment was performed in triplicate.

Side-effect assessmentSide effects of treatments with Mel or Mel-QA were assess-

ed by both animal's weight and hematologic parametermeasurements.

(i) Weight variation was calculated according to the formula:

% ¼ [(weight at day x � weight at day 4)/weight at day 4)� 100.

(ii) Hematologic parameters were assessed at day 18, that is,two days after the end of treatments. The blood (50 mL byretro-orbital puncture) of each animal was removed andprocessed on a Melet Schloesing MS9-5 HematologyAnalyzer (Diamond Diagnostics).

Statistical analysesStatistical analyses were conducted using the GraphPad

Prism 5 software. Data are reported as mean � SD. Resultswere analyzed by ANOVA followed by Tukey post test. Weconsidered P values < 0.05 to indicate statistical significance(�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

ResultsEvaluation of the QA binding to PGs by SPR

Studies were performed for Mel-QA, respectively, to Melinjected in increasing concentrations (0–2 mmol/L). The sensor-grams obtained after signal subtraction of control was displayedversus time. As illustrated in Fig. 1 and regards to Kd values, Mel-QA exhibited affinity to aggrecan (Kd ¼ 2.25 � 0.92 mmol/L) incontrast to Mel, demonstrating that QA vector allows aggrecanbinding.

In vivo biodistribution studies of [3H]-Mel and [3H]-Mel-QARadiosyntheses of [3H]-Mel and [3H]-Mel-QA. The radiosynthesesof [3H]-Mel and [3H]-Mel-QA are shown in Fig. 2A. Both Mel-QA and Mel were radioisotopically labeled on the alkylatingmoiety, also 3H radionuclide was introduced in the chloroethylgroups. [3H]-Mel was synthesized from N-t-Boc-4-amino-L-phenylalanine ethyl ester according to a slightly modifiedsequence previously reported in the literature (24). For the

Kd = 302 mol/L2 = 1.25

Kd = 3.3 × 10–3 mol/L χ χ2 = 0.894

Mel-QA

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Figure 1.

Affinity kinetics between aggrecan and Mel-QA or Mel by SPR. Representative examples of sensorgrams for the affinity kinetic analysis of Mel-QA and Mel. Thedissociation constant (Kd) of each example are given.

Peyrode et al.





preparation of [3H]-labeled Mel-QA, the radiosyntheticsequence starting from [3H]-Mel was adapted from the five-step synthesis developed for the corresponding nonradioactivederivative and 14C-labeled Mel-QA (17, 25). Both [3H]-Mel and[3H]-Mel-QA were successfully obtained with a specific activityof 814 MBq/mmol and a radiochemical purity > 97 %, asdetermined by radio-RP-HPLC analyses.

QA functionalization allows tumor uptake of Mel. The distribu-tion of radioactivity in several tissues after intravenous injectionof 2.5 MBq of [3H]-Mel or [3H]-Mel-QA in SRC animals isexpressed as the percentage of injected dose per gram of tissue(% ID/g; Table 1). After injection of [3H]-Mel-QA, radioactivity

rapidly accumulates in PG-rich tissues such as cartilages andtumor (Fig. 2C). Five minutes postinjection (p.i.) of [3H]-Mel-QA, the T/M value was significantly higher (P < 0.05) comparedwith [3H]-Mel (T/M values 5 minutes postinjection: 5.77� 1.11for [3H]-Mel-QA versus 2.22 � 1.20 for [3H]-Mel; Fig. 2B).

Furthermore, high levels of each drug were found in themetabolizing and elimination organs, such as liver and kid-neys. As expected for small hydrophilic compounds, Mel andMel-QA are cleared in the kidney (i.e., about 16% of cumu-lative renal excretion) and no significant difference wasobserved between the two compounds at 24 hours postinjet-cion (Table 1). However, radioactivity was observed in theliver after [3H]-Mel-QA injection as compared with [3H]-Mel.

T

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a Reagents and conditions: (i) NaBH4 , [3H]-NaBH4 (3.7 × 103 MBq), EtOH, rt, 5 h; (ii) EtOH, 90°C, 17 h;(iii) ethylene oxide, acetic acid, rt, 24 h; (iv) PPh3, CCl4, DCM, rt, 18 h; (v) 6 N HCl, 90°C, 4 h30; (vi) 1)(Boc)2O, TEA, MeOH, rt, 1 h30; 2) NH2(CH2)3N(CH3)2, DCC, HOBt, DCM, rt, 18 h; (vii) CH3I, EtOH, rt,18 h; (viii) 1) HCl/EtOH, rt, 3 h; 2) Dowex(chloride) 1 × 8−200.

Radioactivity distribution of [3H]-Mel-QA at 5 min p.i.40-µm thick sagittal slice

Corresponding anatomic slice

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Radioactivity distribution of [3H]-Mel at 5 min p.i. 40-µm thick sagittal slice

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Figure 2.

Conjugation with QA allows a rapid accumulation of Mel in tumor. A, radiolabeling of [3H]-Mel and its QA conjugate ([3H]-Mel-QA). B, T/M ratios at 5 minutes,1 hour, and 6 hours after intravenous administration of [3H]-Mel and [3H]-Mel-QA. � , P < 0.05. C, representative two-dimensional images obtained ofrat whole-body slices showing the tissue distribution of radioactivity at 5 minutes postinjection (p.i.) after intravenous administration of [3H]-Mel-QA (C1)or [3H]-Mel (C2). T, tumor; AC, articular cartilage.






Finally, radioactivity in the blood was higher after injectionof [3H]-Mel than [3H]-Mel-QA (Table 1).

In vivo antitumor efficacy and side effects of Mel and Mel-QAin SRC modelMel-QA delays chondrosarcoma growth in vivo. Mel-QA and Melwere given at 16 mmol/kg according to a q4d � 3 schedulebeginning on day 8 after tumor implantation. The control groupreceived intravenous saline injection.

On the basis of the tumor volume monitoring, a significantinhibition of tumor growth (P < 0.01) was observed for bothMel-QA- and Mel-treated rats compared with the control group,but no significant difference was observed between the twotreated groups (Fig. 3A).

In vivo antitumor effects were also assessed by 99mTc-NTP15-5 scintigraphic imaging, as this radiotracer was previouslydemonstrated to provide suitable criteria for monitoring chon-drosarcoma (14–16). As illustrated in Fig. 3B, quantitativeanalysis of in vivo 99mTc-NTP 15–5 imaging of tumors evi-denced significant changes in radiotracer uptake in treatedanimals versus controls. A significant increase of 84% of T/Mratio (P < 0.05) was observed in nontreated rats between day 7(1 day before treatment) and day 30 (14 days after the lastdrug injection), whereas this ratio remained overall constant inMel- or Mel-QA–treated rats. Furthermore, to assess the in vivospecificity of 99mTc-NTP 15-5 imaging and to exclude thepossibility of unspecific uptake in tumoral tissue, 99mTc-NTP15-5 and its nontargeted equivalent (99mTc-15-5) were injectedby intravenous route in additional SRC-bearing rats (n ¼ 10).Time–activity curves are shown in Fig. 3C in term of T/M ratio.From 5 minutes postinjection of 99mTc-NTP 15-5, T/M ratiogradually increased and reached a plateau as soon as 30minutes which was maintained at least for 1 hour (T/M values1 hour postinjection: 2.25 � 0.11), allowing tumor visualiza-tion with an excellent contrast (Fig. 3D). On the contrary, T/Mratio for 99mTc-15-5 was around 1 throughout the course ofthe imaging. According to these results, we demonstrated thepotential of 99m Tc-NTP 15-5 imaging as a "methodologycompanion" of targeted therapies of chondrosarcoma.

To characterize the antiproliferative effects, tumors wereremoved one day after the end of the treatments and anatomo-pathologic analyses were performed. For both treated groups,histopathologic analyses showed signs of tumor regression, suchas necrotic cells, fibroinflammatory component, and increasedvascularization. These signs were observed for 57% and 40% oftumors in the Mel-QA and Mel groups, respectively (Fig. 4A).Furthemore, mitotic activity index values showed a significantdecrease (P < 0.01) in the proliferating activity in the two treatedgroups (Fig. 4B). In contrast, the tumors of nontreated rats werecharacterized by hypercellularity with a high mitotic activityindex.

Finally, in a parallel study, SRC-bearing rats were treatedfollowing the same protocol and tumors were excised one weekafter the final injection for 1H-HRMAS NMR spectroscopy anal-yses (Fig. 4C) which confirmed necrosis and apoptosis. Resultsrevealed a significant increase (P < 0.01) in the spectral intensityratio of methylene (CH2) resonance (at 1.28 ppm) to methyl(CH3) resonance (at 0.9 ppm) in the Mel-QA–treated group ascompared with controls (Fig. 4D). This ratio, which is consideredas an apoptosis marker (26, 27), was also significantly higher(P < 0.01) in the Mel-QA–treated group than in the Mel-treatedgroup (Mel-QA: 2.34� 0.22 versusMel: 1.47� 0.08). In the sameway, a significant increase in CH2(lip)/total creatine(tCr) ratio,which is considered as a necrosismarker (28), was observed in theMel-QA group (Mel-QA: 11.5 � 2.07 vs. Mel: 5.0 � 1.26).

Significant reduction of side effects with QA derivative. The bodyweight of each rat was regularly recorded and hematologicparameters were assessed 48 hours after the last dose of bothcompounds (Fig. 5). Mel-treated rats exhibited significantweight loss starting from the second injection which reached15% at day 23 postimplantation (Fig. 5A). Furthermore, theclinical score including diarrhea, rough coat, closed eyes, leth-argy, and bleeding were determined for each animal through-out the time course of the study: we assigned a score of 1(presence) or 0 (absence) for each of these criteria. One dayafter the last dose of treatment, no clinical side effects wereobserved for Mel-QA–treated rats and controls, in contrast to

Table 1. Comparative distribution of radioactivity in tumor, cartilage, liver, kidney, lung, muscle and blood, and cumulative radioactivity excreted in urine afterintravenous administration of [3H]-Mel or [3H]-Mel-QA

Comparative distribution of radioactivity after i.v. administration of [3H]-Mel-QA or [3H]-MelTumora Cartilagea Livera Kidneya Lunga Musclea Bloodb

5 min [3H]-Mel 1.79 � 1.62 0.93 � 0.01 7.87 � 6.64 29.48 � 25.39 6.15 � 3.82 1.10 � 0.55 0.24 � 0.05[3H]-Mel-QA 2.73 � 0.48 1.97 � 0.12c 17.23 � 4.82 41.80 � 3.43 3.43 � 0.81 0.48 � 0.04 0.09 � 0.00d

15 min [3H]-Mel 1.84 � 1.44 ND 6.43 � 3.34 35.13 � 19.07 4.90 � 2.06 1.57 � 0.97 0.20 � 0.07[3H]-Mel-QA 3.35 � 0.01 3.38 � 0.12c 10.28 � 10.54 37.50 � 0.05 19.58 � 0.00 0.85 � 0.58 0.00 � 0.00

1 h [3H]-Mel 2.05 � 0.77 ND 3.66 � 0.42 29.13 � 7.09 3.31 � 0.30 1.26 � 0.17 0.07 �0.05[3H]-Mel-QA 1.62 � 0.62 ND 13.28 � 4.68 21.71 � 4.17 2.05 � 2.05 0.53 � 0.18 0.05 � 0.03

6 h [3H]-Mel 1.26 � 0.01 ND 1.26 � 0.13 12.01 � 0.91 1.09 � 0.01 0.68 � 0.17 0.04 � 0.00[3H]-Mel-QA 0.72 � 0.20 ND 2.70 � 0.02d 9.84 � 0.96 1.56 � 0.17 0.46 � 0.20 0.03 � 0.01

24 h [3H]-Mel 1.04 � 0.01 ND 1.25 � 0.38 9.35 � 2.55 0.88 � 0.01 0.60 � 0.05 0.08 � 0.03[3H]-Mel-QA 0.87 � 0.01 ND 1.37 � 0.05 5.60 � 0.01 0.87 � 0.01 0.43 � 0.11 0.03 � 0.00

Cumulative radioactivity excreted in urine after i.v. administration of [3H]-Mel-QA or [3H]-Mel (% of ID)Time (h) 0–6 6–24 0–24[3H]-Mel 12.34 4.31 16.65[3H]-Mel-QA 10.01 5.90 15.91

Abbreviation: ND, not detectable.a% of ID/g tissue.b% of ID.cP < 0.01.dP < 0.05.

Peyrode et al.





the Mel-treated rats with an average clinical score of 2.7 � 2.1.For Mel-treated rats, these symptoms were associated with asevere leucopenia, a common side effect of alkylating agents,which was marked by a significant decrease in white blood cellsand lymphocytes numbers (Fig. 5B). Furthermore, in compar-ison with Mel-treated animals, an improvement in hematologicparameters was observed for Mel-QA treatment, that is, atten-uation of leucopenia with, more specifically, a significantattenuation of lymphopenia (P < 0.01).

Discussion

Chondrosarcoma, with an estimated incidence of 0.2 in100,000 patients per year, is a rare disease with most patientsbeing cured by a surgical excision. However, in case of unre-sectable locally advanced, inoperable, or metastatic disease,chemotherapy and radiotherapy appear largely ineffective.The poor prognostic and lack of effective treatments marked-

ly highlight a pressing need to develop new therapeuticapproaches (1, 5).

As efficacy of antineoplastic treatments is mainly hampered bytoxic side effects, improving their therapeutic index, by reducingadverse effects and increasing drug accumulation in tumor tissue,remains a challenge in drug development. Among the numerousstrategies aimed at such objectives, "carrier" with preferentialbinding to one or more tumor targets may offer promisingtherapeutic opportunities. This was the strategy employed herefor experimental chondrosarcoma: a QA function (i.e., a carrierexhibiting affinity for the high negative fixed charged density ofPGs) was conjugated to Mel. In this way, we expected to improveits therapeutic index by reducing systemic toxicity while keepingan antitumor activity.

First of all, the role of QA function for PG binding wasdemonstrated in vitro by SPR with aggrecan, the major PGcomponent of chondrosarcoma. As expected, binding ofthe QA-functionalized derivative of Mel to aggrecan was

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Figure 3.

Evaluation of the in vivo antitumor activity of Mel-QA and its nontargeted equivalent (Mel). A, in vivo assessment of tumor growth. Mel and Mel-QA weregiven by intravenous route at 16 mmol/kg according to a q4d � 3 schedule. Tumor volume was monitored regularly. Significance was calculatedversus control group (�). B, quantitative analysis of 99mTc-NTP 15-5 accumulation in tumors versus muscle at day 7 and day 30 postimplantation.Evaluation of the specificity of 99mTc-NTP 15-5 imaging. C, T/M ratio of 99mTc-NTP 15-5 and 99mTc-15-5 at different time points after intravenous injectionin SRC-bearing rats. D, structure of 99mTc-NTP 15-5 and its nontargeted equivalent 99mTc-15-5 with representative in vivo scintigraphic images of thetumor-bearing hind limb obtained 1 hour after intravenous injection of 25 MBq of 99mTc-NTP 15-5 (D1) or 99mTc-15-5 (D2). FC: femoral condyle; TP, tibialplateau; T, tumor.






characterized with a Kd value in the millimolar range (2.25 �0.92 mmol/L) which is not observed for Mel. These resultsconfirmed our hypothesis that the Mel–QA conjugate estab-lishes ionic interactions with the high negative fixed-chargedensity of aggrecan. The next step was then to confirm in vivothe interaction of both Mel-QA and Mel with the ECM ofchondrosarcoma by a biodistribution study using the SRCmodel. This preclinical model reproduces the histologic andclinical behaviors of the human grade II disease (29–32). Forbiodistribution study, 3H radionuclide was introduced withexcellent radiochemical purities (>97%) on the common alky-

lating group to allow comparative biodistribution of bothcompounds. Quantitative whole-body autoradiography show-ed rapid distribution of radioactivity after [3H]-Mel-QA intra-venous administration: radioactivity rapidly accumulated inhealthy cartilaginous tissues and tumor. A high accumulationof radioactivity was also observed in the kidney and liver, aspreviously reported for Mel-QA and Mel in healthy rats (25).This accumulation of radioactivity in liver could be ascribedto metabolism but specific PG binding could not be ex-cluded. As a parenchymal organ, liver contains mainly heparansulfate-GAGs with additional minor amounts of dermatan

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Figure 4.

Anatomopathologic analysis one day after the last injection.A, histopathologic analysis, hematoxylin and eosin, original magnification: �10; A1, control: the tissue shows an important mitotic activity (M) withoutfibroinflammatory component; A2,. melphalan- treated rat; A3, melphalan-QA–treated rat: both tissues show a lot of necrotic cells (NC) associated with afibroinflammatory component. B, mitotic activity index was evaluated as the number of mitotic figures per fields.1H HRMAS profiling analyses one day after the last injection.C, representative 1H NMR spectra of SRC tumors of treated and nontreated rats one day after the end of treatment. Major assignments: a, CH3 signal of fattyacids residues of mobile lipids (0.90 ppm); b, CH2 signal of fatty acids residues of mobile lipids (1.28 ppm); and c, CH3 signal of creatine (3.03 ppm). D, CH2

(lip)/CH3(lip) ratio and CH2(lip)/total creatine (tCr) for each group.

Peyrode et al.





sulfate-GAGs, hyaluronic acid (HA), and chondroitin sulfate-GAGs which are barely detectable (33, 34). According to us, asautoradiography showed radioactivity of all radiolabeled deri-vatives (unchanged compound as well as its metabolites), liversignal could also be mainly attributable to metabolism andbile excretion of Mel-QA since previous distribution, in healthyrats, of [14C]-Mel-QA evidenced fecal biliary excretion ofaround 30% (25). These results are also consistent with pub-lished data of Mel (35, 36).

In the SRC model, there is a tendency for radioactivity toaccumulate at a higher level in the tumor after [3H]-Mel-QAinjection as compared with [3H]-Mel but statistically non signif-icant due to the variability between animals. To correct the inter-rat variability, T/M ratio was calculated for each animal. If radio-activity is expected to accumulate specifically in tumor tissue, andnot only due to blood flow, T/M ratio should be expected tobe higher than 1. The mean T/M ratio for Mel-QA at 5 minutesafter injection (5.77�1.11)was significantly higher than for [3H]-Mel (2.22 � 1.20, P < 0.05). It could be due to an enhancedaccumulation in tumor as well as a lower circulating activity ofthe radiolabeled Mel-QA.

Finally, we confirmed the interest of PG-targeting strategywith the QA function for chondrosarcoma therapy notably interm of therapeutic index, essential in oncology. Combiningtumor volume assessment, in vivo 99mTc-NTP 15-5 scintigraphicimaging of PGs, 1H-HRMAS NMR spectroscopy, and histologicanalyses of biopsies, this work demonstrates that the conjuga-tion of a QA function to Mel does not hamper its in vivoefficiency. More importantly, a significant decrease of sideeffects in terms of body weight and haematologic profile wasobserved for animals treated with Mel-QA as compared withthose receiving Mel. Thus, QA function leads to a significantimprovement of the therapeutic index. The excellent tolerabilityof Mel-QA could enable repeated cycles for the therapeuticeffects in patients with chondrosarcoma.

The tumor growth inhibition observed in the SRC modeltreated by Mel-QA raises the question of the mechanism at theorigin of this effect. Assessment of alkylating capacity using the 4-(4-nitrobenzyl)pyridine assay, as described previously (17),proved that an alkylating activity was maintained for Mel-QAwith an alkylation rate of 52.5�4.9�10�5min�1 comparedwith

20.9 � 2.6 � 10�5 min�1 for Mel (Supplementary Table S1).Nevertheless, is the biological activity observed only attributedto DNA alkylation, like for Mel (37), or to other events, such asthe degratdation of PGs? Indeed, we think that the mechanismof action ofMel-QA ismore complex than a simple alkylation andmay be due to a remodeling of the PGs of ECM. We previouslydemonstrated that treatments withMel-QA induce changes in theexpression and degradation of the PGs (13). PGs are an importantcomponent of the tumor microenvironment, which represents acomplex and highly dynamic media basically composed of non-malignant cells as well as a ECM consisting of fibrous structuralproteins, fibrous adhesive proteins, and PGs. Because of theircomplex structure, PGs play an important role in cell–cell andcell–ECM interactions and signaling in a variety of cellular func-tions (motility, adhesion, growth; refs. 38, 39). Modifications ofPGs contribute to altered composition of ECM and also couldexplain the reduced tumor growth observed withMel-QA in SRC-bearing rats. Concerning chondrosarcoma, the importance of PGsin SRC growth was firstly suggested by Oegema and colleagueswho hypothesized that reducing the PG content was a way todecrease accessibility of growth factors and to increase the immu-nologic cell infiltration (40).

Recently, tumor microenvironment has been gradually recog-nized as a key contributor for cancer progression and drugresistance (41, 42). PGs appear as major partners for multipleorgan integrity and function, and might represent interestingtargets in other malignant pathologic processes, such as headand neck carcinomas (43, 44). As QA strategy has demonstratedits potential for chondrosarcoma management through ECMinteraction, it will be of great interest to take advantage of anotherphenotypic feature of chondrosarcomamicroenvironment that ishypoxia (7, 45, 46). Exploiting hypoxia is well-documented incancer therapy through hypoxia-activated prodrugs and inhibi-tors of molecular targets upon which hypoxic cell survivaldepends. Therefore, combining PG targeting and hypoxia-acti-vated prodrugs could serve as basis for innovative and selective,microenvironmentally targeted chondrosarcoma therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

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QA conjugation to Mel strongly attenuates side effects. A, percent change in body weight from day 4 postimplantation. While there is no weight loss in theMel-QA–treated group, a significant weight loss is observed in the Mel-treated group starting from the second injection. B, hematologic parameters48 hours after the end of the treatment (day 18). QA conjugation allows a significant attenuation of lymphopenia.






Authors' ContributionsConception and design: C. Peyrode, V. Weber, A. Vidal, P. Auzeloux,J.-M. Chezal, E. Miot-NoiraultDevelopment of methodology: C. Peyrode, V. Weber, A. Voissi�ere, A. Vidal,P. Auzeloux, M. Borel, E. Miot-NoiraultAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Peyrode, V. Weber, A. Voissi�ere, M. Borel,M.-M. Dauplat, F. R�edini, E. Miot-NoiraultAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Peyrode, V. Weber, M. Borel, E. Miot-NoiraultWriting, review, and/or revision of the manuscript: C. Peyrode, V. Weber,A. Maisonial-Besset, P. Auzeloux, J.-M. Chezal, E. Miot-NoiraultAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V. Gaumet, M. Quintana, F. DegoulStudy supervision: C. Peyrode, V. Weber, J.-M. Chezal, E. Miot-NoiraultOther (radiochemistry): A. Maisonial-Besset

AcknowledgmentsThe authors thank Delphine Skrzydelski and Romain Vives and especially

Sophie Besse for her technical expertise and help for in vivo treatments andhematologic sampling.

Grant SupportResearchers assigned to our laboratory UMR 990 Inserm/UdA received

financial support from Ligue contre le cancer Auvergne, CPER, and PRTK(INCa/DGOS 2015-051) for coordination of all the work presented here.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 9, 2016; revised August 1, 2016; accepted August 3, 2016;published OnlineFirst August 29, 2016.

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2016;15:2575-2585. Published OnlineFirst August 29, 2016.Mol Cancer Ther Caroline Peyrode, Valérie Weber, Aurélien Voissière, et al. Chondrosarcoma: Preclinical Proof of ConceptProteoglycans as Target for an Innovative Therapeutic Approach in

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