Temsirolimus combined with cisplatin or bevacizumab is active in osteosarcoma models

Temsirolimus combined with cisplatin or bevacizumab is activein osteosarcoma models

Emmy D.G. Fleuren1, Yvonne M.H. Versleijen-Jonkers1, Melissa H.S. Roeffen1, Gerben M. Franssen2, Uta E. Flucke3,

Peter J. Houghton4, Wim J.G. Oyen2, Otto C. Boerman2 and Winette T.A. van der Graaf1

1 Department of Medical Oncology, Radboud University Medical Centre, Nijmegen, the Netherlands2 Department of Nuclear Medicine, Radboud University Medical Centre, Nijmegen, the Netherlands3 Department of Pathology, Radboud University Medical Centre, Nijmegen, the Netherlands4 Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, Columbus, OH

Mammalian target of rapamycin (mTOR) is a new promising oncological target. However, most clinical studies reported only

modest antitumor activity during mTOR-targeted monotherapies, including studies in osteosarcomas, emphasizing a need for

improvement. We hypothesized that the combination with rationally selected other therapeutic agents may improve response.

In this study, we examined the efficacy of the mTOR inhibitor temsirolimus combined with cisplatin or bevacizumab on the

growth of human osteosarcoma xenografts (OS-33 and OS-1) in vivo, incorporating functional imaging techniques and micro-

scopic analyses to unravel mechanisms of response. In both OS-33 and OS-1 models, the activity of temsirolimus was signifi-

cantly enhanced by the addition of cisplatin (TC) or bevacizumab (TB). Extensive immunohistochemical analysis demonstrated

apparent effects on tumor architecture, vasculature, apoptosis and the mTOR-pathway with combined treatments. 30-Deoxy-

30-18F-fluorothymidine (18F-FLT) positron emission tomography (PET) scans showed a remarkable decrease in 18F-FLT signal in

TC- and TB-treated OS-1 tumors, which was already noticeable after 1 week of treatment. No baseline uptake was observed in

the OS-33 model. Both immunohistochemistry and 18F-FLT-PET demonstrated that responses as determined by caliper meas-

urements underestimated the actual tumor response. Although 18F-FLT-PET could be used for accurate and early response

monitoring for temsirolimus-based therapies in the OS-1 model, we could not evaluate OS-33 tumors with this molecular

imaging technique. Further research on the value of the use of 18F-FLT-PET in this setting in osteosarcomas is warranted. Over-

all, these findings urge the further exploration of TC and TB treatment for osteosarcoma (and other cancer) patients.

Osteosarcoma is the most commonly diagnosed primarymalignant tumor of the bone mainly affecting children andadolescents.1 Current standard treatment regimens consist ofsurgery and polychemotherapy. Unfortunately, despite multi-modal treatment the final outcome has not improved signifi-cantly during the last decade and severe side effects ofintensive chemotherapy treatment schedules are observed fre-quently. On top of that, research on targeted treatments inosteosarcomas lags behind. This underscores the absolute

need for novel, targeted therapies to treat these often youngpatients.

Blocking of mammalian target of rapamycin (mTOR) sig-naling emerged as a promising approach to target osteosarco-mas. mTOR, also known as sirolimus effector protein, is anintracellular serine/threonine kinase involved in the phospha-tidylinositol 3-kinase (PI3K)/Akt signaling cascade and sig-nals downstream of several receptor tyrosine kinases (RTKs),including but not limited to the insulin-like growth factor 1

Key words: temsirolimus, cisplatin, bevacizumab, osteosarcoma, 18F-FLT-PET

Abbreviations: ECM: extracellular matrix; Erk: extracellular signal regulated kinase; EGFR: Epidermal Growth Factor Receptor; 4EBP1:

eukaryotic initiation factor 4E binding protein 1; 18F-FLT: 30-deoxy-30-18F-fluorothymidine; 18F-FDG: 18F-fluorodeoxyglucose; HIF-1a:

hypoxia inducible factor 1 a; IGF-1R: Insulin-like Growth Factor 1 Receptor; IHC: immunohistochemistry; mTOR: mammalian target

of rapamycin; PET: positron emission tomography; PI3K: phosphatidylinositol-kinase 3; RTK: receptor tyrosine kinase; RPS6K1: ribo-

somal protein S6 kinase 1; RTV: relative tumor volume; ROI: region of interest; (r)SUVmean/max: (relative) mean/maximum standar-

dized uptake value; TC: temsirolimus 1 cisplatin; TB: temsirolimus 1 bevacizumab; VEGF: vascular endothelial growth factor.

Additional Supporting Information may be found in the online version of this article.

Grant sponsor: Pfizer (to W.T.A.G.); Grant number: WS979256; Grant sponsor: Radboud AYA Foundation

DOI: 10.1002/ijc.28933

History: Received 29 Nov 2013; Accepted 14 Apr 2014; Online 26 Apr 2014

Correspondence to: Emmy D.G. Fleuren, Department of Medical Oncology (Internal Postal Code: 452), Radboud University Medical

Centre, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands, Tel.: 0031-24-3618897, Fax: 0031-24-3540788,

E-mail: [email protected]

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Int. J. Cancer: 135, 2770–2782 (2014) VC 2014 UICC

International Journal of Cancer

IJC

receptor (IGF-1R) and epidermal growth factor receptor(EGFR).2 The two best-characterized targets of mTOR areribosomal protein (RP) S6 kinase 1 (S6K1) and eukaryoticinitiation factor 4E (elF-4E) binding protein 1 (4EBP1).3

Activation of the mTOR-pathway results in ribosomal trans-lation of mRNA into proteins necessary for cell growth, cellcycle progression and cell metabolism.4 mTOR is alsoinvolved in angiogenesis via its downstream effects on thehypoxia inducible factor 1 a (HIF-1a)/vascular endothelialgrowth factor (VEGF) signaling cascade.

The mTOR-signaling pathway has been implicated inmany human cancers, and there are indications that mTORplays a role in osteosarcomas as well.4 Several osteosarcomacell lines and most osteosarcoma patient samples expressmTOR.5–8 More importantly, the mTOR/p70S6K-pathwaycontributes to disease progression and is linked to a poorprognosis in osteosarcoma patients.7 Several mTOR inhibitorshave been developed, including rapamycin (sirolimus), evero-limus, ridaforolimus (deforolimus) and temsirolimus.9,10

Rapamycin already proved to be very effective in inhibitingosteosarcoma cell growth, migration and invasion in vitro aswell as delaying tumor growth in several in vivo osteosar-coma xenografts.5,11,12 It even led to significant inhibition ofosteosarcoma lung metastases in an experimental model.13 Inosteosarcoma patients, two confirmed and one unconfirmedpartial response were observed during ridaforolimus treat-ment out of 54 not further specified bone sarcoma patients.14

A recent pediatric phase I study, however, reported no objec-tive responses in the three included osteosarcoma patientsduring temsirolimus treatment.15 Since currently at best par-tial responses were seen in osteosarcomas during mTOR-mediated treatment in the clinic, the current opinion is thatmTOR inhibitors as monotherapy lack sufficient efficacy inthese patients. Combination with rationally selected othertherapeutic agents may improve response.

In the present study, we focused on the combination ofthe mTOR inhibitor temsirolimus with other drugs andaimed to investigate two different combined therapeuticapproaches. Because temsirolimus induces cell cycle arrestrather than apoptosis, one rational approach would be theaddition of chemotherapeutic agents. Given the sensitivity ofosteosarcoma for cisplatin, this chemotherapeutic agent wasselected. Furthermore, since the mTOR-pathway is involvedin angiogenesis and temsirolimus inhibits tumor growth by

targeting the mTOR/HIF-1a/VEGF-signaling pathway, thecombination with specific VEGF inhibitors may improvetumor response as well.16 Therefore, we selected bevacizu-mab. Angiogenesis inhibition has been proven active in soft-tissue sarcomas for which pazopanib has been registered.17

Moreover, the role of angiogenesis inhibition is currentlybeing tested in osteosarcoma studies. Bevacizumab combinedwith standard chemotherapy is subject of a currently ongoingclinical trial (NCT00667342) and clinical activity wasreported with the angiogenesis inhibitors pazopanib, sorafe-nib and regorafenib in osteosarcomas.18

In summary, we combined temsirolimus with cisplatin orbevacizumab and examined the efficacy of these treatmentson the growth of human osteosarcoma xenografts in vivo.We also acquired 18F-fluorodeoxyglucose (18F-FDG) and 30-deoxy-30-18F-fluorothymidine (18F-FLT) positron emissiontomography (PET) scans before, during and after treatmentto closely monitor tumor metabolism and proliferation,respectively, because in bone sarcomas tumor volume notnecessarily correlates with the actual tumor response.19 Atthe end of the experiment, all tumors were collected and ana-lyzed extensively with immunohistochemistry (IHC) to studychanges in tumor microenvironment and the mTOR-pathway.

Material and MethodsMouse models

For all experiments, female BALB/c nude mice (6–8 weeksold, weighing 20–26 g) were used. Mice were housed underclean, sterile standard conditions in filter-topped cages (5–6mice per cage), with free access to standard animal chow andwater. We specifically chose to use two human osteosarcomaxenografts (OS-1 and OS-33) to mimic osteosarcomas asclosely as possible. The xenografts were created and gener-ously provided by the Pediatric Preclinical Testing Program(PPTP; Columbus, OH). Because xenografts are created bydirect transplantation of a patient tumor fraction in animmunodeficient mouse, without any previous in vitro cul-ture or clonal selection, the genetic background and heteroge-neity of human tumor tissue is preserved in a better waythan with cell culture and retains several characteristics moreclosely reflecting the patient situation. OS-1 and OS-33 mod-els were selected because of their different responses to rapa-mycin monotherapy.11 Both models are p53-wildtype.

What’s new?

Blockade of mammalian target of rapamycin (mTOR) represents a promising approach in the treatment of osteosarcoma,

although mTOR monotherapy has met with mixed results in patients. This study suggests that combination therapy may be

the key to success. Using in vivo osteosarcoma models, the authors show that the activity of the mTOR-inhibitor temsirolimus

is significantly enhanced by the addition of cisplatin or bevacizumab. In addition, immunohistochemical and 18F-FLT-PET analy-

ses of tumor response indicate that tumor volumes underestimate treatment efficacy, highlighting the importance of incorpo-

rating functional imaging techniques for accurate tumor monitoring in osteosarcoma.

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Xenografts (solid tumor pieces of 10–50 mm3) were subcuta-neously (s.c.) implanted on the flank near the hind limb fortherapy experiments. Experiments were performed whentumors reached a size of 0.1–0.3 cm3, which was 2 weeks(OS-33) or 3 weeks (OS-1) after initial implantation. Allexperiments were approved by and carried out in accordancewith the guidelines of the institutional Animal Welfare Com-mittee of the Radboud University Nijmegen.

Dose determination and schedule

In order to obtain clinically relevant and applicable results,we decided to use a dose close to the maximum tolerateddose (MTD) as determined in humans instead of testing eachcompound at the MTD determined in mice. Numerous dataindicate that the MTD found in mice is often higher than inhumans, possibly because some agents specifically target thehuman (and not mouse) antigen (e.g., VEGF-targeting withbevacizumab), which can lead to overestimation of drugeffectiveness (higher dose administration) and underestima-tion of toxicity in preclinical models.20 Additionally, combin-ing different therapies may increase toxicity. To avoid theseproblems and ultimately facilitate translation of our results tothe clinic, we performed an extensive literature search todeduce a clinically tolerable dose for each compound, whichcan likely be used safely in our anticipated combination regi-mens, and tested these doses in our mouse models (Support-ing Information Tables 1 and 2).

Animal dosing schedules were based on previously pub-lished studies.16,21 Since concurrent administration of chemo-therapy and temsirolimus was equally or more effective thansequential administration in preclinical tumor models, weadministered cisplatin and temsirolimus concurrently.22

Combination therapies with bevacizumab can be trickybecause bevacizumab significantly reduced tumor targeting of(EGFR- and IGF-1R-) antibodies in vivo, emphasizing theimportance of timing and sequencing of bevacizumab incombination with other antibodies.23 Because temsirolimus isa small molecule inhibitor and therefore much smaller thanthe antibody bevacizumab, we decided to administer bevaci-zumab and temsirolimus concurrently. It is expected thattemsirolimus will reach the tumor before bevacizumab thusbevacizumab will not affect the concentration of temsirolimusin the tumor.

In vivo therapy experiments

Temsirolimus (CCI-799; Pfizer B.V., Capelle a/d IJssel, theNetherlands) was administered intraperitoneally (i.p.) at adose of 1 mg/kg, every 3 days. Cisplatin (Pharmachemie,Haarlem, the Netherlands) was administered i.p. at a dose of3.5 mg/kg every 21 days and bevacizumab (Avastin, RocheDiagnostics, Grenzach-Wyhlen, Germany) was administeredi.p. at a dose of 5 mg/kg twice weekly. Each therapy groupconsisted of 8–10 mice and mice were treated for 4 weeks.Tumor growth was closely monitored by caliper measure-ments twice weekly in three dimensions (length (l), width

(w) and height (h); all maximum diameter). Tumor sizeswere calculated using the formula: 4/3 3 p 3 l/2 3 w/2 3

h/2. On Day 28, animals were euthanized. If mice werescheduled for treatment on Day 28, animals were euthanized4 hr postinjection. Tumor sizes are depicted as relative tumorvolumes (RTV 5 tumor volume at any time (Vt)/tumor vol-ume at t 5 0 (Vt0)).

Small animal PET scans

Small animal PET scans were performed before, during andafter therapy experiments (Days 22, 7 and 28, respectively)using the Siemens Inveon PET/CT scanner (Siemens, theNetherlands). OS-1 and OS-33 tumor-bearing micereceived an intravenous injection of approximately 10 MBq18F-FDG or 18F-FLT (n 5 2 per group). The actual injectedactivity of 18F-FDG or 18F-FLT was calculated by subtract-ing the activity in the syringe after injection of the radiola-beled compound from the activity in the syringe beforeinjection of the radiolabeled compound. Directly after 18F-FDG injection, mice were anesthetized using isoflurane/O2

inhalation. For 18F-FLT analysis, direct anesthesia is notrequired. After 45 min, mice (four at a time) were scannedfor 20 min under isoflurane/O2 anesthesia using the Sie-mens Inveon PET scanner. Scans were subsequently recon-structed with Siemens Inveon reconstruction software(Siemens). Images were then analyzed with AMIDE (2D;version 0.9.1) or Siemens Inveon Research Workplace soft-ware (3D; Siemens).

For quantitative analysis of the PET images, regions ofinterest (ROIs) were manually drawn over the tumors usingSiemens Inveon Research Workplace software (Siemens).Because no baseline 18F-FDG was observed in both models,only 18F-FLT uptake was quantitatively analyzed. The 18F-FLT signal intensity for each voxel within the ROI was regis-tered as counts, and mean and maximum counts per ROIwere recorded. Subsequently, mean and maximum standar-dized uptake values (SUVmean and SUVmax) were calcu-lated by correcting for the injected 18F-FLT dose, timeelapsed between injection and scan and body weight. RelativeSUVmean and SUVmax (rSUVmean and rSUVmax) valueswere calculated by dividing the SUVmean or SUVmax at anytime by the SUVmean or SUVmax at baseline (Day 22), andwere used to compare differences in 18F-FLT uptake before,during and after treatment. Differences in 18F-FLT uptake atDays 7 and 28 are depicted as percentage reduction of rSUV-mean or rSUVmax compared to baseline uptake (Day 22).

CT scan

When possible, CT scans were acquired directly after PETimaging. Mice were scanned for 8 min using the Inveon CTscanner (Siemens, the Netherlands), and images were recon-structed using Siemens reconstruction software. To determinethe exact location of 18F activity, CT and PET scans werecoregistered. 3D images were created using Siemens InveonResearch Workplace software (Siemens).

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Immunohistochemistry

Tumor xenografts were stained immunohistochemically toevaluate general tumor characteristics (vascularization, CD34;proliferation, Ki-67; and apoptosis, caspase 3) and effects onthe mTOR-pathway (phosphorylated mTOR (pmTOR), pS6RPand p4EBP1). To keep conditions identical, all tumors fromeach xenograft were stained in the same run. Xenografts werefixed in 4% formalin and subsequently embedded in paraffin.Tumor sections (4 lm) were deparaffinized in xylol and rehy-drated through a graded ethanol into water series. Antigenretrieval was performed by microwave heating of slides in a 10mM sodium citrate buffer, pH 6 for 10 min (Ki-67, caspase 3,pmTOR, pS6RP and p4EBP1) or 20 min (CD34) at 100�C.Endogenous peroxidase activity was blocked with 3% H2O2 for10 min at room temperature (RT), and nonspecific bindingwas prevented by blocking with 20% normal goat serum ornormal rabbit serum in phosphate-buffered saline (PBS) ortris-buffered saline (TBS; phospho-antibodies) for 30 min atRT. For caspase 3 staining, an additional avidin-biotin-block(Vector Laboratories, Burlingame) was incorporated. Subse-quently, sections were incubated with polyclonal rat anti-CD34(1:20, Monosan), monoclonal rabbit anti-Ki-67 (1:200, Neo-markers), monoclonal rabbit antiactive caspase 3 (1:1,000, BDPharmingen), monoclonal rabbit anti-pmTOR (1:50, Cell Sig-naling Technology), polyclonal rabbit anti-pS6RP (1:500, CellSignaling Technology) or monoclonal rabbit anti-p4EBP1(1:1,000, Cell Signaling Technology) overnight at 4�C. Substi-tution of the primary antibody by PBS served as negative con-trol. Sections were then incubated with goat-anti-rabbit orrabbit-anti-rat biotinylated secondary antibody (1:200, VectorLaboratories) for 30 min at RT. Finally, avidin-biotin-enzymecomplex (1:50, Vector Laboratories) was added for 30 min atRT, followed by an incubation for 7 min at RT in 3,30-diami-nobenzidine (Bright-DAB) to visualize antigen expression.Slides were counterstained with hematoxylin, dehydrated andcoverslipped. CD34, Ki-67 and caspase 3 stained slides weresubsequently digitally analyzed with the KS400-Axiophot lightmicroscope (Carl Zeiss, Germany) and scored quantitativelyusing KS400 software (Carl Zeiss) and custom-written macrosto obtain objective and reproducible results. Fifteen (CD34and caspase 3) or six (Ki-67) nonoverlapping fields per sectionwere randomly selected at 200x magnification. For CD34 anal-ysis, both the number of blood vessels and blood vessel sizewere calculated and analyzed. For Ki-67 and caspase 3 stain-ings, the percentage of positive cells as proportion of allcounted cells was calculated and used for further analysis.Changes are expressed as percentage difference compared tocontrol tumors at the end of the experiment (CD34 and Ki-67:% reduction; caspase 3: % increase). pmTOR, pS6RP andp4EBP1 stainings were scored semiquantitatively as follows: 0,no positive cells; 1, low; 2, medium; 3, high staining intensity(>10% of tumor cells; Supporting Information Fig. 1). Meanintensity scores were used to compare differences betweengroups. We also determined the percentage of necrotic lesionsin all tumors.

Statistical methods

A Student’s t-test was used to compare differences betweentreatment groups. Linear correlation between two continuousparameters was evaluated using Spearman correlation. p-Val-ues <0.05 were considered statistically significant.

ResultsEfficacy of temsirolimus and cisplatin on tumor growth

The efficacy of temsirolimus (1 mg/kg) and cisplatin (3.5 mg/kg), alone and in combination (TC), was evaluated in micebearing OS-33 and OS-1 osteosarcoma xenografts. All singleand combined doses were tolerated well, and no mice died orwere excluded from analysis as a result of treatment-relatedtoxicity. Due to rapid tumor growth, OS-33 control micewere sacrificed before 4 weeks. In all other treatment groups,no mice died or had to be sacrificed during the experiment.Tumor responses, expressed as percentage reduction com-pared to controls at the end of the experiment, were primar-ily assessed by RTVs of treated and control mice. In OS-33tumors, both temsirolimus (60%, p 5 0.01) and cisplatin(61%, p 5 0.01) monotherapies significantly inhibited tumorgrowth as compared to controls. Combined TC treatment(85%, p < 0.01) resulted in even more tumor growth retarda-tion, which was significantly better than each monotherapy(both p < 0.01; Fig. 1). In mice with OS-1 xenografts, temsir-olimus monotherapy also significantly inhibited tumorgrowth as compared to control mice (24%, p 5 0.03),although less pronounced than in OS-33 tumors. Interest-ingly, although cisplatin monotherapy (16%, p 5 0.13) didnot significantly affect OS-1 tumor growth, combined TCtreatment (49%, p < 0.01) resulted in an improved tumorresponse with significantly more tumor growth inhibitioncompared to each monotherapy (both p < 0.05; Fig. 1).

Efficacy of temsirolimus and bevacizumab on tumor growth

The efficacy of temsirolimus (1 mg/kg) and bevacizumab (5mg/kg), alone and in combination (TB), was evaluated inOS-33 and OS-1 models as well. All single and combineddoses were well tolerated. As a single agent, bevacizumab sig-nificantly inhibited OS-33 (70%, p 5 0.01) and OS-1 (35%, p< 0.01) tumor growth as compared to controls (Fig. 1).Combined TB treatment, however, resulted in significantlygreater efficacy compared to the monotherapies in both OS-33 (91%, p < 0.01) and OS-1 (57%, p < 0.01) xenografts(Fig. 1). The efficacy of all treatments on osteosarcoma xeno-grafts are summarized in Table 1.

Treatment effects on general tumor characteristics

At the end of the experiment, all tumors were dissected andanalyzed immunohistochemically to assess and quantify themicroscopic effects of mono and combined therapies on gen-eral tumor characteristics (hematoxylin and eosin (H&E),CD34, Ki-67 and caspase 3) and to identify possible mecha-nisms for the observed differences in growth inhibition.

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Representative images of general tumor characteristics andcorresponding quantitative data are shown in Figures 2 and3. Conventional H&E analysis revealed numerous viabletumor cells in untreated OS-1 and OS-33 tumors, with virtu-ally no necrotic areas and some extracellular matrix (ECM).Although in OS-33 tumors none of the treatments resulted in

an increase of necrotic lesions, TC and TB treatment didhave an apparent effect on tumor architecture as large areasof ECM components were observed after treatment. Micro-scopic effects were even more pronounced in OS-1 tumors.Bevacizumab- and TB-treated OS-1 tumors showed anincreased number of necrotic lesions. In addition, TC- and

Figure 1. Treatment efficacy on the growth of OS-33 and OS-1 tumors. Nude mice bearing OS-33 (a) or OS-1 (b) human osteosarcoma xeno-

grafts were randomized into six treatment groups and were subsequently treated with either vehicle (control), temsirolimus (1 mg/kg), cis-

platin (3.5 mg/kg), bevacizumab (5 mg/kg), T 1 C or T 1 B (n 5 8–10 per group). Mice were treated for 4 weeks. Values are presented as

mean relative tumor volume 6 SD. Expanded figures are given in Supporting Information Figure 2. (c and d) Kaplan–Meier curves for

progression-free survival (PFS; defined as the first measured event of triplication of tumor volume as compared to the initial tumor volume)

for OS-33 (c) and OS-1 (d) tumors. Color schemes are identical to (a) and (b). (e) and (f) Growth of individual OS-33 (e) and OS-1 (f)

tumors. Values are presented as tumor volume in mm3. [Color figure can be viewed in the online issue, which is available at wileyonlineli-

brary.com.]

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http://wileyonlinelibrary.com


TB-treated OS-1 tumors both showed apparent ECM featuresand large pseudocysts (Fig. 2c). Although most apparentmicroscopic effects were observed in the best respondingtumors as determined by caliper measurements, these dataalso indicate that the tumor volume could underestimate theactual tumor response. During TC and TB treatment, OS-1and OS-33 tumor volumes remained stable or even increased(Fig. 1), suggesting tumor growth, but IHC clearly demon-strated the presence of large ECM regions and even largepseudocysts in some tumors. Fluids in the pseudocystsseemed to push them further outwards (Fig. 2c), which couldresult in an increase in tumor volume, though this is obvi-ously not due to an increase in the number of tumor cells.

CD34 analysis showed numerous, large blood vessels inuntreated OS-33 tumors. Bevacizumab, TC and TB treatmentsignificantly decreased the number of blood vessels as com-pared to controls (88%, 54% and 97%, respectively, all p <0.01), with hardly any blood vessels left in TB-treatedtumors. Interestingly, temsirolimus, cisplatin and TC treat-ment significantly reduced the size of blood vessels in OS-33tumors (33%, 26% and 41%, respectively, all p < 0.01). InOS-1 tumors, bevacizumab (37%, p 5 0.03) and TB (56%, p< 0.01) treatment also significantly affected the number ofblood vessels, though not as apparent as in OS-33 tumorsand no differences were observed for any treatment concern-ing blood vessel size in OS-1 tumors.

Ki-67 staining revealed numerous proliferating tumor cellsin untreated OS-1 and OS-33 tumors. Although all treatmentsresulted in a lower fraction of proliferating cells in both mod-els, this effect was only significant in OS-1 tumors treated withbevacizumab (26%, p 5 0.01) or TC (24%, p 5 0.03).

Caspase 3 staining demonstrated an increased number ofapoptotic cells in both models upon cisplatin (OS33: 48%, p 5

0.03; OS-1: 36%, p 5 0.03) and TB (OS33: 136%, p < 0.01;OS-1: 143%, p 5 0.01) treatment. In OS-1 tumors, also TC(101%, p < 0.01) treatment led to a significant increase of

apoptotic cells. The increased number of apoptotic cells incisplatin-treated OS-1 tumors furthermore indicates that thesetumors are not completely cisplatin-resistant. The fact thatonly relatively viable, thus unaffected, tumor areas could beselected for quantitative IHC analysis, might underestimate theactual response because necrotic lesions are not included.

Treatment effects on the mTOR-pathway

In addition to general tumor characteristics, we alsoaddressed specific effects on the mTOR-pathway by evaluat-ing pmTOR, pS6RP and p4EBP1 expression in all xeno-grafts (Fig. 4). When present, pmTOR and p4EBP1 weremore or less homogeneously expressed (>70% of tumorcells), while the pS6RP expression pattern was very hetero-geneous throughout tumors. There was a significantdecrease in pmTOR expression upon TB treatment in bothxenografts (OS33: p 5 0.01; OS-1: p 5 0.02) and upon cis-platin and TC treatment in OS-1 tumors (p < 0.01 and p 5

0.01, respectively). TC treatment significantly decreasedpS6RP expression levels in the majority of tumor cells inboth models (OS-33: p < 0.001, OS-1: p 5 0.03), as didtemsirolimus in OS-33 models (p < 0.001). Interestingly, intemsirolimus, TC- and TB-treated OS-33 tumors, there wasa subpopulation of tumor cells that remained highly pS6RPpositive. Closer examination of these cells revealed anunusual cellular morphology making it difficult to determinethe exact cell phase. The lack of an apparent nuclear mem-brane, however, points toward an apoptotic process, andthere was indeed some overlap with the caspase 3 expres-sion pattern, suggesting that at least some of the highlypS6RP-positive cells are apoptotic (Figs. 2a and 4c).Bevacizumab-treated OS-33 tumors showed a slight, but sig-nificant, increase in pS6RP expression (p < 0.01). No differ-ences in p4EBP1 expression levels were found in OS-33tumors, but a slight decrease was observed in cisplatin-treated OS-1 tumors (p 5 0.04).

Table 1. Therapy response of OS-33 and OS-1 tumors

OS-33 (t 5 3.5 weeks)1 OS-1 (t 5 4 weeks)

Treatment RTV 6 SD % Reduction RTV2 p-Value3 Response4 RTV 6 SD % Reduction RTV p-Value Response

Control 11.5 6 3.4 0 x No 3.7 6 0.8 0 x No

Temsirolimus (T) 4.6 6 1.1 60 0.01 Int 2.8 6 0.9 24 0.03 Low

Cisplatin (C) 4.5 6 1.4 61 0.01 Int 3.1 6 1.2 16 0.13 Low

Bevacizumab (B) 3.4 6 1.1 70 0.01 Int 2.4 6 0.7 35 <0.01 Low

T 1 C 1.7 6 0.8 85 <0.01 High 1.9 6 0.6 49 <0.01 Low

T 1 B 1.0 6 0.5 91 <0.01 High 1.6 6 0.4 57 <0.01 Int

1For OS-33 tumors, all RTVs are given for t 5 3.5 weeks because the majority of control mice was sacrificed before t 5 4 weeks due to rapid tumorgrowth.2% reduction RTV as compared to control group.3All p-values in this table are as compared to control tumors. A p-value <0.05 was considered statistically significant.4In vivo responses were defined as described by Houghton et al.51 In short, the mean relative tumor volume (RTV) of the treatment group (T) wasdivided by the mean RTV of the control group (C): T/C. Criteria: T/C � 15% high response; T/C � 45% but >15% intermediate response; T/C >45% low response. Note that in the present study, growth differences are depicted as % reduction RTV, which is the opposite of T/C (% reductionRTV 5 1 2 T/C).Abbreviations: RTV: relative tumor volume; SD, standard deviation; int, intermediate.

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18F-FDG-PET and 18F-FLT-PET imaging

Since it was recently demonstrated that the reduction intumor volume not necessarily reflects the actual tumorresponse, especially in bone sarcomas, we wanted to obtain

more information concerning tumor metabolism and tumorproliferation before, during and after therapy with 18F-FDG-PET and 18F-FLT-PET scans, respectively19. No baseline 18F-FDG uptake was observed in OS-1 and OS-33 models, while

Figure 2. Treatment effects on general tumor characteristics of OS-33 and OS-1 models. Representative images of HE staining, CD34, Ki-67

and caspase 3 expression in OS-33 (a) and OS-1 (b and c) tumors. (c) shows an example of a large cyst that appears to push outwards in

a TC-treated OS-1 tumor at 325 magnification. All tumors (8–10 per group) were stained and analyzed. Images are 3100 magnification

unless stated otherwise, hematoxylin counterstain. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.

com.]

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baseline 18F-FLT uptake was solely demonstrated in OS-1tumors (Fig. 5a). Therefore, only 18F-FLT-PET scans of OS-1tumors were further analyzed. At baseline (Day 22), OS-1tumors from all treatment groups showed apparent 18F-FLTactivity (SUVmean 0.9 6 0.2 and SUVmax 1.3 6 0.3), butthe distribution of 18F-FLT was heterogeneous in sometumors (Fig. 5b). Although all treatments (including controls)induced a decrease to some extent in 18F-FLT-signal at Day7, this effect was not sustained in the monotherapy and con-trol groups at Day 28 (Fig. 5c). Only in TC- and TB-treatedtumors, the decrease in 18F-FLT-signal at Day 7 (TC SUV-mean: 35% SUVmax: 33%; TB SUVmean: 36% SUVmax:36%) was still present or even more pronounced at the endof the experiment (TC SUVmean: 33% SUVmax: 33%; TBSUVmean: 48% SUVmax: 43%). 18F-FLT-responses were pre-dominantly observed in the central part of OS-1 tumors (Fig.5b). Although the 18F-FLT-PET response correlated with

therapy-response in general (most apparent response in bestresponding tumors), these data also show that the tumor vol-ume might underestimate the actual tumor response, similaras observed with IHC. In both TC- and TB-treated tumors,tumor volumes remained similar or slightly increased duringtreatment, while 18F-FLT uptake decreased. In addition, 18F-FLT-PET could be an early predictor of treatment responsewith these combined therapies, because the decrease in 18F-FLT-signal at Day 28 was already present after 1 week oftreatment. Although at Day 7, the (relative) tumor volume inTC (1.3 6 0.3) or TB (1.1 6 0.3) tumors as determined bycaliper measurement already differed significantly from con-trols (1.9 6 0.6; both p < 0.05), this difference was onlyminimal compared to the differences seen on 18F-FLT-PETscans. Thus the reduction in 18F-FLT-signal precedes volu-metric changes, distinguishing responders fromnonresponders.

Figure 3. Quantitative analysis of general tumor characteristics in OS-33 and OS-1 tumors. (a) Example of quantitative CD34 analysis show-

ing both the regular IHC image (top) and the corresponding digitalized image (bottom). Both images are 3200 magnification. (b) Quantita-

tive CD34 analysis in OS-33 and OS-1 tumors demonstrating both the number of blood vessels (left) and blood vessel size (right) per

treatment group. Values are presented as mean vessel count per mm2 or vessel surface in mm2 6 SD. *p < 0.05, **p < 0.01. (c) Example

of quantitative Ki-67 (upper panel) and caspase 3 (lower panel) analysis showing the regular IHC image (left), corresponding digitalized

image (middle; 1 cells in green) and the digitalized image showing both positive and total number of cells (right; 1 cells in red; all cells

in green). All images are 3200 magnification. (d) Quantitative Ki-67 and caspase 3 analysis in OS-33 (left panel) and OS-1 (right panel)

tumors. All tumors (8–10 per group) were quantitatively analyzed. Values are presented as the mean percentage of positive cells as propor-

tion of all counted cells per section (200x) 6 SD. *p < 0.05, **p < 0.01. [Color figure can be viewed in the online issue, which is available

at wileyonlinelibrary.com.]

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Because 18F-FLT uptake is known to reflect tumor prolif-eration, we also investigated possible relations between tumor18F-FLT uptake and Ki-67 expression in OS-1 tumors. Ki-67

IHC expression, SUVmax, SUVmean, rSUVmax and rSUV-mean values at Day 28 for all 18F-FLT-PET-scanned mice (n5 12) can be found in Supporting Information Table 3.

Figure 4. Treatment effects on the mTOR-pathway in OS-33 and OS-1 tumors. Representative images of pmTOR, ps6RP and p4EBP1 expres-

sion in OS-33 (a) and OS-1 (b) tumors. (c) zooms in on the odd-looking ps6RP-positive cells in a temsirolimus-treated OS-33 tumor at

3450 magnification (HE). All tumors (8–10 per group) were stained and analyzed. Images are 3100 magnification unless stated otherwise,

hematoxylin counterstain. (d) Semiquantitative analysis for pmTOR, pS6RP and p4EBP1 expression in OS-33 (left) and OS-1 (right) tumors.

Values are presented as mean IHC intensity score 6 SD. Because in OS-1 tumors the pS6RP expression pattern was very heterogeneous

(but when present, intensity levels were equal), we scored these tumors based on the percentage of pS6RP-positive cells (range 0–1; mean

percentage of pS6RP-positive cells 6 SD are given). *p < 0.05, **p < 0.01. [Color figure can be viewed in the online issue, which is avail-

able at wileyonlinelibrary.com.]

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Spearmans q analysis revealed no significant correlationsbetween Ki-67 expression and all tested 18F-FLT uptakeparameters (SUVmax: r 5 20.032, SUVmean: r 5 20.094,rSUVmax: r 5 0.309, rSUVmean: r 5 0.249; all p > 0.05; n5 12; Supporting Information Table 4).

Both OS-1 and OS-33 models are highly proliferativetumors with similar percentages of Ki-67-positive tumor cells(Figs. 2 and 3d), but only the OS-1 model showed 18F-FLTuptake. This further indicates that in our models there is nostraightforward correlation between Ki-67 expression and18F-FLT uptake.

DiscussionHere, we show for the first time that the efficacy of temsiroli-mus is significantly enhanced by the addition of either cispla-tin or bevacizumab in osteosarcoma xenografts, and alltherapies were tolerated well. We also demonstrated thattumor responses as determined by caliper measurementsunderestimated the actual tumor response as shown withboth IHC and 18F-FLT-PET imaging, highlighting the

importance of incorporating molecular imaging techniquesfor accurate response monitoring. In addition, our resultsindicate that 18F-FLT-PET can potentially be used to measureresponse in the early phases of treatment.

Previously, rapamycin was also shown to inhibit osteosar-coma xenografts growth.11 Moreover, rapamycin combinedwith cyclophosphamide or vincristine was significantly moreactive than either agent alone in several sarcoma xenografts.Additive efficacy was reported in one out of two osteosar-coma models, but excessive toxicity was observed in thesemodels as well.24 Rapamycin combined with cisplatin alsoresulted in excessive toxicity. However, at lower doses of cis-platin, rapamycin potentiated cisplatin activity.24 To avoidtoxicity problems in our in vivo therapy experiments andeventually in the clinic, we used relatively low and clinicallyapplicable doses of temsirolimus and cisplatin, and this com-bination turned out to be well tolerated in mice. Even atthese relatively low doses, significant growth inhibition wasdemonstrated in both monotherapy groups (with the excep-tion of cisplatin in OS-1 tumors), and superior efficacy was

Figure 5. Treatment effects on 18F-FLT-PET response in OS-1 tumors. Representative 3D PET/CT (left panel) and CT (right panel) scans show-

ing baseline 18F-FLT uptake of OS-1 and OS-33 tumors. Arrows indicate tumor localization. (b) Representative 2D PET scans of 18F-FLT

uptake in OS-1 tumors before (t 5 22 days), during (t 5 7 days) and after (t 5 28 days) treatment. Scans are focused on the OS-1 tumors.

(c) Relative SUVmean and SUVmax of 18F-FLT in OS-1 tumors before (t 5 22), during (t 5 7) and after (t 5 28) treatment. Values are pre-

sented as group averages (n 5 2 per group). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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demonstrated with the combination in both osteosarcomamodels. In other studies, mTOR inhibition was also shown tosensitize cancer cells to chemotherapy, including pediatricsarcoma models.24,25 Moreover, the mTOR inhibitor everoli-mus dramatically enhanced cisplatin-induced apoptosis inwildtype p53, but not mutant p53, tumor cells.26 Both OS-1and OS-33 models are p53-wildtype, as is the majority (62%)of clinical osteosarcoma samples.27 These data indicate thatthe combination of temsirolimus and cisplatin is worthexploring in osteosarcoma patients, even at relatively lowdoses. It is suggested that this combination can safely be usedin patients based on the fact that the combination of cisplatinwith other mTOR inhibitors is tolerable.28,29

We also investigated the efficacy of temsirolimus com-bined with bevacizumab on osteosarcoma xenografts.Although little was known about the efficacy of this combina-tion in osteosarcomas, rapamycin combined with bevacizu-mab previously demonstrated synergistic effects inhepatocellular and ovarian carcinoma xenografts.30,31 In thepresent study, we demonstrate that also in osteosarcomamodels, temsirolimus combined with bevacizumab is superiorto each monotherapy, warranting further testing of this com-bination in osteosarcoma patients. Previously, this combina-tion raised some tolerability concerns. Although rapamycincombined with bevacizumab was well tolerated in the major-ity of patients with advanced malignancies, some studiescombining temsirolimus and bevacizumab demonstratedhigher than anticipated toxicity which limited treatmentadherence over time.32–34 On the other hand, it has also beenreported that the same dosing schedule was well tolerated inthe majority of cancer patients.35 Even the addition of chem-otherapy to temsirolimus and bevacizumab appeared tolera-ble in patients with advanced malignancies.36,37 Because thereis an absolute need for new, targeted treatments in osteosar-coma patients, it would be worthwhile to explore the TBcombination in osteosarcoma patients. Obviously, patientsshould be closely monitored for treatment-related toxiceffects, especially since it concerns in general young patients,which was not the case in prior clinical studies.

In the present study, we microscopically examined alltumors to unravel mechanisms by which tumor growth isinhibited together with an expert sarcoma pathologist(U.E.F.). General H&E analysis revealed treatment-relatedarchitectural changes which were most pronounced uponTC- and TB-treatment in both models. Although growthcurves implied generally better tumor responses for OS-33tumors than for OS-1 tumors, the most obvious architecturaleffects were observed with combined treatments in OS-1tumors in which necrosis and large cystic changes wereobserved, both strong indicators of response. In clinical prac-tice, the histological response to chemotherapy in (osteosar-coma) patients is based on the amount of tumor necrosis(>90% good response).38 Studies in gastrointestinal stromaltumors (GIST) showed that the imatinib-induced cyst-likeappearance of GIST liver metastases indicates a significant

response to imatinib resulting in disease stabilization. Thesecystic lesions in GIST can even be larger than the initialtumor, which would correspond to progressive diseaseaccording to standard criteria, although the exact oppositeoccurs. Lesions that increase in size but have a cystic appear-ance should therefore not be judges as a sign of progres-sion.39 Therefore, the significance of treatment-induced cysticchanges in osteosarcomas is interesting but its exact relevanceremains to be investigated in a larger treatment sample. Thedecrease in 18F-FLT activity in TC- and TB-treated OS-1tumors suggests that a phenomenon like we know in GIST,with regard to the cystic transformation in respondingtumors despite lack of decrease in tumor volume, may occur.This supports previous data stating that tumor volume notnecessarily reflects the actual tumor response.19,40

IHC demonstrated apparent effects on (micro)vessel den-sity in both models upon bevacizumab and TB treatment,with hardly any blood vessels left in TB-treated tumors.Smaller blood vessels were observed in temsirolimus-treatedOS-1 tumors. Interestingly, TC treatment resulted in both adecrease of the number of blood vessels and blood vessel sizein OS-33 tumors. Other studies also reported apparent vascu-lar effects upon temsirolimus, bevacizumab and TB treat-ment, but effects of TC on tumor vasculature have not beenaddressed before.16,30,31 In both models, TC and TB treat-ment resulted in less proliferating cells, although this wasonly significant in OS-1 tumors upon bevacizumab and TCtreatment, possibly due to a high variability in growth ofcontrol tumors. In addition, temsirolimus is known to induceG1-phase arrest, and the Ki-67 antibody is capable of recog-nizing cells in this phase, although the presence of Ki-67 inG1 is irregular.41 A significantly increased number of apopto-tic cells was observed in cisplatin- and TB-treated OS-1 andOS-33 models and in TC-treated OS-1 tumors. This indicatesthat OS-1 tumors, although thought to be cisplatin-resistantbased on growth curves, are in fact not completely cisplatin-resistant. This is also supported by the decrease in pmTORand p4EBP1 expression, and is in line with other publisheddata.21 Other studies also demonstrated increased apoptosiswhen an mTOR inhibitor was combined with cisplatin.26,42,43

In addition to general tumor characteristics, we alsoaddressed specific effects on the mTOR-pathway. Althoughsome variations existed between treatment efficacy on OS-1and OS-33 xenografts, in both models TB treatment reducedpmTOR expression and TC treatment reduced pS6RP expres-sion. Minimal effects were observed on p4EBP1 expression.Other studies also reported more effective blocking ofpS6RP-signalling than of p4EBP1-signalling by mTOR inhibi-tors.44–46 It has been described that temsirolimus alonealready showed pronounced effects on Ki-67, caspase-3, ves-sel and mTOR-target protein expression, but it should betaken into account that doses of temsirolimus in these studieswere about 20 times higher than in our present study.16,45

Our (quantitative) IHC data might also underestimate theactual tumor response because only relatively viable tumorareas were selected for scoring.

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The 18F-FLT-PET scans further support the idea thattumor volumes could underestimate the actual tumorresponse by showing an apparent decrease in 18F-FLT-signalin TC- and TB-treated OS-1 tumors. This response wasalready present after 1 week of treatment, indicating that 18F-FLT-PET imaging could be an attractive method for earlyresponse monitoring. Because we only imaged two mice pergroup, a more extensive study addressing this topic is war-ranted. In other preclinical studies, however, 18F-FLT-PETwas also capable to monitor (early) temsirolimus responseand 18F-FLT-PET is now being evaluated for monitoringmTOR-targeted therapies in the clinic (NCT01246817 andNCT01143779).47,48 Although the exact value of 18F-FLT-PET imaging during temsirolimus-targeted therapies in osteo-sarcoma patients remains to be established, a recently initi-ated clinical study in osteosarcomas aims to evaluate theindication of 18F-FLT-PET imaging in determining tumorresponse after one cycle of neoadjuvant chemotherapy(NCT01882231). At present however, the use of 18F-FLT-PET in osteosarcomas is still poorly understood, also exem-plified in the present study because the OS-33 model wasexcluded from 18F-FLT-PET analysis due to lack of baseline18F-FLT uptake. Although in some studies 18F-FLT uptakecorrelated with Ki-67 expression, we did not find such a cor-relation. Both OS-1 and OS-33 xenografts are highly prolifer-ative tumors, as shown by both Ki-67-positive cells andapparent tumor growth, but only the OS-1 model showed18F-FLT uptake. Also in treated OS-1 tumors, there was nosignificant correlation between 18F-FLT uptake and Ki-67expression. A possible correlation may be influenced by thefact that for Ki-67 quantification one slide per tumor wasanalyzed, whereas for 18F-FLT quantification the wholetumor was examined. Nevertheless, recent research supportsour findings that there is not always a straightforward corre-lation between 18F-FLT uptake and Ki-67. 18F-FLT-PETreflects tumor proliferation as a function of thymidine salvagepathway utilization, whereas Ki-67 is a more general prolifer-ation marker.49,50 Since Ki-67 staining is positive for any cellnot in the G0-phase, and activity of the thymidine pathwayis typically confined to the S-phase, 18F-FLT uptake seems toreflect tumor proliferation in a more specific way. The lackof correlation between Ki-67 and 18F-FLT uptake could also

be due to the fact that some tumors could have a high levelof de novo thymidine synthesis (via thymidine synthetase),and therefore are less dependent on the salvage pathway.49

Nevertheless, because temsirolimus is known to induce G1-phase arrest, 18F-FLT uptake may more accurately reflecteffects on tumor proliferation during temsirolimus-basedtreatments than Ki-67 staining, further supporting incorpora-tion of 18F-FLT-PET imaging for accurate temsirolimus-based response monitoring. Because the exact use of 18F-FLT-PET in osteosarcomas is still poorly understood, addi-tional studies addressing this topic are warranted.

In our study we chose to use s.c. human osteosarcomaxenografts instead of injecting cultured cell lines, to mimicosteosarcomas as closely as possible. These xenografts retainfor instance ECM components. Although we believe thatthese models are appropriate for initially assessing respon-siveness of temsirolimus-based therapies, which was the goalof this study, the use of orthotopic and/or metastatic modelswould be more clinically relevant. Future studies shouldaddress this issue.

At present, at best partial responses were observed inosteosarcoma patients during mTOR-targeted monotherapies.Our study clearly shows that the efficacy of the mTOR inhib-itor temsirolimus can be significantly enhanced by the addi-tion of either cisplatin or bevacizumab. Importantly, IHCand 18F-FLT-PET analysis showed that the efficacy of com-bined treatments is even greater than tumor volumes suggest.This urges the further exploration of these combined thera-pies for the treatment of osteosarcoma patients and highlightsthe importance of incorporating molecular imaging techni-ques, such as 18F-FLT-PET, for accurate and possibly alsoearly response monitoring.

AcknowledgementsThe authors thank the PPTP for generously providing theosteosarcoma xenografts. Jeroen van der Laak, Debby Smits,Bianca Lemmers-van de Weem, Iris Lamers-Elemans, KittyLemmens-Hermans, Nicole Bakker, Henk Arnts, ClaudiaLagarde (all from the Radboud University Medical Centre,Nijmegen, the Netherlands) and Doris Phelps (The ResearchInstitute at Nationwide Children’s Hospital, Columbus, OH)provided excellent technical assistance.

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Temsirolimus combined with cisplatin or bevacizumab is active in osteosarcoma models