Proteoglycans as Target for an Innovative
Therapeutic Approach in Chondrosarcoma:
Preclinical Proof of Concept
Caroline Peyrode
1,2, Valerie Weber
1,2, Aurelien Voissiere
1,2, Aurelie Maisonial-Besset
1,2,
Aur
elien Vidal
3, Philippe Auzeloux
1,2, Vincent Gaumet
1,2, Mich
ele Borel
1,2,
Marie-Melanie Dauplat
4, Mercedes Quintana
1,2, Fran
¸coise Degoul
1,2,
Fran
¸coise Redini
5, Jean-Michel Chezal
1,2, and Elisabeth Miot-Noirault
1,2Abstract
To date, surgery remains the only option for the treatment of chondrosarcoma, which is radio- and chemoresistant due in part to its large extracellular matrix (ECM) and poor vascularity. In case of unresectable locally advanced or metastatic diseases with a poor prognosis, improving the management of chon-drosarcoma still remains a challenge. Our team developed an attractive approach of improvement of the therapeutic index of chemotherapy by targeting proteoglycan (PG)-rich tissues using a quaternary ammonium (QA) function conjugated to mel-phalan (Mel). First of all, we demonstrated the crucial role of the QA carrier for binding to aggrecan by surface plasmon resonance. In the orthotopic model of Swarm rat chondrosar-coma, an in vivo biodistribution study of Mel and its QA derivative (Mel-QA), radiolabeled with tritium, showed rapid
radioactivity accumulation in healthy cartilaginous tissues and tumor after [3H]-Mel-QA injection. The higher T/M ratio of the
QA derivative suggests some advantage of QA-active targeting of chondrosarcoma. The antitumoral effects were characterized by tumor volume assessment, in vivo 99mTc-NTP 15-5 scinti-graphic imaging of PGs, 1H-HRMAS NMR spectroscopy, and
histology. The conjugation of a QA function to Mel did not hamper 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, QA conjugation 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.
Introduction
One of the key problems in chondrosarcoma, the second most common type of skeletal malignancy after osteosarcoma, is the lack of response to both chemotherapy and radiotherapy, giving rise to poor patient outcomes and leaving surgery as the only effective curative treatment. The type of required surgical proce-dure may vary according to the grade, the initial location, and the extent of the disease. Large en bloc dramatic resection or amputa-tion, often necessary to allow the local control of the tumor and to avoid metastases, are at the origin of strong disability and mor-bidity (1). Nevertheless, wide resection is not always possible in large tumors and critical anatomic locations such as pelvis, axial
skeleton, and also in many cases of local recurrence. The clinical behavior and the prognosis for patients with chondrosarcoma vary 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 prevent recurrences and tofind better treatment options for patients with inoperable 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. A recent study has shown that some chondrosarcoma cell lines may display moderate sensitivity to doxorubicin and cisplatin, the most 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 thefirst-line (2, 3). The study of Van Maldegen and colleagues also demonstrated that the survival rate in patients with unresectable chondrosarcoma was also significantly higher in the case of systemic treatments (doxo-rubicin-based chemotherapy, and nonchemotherapy-based agents such as imatinib and sirolimus) compared with patients managed with best supportive care only (overall survival at 3 years: 26% vs. 8% respectively, P < 0.05; ref. 5). According to such poor outcome and no global improvement in the survival of patients with chondrosarcoma over the past 30 years, personal-ized medicine including a combination of targeted therapies will most likely be the best approach to tackle the resistance of human chondrosarcoma.
1Clermont Universite, Universite d'Auvergne, Imagerie Moleculaire et Therapie Vectorisee, Clermont-Ferrand, France.2INSERM, UMR990, Clermont-Ferrand, France.3Arronax, Saint-Herblain, France. 4CLCC Jean-Perrin, Clermont-Ferrand, France.5INSERM, UMR S957, Nantes Atlantique Universite, Nantes, France.
Note: Supplementary data for this article are available at Molecular Cancer Therapeutics 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: caroline.peyrode@udamail.fr
doi: 10.1158/1535-7163.MCT-16-0003
Treatments targeting tumor microenvironment have therefore been 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 anticancer agents have to diffuse over a relatively long distance to reach tumor 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 approach of targeting the proteoglycan (PG)-rich cartilaginous tissues using a quaternary ammonium (QA) function as a carrier for drugs or radionuclides. The positively charged QA interacts with the sulfate and carboxylate groups present in PGs highly expressed in the chondrogenic ECM (8). With this strategy, we successfully delivered imaging agents as well as therapeutic drugs (such as anti-inflammatory drugs, metalloproteinase inhibitors, cytotoxics, and more recently radiosensitizing nano-particles; refs. 8–13) selectively to PG-rich tissues such as cartilage and chondrosarcoma. In particular, the suitability and high sensitivity of the radiotracer N-[triethylammonium]-3-propyl-1,4,7,10,13-pentaazacyclopentadecane radiolabeled with99mTc (99m
Tc-NTP 15-5) was validated for in vivo diagnosis and follow up of chondrosarcoma via scintigraphic imaging (14–16). This radiotracer is currently transferred into clinic for afirst 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 vitro and in vivo preclinical experiments (13). The aim of the current study was to characterize this therapeutic strategy.
First of all, the binding ability of Mel-QA to aggrecan (major PG in 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 of the tritium-radiolabeled Mel-QA, as compared with Mel. The main interest of the QA vector was to strongly attenuate side effects, monitored by hematologic analyses and body weight, while maintaining a tumor growth inhibition assessed by in vivo99mTc-NTP 15-5 scintigraphic imaging,1H-HRMAS NMR spectroscopy, and histologic analyses of biopsies.
The work reported here clearly validates the relevance of PGs as target for an innovative therapeutic approach in chondrosarcoma.
Materials and Methods
Chemical synthesis
Mel was used as its hydrochloride form after treatment of Mel (Interchim) with a solution of 2 N hydrochloric acid in diethyl ether. The QA conjugate of Mel (Mel-QA) was prepared as already 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 described previously (18).
SPR binding assays
To demonstrate the binding affinity between the QA entity and aggrecan, we used the SPR method with aggrecan immobilized onto the surface of the sensor chip. When studied compounds interact with aggrecan, they form a complex that modifies the
refractive index. This change is measured in real-time and the signal obtained is plotted in resonance unit (RU) versus time (19, 20). SPR assays were carried out on a Biacore T200 instrument with CM4 sensor chips (GE Healthcare). Sensor chip CM4 has a dextran matrix with a low degree of carboxylation which may be of value for reducing nonspecific binding with highly positive charged analytes and may be useful for kinetic analysis. To immobilize the ligand, sensor chip wasfirstly activated using a 1:1, v:v, mixture of a 0.2 mol/L aqueous 1-ethyl-3-(3-dimethyla-minopropyl) carbodiimide hydrochloride salt solution and a 0.5 mol/L aqueous N-hydroxysuccinimide (Amine Coupling Kit, GE Healthcare) solution at aflow rate of 5 mL/minute for 10 minutes. After that, aggrecan from bovine articular cartilage (Sigma Aldrich) was coated to the sensor chips at 400 mg/mL in running buffer HBS-Pþ1 (0.01 mol/L HEPES, 0.15 mol/L NaCl, 0.05%, v/v, surfactant p20, pH 7.4) containing 6 mmol/L of hexadecyltrimethylammoniumbromide (CTAB) with aflow rate of 5 mL/minute to a level of approximately 500 RU. Unoccupied binding sites were blocked using an aqueous 1 mol/L ethanol-amine solution (pH 8.5; Amine Coupling Kit, GE Healthcare) with aflow rate of 5 mL/minute for 10 minutes. Mel and Mel-QA were injected with a flow rate of 30 mL/minute in increasing concentrations (0–2 mmol/L) using five 600-second injections and a 200-second dissociation time. After each experiment,flow cells were regenerated twice with NaCl 2 mol/L for 150 seconds. The interactions were recorded as the difference in response between the immobilized aggrecanflow cell and a corresponding controlflow cell (activated and blocked, but without immobi-lized aggrecan). Kinetic and statistic parameters (Kdand c2) were
determined by Biacore T200 Evaluation software byfitting the steady-state values at equilibrium (Req). Experiments were per-formed in triplicate.
Orthotopic model of Swarm rat chondrosarcoma
The 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 under the authorization of the French Directorate of Veterinary Ser-vices and in accordance with the 2010/63/UE Directive and were conducted as described previously (13) using male Spra-gue–Dawley rats (Charles River Laboratories). All experiments were conducted after ethics committee approval (C2E2A, nCE 82-12). Allograft transplantation of a 10-mm3SRC fragment was performed on the right paw, the other paw being used as the contralateral control. These SRC fragments were collected from well-developed tumors on the paratibial area of donor Sprague–Dawley rats.
Radiosyntheses of [3H]-Mel and [3H]-Mel-QA
An 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 unlabeled
NaBH4). Then the crude reaction mixture containing amine 1 and
N-t-Boc-4-(2-hydroxy-2-[3H]ethyl)amino-L-phenylalanine ethyl ester ([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) in dichloromethane (20 mL) gave, after silica gel chromatographic
purification, the corresponding tritiated dichloro derivative [3 H]-6 (0.9H]-6 mmol, 9H]-62 MBq) in 48% chemical yield. Then, the ester and the t-Boc protecting group of compound [3H]-6 were
hydro-lyzed with an aqueous 6 N hydrochloric acid solution (5 mL) to provide the hydrochloride salt of tritiated Mel ([3H]-Mel, 0.96 mmol, 740 MBq) in quantitative chemical yield. On the basis of this radiolabeling sequence, [3H]-Mel was isolated with a specific activity of 777 MBq/mmol and a radiochemical purity > 98%, as determined by HPLC analyses (Supplementary Fig. S2). The overall yield of this radiochemical procedure was 20%.
For the preparation of tritiated Mel-QA, afive-step radiosynth-esis starting from hydrochloride salt of [3H]-Mel was developed.
Thus, after Boc protection of the amino group of [3H]-Mel (0.69
mmol, 573 MBq), N-protected Mel was immediately coupled with 3-(dimethylamino)propylamine (0.83 mmol) in the pres-ence of dicyclohexylcarbodiimide (DCC, 1.03 mmol) and 1-hydroxybenzotriazole (HOBt, 0.74 mmol) to give the corre-sponding amide intermediate [3H]-7 in 75% chemical yield after purification by column chromatography. Treatment with methyl iodide (2.3 mmol) in ethanol gave the QA compound [3H]-8 in quantitative yield. Finally, the t-Boc–protecting group was removed 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 QA derivative 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 of 814 MBq/mmol and a radiochemical purity > 97%, as deter-mined by HPLC analyses (Supplementary Fig. S2). The overall yield of this radiosynthesis was 15% based on [3H]-NaBH4.
Tissue distribution study of [3H]-Mel and [3H]-Mel-QA conjugate by whole-body autoradiography and urinary excretion
This study was performed on thirty SRC-bearing rats at stage day 20 postimplantation. Animals were randomized into two groups (n ¼ 15 per group) and received intravenous injection of 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, then 1, 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 carbon dioxide inhalation, frozen in liquid nitrogen, and embedded in blocks of carboxymethyl cellulose 2%, that were sagittally sectioned into 40-mm slices at22C with a cryomicro-tome (Reichert-Jung Cryopolycut). Each slice was dehydrated for 48 hours in a cryochamber. Whole-body slices (25 slices per animal and per time point) were then exposed for 1,000 minutes in a digital autoradiographic analyzer (bImager, Biospace Measures). Surfacic activity (counts in cpm/mm2) was quantified in regions of interest (ROI) delineated over organs of interest including tumors, corrected for radioactive decay, 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/mm2in 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 urine
collection from 6 to 24 hours after injection.
Radioactivity of urine or blood samples was directly measured after addition of Packard Ultima Gold cocktail (PerkinElmer) in a Wallac Winspectral 1414 Liquid Scintillation Spectrometer (PerkinElmer).
Tumor growth inhibition assessment
Experiments 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 received three doses of Mel or Mel-QA (16 mmol/kg per injection) by intravenous 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 the formula: 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 vivo99mTc-NTP 15-5 scintigraphic imaging targeting PGs
Antitumor efficacy was monitored by scintigraphic imaging, using the radiotracer99mTc-NTP 15-5 developed by our group at day 7 (one day before treatment) and day 30 (2 weeks after treatment; ref. 15). Acquisitions were performed 30 minutes after intravenous tracer administration (25 MBq/animal), with a 10-minute planar acquisition for each posterior paw posi-tioned over the parallel-hole collimator of a small-animal g camera (GammaImager, Biospace) and with a 15% window centered on the 140-keV photopeak of99mTc. All the scans were evaluated by the same experienced investigator, using fixed-size ROIs delineated over tumor and muscle patterns. For each ROI, total activity, count in cpm (counts per minutes) per mm2 were obtained. Tumor-to-muscle ratio was calculated: T/M¼ (counts per minute/mm2in 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 with99mTc-NTP
15-5 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 to
determine the kinetics of the tracer distribution in tumor. SRC animals were intravenously injected via the tail vein with 25 MBq of99mTc-NTP 15-5 or99mTc-15-5 simultaneously to the starting of a 120-minute duration list mode acquisition using a 5-minute sampling time. Time–activity curves were obtained from ROIs drawn around tumor and muscle with all measured activities corrected for radioactive decay and T/M ratio calculated at each time point.
Antitumor efficacy analysis by1H HRMAS NMR spectroscopy profile
1
H HRMAS spectroscopy profile analyses were performed in a parallel study following the same protocol with additional ani-mals (4 aniani-mals per group). Tumors were excised one week after thefinal injection, rapidly frozen in liquid nitrogen and stored at 80C until analysis.
All experiments were performed on frozen tumor samples flushed for a few seconds in cooled D2O.1H NMR spectroscopy
was performed on a small-bore Bruker DRX 500 magnet (Bruker) equipped with an HRMAS probe. Samples were then set into 4-mm diameter, 50-mL free volume ZrO2 rotor tubes without
upper spacer. A total of 3 mL of D2O containing 0.75%
3-(tri-methylsilyl)propionic-2,2,3,3-d4 acid (TSP; Sigma), used for
peak referencing, was added to the rotor tubes to lock the spectrometer. Rotors were spun at 4 kHz to keep the rotation sidebands out of the acquisition window. A Bruker cooling unit was used to maintain the sample temperature at 277 K to min-imize tissue degradation. One-dimensional1H NMR sequence
was a saturation recovery sequence. The resonances were assigned on the basis of the known chemical shifts of the major structural groups using tissue data (21). The relative content of metabolites was estimated by peak area integration. To compare the metabolic profiles between experimental groups, each peak was normalized with the respect of the total integral of the spectrum in the 0.5 to 4.7 ppm region (21–23). Sample stability inside the rotor was assessed at a temperature of 277 K over several hours by a series of analyses. During the 48-minute overall acquisition period, no significant changes in NMR signals of biological material were observed. Each experiment was performed in triplicate. Side-effect assessment
Side effects of treatments with Mel or Mel-QA were assess-ed by both animal's weight and hematologic parameter measurements.
(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 by retro-orbital puncture) of each animal was removed and processed on a Melet Schloesing MS9-5 Hematology Analyzer (Diamond Diagnostics).
Statistical analyses
Statistical analyses were conducted using the GraphPad Prism 5 software. Data are reported as mean SD. Results were analyzed by ANOVA followed by Tukey post test. We considered P values < 0.05 to indicate statistical significance (, P < 0.05;, P < 0.01;, P < 0.001).
Results
Evaluation of the QA binding to PGs by SPR
Studies were performed for Mel-QA, respectively, to Mel injected in increasing concentrations (0–2 mmol/L). The sensor-grams obtained after signal subtraction of control was displayed versus time. As illustrated in Fig. 1 and regards to Kdvalues,
Mel-QA exhibited affinity to aggrecan (Kd¼ 2.25 0.92 mmol/L) in
contrast to Mel, demonstrating that QA vector allows aggrecan binding.
In vivo biodistribution studies of [3H]-Mel and [3H]-Mel-QA
Radiosyntheses of [3H]-Mel and [3H]-Mel-QA. The radiosyntheses
of [3H]-Mel and [3H]-QA are shown in Fig. 2A. Both
Mel-QA and Mel were radioisotopically labeled on the alkylating moiety, also3H radionuclide was introduced in the chloroethyl
groups. [3
H]-Mel was synthesized from N-t-Boc-4-amino-L-phenylalanine ethyl ester according to a slightly modified sequence previously reported in the literature (24). For the
Kd = 302 mol/L 2 = 1.25 Kd = 3.3 × 10–3 mol/L χ2 = 0.894 χ Mel-QA Time (s) Response (RU) 200 400 600 800 20 40 60 80 2 mmol/L 1.5 mmol/L 1 mmol/L 0.8 mmol/L 0.6 mmol/L 0.5 mmol/L 0.4 mmol/L 0.2 mmol/L 0 mmol/L Mel Time (s) Response (RU) 200 400 600 800 20 40 60 80 2 mmol/L 1.5 mmol/L 1 mmol/L 0.8 mmol/L 0.6 mmol/L 0.5 mmol/L 0.4 mmol/L 0.2 mmol/L 0 mmol/L 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. The dissociation constant (Kd) of each example are given.
preparation of [3H]-labeled Mel-QA, the radiosynthetic sequence starting from [3H]-Mel was adapted from the five-step synthesis developed for the corresponding nonradioactive derivative and14C-labeled Mel-QA (17, 25). Both [3H]-Mel and [3H]-Mel-QA were successfully obtained with a specific activity of 814 MBq/mmol and a radiochemical purity > 97 %, as determined by radio-RP-HPLC analyses.
QA functionalization allows tumor uptake of Mel. The distribu-tion of radioactivity in several tissues after intravenous injecdistribu-tion of 2.5 MBq of [3H]-Mel or [3H]-Mel-QA in SRC animals is expressed 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 and tumor (Fig. 2C). Five minutes postinjection (p.i.) of [3 H]-Mel-QA, the T/M value was significantly higher (P < 0.05) compared with [3H]-Mel (T/M values 5 minutes postinjection: 5.77 1.11 for [3H]-Mel-QA versus 2.22 1.20 for [3H]-Mel; Fig. 2B).
Furthermore, high levels of each drug were found in the metabolizing and elimination organs, such as liver and kid-neys. As expected for small hydrophilic compounds, Mel and Mel-QA are cleared in the kidney (i.e., about 16% of cumu-lative renal excretion) and no significant difference was observed between the two compounds at 24 hours postinjet-cion (Table 1). However, radioactivity was observed in the liver after [3H]-Mel-QA injection as compared with [3H]-Mel.
T T AC AC AC AC
A
C
B
a Reagents and conditions: (i) NaBH
4 , [3H]-NaBH4 (3.7 × 103MBq), EtOH, rt, 5 h; (ii) EtOH, 90°C, 17 h; (iii) ethylene oxide, acetic acid, rt, 24 h; (iv) PPh3, CCl
4, 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
*
*, P < 0.05 T AC T AC C1 C2Radioactivity distribution of [3H]-Mel at 5 min p.i.
40-µm thick sagittal slice
Corresponding anatomic slice
*
0 1 2 3 4 5 6 7 85 min 1 heure 6 heures
T/M uptake ratios
Time after i.v. administration (min) Mel-QA Mel
A
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 of rat 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 injection of [3H]-Mel than [3H]-Mel-QA (Table 1).
In vivo antitumor efficacy and side effects of Mel and Mel-QA in SRC model
Mel-QA delays chondrosarcoma growth in vivo. Mel-QA and Mel were given at 16 mmol/kg according to a q4d 3 schedule beginning on day 8 after tumor implantation. The control group received intravenous saline injection.
On the basis of the tumor volume monitoring, a significant inhibition of tumor growth (P < 0.01) was observed for both Mel-QA- and Mel-treated rats compared with the control group, but no significant difference was observed between the two treated groups (Fig. 3A).
In vivo antitumor effects were also assessed by 99mTc-NTP 15-5 scintigraphic imaging, as this radiotracer was previously demonstrated to provide suitable criteria for monitoring chon-drosarcoma (14–16). As illustrated in Fig. 3B, quantitative analysis of in vivo 99mTc-NTP 15–5 imaging of tumors
evi-denced significant changes in radiotracer uptake in treated animals versus controls. A significant increase of 84% of T/M ratio (P < 0.05) was observed in nontreated rats between day 7 (1 day before treatment) and day 30 (14 days after the last drug injection), whereas this ratio remained overall constant in Mel- or Mel-QA–treated rats. Furthermore, to assess the in vivo specificity of 99mTc-NTP 15-5 imaging and to exclude the
possibility of unspecific uptake in tumoral tissue,99mTc-NTP 15-5 and its nontargeted equivalent (99mTc-15-5) were injected by 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 ratio gradually increased and reached a plateau as soon as 30 minutes which was maintained at least for 1 hour (T/M values 1 hour postinjection: 2.25 0.11), allowing tumor visualiza-tion with an excellent contrast (Fig. 3D). On the contrary, T/M ratio for 99mTc-15-5 was around 1 throughout the course of
the imaging. According to these results, we demonstrated the potential of 99m Tc-NTP 15-5 imaging as a "methodology companion" of targeted therapies of chondrosarcoma.
To characterize the antiproliferative effects, tumors were removed 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, such as necrotic cells,fibroinflammatory component, and increased vascularization. These signs were observed for 57% and 40% of tumors in the Mel-QA and Mel groups, respectively (Fig. 4A). Furthemore, mitotic activity index values showed a significant decrease (P < 0.01) in the proliferating activity in the two treated groups (Fig. 4B). In contrast, the tumors of nontreated rats were characterized by hypercellularity with a high mitotic activity index.
Finally, in a parallel study, SRC-bearing rats were treated following the same protocol and tumors were excised one week after thefinal injection for1H-HRMAS NMR spectroscopy anal-yses (Fig. 4C) which confirmed necrosis and apoptosis. Results revealed a significant increase (P < 0.01) in the spectral intensity ratio of methylene (CH2) resonance (at 1.28 ppm) to methyl
(CH3) resonance (at 0.9 ppm) in the Mel-QA–treated group as
compared with controls (Fig. 4D). This ratio, which is considered as an apoptosis marker (26, 27), was also significantly higher (P < 0.01) in the Mel-QA–treated group than in the Mel-treated group (Mel-QA: 2.34 0.22 versus Mel: 1.47 0.08). In the same way, a significant increase in CH2(lip)/total creatine(tCr) ratio,
which is considered as a necrosis marker (28), was observed in the Mel-QA group (Mel-QA: 11.5 2.07 vs. Mel: 5.0 1.26). Significant reduction of side effects with QA derivative. The body weight of each rat was regularly recorded and hematologic parameters were assessed 48 hours after the last dose of both compounds (Fig. 5). Mel-treated rats exhibited significant weight loss starting from the second injection which reached 15% at day 23 postimplantation (Fig. 5A). Furthermore, the clinical 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 day after the last dose of treatment, no clinical side effects were observed 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 after intravenous administration of [3H]-Mel or [3H]-Mel-QA
Comparative distribution of radioactivity after i.v. administration of [3H]-Mel-QA or [3H]-Mel
Tumora 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 0.070.05 [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. c P < 0.01. d P < 0.05.
the Mel-treated rats with an average clinical score of 2.7 2.1. For Mel-treated rats, these symptoms were associated with a severe leucopenia, a common side effect of alkylating agents, which was marked by a significant decrease in white blood cells and lymphocytes numbers (Fig. 5B). Furthermore, in compar-ison with Mel-treated animals, an improvement in hematologic parameters was observed for Mel-QA treatment, that is, atten-uation of leucopenia with, more specifically, a significant attenuation of lymphopenia (P < 0.01).
Discussion
Chondrosarcoma, with an estimated incidence of 0.2 in 100,000 patients per year, is a rare disease with most patients being 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 therapeutic approaches (1, 5).
As efficacy of antineoplastic treatments is mainly hampered by toxic side effects, improving their therapeutic index, by reducing adverse effects and increasing drug accumulation in tumor tissue, remains a challenge in drug development. Among the numerous strategies aimed at such objectives, "carrier" with preferential binding to one or more tumor targets may offer promising therapeutic opportunities. This was the strategy employed here for experimental chondrosarcoma: a QA function (i.e., a carrier exhibiting affinity for the high negative fixed charged density of PGs) was conjugated to Mel. In this way, we expected to improve its therapeutic index by reducing systemic toxicity while keeping an antitumor activity.
First of all, the role of QA function for PG binding was demonstrated in vitro by SPR with aggrecan, the major PG component of chondrosarcoma. As expected, binding of the QA-functionalized derivative of Mel to aggrecan was
0 0.5 1 1.5 2 2.5 3
Vehicle Mel-AQ Mel
T/M v a lu e Day 7 Day 30
A
*B
N N N N N N Cl -+ Tc O O 99m 99mTc-NTP 15-5 N N HN N N Tc O O 99m 99mTc-15-5 D1 D2 T TC
D
Injections Mel Mel-QA Vehicle 4,000 3,500 3,000 2,500 2,000 0 0 0 10 20 Time (min) T/M ratio 30 40 50 60 0 0.5 1 1.5 2 2.5 3 10 20 30 500 1,000 1,500 Tumor volume (mm 3)Days after implantation
* , P < 0.05 * , P < 0.05 ** , P < 0.01 * , P < 0.05 ** , P < 0.01
**
**
**
**
*
**
**** ** ** **** ** ** ** FC TP 99mTc-NTP 15-5 99mTc-15-5 Figure 3.Evaluation of thein vivo antitumor activity of Mel-QA and its nontargeted equivalent (Mel). A, in vivo assessment of tumor growth. Mel and Mel-QA were given by intravenous route at 16mmol/kg according to a q4d 3 schedule. Tumor volume was monitored regularly. Significance was calculated versus control group (). B, quantitative analysis of99mTc-NTP 15-5 accumulation in tumors versus muscle at day 7 and day 30 postimplantation. Evaluation of the specificity of99mTc-NTP 15-5 imaging. C, T/M ratio of99mTc-NTP 15-5 and99mTc-15-5 at different time points after intravenous injection in SRC-bearing rats. D, structure of99mTc-NTP 15-5 and its nontargeted equivalent99mTc-15-5 with representative
in vivo scintigraphic images of the tumor-bearing hind limb obtained 1 hour after intravenous injection of 25 MBq of99mTc-NTP 15-5 (D1) or99mTc-15-5 (D2). FC: femoral condyle; TP, tibial plateau; T, tumor.
characterized with a Kdvalue in the millimolar range (2.25
0.92 mmol/L) which is not observed for Mel. These results confirmed our hypothesis that the Mel–QA conjugate estab-lishes ionic interactions with the high negative fixed-charge density of aggrecan. The next step was then to confirm in vivo the interaction of both Mel-QA and Mel with the ECM of chondrosarcoma by a biodistribution study using the SRC model. This preclinical model reproduces the histologic and clinical behaviors of the human grade II disease (29–32). For biodistribution study, 3H radionuclide was introduced with excellent radiochemical purities (>97%) on the common
alky-lating group to allow comparative biodistribution of both compounds. Quantitative whole-body autoradiography show-ed rapid distribution of radioactivity after [3H]-Mel-QA intra-venous administration: radioactivity rapidly accumulated in healthy cartilaginous tissues and tumor. A high accumulation of radioactivity was also observed in the kidney and liver, as previously reported for Mel-QA and Mel in healthy rats (25). This accumulation of radioactivity in liver could be ascribed to metabolism but specific PG binding could not be ex-cluded. As a parenchymal organ, liver contains mainly heparan sulfate-GAGs with additional minor amounts of dermatan
0 2 4 6 8 10 12 14 16 18 CH2(lip)/tCr M M NC NC NC NCNC F F M 1. Control 2. Mel 3. Mel-QA c: tCreatine c c a: CH3(lip) a a b: CH2(lip) b b 0 0.5 1 1.5 2 2.5 3 0.00 4 3 2 1 [ppm] 0.01 0.02 0.03 CH2(lip)/CH3(lip) A1: Control * ** ** **** *, P < 0.05 **,P < 0.01
A
A2: Mel-treated ratA3: Mel-QA−treated rat
B
0.18 0.02 2.34 0 0.5 1 1.5 2 2.5 3 3.5Mitotic activity index
Mel-QA Mel Vehicle ** ** *,P < 0.05 **,P < 0.01
C
D
Vehicle Mel Mel-QA
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) without fibroinflammatory component; A2,. melphalan- treated rat; A3, melphalan-QA–treated rat: both tissues show a lot of necrotic cells (NC) associated with a fibroinflammatory component. B, mitotic activity index was evaluated as the number of mitotic figures per fields.
1H HRMAS pro
filing analyses one day after the last injection.
C, representative1H NMR spectra of SRC tumors of treated and nontreated rats one day after the end of treatment. Major assignments: a, CH3signal of fatty acids residues of mobile lipids (0.90 ppm); b, CH2signal of fatty acids residues of mobile lipids (1.28 ppm); and c, CH3signal of creatine (3.03 ppm). D, CH2 (lip)/CH3(lip) ratio and CH2(lip)/total creatine (tCr) for each group.
GAGs, hyaluronic acid (HA), and chondroitin sulfate-GAGs which are barely detectable (33, 34). According to us, as autoradiography showed radioactivity of all radiolabeled deri-vatives (unchanged compound as well as its metabolites), liver signal could also be mainly attributable to metabolism and bile excretion of Mel-QA since previous distribution, in healthy rats, of [14C]-Mel-QA evidenced fecal biliary excretion of around 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 to accumulate at a higher level in the tumor after [3H]-Mel-QA
injection as compared with [3H]-Mel but statistically non
signif-icant due to the variability between animals. To correct the inter-rat variability, T/M inter-ratio was calculated for each animal. If radio-activity is expected to accumulate specifically in tumor tissue, and not only due to bloodflow, T/M ratio should be expected to be higher than 1. The mean T/M ratio for Mel-QA at 5 minutes after injection (5.77 1.11) was significantly higher than for [3
H]-Mel (2.22 1.20, P < 0.05). It could be due to an enhanced accumulation in tumor as well as a lower circulating activity of the radiolabeled Mel-QA.
Finally, we confirmed the interest of PG-targeting strategy with the QA function for chondrosarcoma therapy notably in term of therapeutic index, essential in oncology. Combining tumor volume assessment, in vivo99mTc-NTP 15-5 scintigraphic imaging of PGs,1H-HRMAS NMR spectroscopy, and histologic analyses of biopsies, this work demonstrates that the conjuga-tion of a QA funcconjuga-tion to Mel does not hamper its in vivo efficiency. More importantly, a significant decrease of side effects in terms of body weight and haematologic profile was observed for animals treated with Mel-QA as compared with those receiving Mel. Thus, QA function leads to a significant improvement of the therapeutic index. The excellent tolerability of Mel-QA could enable repeated cycles for the therapeutic effects in patients with chondrosarcoma.
The tumor growth inhibition observed in the SRC model treated by Mel-QA raises the question of the mechanism at the origin 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-QA with an alkylation rate of 52.5 4.9 105min1compared with
20.9 2.6 105min1for Mel (Supplementary Table S1). Nevertheless, is the biological activity observed only attributed to DNA alkylation, like for Mel (37), or to other events, such as the degratdation of PGs? Indeed, we think that the mechanism of action of Mel-QA is more complex than a simple alkylation and may be due to a remodeling of the PGs of ECM. We previously demonstrated that treatments with Mel-QA induce changes in the expression and degradation of the PGs (13). PGs are an important component of the tumor microenvironment, which represents a complex and highly dynamic media basically composed of non-malignant cells as well as a ECM consisting offibrous structural proteins,fibrous adhesive proteins, and PGs. Because of their complex structure, PGs play an important role in cell–cell and cell–ECM interactions and signaling in a variety of cellular func-tions (motility, adhesion, growth; refs. 38, 39). Modifications of PGs contribute to altered composition of ECM and also could explain the reduced tumor growth observed with Mel-QA in SRC-bearing rats. Concerning chondrosarcoma, the importance of PGs in SRC growth wasfirstly suggested by Oegema and colleagues who hypothesized that reducing the PG content was a way to decrease 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 drug resistance (41, 42). PGs appear as major partners for multiple organ integrity and function, and might represent interesting targets in other malignant pathologic processes, such as head and neck carcinomas (43, 44). As QA strategy has demonstrated its potential for chondrosarcoma management through ECM interaction, it will be of great interest to take advantage of another phenotypic feature of chondrosarcoma microenvironment that is hypoxia (7, 45, 46). Exploiting hypoxia is well-documented in cancer therapy through hypoxia-activated prodrugs and inhibi-tors of molecular targets upon which hypoxic cell survival depends. 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 Interest
No potential conflicts of interest were disclosed. 0 10 20 30 40 50 60 70 80 90 % of Lymphocytes -20 -10 0 10 20 30 40 50 60 70 24 19 14 9 4 Relative body weight (%)
Days after implantation
Mel-QA–treated rats Mel-treated rats Controls ** ** ** ** ** ** Injection
A
* , P < 0.05 * , P < 0.05 ** , P < 0.01***, P < 0.001 ** , P < 0.01 0 2 4 6 8 10 12White blood cells (×103/mL)
Mel-treated rats Mel-QA–treated rats Controls
0 1 2 3 4 5 6 7 8
Red blood cells (×106/mL) 0 2 4 6 8 10 12 14 16 Hemoglobin (g/dL) ** *** **
B
* ** Figure 5.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 the Mel-QA–treated group, a significant weight loss is observed in the Mel-treated group starting from the second injection. B, hematologic parameters 48 hours after the end of the treatment (day 18). QA conjugation allows a significant attenuation of lymphopenia.
Authors' Contributions
Conception and design: C. Peyrode, V. Weber, A. Vidal, P. Auzeloux, J.-M. Chezal, E. Miot-Noirault
Development of methodology: C. Peyrode, V. Weber, A. Voissiere, A. Vidal, P. Auzeloux, M. Borel, E. Miot-Noirault
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Peyrode, V. Weber, A. Voissiere, M. Borel, M.-M. Dauplat, F. Redini, E. Miot-Noirault
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Peyrode, V. Weber, M. Borel, E. Miot-Noirault Writing, review, and/or revision of the manuscript: C. Peyrode, V. Weber, A. Maisonial-Besset, P. Auzeloux, J.-M. Chezal, E. Miot-Noirault
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): V. Gaumet, M. Quintana, F. Degoul Study supervision: C. Peyrode, V. Weber, J.-M. Chezal, E. Miot-Noirault Other (radiochemistry): A. Maisonial-Besset
Acknowledgments
The authors thank Delphine Skrzydelski and Romain Vives and especially Sophie Besse for her technical expertise and help for in vivo treatments and hematologic sampling.
Grant Support
Researchers 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 article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance 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.
References
1. Angelini A, Guerra G, Mavrogenis AF, Pala E, Picci P, Ruggieri P. Clinical outcome of central conventional chondrosarcoma. J Surg Oncol 2012; 106:929–37.
2. Italiano A, Le Cesne A, Bellera C, Piperno-Neumann S, Duffaud F, Penel N, et al. GDC-0449 in patients with advanced chondrosarcomas: a French Sarcoma Group/US and French National Cancer Institute Single-Arm Phase II Collaborative Study. Ann Oncol 2013;24:2922–6.
3. Italiano A, Mir O, Cioffi A, Palmerini E, Piperno-Neumann S, Perrin C, et al. Advanced chondrosarcomas: role of chemotherapy and survival. Ann Oncol 2013;24:2916–22.
4. van Oosterwijk JG, Herpers B, Meijer D, Bruijn IHB, Cleton-Jansen AM, Gelderblom H, et al. Restoration of chemosensitivity for doxorubicin and cisplatin in chondrosarcoma in vitro: BCL-2 family members cause che-moresistance. Ann Oncol 2012;23:1617–26.
5. van Maldegem AM, Gelderblom H, Palmerini E, Dijkstra SD, Gambarotti M, Ruggieri P, et al. Outcome of advanced, unresectable conventional central chondrosarcoma. Cancer 2014;120:3159–64.
6. van Oosterwijk JG, Anninga JK, Gelderblom H, Cleton-Jansen A-M, Bovee JVMG. Update on targets and novel treatment options for high-grade osteosarcoma and chondrosarcoma. Hematol Oncol Clin North Am 2013;27:1021–48.
7. Chen C, Zhou H, Wei F, Jiang L, Liu X, Liu Z, et al. Increased levels of hypoxia-inducible factor-1a are associated with Bcl-xL expression, tumor apoptosis, and clinical outcome in chondrosarcoma. J Orthop Res 2011;29:143–51.
8. Madelmont J-C, Giraud I, Nicolas C, Maurizis J-C, Rapp M, Ollier M, et al. , inventors; Institut National De La Sante Et De La Recherche Medicale, assignee. Novel quaternary ammonium derivatives, method for preparing same and pharmaceutical use. Patent WO2001000621 A1. 2001 Jan 4.
9. Miot-Noirault E, Vidal A, Morlieras J, Bonazza P, Auzeloux P, Besse S, et al. Small rigid platforms functionalization with quaternary ammonium: targeting extracellular matrix of chondrosarcoma. Nanomedicine 2014;10: 1887–95.
10. Vidal A, Sabatini M, Rolland-Valognes G, Renard P, Madelmont J-C, Mounetou E. Synthesis and in vitro evaluation of targeted tetracycline derivatives: effects on inhibition of matrix metalloproteinases. Bioorg Med Chem 2007;15:2368–74.
11. Nicolas C, Verny M, Giraud I, Ollier M, Rapp M, Maurizis JC, et al. New quaternary ammonium oxicam derivatives targeted toward cartilage: syn-thesis, pharmacokinetic studies, and antiinflammatory potency. J Med Chem 1999;42:5235–40.
12. Giraud I, Rapp M, Maurizis JC, Madelmont JC. Application to a cartilage targeting strategy: synthesis and in vivo biodistribution of [14C]-labeled quaternary ammonium-glucosamine conjugates. Bioconjug Chem 2000; 11:212–8.
13. Peyrode C, Weber V, David E, Vidal A, Auzeloux P, Communal Y, et al. Quaternary ammonium-melphalan conjugate for anticancer therapy of chondrosarcoma: in vitro and in vivo preclinical studies. Invest New Drugs 2012;30:1782–90.
14. Miot-Noirault E, Vidal A, Auzeloux P, Peyrode C, Madelmont C, Chezal J-M. Invivo scintigraphic imaging of proteoglycans. Methods Mol Biol 2012;836:183–98.
15. Peyrode C, Gouin F, Vidal A, Auzeloux P, Besse S, Dauplat M-M, et al. A "Proteoglycan targeting strategy" for the scintigraphic imaging and mon-itoring of the Swarm rat chondrosarcoma orthotopic model. Sarcoma 2011;2011:691608.
16. Miot-Noirault E, David E, Vidal A, Peyrode C, Besse S, Dauplat M-M, et al. 99mTc-NTP 15-5 assessment of the early therapeutic response of chon-drosarcoma to zoledronic acid in the Swarm rat orthotopic model. EJNMMI Res 2013;3:1–40.
17. Giraud I, Rapp M, Maurizis J-C, Madelmont J-C. Synthesis and in vitro evaluation of quaternary ammonium derivatives of chlorambucil and melphalan, anticancer drugs designed for the chemotherapy of chondro-sarcoma. J Med Chem 2002;45:2116–9.
18. Vidal A, Gaumet V, Galmier M-J, Besse S, Leal F, Gachon F, et al. Development of a freeze-dried kit formulation for the preparation of 99mTc-NTP 15-5, a radiotracer for scintigraphic imaging of proteogly-cans. Appl Radiat Isot 2015;101:1–9.
19. Ghosh T, Williams L, Mastrangelo CH. Label-free detection of protein binding with multisine SPR microchips. Lab Chip 2011;11: 4194–9.
20. Taguchi Y, Takano E, Takeuchi T. SPR sensing of bisphenol A using molecularly imprinted nanoparticles immobilized on slab optical waveguide with consecutive parallel Au and Ag deposition bands coexistent with bisphenol A-immobilized Au nanoparticles. Langmuir 2012;28:7083–8.
21. Borel M, Degoul F, Communal Y, Mounetou E, Bouchon B, C-Gaudreault R, et al. N-(4-iodophenyl)-N'-(2-chloroethyl)urea as a microtubule dis-rupter: in vitro and in vivo profiling of antitumoral activity on CT-26 murine colon carcinoma cell line cultured and grafted to mice. Br J Cancer 2007;96:1684–91.
22. Degoul F, Borel M, Jacquemot N, Besse S, Communal Y, Mishellany F, et al. In vivo efficacy of melanoma internal radionuclide therapy with a 131I-labelled melanin-targeting heteroarylcarboxamide molecule. Int J Cancer 2013;133:1042–53.
23. Ben Sellem D, Elbayed K, Neuville A, Moussallieh F-M, Lang-Averous G, Piotto M, et al. Metabolomic characterization of ovarian epithelial carcinomas by HRMAS-NMR spectroscopy. J Oncol 2011;2011: 174019.
24. Verny M, Nicolas C. [3H]-labelling of hydroxyethyl groups– synthesis of s-2-hydroxy [2-3H] ethyl) glutathione and of [3H]-melphalan. J Label Compd Radiopharm 1988;25:949–55.
25. Rapp M, Giraud I, Maurizis J-C, Galmier M-J, Madelmont J-C. Synthesis and in vivo biodisposition of [14C]-quaternary ammoniummelphalan conjugate, a potential cartilage-targeted alkylating drug. Bioconjug Chem 2003;14:500–6.
26. Shih C-M, Ko W-C, Yang L-Y, Lin C-J, Wu J-S, Lo T-Y, et al. Detection of apoptosis and necrosis in normal human lung cells using 1H NMR spectroscopy. Ann N Y Acad Sci 2005;1042:488–96.
27. Blankenberg FG, Katsikis PD, Storrs RW, Beaulieu C, Spielman D, Chen JY, et al. Quantitative analysis of apoptotic cell death using proton nuclear magnetic resonance spectroscopy. Blood 1997;89: 3778–86.
28. Tzika AA, Cheng LL, Goumnerova L, Madsen JR, Zurakowski D, Astrakas LG, et al. Biochemical characterization of pediatric brain tumors by using in vivo and ex vivo magnetic resonance spectroscopy. J Neurosurg 2002;96: 1023–31.
29. Hamm CA, Stevens JW, Xie H, Vanin EF, Morcuende JA, Abdulkawy H, et al. Microenvironment alters epigenetic and gene expression profiles in Swarm rat chondrosarcoma tumors. BMC Cancer 2010; 10:471.
30. Kenan S, Steiner GC. Experimental transplantation of the Swarm rat chondrosarcoma into bone: radiological and pathological studies. J Orthop Res 1991;9:445–51.
31. Grimaud E, Damiens C, Rousselle AV, Passuti N, Heymann D, Gouin F. Bone remodelling and tumour grade modifications induced by interac-tions between bone and Swarm rat chondrosarcoma. Histol Histopathol 2002;17:1103–11.
32. Stevens JW. Swarm chondrosarcoma: a continued resource for chondro-blastic-like extracellular matrix and chondrosarcoma biology research. Connect Tissue Res 2013;54:252–9.
33. Roskams T, Moshage H, de Vos R, Guido D, Yap P, Desmet V. Heparan sulfate proteoglycan expression in normal human liver. Hepatology 1995;21:950–8.
34. Baghy K, Tatrai P, Regos E, Kovalszky I. Proteoglycans in liver cancer. World J Gastroenterol 2016;22:379–93.
35. Byington KH, Bowe CC, McKinsey DS. Biliary excretion of mel-phalan by control and anuric rats. Biochem Pharmacol 1980;29: 2518–20.
36. Brown RK, Duncan G, Hill DL. Distribution and elimination of melphalan in rats and monkeys and distribution in tumors of mice bearing L1210 or P388 leukemias sensitive and resistant to this agent. Cancer Treat Rep 1980;64:643–8.
37. Polavarapu A, Stillabower JA, Stubblefield SGW, Taylor WM, Baik M-H. The mechanism of guanine alkylation by nitrogen mustards: a computational study. J Org Chem 2012;77:5914–21.
38. Theocharis AD, Skandalis SS, Tzanakakis GN, Karamanos NK. Pro-teoglycans in health and disease: novel roles for proPro-teoglycans in malignancy and their pharmacological targeting. FEBS J 2010;277: 3904–23.
39. Mihov D, Spiess M. Glycosaminoglycans: Sorting determinants in intracellular protein traffic. Int J Biochem Cell Biol 2015;68: 87–91.
40. Oegema TR, Parzych SM. Effect of the retinoic acid analog Ro 11-1430 on proteoglycans of Swarm rat chondrosarcoma. J Natl Cancer Inst 1981;67:99–106.
41. Heinrich EL, Walser TC, Krysan K, Liclican EL, Grant JL, Rodriguez NL, et al. The inflammatory tumor microenvironment, epithelial mesen-chymal transition and lung carcinogenesis. Cancer Microenviron 2012; 5:5–18.
42. Harisi R, Jeney A. Extracellular matrix as target for antitumor therapy. OncoTargets Ther 2015;8:1387–98.
43. Skandalis SS, Theocharis AD, Vynios DH, Papageorgakopoulou N, Hjerpe A, Karamanos NK, et al. Cartilage aggrecan undergoes significant compositional and structural alterations during laryn-geal cancer. Biochim Biophys Acta 2006;1760:1046–53. 44. Vynios DH, Theocharis DA, Papageorgakopoulou N, Papadas TA,
Mastronikolis NS, Goumas PD, et al. Biochemical changes of extracellular proteoglycans in squamous cell laryngeal carcinoma. Connect Tissue Res 2008;49:239–43.
45. Kubo T, Sugita T, Shimose S, Matsuo T, Arihiro K, Ochi M. Expression of hypoxia-inducible factor-1alpha and its relationship to tumour angiogen-esis and cell proliferation in cartilage tumours. J Bone Joint Surg Br 2008;90:364–70.
46. Boeuf S, Bovee JVMG, Lehner B, Hogendoorn PCW, Richter W. Correlation of hypoxic signalling to histological grade and outcome in cartilage tumours. Histopathology 2010;56:641–51.
