L-MTP-PE and zoledronic acid combination in : preclinical evidence of positive therapeutic combination for clinical transfer Kevin Biteau, Romain Guiho, Mathias Chatelais, Julien Taurelle, Julie Chesneau, Nadège Corradini, Dominique Heymann, Françoise Rédini

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Kevin Biteau, Romain Guiho, Mathias Chatelais, Julien Taurelle, Julie Chesneau, et al.. L-MTP- PE and zoledronic acid combination in osteosarcoma: preclinical evidence of positive therapeutic combination for clinical transfer. American Journal of Research, e-Century Publishing, 2016, 6, pp.677 - 689. ￿inserm-01703301￿

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Original Article L-MTP-PE and zoledronic acid combination in osteosarcoma: preclinical evidence of positive therapeutic combination for clinical transfer

Kevin Biteau1,2, Romain Guiho1,2, Mathias Chatelais1,2, Julien Taurelle1,2, Julie Chesneau1,2, Nadège Corradini1,2,3, Dominique Heymann1,2, Françoise Redini1,2,4

1INSERM, UMR-957, Equipe Ligue Contre le Cancer 2012, Nantes, F-44035, France; 2Université de Nantes, Fac- ulté de Médecine, Laboratoire De Physiopathologie De La Résorption Osseuse Et Thérapie Des Tumeurs Osseus- es Primitives, Nantes, F-44035, France; 3Pediatric Oncology Unit, Hôpital Mère-Enfant, Nantes, F-44035, France; 4CHU Hôtel Dieu, Nantes, F-44035, France Received January 29, 2016; Accepted February 1, 2016; Epub February 15, 2016; Published March 1, 2016

Abstract: Osteosarcoma, the most frequent malignant primary bone tumor in pediatric patients is characterized by osteolysis promoting tumor growth. Lung metastasis is the major bad prognosis factor of this disease. Zoledronic Acid (ZA), a potent inhibitor of bone resorption is currently evaluated in phase III randomized studies in Europe for the treatment of osteosarcoma and Ewing sarcoma. The beneficial effect of the liposomal form of Muramyl-TriPep- tide-Phosphatidyl Ethanolamine (L-mifamurtide, MEPACT®), an activator of populations has been dem- onstrated to eradicate lung metastatic foci in osteosarcoma. The objective of this study was to evaluate the potential therapeutic benefit and the safety of the ZA and L-mifamurtide combination in preclinical models of osteosarcoma, as a prerequisite before translation to patients. The effects of ZA (100 µg/kg) and L-mifamurtide (1 mg/kg) were investigated in vivo in xenogeneic and syngeneic mice models of osteosarcoma, at clinical (tumor proliferation, spontaneous lung metastases development), radiological (bone microarchitecture by microCT analysis), biological and histological levels. No interference between the two drugs could be observed on ZA-induced bone protection and on L-mifamurtide-induced inhibition of lung metastasis development. Unexpectedly, ZA and L-mifamurtide asso- ciation induced an additional and in some cases synergistic inhibition of primary tumor progression. L-mifamurtide has no effect on tumor proliferation in vitro or in vivo, and macrophage population was not affected at the tumor site whatever the treatment. This study evidenced for the first time a significant inhibition of primary osteosarcoma progression when both drugs are combined. This result constitutes a first proof-of-principle for clinical application in osteosarcoma patients.

Keywords: Osteosarcoma, liposomal mifamurtide, zoledronic acid, inhibition of lung metastases development, bone protection

Introduction and overall survival have only slightly improv- ed during the last decades remaining around Osteosarcoma is the most frequent malignant 65-70% at 5 years for localized forms and only primary bone tumor in young people with 25% for patients who present pulmonary metas- around 1000 new cases per year in Europe [1, tases at diagnosis. The current drug regimens 2]. It arises mainly in adolescents and young used for osteosarcoma pediatric patients as- adults (median age 18 years), with a preferen- sociate MTX-VP-IFO (High dose , tial location in the metaphysis of long bones. Vincristin, platinium and ) or API-AI The standard treatment of osteosarcoma con- for adults (adriamycin-platinium-ifosfamide). sists in multi-drug as neo-adju- However, patients who do not respond to che- vant and adjuvant settings associated with sur- motherapy have an overall survival of only gical local control [3, 4]. Despite recent ad- 20-25% at 5 years [5, 6]. Therefore, new thera- vances in limb-salvage surgery and poly-che- peutic targets are urgently needed for these motherapy combinations, event-free survival patients at high risk. L-MTP-PE and zoledronic acid combination in osteosarcoma

Recently, L-mifamurtide (Liposomal-Muramyl produce osteoclast activating factors such TriPeptide-PhosphatidylEthanolamine: L-MTP- as PTH-rP, IL-11, IL-6 or the main regulator PE) has been proposed as adjuvant therapy of osteoclast function: Receptor Activator of for osteosarcoma patients in the United States. NF-кB Ligand (RANKL). When activated, osteo- It is a synthetic analog of the muramyl dipep- clasts are able to degrade bone, and allow the tide (MDP), resulting from the covalent addi- release of growth factors stored in the bone tion of alanin and dipalmitoylphosphatidyl matrix such as Transforming Growth Factor-b, ethanolamine to MDP, a peptidoglycan found in Insulin-like Growth Factor-1, Fibroblast Growth bacterial cell wall [7]. L-mifamurtide acts as Factor… that in turn activate tumor cell prolif- a nonspecific immunomodulator by activating eration [22]. and related to the The third generation nitrogen-containing Bis- upregulation of tumoricidal activity and secre- phosphonates (N-BPs) and among them zole- tion of pro-inflammatory including dronic acid are potent inhibitors of osteoclast (TNF)-a, (IL)-1, functions resulting in inhibition of bone resorp- IL-6, IL-8, IL-12, nitric oxide (NO), prostaglandin tion [23, 24]. In addition, N-BPs slow down E2 (PGE2) and PGD2 [8-10]. The lipid composi- tumor growth in bone, both indirectly by inhibit- tion of mifamurtide has been developed to tar- ing osteoclast activation and directly by inhibi- get delivery of the drug selectively to mono- tion of tumor cell proliferation, angiogenesis, cytes and macrophages in liver, and migration and activation of γδT lymphocytes lungs [7, 11, 12]. It also facilitates signaling via [25, 26]. Therefore, these drugs are currently phosphatidyl serine recognition by macroph- used as therapeutic agents in pathologies ages [13]. Moreover, the liposomal formulation associated with bone degradation from tumor can improve the safety profile of several drugs origin or not (osteoporosis, multiple myeloma, by modifying parent drug or solubilization agent bone metastases and primary bone tumors) toxicity [14]. In 2011, Buddingh and al. report- [27, 28]. ed that tumor-associated-macrophages (TAMs) are associated with good prognosis in high Considering that osteolysis promoting tumor grade osteosarcoma [15]. In contrast to most growth and lung metastasis dissemination are other tumor types, TAMs are associated with the two key points in osteosarcoma progres- reduced metastasis and improved survival for sion, it is tempting to propose ZA and L-mi- those patients. A phase III randomized clinical famurtide combination in osteosarcoma treat- trial was conducted by the Children‘s oncology ment, each of these drugs already proving their group (COG) from 1993 to 1997 [16]. Significant efficacy. Before clinical application, preclinical improvement in EFS and overall survival were studies are needed to see whether L-mifa- observed in patients randomized to receive murtide could interfere with ZA induced preven- L-mifamurtide [7, 17, 18]. In the European Un- tion of associated bone lesions, and conversely ion, except in France, L-mifamurtide is indicat- whether ZA may interfere with L-mifamurtide ed in patients aged between 2 and 30 years induced inhibition of lung metastasis disse- with high-grade, resectable, nonmetastatic os- mination. teosarcoma after the complete surgical wide Material & methods resection. Cell culture In France, OS2006 is the current clinical proto- col for pediatric and adult osteosarcoma pa- Human KHOS and murine K7M2 osteosarco- tients, a phase III randomized study associat- ma cell lines were purchased from the Ameri- ing Zometa® (ZA, zoledronic acid) with con- can Type Culture Collection (ATCC, LGC Pro- ventional poly-chemotherapy and surgery. This mochem, Molsheim, France) and maintained in protocol is based on preclinical studies from Dulbecco’s Modified Eagle’s Medium (DMEM, our group demonstrating the advantage of Lonza, Switzerland). Murine MOS-J (Jackson combination of antitumor therapy with drugs Laboratory) osteosarcoma cell line was cul- that target the bone microenvironment [19, tured in Roosevelt Park Memorial Institute 20]. Indeed, interactions between tumor cells (RPMI) medium (Lonza). Media were supple- and bone cells are closely regulated in the so mented with 5% fetal calf serum (Hyclone, called “vicious cycle” as initially proposed by USA). All cell lines were cultured in a humidified

Paget [21]. Tumor cells proliferating in bone 5% CO2/air atmosphere at 37°C.

678 Am J Cancer Res 2016;6(3):677-689 L-MTP-PE and zoledronic acid combination in osteosarcoma

Table 1. Primer sequences used in quantitative real-time PCR and the comparative cycle thresh- analysis old (Ct) method was used to calcu- Gene Forward primer Reverse primer late relative quantification of tar- hGAPDH TGGGTGTGAACCATGAGATATG GGTGCAGGAGGCATTGCT get mRNAs. Each assay was per- formed in triplicate. mHPRT TCCTCCTCAGACCGCTTTT CCTGGTTCATCATCGCTAATC mF4/80 TCCTCCTTGCCTGGACACT GCCTTGAAGGTCAGCAACC Animal models of osteosarcoma mFas TGAATGGGGGTACACCAACC TGCTCTTCATCGCAGAGTGT hFas GAAGGGAAGGAGTACACAGACA TGGTATTCTGGGTCCGGGTG Five week-old female Rj: NMRI CD64 AGGGCGGAAAGAGAAGATGC ATGACTGGGGACCAAGCACT nude mice for xenogeneic models or C57BL/6, BALB/c mice for syn- geneic models were purchased Drugs from Janvier Breedings (Le Genest Saint Isle, France). Mice were anesthetized by inhalation L-mifamurtide was provided by TAKEDA (IDM of an isoflurane/air mixture (2%, 1 L/min). PHARMA SAS). Each vial containing 4 mg of Primitive osteosarcoma was induced by intra- mifamurtide powder in 1 g excipient was divid- muscular paratibial injection of 106 human ed into 8 tubes and stored at 4°C. Powder was KHOS osteosarcoma cells (xenogeneic model) reconstituted extemporaneously in sodium ch- or 3.106 MOS-J or K7M2 mice osteosarcoma loride 0.9% and used within 6 hrs. ZA, 1-hydroxy- cells respectively in C57BL/6 and BALB/c mice 2-(1H-imidazole-1-yl) ethylidene-bisphosphonic (syngeneic models). Tumor development is acid supplied as the disodium salt by associated to osteolytic lesions and with dis- Pharma AG (Basel, Switzerland), was dissolved semination of spontaneous lung metastasis in PBS as 10 mM stock solution and stored mimicking the human pathology. The day after at -20°C. -PBS (clodronateliposome. tumor cell injection, mice were randomly com, Netherlands) was used as control. assigned (n=8-10) to vehicle (NaCl 0.9%), ZA, L-mifamurtide or ZA+L-mifamurtide (bitherapy) Cell proliferation groups. ZA (100 µg/kg; s.c.) or L-mifamurtide (1 mg/kg; i.v.) was injected twice per week. Tumor Osteosarcoma cell lines were plated in respec- volume was measured three times weekly tive media and treated with L-mifamurtide or (length*width*depth*0.5). Data points were zoledronic acid at indicated concentration for expressed as mean tumor volume ± SEM. 48 hours, cell growth being measured using crystal violet assay as described previously Mice were sacrificed when the tumor volume [29]. reached 10% of body weight for ethical rea- sons. Primary bone tumor was harvested for Quantitative reverse transcription-PCR immunohistochemistry analysis, lung metasta- ses were macroscopically counted under a bin- Total RNA was extracted from mice osteosar- ocular loupe. Animal care and experimental coma biopsies (24 h after the last drug injec- protocols were approved by the French Ministry tion) using Direct-zol RNA MiniPrep (Zymo Re- of Agriculture (agreement n°D-44015 for the search) with DNase treatment to remove resid- Experimental Therapy Unit located at the Me- ual genomic DNA. RNA quantity and quality was dical School of Nantes) and were realized in evaluated by determining A260/A280 ratio accordance with the institutional guidelines of using NanoDrop. Complementary DNA was syn- thesized from isolated RNA using ThermoScript the regional ethical committee (CREEA Pays real-time polymerase chain reaction (RT-PCR) de la Loire, PdL06, France), with the protocol System (Invitrogen, Carlsbad, CA, USA). Qu- agreement n°1280.01 and 1281.01, and un- antitative-PCR (qPCR) was performed using der the supervision of authorized investiga- primers (Table 1) on Chromo4 instrument (Bi- tors. orad, Richmond, CA, USA) using SYBR Green Immunohistochemistry Supermix reagents (Biorad). Target gene ex- pression was normalized to GAPDH levels in Immunohistochemical stainings were perfor- respective samples as an internal standard, med on formalin-fixed and paraffin-embedded

679 Am J Cancer Res 2016;6(3):677-689 L-MTP-PE and zoledronic acid combination in osteosarcoma

Figure 1. L-mifamurtide does not interfere with ZA-induced bone protection. A. Representative microCT and 3D reconstruction of the tumor-bearing tibia taken ex-vivo, from KHOS xenogeneic (upper panel) and MOS-J syngeneic (lower panel) models treated or not (Ctrl) with L-mifamurtide (1 mg/kg), ZA (100 µg/kg), or both (bitherapy) at day 1, twice a week, until week 4-5. B. MicroCT quantification of tibia bone volume (BV, mm3) and C. Tibia cortical thickness (Co.Th, mm) were calculated on the tibia of tumor bearing mice of the different groups. Median were represented for each group (n=4). *p<0.05.

3 µm sections of tumor samples using ade- close contact with osteosarcoma and excludes quate primary antibody. Immunodetection per- trabecular bone. Cortical thickness (Ct.Th) was formed using secondary biotinylated antibod- defined as the mean cortical volume divided by ies and streptavidin HRP-complex was reveal- the outer bone surface as previously described ed with 3,3’-diaminobenyidine (DAB-Dako) fol- [30]. Trabecular bone parameters were also lowed by counterstaining with hematoxylin. analyzed. Negative control was analyzed using a similar procedure without primary antibody. Same Statistical analysis slides were used to estimate the percentage of GraphPad InStat v3.02 software (La Jolla, CA, necrosis area among the total tumor. USA) was used. In vivo experimentation results Micro-CT were analyzed with the unpaired nonparamet- ric method and Dunn’s multiple comparisons Analyses of the bone microarchitecture were following the Kruskal-Wallis test. A p value of performed using a SkyScan 1076 in vivo micro- less than 0.05 was considered statistically CT scanner (SkyScan, Kontich, Belgium). Tests significant. were performed at early time (d21, tumor vol- ume around 500 mm3) or at necropsy (tumor Results volume around 2000 mm3). All tibiae/fibulae L-mifamurtide does not interfere with ZA- were scanned using the same parameters induced bone protection (pixel size 18 µm, 50 kV, 0.5-mm Al filter and 0.7 degree of rotation step). Three-dimensional The first objective of the study was to investi- reconstructions and analysis of bone parame- gate the potential interference of L-mifamurtide ters were performed using NRecon and CTan treatment with the protective zoledronic acid softwares (SkyScan). Calculation of cortical effects on bone during osteosarcoma progres- bone volume (BV) following 3D morphometric sion. Two syngeneic and one xenogeneic mod- parameters (bone ASBMR nomenclature) was els of osteosarcoma were used: respectively performed on 5.5-7.2 mm of tibia length K7M2 and MOS-J induced in the BALB/c and (depending on mice model) from the fibula C57BL/6 mouse strains, and KHOS induced in insertion. This area corresponds to bone in NMRI-nude mice. The day after tumor cell injec-

680 Am J Cancer Res 2016;6(3):677-689 L-MTP-PE and zoledronic acid combination in osteosarcoma

Figure 2. Treatment of osteosarcoma bearing mice with L-Mifamurtide alone or in association with ZA inhibits lung metastases development in both xenogeneic (KHOS) and syngeneic (MOS-J) models. A. NMRI-nude or C57BL/6 mice were injected respectively with KHOS (xenogeneic) or MOS-J (syngeneic) osteosarcoma cells and treated with vehicle (Ctrl), L-mifamurtide, ZA or both (bitherapy) as described in Figure 1. B. Graphs indicate individual (dots) and median (lines) numbers of lung metastases macroscopically counted in mice lungs from each group at equivalent primary bone tumor volume (2000 mm3). tion, mice were treated with ZA (100 µg/kg) the cortical thickness (Co.Th) (from 0.14 to and/or L-mifamurtide (1 mg/kg) twice a week. 0.21 and 0.18 mm; Figure 1C), and trabecular The bone microarchitecture parameters of the parameters (not shown). Similar observations tumor-bearing tibia have been measured in all were made with the syngeneic mouse model of models and treatment conditions using X-ray, osteosarcoma MOS-J (Figure 1A-C lower panel), micro-CT and 3D reconstruction analysis (Fig- in which L-mifamurtide treatment did not affect ure 1A). Data revealed a decrease of the tumor- the tumor-associated bone preservation as associated osteolysis in the ZA-treated mice as compared to control group. compared to the untreated control group. Addition of L-mifamurtide did not modulate ZA Zoledronic acid does not interfere with L- induced bone protection in all models tested mifamurtide-induced inhibition of lung metas- (Figure 1). An extensive analysis of multiple tasis dissemination and development bone parameters revealed an increase of tumor-associated bone quality in the ZA treated The second objective was to investigate the groups as compared to control group. In the effect of L-mifamurtide and ZA on spontaneous xenogeneic KHOS mouse model, ZA treatment metastasis dissemination from primary bone alone and combined with L-mifamurtide in- tumors induced in xenogeneic and syngeneic creased the cortical bone volume (BV) (from osteosarcoma models. Mice were treated by ZA 4.88 to 7.79 and 7.62 mm3 respectively as and L-mifamurtide during ~5 weeks (Figure 2A) compared to control group, p<0.05; Figure 1B), and metastases were counted at equivalent

681 Am J Cancer Res 2016;6(3):677-689 L-MTP-PE and zoledronic acid combination in osteosarcoma

Figure 3. L-mifamurtide acts with ZA to reduce tumor growth both in syngeneic (MOS-J) and xenogeneic (KHOS) mouse models of osteosarcoma. Mice were treated with L-mifamurtide (1 mg/kg) and ZA (100 µg/kg) or both starting at day 1 after tumor cell injection as described in Figure 1. The mean tumor volume from (A) xenogeneic KHOS and (B) syngeneic MOS-J mice models, and individual tumor volumes (C) from xenogeneic KHOS model were compared between the 4 groups ± SEM (n=8). *p<0.001. tumor volume. Whatever the model, a trend of growth in syngeneic (MOS-J) and xenogeneic diminished spontaneous lung metastasis dis- (KHOS) osteosarcoma models. In both cases, semination was observed in L-mifamurtide the tumor cells were injected in close contact treated groups (Figure 2B), and still when ani- to the tibia, after periosteum activation. The mals were treated with ZA combined with tumors were apparent within 10 days, and L-mifamurtide. No significant differences could develop for 4-5 weeks. In each model, L-mi- be observed probably because of the high vari- famurtide or ZA alone did not induce any signifi- ability and the small animal number in each cant effect on primary tumor development group. No difference was observed between (Figure 3A, B). However, the combination of control and zoledronic acid treated groups. both drugs significantly inhibited tumor growth in xenogeneic and syngeneic models. The mean The combination of ZA and L-mifamurtide tumor size at 22 days for the xenogeneic KHOS shows no interference on ZA induced bone pro- model was 1285 ± 143 mm3 in the control tection effects and L-mifamurtide induced inhi- group, compared to 872 ± 128 mm3 in mice bition of lung metastasis development both in syngeneic and xenogeneic models of osteosar- treated with bitherapy (means ± SEM, p<0.001; coma in mice. ZA is known to have direct anti- Figure 3A). The mean tumor size at 35 days for the syngeneic MOS-J model was 2284 ± 176 cancer effects in vitro and in vivo in primary 3 and secondary bone tumors [20]. To go further, mm in the control group, compared to 1359 ± 3 we investigated whether L-mifamurtide would 177 mm in the group treated with bitherapy affect the antitumor effect of ZA on osteosar- (means ± SEM, p<0.001; Figure 3B). Similar coma primary bone tumor. results were obtained in the K7M2 syngeneic mice model (not shown). In summary, combina- In vivo significant inhibitory effect ofL- mi- tory therapy induced 41% and 55% inhibition of famurtide associated with zoledronic acid on mean tumor volume in syngeneic models (MOS- primary bone tumor progression in syngeneic J and K7M2 respectively) and 37% in the xeno- and xenogeneic models of osteosarcoma geneic model (KHOS) at final time point.

The effect of L-mifamurtide and ZA therapeutic In both syngeneic models, the mean decrease association was analyzed on primary tumor in tumor volume of mice treated with ZA and

682 Am J Cancer Res 2016;6(3):677-689 L-MTP-PE and zoledronic acid combination in osteosarcoma

Figure 4. L-mifamurtide alone or associated with ZA does not directly affect tumor cell proliferation in vivo and in vitro. A. Osteosarcoma cells were treated with ZA, L-mifamurtide or both at indicated doses for 48 h. Cell growth was determined by crystal violet analysis and compared to control untreated cells. B. KHOS tumor biopsies were col- lected and Ki67 staining was evaluated by immunohistochemical analysis. Specimens were scored and estimated in percentage mean of Ki67+-cells compare to the control group, which corresponds to the mice bearing tumor that did not receive any treatment but vehicle. C. Representative picture of Ki67 staining for control and bitherapy treated groups.

L-mifamurtide reflects a slowing tumor growth in combination was studied on osteosarcoma in some mice, and no response in others cell proliferation in vitro, and in vivo by Ki67 (Figure 3C). These unexpected results demon- staining on tumor biopsies. Results show that strate that L-mifamurtide acts with ZA to reduce L-mifamurtide alone has no direct effect on the primary tumor growth in vivo. proliferation rate of the two osteosarcoma cell lines MOS-J and KHOS in vitro (Figure 4A). In L-mifamurtide does not affect osteosarcoma addition, L-mifamurtide does not significantly cell proliferation in vivo or in vitro modify the ZA-induced inhibition of osteosar- coma cell proliferation (Figure 4A). Considering In order to analyze the mechanisms involved in the in vivo mechanisms of action, no significant the tumor growth inhibition observed in vivo difference could be observed by Ki67 immuno- with the L-mifamurtide + ZA therapeutic asso- histochemical (IHC) staining of KHOS osteosar- ciation, the direct effect of each drug alone and coma in situ cell proliferation between treated

683 Am J Cancer Res 2016;6(3):677-689 L-MTP-PE and zoledronic acid combination in osteosarcoma

Figure 5. L-mifamurtide and/or zoledronic acid treatments did not affect /macrophage markers in osteo- sarcoma syngeneic tumor biopsies. A. Staining of osteosarcoma tissues from MOS-J syngeneic mouse model for F4/80 22 days after tumor induction. B. Expression of F4/80, CD64, Fas mRNA by MOS-J tumor biopsies. NS: no statistic differences between groups. Mean ± SD. and untreated mice (Figure 4B, 4C). Moreover, tumors of the different groups in the syngeneic necrosis area percentage is not affected by MOS-J mouse model (Figure 5A). QPCR analy- any treatment and similar observations were ses from tumor biopsies confirmed that any obtained for xenogeneic models (not shown). It treatment affected the F4/80 (macrophages) should be mentioned that osteosarcoma pres- or CD64 (monocytes/macrophages) mRNA ex- ents high heterogeneity inter- and intra-groups pression (Figure 5B). Similar observations were leading to difficult interpretation of IHC results made with the KHOS xenogeneic mouse model at significant level. (not shown). Moreover, IHC scoring of iNOS (M1 macrophages) and ArgI (M2 macrophages) did Therefore, based on these in vitro results, the not show any differences between groups (not additive effect of both therapies cannot be shown). Therefore, Fas mRNA expression was explained by a direct anti-tumor effect of L- evaluated at the primary tumor site in osteo- mifamurtide. sarcoma models under ZA and L-mifamurti- de treatments. Results showed no significant Mechanisms of action of L-mifamurtide and ZA modification of Fas mRNA expression in synge- combination in vivo neic MOS-J (Figure 5B) or xenogeneic KHOS (not shown) bone tumor biopsies. Because L-mifamurtide is known to activate macrophage- and monocyte-based immune In summary, neither ZA nor L-mifamurtide sig- response, the significant inhibitory effect of nificantly alters the monocyte/macrophage po- bitherapy that was observed on primary tumor pulations in the osteosarcoma models used in progression may be linked to the modulation of this study. Furthermore, our results suggest these immune cell populations by the associa- that L-mifamurtide does not affect macrophage tion of both drugs. Therefore, monocyte/macro- activation in the primary tumor site as analyzed phage populations were analyzed at primary by iNOS/ArgI IHC staining. site by the relative proportion of their markers at transcript (qPCR) and protein levels (IHC) on Discussion mouse osteosarcoma biopsies of each group. The absence of overall survival improvement IHC analyses showed no differences in the for more than 40 years since the application macrophages F4/80+ infiltrates in primary bone of Rosen poly-chemotherapy protocols [31] is

684 Am J Cancer Res 2016;6(3):677-689 L-MTP-PE and zoledronic acid combination in osteosarcoma associated with the lack of relevant therapeutic Even if a strong trend (but not significant) options for high-grade osteosarcoma patients toward an anti-metastatic activity of L-mifa- (especially patients with lung metastases). In murtide was shown in our mouse models, we addition to conventional chemotherapy, multi- could not observe any increase of the overall ple investigational agents have being studied. survival in these groups, as it has been previ- Among them, zoledronic acid has already dem- ously described in dogs [35] and patients [18]. onstrated its therapeutic interest for bone Overall, analyses of tumor behavior are ren- malignancies including osteosarcoma [19, 20]. dered complicated due to the high tumor het- L-mifamurtide is also one of the more promis- erogeneity in osteosarcoma. The general so- ing drugs for osteosarcoma acting as immune urce of heterogeneity is caused by specific stimulatory agent. At clinical level, Meyer’s tumor microenvironment, composed of stromal study was designed to assess whether the cells, the availability of vascular network, and addition of L-MTP-PE and/or ifosfamide to a the host’s immune system [36]. Primary osteo- standard chemotherapeutic regimen (doxorubi- sarcoma progression was so rapid in our mod- cin--high dose methotrexate) would els that pulmonary metastasis dissemination increase both EFS and overall survival in newly was systematically observed, prompting us to diagnosed patients with high-grade osteosar- sacrifice the mice early. In these cases, it could coma. ZA is currently proposed in clinical trials not be possible to observe any significant effect for osteosarcoma (OS2006 - NCT00470223) of L-mifamurtide, but only harm trends. The effi- and Ewing sarcoma (EWING2008 - NCT00- cacy of the L-mifamurtide should be investigat- 987636, EuroEWING2012 - ISRCTN92192408) ed in models with slower metastasis develop- patients, and L-mifamurtide therapy has been ment. conducted in phase II (MEMOS - ISRCTN82- 138287) and phase III trials in patients with In the second part of our study, we wanted to newly diagnosed OS [18, 32]. Therefore, the understand why the L-mifamurtide and ZA com- combination of both drugs could represent a bination induced a synergistic and significant promising therapeutic option as both drugs inhibition of primary osteosarcoma progression target different complementary pathways: L-mi- in our mice models whereas each drug showed famurtide activates macrophages and mono- no significant effect. This unexpected result cytes which help to eradicate lung metastasis was observed both in xenogeneic and synge- development, and ZA inhibits osteoclast func- neic models with 32% and 41% inhibition of tion by inducing their apoptosis [33], and there- primary tumor growth respectively. Previous fore indirectly induced tumor growth inhibition. results from the literature showed a direct and In the present study, we described for the first indirect anti-tumor activity of L-mifamurtide on time a strong trend for L-mifamurtide to inhibit osteosarcoma cells in vitro or in metastases lung metastasis dissemination in both synge- development after resection of the primary neic and xenogeneic osteosarcoma models in tumor [8, 37]. Moreover, this inhibitory effect mice. Moreover, its combination with ZA sh- on primary tumor growth is even more unex- owed no interference of both drugs on each pected as ZA and L-mifamurtide were not asso- other. Relevant complementary orthotopic mo- ciated with conventional chemotherapy as usu- dels of osteosarcoma with spontaneous pulmo- ally in clinics. Several hypotheses could be nary metastasis development were used to proposed to explain those results. better reproduce the human clinical features and to demonstrate the value of this therapeu- First, both drugs act synergistically by directly tic approach [34]. Preclinical studies using inhibiting tumor proliferation. However, when xenogeneic and syngeneic models of osteosar- we studied the potential direct anti-tumor activ- coma demonstrated that ZA did not interfere ity of L-mifamurtide alone and associated with with L-mifamurtide-inducing lung metastases ZA in vitro, no inhibition of osteosarcoma cell reduction, and in parallel, L-mifamurtide did growth could be observed. In addition, tumor not modulate the ZA-induced bone protection. biopsies were collected in mice for histological These results in murine models are in agree- analysis 24 hours after treatment at early ment with previous preclinical data reported by (week 3) or end time point (week 5). We were only one team who described the benefit of unable to show a decreased proliferation rate L-mifamurtide on lung metastasis formation in as analyzed by Ki67 staining in the bitherapy dog models of osteosarcoma [35]. group in our different models. However, we may

685 Am J Cancer Res 2016;6(3):677-689 L-MTP-PE and zoledronic acid combination in osteosarcoma wonder whether the effects of ZA and L-mi- side effect of ZA on TAMs. Different approach- famurtide are transient as a consequence of es by IHC, qPCR and flow cytometry were used and pro-inflammatory cytokines up regu- to study macrophage sub-populations in mice lation for example. In that case, these parame- tumor biopsies from xenogeneic and syngeneic ters have to be analyzed earlier. In fact, the syn- osteosarcoma models. However, no differenc- ergic interaction of ZA and L-mifamurtide on es in macrophage sub-populations infiltrating tumor progression may be due to a transient primary bone tumor could be observed what- but sufficient effect on pro-inflammatory cyto- ever the methodological approaches used. kine upregulation. In conclusion, no interference between zole- Various studies have reported the stimulation dronic acid and the liposomal form of mifamur- of macrophages by L-mifamurtide + IFN-γ in tide was observed for the studied parameters. vitro [38, 39]. ‘Priming’ of macrophages by Combination of both drugs showed no signifi- IFN-γ may enhance liposome up-take and im- cant difference on ZA induced bone protection prove the response to bacterial components effects and on L-mifamurtide induced inhibition such as MDP or presumably mifamurtide. We of lung metastasis development. Unexpectedly, therefore hypothesized that ZA treatment may an additive and in some cases synergistic inhib- increase the IFN-γ level at plasma or local loca- itory effect was observed on primary osteosar- tion high enough to ‘prime’ macrophages. It has coma progression that could not be explained been reported by clinicians that zoledronic acid by a direct synergy between drugs on osteosar- therapy leads to fever symptoms in patients for coma cell proliferation, neither by a modifica- 24-48 h, probably due to transient TNF-α and tion of TAMs population at the primary bone IL-6 increases, but not IFN-γ [40]. We indeed site. Even if the mechanism remains unclear up confirm these data, as we were unable to show to now, those results constitutes a promising a detectable IFN-γ plasma level in the mice 24 demonstration for clinical application in osteo- hours after zoledronic acid treatment (not sarcoma patients. shown). It is known that L-mifamurtide binds Acknowledgements to the cytosolic NOD2 “receptor” to stimulate NF-ĸB signaling pathways, activating the ex- This work was supported by a grant from pression of inflammatory cytokines including Takeda France (Paris, France) and by Region IL-12 [41]. It is admitted that plasma IL-12 Centre-Val de Loire (MeTaPulm-R; Tours, increases after administration of L-mifamurtide, France). The authors wish thanks G. Hamery, J. and that IL-12 upregulates FAS expression in Pajot and P. Monmousseau for their kind assis- human osteosarcoma lung metastases. It also tance at the animal facility care platform known that Fas expression inversely correlates (Faculté de Médecine, Nantes, France). with metastatic activity in osteosarcoma [42]. However, we were unable to observe any en- Declaration of conflict of interest hancement of Fas mRNA expression (follow- ing L-mifamurtide treatment) in primary bone None. tumor in both xenogeneic and syngeneic mouse models of osteosarcoma (not shown). Address correspondence to: Françoise Redini, IN- SERM, UMR-957, Equipe Ligue Contre Le Cancer Because L-mifamurtide acts as a potent stimu- 2012, Nantes, F-44035, France. E-mail: francoise. lator of macrophage activity preferentially at [email protected] lung site [43], the question raised about its action at the primary osteosarcoma site. In par- References allel, the role of ZA in tumor macrophages pop- ulation is controversial. Because ZA induced [1] Meyers PA and Gorlick R. Osteosarcoma. Pedi- osteoclast apoptosis (via the inhibition of the atric Clinics of North America 1997; 44: 973- small G protein prenylation), it could be sug- 989. [2] Stiller CA, Bielack SS, Jundt G and Steliarova- gested that ZA also affects macrophages, as Foucher E. Bone tumours in European children previously described in breast cancer [44], and adolescents, 1978-1997. Report from the especially those present in the bone tumor Automated Childhood Cancer Information Sys- microenvironment. We hypothesize that L-mi- tem project. Eur J Cancer 2006; 42: 2124- famurtide may counterbalance the potential 2135.

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