Preclinical evaluation of Sunitinib as a single agent in the prophylactic setting in a mouse model of bone metastases.

2.5 MB·English

by  Schem, Christian

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RESEARCH ARTICLE Open Access
Preclinical evaluation of Sunitinib as a single
agent in the prophylactic setting in a mouse
model of bone metastases
Christian Schem3†, Dirk Bauerschlag4†, Sascha Bender3, Ann-Christin Lorenzen3, Daniel Loermann3, Sigrid Hamann3,
Frank Rösel3, Holger Kalthoff2, Claus C Glüer1, Walter Jonat3 and Sanjay Tiwari1*
Abstract
Background: Asubstantial number ofbreast cancer patients are identified as being athigh risk of developing
metastatic disease.With increasing number oftargeted therapeutics entering clinical trials, chronic administration of
theseagents may be a feasible approach for theprevention of metastases within this subgroup of patients. Inthis
preclinical study we examined whether Sunitinib,a multi-tyrosine kinase inhibitor which has anti-angiogenic and
anti-resorptive activity,is effective inthe prevention ofbonemetastases.
Method: Sunitinib was administered daily withthefirst dose commencing prior to tumor cell inoculation.
Intracardiac injection was performed with MDA-MB23bone-seeking cells, which were stably transfected with
DsRed2. Invivo plain radiography and fluorescent imaging (Berthold NightOwl)was used intheanalysis of bone
metastases. Histomorphometrywas used for the quantification of TRAP+ cellsfrom bone sections and
immunohistochemistry was performed using anantibody reactive to CD34 for quantification ofmicrovessel density.
Results: Preventive dosing administration ofSunitinibdoes not inhibit colonization oftumor cells to bone or
reduce the size of osteolytic lesions. There was a decrease in thenumberof TRAP+ cells withSunitinibtreatment
butthis did notreach significance. Sunitinib inhibited tumor growth as determined by imaging offluorescent
tumor area. Immunohistochemical analyses ofmicrovessel density revealed a concomitant decrease in thenumber
oftumor blood vessels.
Conclusions: The findingssuggest that Sunitinibcan be used as a therapeutic agent for the treatment of bone
metastases but as a single agent it is not effective interms of prevention. Thereforea combination approach with
other cytostatic drugs should be pursued.
Keywords: Sunitinib, Bone metastases,Breast cancer, Imaging
Background A number of preclinical studies have demonstrated that
Upto40%ofpatientswithearlystagebreastcancerhave heightened bone resorption creates an environment
disseminated tumors cells in the bone marrow [1]. Fur- that promotes growth of breast and prostate cancer
thermore, bone is the most common site for breast cells [2-7]. Therefore, agents that are anti-resorptive
cancer metastasis with 50% of metastatic breast cancer may prevent the development of bone lesions and
patients presentingwith bone metastasis. Increased bone reduce the risk of relapse in bone. Indeed, clinical trials
resorption is becoming increasingly recognized as a risk have shown that adjuvant anti-osteoclast therapy with
factor for development of metastatic tumor in the bone. the bisphosphonates Zoledronic Acid results in decreased
numbersofdisseminated tumor cells inthe bone marrow
[8,9]. Furthermore, adjuvant Zoledronic Acid therapy in
*Correspondence:[email protected] post-menopausal women with early stage breast cancer
†Equalcontributors
1MolecularImagingNorthCompetenceCenter,DepartmentofDiagnostic resultsinanincreaseinrelapsefreesurvival[10,11].
Radiology,UniversityHospitalSchleswig-Holstein,CampusKiel,Germany
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Schemetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Preclinical studies indicate that angiogenic inhibitors institutional authorities and approved by local author-
are effective in reducing tumor burden in bone [12-15]. ities(number,V312–72241.121-10(53-5/06).
The mode of action is through dual targeting of tumor
blood vessels and osteoclast activity [12,14,16-18]. Inhib- Treatmentgroups
ition of VEGF signaling can markedly affect bone All treatment began two days prior to tumor cell inocu-
remodeling since VEGF directly affects the proliferation lation. One group of mice (n=7) were treated with
and maturation of osteoclasts, osteoblasts, and their pre- 40 mg/kg Sunitinib (orally/daily), a second group served
cursors [19-24]. However, their effectiveness in reducing as a control group and was administered with carboxy-
metastatic risk from disseminated tumor cells is un- methylcellulose vehicleformulation(orally/daily)(n=7).
known. Amongst the patients who may benefit from
prophylactic treatment are those who have higher num- Fluorescentimaging
bers of involved lymph nodes, larger tumor size, a triple Anesthetized mice were imaged for DsRed2 fluores-
negative phenotype and/or those with a low estrogen, cence using a Peltier cooled charged-coupled device
high boneresorptiveenvironment. camera (NightOWL LB 983; Berthold Technologies,
For a therapeutic approach in the prophylactic setting Bad Wildbad, Germany) to assess tumor growth at
to be rational, the angiogenic inhibitor should be cyto- weeks3,4and5post-inoculation.Theexcitationsource
static since non-tumorigenic endothelial cells and osteo- is a ring light used for epi-illumination, mounted 12 cm
clasts are targeted. In addition, the toxicity profile should abovethemice. Filtersof550nm (±10nm)and605nm
be supportive of long-term chronic administration of the (±55 nm) were used to assess excitation and emission
drug. Sunitinib malate is a small tyrosine kinase inhibitor signals respectively. The exposure time was set to 5 s.
with antiangiogenic activity and a safety profile which has Using the WinLight 32 software (Berthold), fluorescent
beenreportedtobeacceptableforchronicoutpatientther- signals (expressed in counts/s) from the images were
apy [25]. The predetermined efficacious dose of 40 mg/kg calculated by selecting a rectangular region of interest
daily in mice maintains plasma Sunitinib concentration around the tumor and integrating the signal of each
above 50 ng/mL, selectively inhibiting VEGR2 and PDGF pixel over the chosen area. To account for variations in
receptorphosphorylation[26].Inclinicaltrials,dosingthat autofluorescence over time and between mice, the rect-
gives rise to Sunitinib plasma concentration of equal or angular region of interest was placed over an adjacent
greater than 50 ng/mL was determined to be 50 mg/day non-boneareatodeterminethebackgroundsignal.This
[27,28]. Recent phase III clinical trials have revealed that signal was then subtracted from the tumor signal. The
administration of Sunitinib to patients with advanced tumor area was calculated using the Winlight32
breast cancer did not increase progression free survival or software and expressed as mm2. The threshold of fluor-
overall survival when used either as a single agent or in escence emission was set to the level at which non-
combination with chemotherapeuticagents[29,30]. Inthis specific fluorescent signal was no longer detected in
study we tested the hypothesis that administration of a adjacent skin. This operated only on the periphery of
clinically relevant dose of Sunitinib malate in the prophy- the tumor, after which an automated peak search func-
lactic setting will decrease the number and extent of tion was utilized to delineate the area of the fluorescent
osteolytic bone lesions. We show efficacy with Sunitinib tumor.
monotherapyininhibitingtumorgrowthof bonemetasta-
sesbutnotthenumberandsizeofosteolyticlesions. Radiographs
Anesthetized mice were radiographed using a LoRad
Methods Selenia digital mammography unit (Hologic GmbH,
Tumorcellinoculation Frankfurt/Main, Germany). Radiographs were taken at
Bone-seekingMDA-MB231cellline(MDA-231BO)was weeks 3, 4 and 5 following intracardiac injection and
obtained from Dr Toshiyuki Yoneda (University of each radiograph was blindly evaluated by CS and A-CL.
Texas Health Science Center). The cell line was stably The area of osteolytic lesions was measured using a
transfected with the fluorescent reporter gene DsRed2 computerized image analysis system (IMPAX; AGFA,
(Clontech) and selected using 800 μg/ml of neomycin. Cologne, Germany) and results were expressed in square
Following establishment of the stably transfected cell millimeters.
line (MDA-231BO-DsRed2), three further in vivo pas-
sages to the bone were performed. One hundred thou- Histomorphometry
sand MDA-231BO-DsRed2 cells were injected by Hind limbs from each animal were dissected and fixed
ultrasound guidance into the left cardiac ventricle of 6 inneutral buffered formalin overnightat +4°C.The bone
week old female Fox nu/nu mice. Animal experiments samples were washed for two hours in cold PBS and
and care were in accordance with the guidelines of decalcified in acetic acid for 6 hours at +4°C. Following
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decalcification the biopsy sample was embedded in All statistical analyses were performed using GraphPad
paraffin wax cut into 7 μM sections using a microtome. Prism4.03.
Histomorphological analyses of the proximal tibia or the
distal femur were performed by cutting a series of 70 μm
sagittal sections every 200 μm, corresponding to the Result
upper,midandlowerregionsand3sectionswereanalysed PreventivedosingadministrationofSunitinibdoesnot
for each animal. The sections were then de-paraffinized inhibitcolonizationoftumorcellstobone
and stained with H&E, TRAP (tartrate-resistant acid Intra cardiac injection of tumor cells recapitulates the
phosphatase), and CD34. TRAP staining was performed later stages of metastases whereby tumor cells that
according to van de Wijngaert and Burger [31] following metastasize to the skeleton adhere to the endosteal sur-
de-paraffinizationandrehydrationofthesectionsinserial face and colonize bone. To determine ifpreventive Suni-
alcohol dilutions. Briefly, the sections were washed in tinib treatment reduced establishment of colonized sites,
distilledwaterandincubatedfor20mininasolutioncon- mice were administered with Sunitinib at the previously
taining 0.2 M Sodium Acetate (Merck, Catalog Nr. 6268) established efficacious dose of 40 mg/kg/day. Dosing
and50mMTartaricAcid,pH5.0(Sigma-Aldrich,Catalog commenced two days before the mice were inoculated
Nr. T10-9). The sections were then incubated in the with tumor cells. Since tumor cells stably expressed the
TRAP running buffer containing 0.1mg/ml napthol AS- red fluorescent protein DsRed2, the number of fluores-
MX phosphate (Sigma N4875) and 1.1 mg/ml Fast Red cent spots corresponding to metastatic boney sites of
TR(Sigma,F8764-16)for1–3hat37°Cuntilcolourreac- the mandible, hind legs, spine and ribs were counted.
tion was complete. The sections were washed in distilled Imaging at three weeks revealed no detectable bone
water, stained with haematoxylin and mounted. TRAP metastases but by four weeks a number of metastases
enumerationwasperformedbyselectingthetumorregion were visible. No significant difference in the number of
in the distal femur or proximal tibia and counting the bone metastases was observed with Sunitinib treatment
number of TRAP-positive cells in contact with endosteal comparedtothe control group (Table1).
surface. Five random regions were selected for counting
and data are expressed as the number of TRAP+ / bone
surface / mm2. CD34 staining was performed using the Sunitinibdoesnotreducethesizeofosteolyticlesions
Anti-Rat HRP-DAB staining kit (R&D Systems, Catalog To determine if preventive treatment with Sunitinib
Nr. CTS017) with a rat monoclonal anti-CD34 antibody reduced the size of osteolytic lesions, plain radiography
(Gene Tex, Catalog Nr. GTX 28158) at 1:500 dilution imaging of leg and rib metastases was performed at week
(50 μg/ml). Microvessel density (MVD) was evaluated by 5 following tumor cell inoculation and the size of the
calculating the selecting three highest areas of vascularity osteolyticlesionswasmeasured(Figure1).Sunitinibtreat-
across the tumor region and the number of angiogenic ment did not significantly decrease the size of osteolytic
vesselswerecountedineachfieldof view.Thenumberof lesionscomparedtothecontrolgroup.
vessels counted was divided by the field of view to yield To determine the effect of treatment on osteoclast
the MVD, expressed as MVD/mm2. Histomorphometric number, bone sections of the hind legs were stained for
measurementoftumorareawasperformedusingthelon- TRAP to determine presence of osteoclasts at the
gitudinal section ofthe distalfemur.Sections through the tumor-bone interface. Histological examination showed
tumor comprising of the largest tumor area were stained lower TRAP-positive osteoclasts in the metastatic hind
withGoldnerstrichrometoidentifytumorareaandstruc- legs of mice treated with Sunitinib but this difference
tural organizationofthebone.Tumor areawasmeasured was not significant (Figure 2A). Histological appearances
fromtheepiphyseallineofthegrowthplateandextending of representative sections stained for TRAP are shown
intothediaphysisandbilaterallybetweentheendocortical inFigure2B.
surfaces.Alinewasdrawnaroundthetumormarginusing
Image J software and a scale bar was used to measure
Table1Totalnumberofbonemetastasesincontroland
lengthsandcalculatethecross-sectionalarea.Atregionsof
Sunitinibgroups
extensive cortical destruction, the tumor area included
growthtotheboundaryoftheperiosteum. Group MiceNr. TotalNr.of TotalNr.of
metastasis metastasis
at4weeks at5weeks
Control 7 14 21
Statisticalanalyses
Two-tailed unpaired Student’s t tests were used to assess Sunitinib(40mg/kg/day) 7 12 21
Fluorescentbonemetastaticlesionswerecountedatweek4andweek5
differences between treated and control groups. P values
followingtumorcellinoculation.Nosignificantdifferencesinthemean
less than 0.05 were considered statistically significant. numberofmetastaseswasobservedbetweengroups.
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Sunitinibasamonotherapyinhibitstumorgrowth
The effect of Sunitinib on tumor growth as a single
agent was evaluated. Fluorescent tumor area was deter-
mined as this parameter has been reported to have a
better correlation with micro-CT-based osteolytic lesion
grade than fluorescent intensity [32]. The fluorescent
area of metastatic tumors was measured when tumors
were first detected at 4 weeks following tumor cell
inoculation and again one week later. Mice treated with
Sunitinib alone had significantly lower tumorfluorescent
areaat4weeksthanmiceinthe controlgroup(Figure 3,
Mean±SD: 8.62±5.13 vs 4.99±3.00, p<0.05). Further-
Figure1Sizeofosteolyticlesions(mm2)innudemicewith more, imaging at 5 weeks also revealed significantly
bonemetastases.Osteolyticlesionswereanalysedbyplain smaller tumors (Mean±SD: 14.75±4.97 vs 9.30±5.42,
radiography5weeksfollowingtumorcellinoculation;Control p<0.05). Therefore Sunitinib preventive treatment
(n=7),Sunitinib(n=7).Boxplotshows25–75percentiles,whiskers results in inhibition of growth of smaller tumors in bone
05–95percentiles,+indicatesmeanandlineindicatesmedian.
and is also effective in inhibiting tumor growth of estab-
Significancewasdeterminedby2-tailedStudentt-test(*p<0.05,op
lished tumors in the bone microenvironment. Histo-
<0.1).Nodifferenceisobservedinthesizeofosteolyticlesionswith
Sunitinibtherapy(mean±SD:Ctrl0.65±0.33;Sunitinib0.59±0.35). logical measurement of cross-sectional tumor area from
the epiphyseal line of the distal femur and extending
into the diaphysis confirmed a decrease in size of tumor
inthebonewith Sunitinib treatment(Figure3B&C).
Furthermore, histological evaluation of tumor angiogen-
esis revealed that Sunitinib significantly inhibited tumor
neovascularization. The mean vessel density (MVD) of
A
B Control Suntininb
Figure2(A)NumberofTRAP-positiveosteoclastsatthebone-tumorinterface.Sixweeksaftertumorcellinoculationboneswerefixedin
formalin,decalcifiedinaceticacidandembeddedinparaffin.SectionswerestainedforTRAPandcounterstainedforHaematoxylin.Fivefieldsof
tumorforeachspecimenwererandomlyselectedandcountedtodeterminethenumberofTRAP-positivemultinucleatedcells.Columnbar
depictsmean±SDof5animalspergroup.op<0.1(B)RepresentativesectionsofbonemetastasesstainedforTRAP(Red)fordetectionof
osteoclastandcounterstainedwithHaematoxylin(blue)fordetectionoftumorcells.Magnification20x.
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A
B C
Control Sunitinib
Figure3(A)Invivoimagingoffluorescenttumorareatomonitortumorgrowth.Nudemicewereinjectedwith1x105MDA-231BO-
DsRed2cellsintotheleftventricleandfluorescentimagingwasperformedatregularintervals.Analysesoffluorescenttumorareawasperformed
forControl(n=7),Sunitinib(n=7).Boxplotshows25–75percentiles,whiskers05–95percentiles,+indicatesmeanandlineindicatesmedian.
Significancewasdeterminedby2-tailedStudentt-test(*p<0.05).GrowthinhibitionwithSunitinibtreatmentissignificantlyreducedat4and5
weeksfollowingtumorcellinoculation(mean±SD:Week4.Ctrl8.62±5.13;Sunitinib4.99±3.00.Week5.Ctrl14.75±4.97;Sunitinib9.30±5.42).
(B)Histomorphometricdeterminationofcross-sectionaltumorsize.LongitudinalsectionsofdistalfemurwerestainedwithGoldnerstrichrome
andthetumorareameasuredfromtheepiphyseallineassociatedwiththegrowthplateandextendingintothediaphysisandbilaterally
betweentheendocorticalenvelope.Thetumormarginmeasuredismarkedbyagreenline.Atregionsofextensivecorticaldestruction,theline
wasdrawntotheboundaryoftheperiosteum.RepresentativesectionsofGoldner’strichromestainedfemurfromcontrolandSunitinibtreated
miceareshown.Magnification5x(C)Cross-sectionaltumorsizeinboneasmeasuredbyhistomorphometryissignificantlydecreasedwith
Sunitinibtreatment(Controlmean±SD2.19±0.39,n=5;Sunitinibmean±SD1.59±0.43;n=5,p<0.05).
hind legs with bone metastases was 63±17 with Sunitinib "conditioning effect" which promote the formation of
treatmentcomparedto139±11forcontrolmice(p<0.01, metastasis by circulating tumor cells [33]. In this study
Figure 4A). The data suggest that Sunitinib is effective in pre-treatment of mice with a dose of 40 mg/kg/day did
inhibitinggrowthof bonemetastasesthroughinhibitionof not lead to enhanced metastasis. This observation is
new blood vessel formation. Histological appearances of consistent with the findings of others that have used
representative sections stained for CD34 are shown in lower therapeutically efficacious doses [34,35] and sup-
Figure4B. port a rationale for application of lower doses of Suniti-
nibtoavoid augmentedinvasiveormetastatic potential.
Discussion The concept of the vicious cycle of bone metastases
Inthisstudyweevaluatedwhetherpreventivedoseadmin- emphasizes the cross-talk between tumor cells and the
istration of the multi-tyrosine kinase Sunitinib is effective bone microenvironment in the process of tumor growth
in inhibiting bone metastases arising from disseminated and bone destruction [36,37]. Following colonization to
tumorcells.WeshowthatSunitinibusedintheprophylac- the bone, secretion of factors by tumor cells stimulate
tic setting does not decrease the number of metastatic osteoclastogenesis via upregulation of receptor activator
colonized sites or inhibit subsequent progression of osteo- of nuclear factor kappa B ligand (RANKL) on osteoblast
lyticlesions.Thereishoweverareductionintumorgrowth and stromal cells. The increased bone resorptive activity
which is associated with a significant decrease in tumor releases growth factors from the bone matrix which in
blood vessels. The findings suggest that Sunitinib alone is turn stimulate tumor cell growth giving rise to further
notabletopreventcolonizationandexpansionofdissemi- bone destruction. The observation that Sunitinib did not
nated tumor cells to bone but may be a useful adjunctive show any therapeutic efficacy in inhibiting osteolytic
therapyinreducingmetastatictumorburden. lesions despite a reduction in tumor size suggest that
It has been reported that prior administration of osteoclast proliferation and activity was sufficiently
Sunitinib at elevated doses (120 mg/kg/day) induce a stimulated through secretions of growth factors by the
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A
Control Sunitinib
B
Figure4(A)Immunohistochemicalstainingofbloodvesseldensitywasperformedonbonesectionsusingthepan-endothelialmarker
CD34andquantified.Threeregionswithhighestbloodvesseldensitywereselectedandthenumberofangiogenicvesselscounted.Boxplot
shows25–75percentiles,whiskers05–95percentiles,lineindicatesmedianof5animalspergroup.Significancewasdeterminedby
2-tailedStudentt-test(**p<0.01).BloodvesselcountissignificantlyreducedwithSunitinibtreatment(mean±SD:Ctrl138.8±27.3;Sunitinib63.4
±45.7).(B)RepresentativestainingofCD34+bloodvessels(brown)andcounterstainedwithHaematoxylin(blue).Magnification200x.
tumor.Increasedosteolysesassociatedwithtumorshrink- evasive response to disturbed tumor vasculature may be
age and decreased vascularization was described previ- manifestedbyincreasedsecretionofcertainfactorswhich
ously for the treatment of bone metastases using the induce osteolysis. Adaptive evasive responses leading to
histone deacetylase inhibitor Vorinostat [38]. The effect increased invasiveness and distant metastases have been
was attributed to off-target effects of the drug on a observed in mouse models of pancreatic neuroendocrine
sub-population of resistant cells giving rise to increased carcinoma and glioblastomas following antiangiogenic
secretion of factors promoting osteolysis. Sunitinib has targeting of VEGF signaling [49,50]. The mechanism
recognized off-target activity with one report indicating by which this occurs is not understood but the hypoxia/
binding to at least 5 off-target kinases with high affinity HIF-1αpathwayisimplicated.
[39]. A possible off-target effect following Sunitinib ther- With an observed decrease in tumor growth in bone
apy is the tumor-independent increase in systemic levels thereisacompellingbiologicalrationalefortheuseoftar-
of factors such as granulocyte colony-stimulating factor getedcombinationtherapywithananti-resorptiveagent.A
andosteopontin[40],factorswhichareknowntopromote retrospective study of patients with bone metastases from
bone resorption [41-46]. In this regard, the application of renal cell carcinoma revealed an increase in progression
second-generation tyrosine kinase inhibitors (TKI's), such freesurvivalandresponserateinpatientstreatedconcomi-
as pazopanib and tivozanib, with improved potency and tantly with bisphosphonate and Sunitinib [51]. Amongst
selectivity may provide more effective treatment options targeted agents which inhibit osteoclast activity and are
[47,48]. Another possibility which may explain our obser- currently subject to clinical evaluation in breast cancer
vation is the adaption of an evasive response by MDA- bonemetastasesareinhibitorstomTOR,RANKL,Srcand
MB231 cells. The cell line is of metastatic origin and an Cathepsin K. A recent clinical trial administrating the
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mTOR inhibitor Everolmus, a rapamycin derivative which Conclusion
inhibits mTORC1, showed beneficial effects on bone turn- We present the first application of Sunitinib in a pre-
overandboneprogressioninpostmenopausalwomenwith clinical mouse model for the prevention of bone metas-
oestrogen-receptor-positivebreastcancer[52].Denosumab, tases and show efficacy in reducing tumor growth. We
ahumanneutralizingantibodytoRANKL,hasbeenshown advocate testing combination of targeted agents in a
tohaveastrongerinhibitoryeffectonboneresorptionthan therapeutic strategy that move to prevention to address
bisphosphonates[53-55].SincelongtermDenosumabther- unmetclinicalneeds.
apy is generally well tolerated, introduction of Denosumab
as a second agent for the inhibition of osteoclast activity, Competinginterests
Theauthorsdeclarenoconflictofinterest.
may further deprive tumor cells of growth factors seques-
tered in the bone matrix. In view of the fact that bone Authors’contributions
remodeling is a complex process requiring orchestrated CS:projectconception,planneddesignandcoordinationoftheproject,
inoculatedthemicewithtumorcells,performedradiologicalimagingand
differentiationofdifferentcelltypesaswellasangiogenesis,
helpeddraftthemanuscript.DB:projectconception,planneddesignand
establishmentoftreatmentorderofagentsanddosagemay coordinationoftheproject,performedradiologicalimagingandhelpeddraft
influencetherapeuticeffectsofcombinationtreatment[56]. themanuscript.SB:performedimmunohistochemistryforTRAPandCD34,
analysedthedata.A-C.L:performedanalysesofplainradiographyimages
Certain limitations of this study are outlined. One is
andtabulateddata.DL:performedimmunohistochemistryforTRAP,analysed
that only a single cell line was employed which is un- thedata.SH:performedsectioningofbone,immunohistochemistryfor
likely to reflect the phenotypic properties of all dissem- CD34,histomorphometry,dataanalysesandtabulation.FR:performed
opticalimaging,assistedinanimalhandlinganddissection.HK:participated
inating cells especially those disseminating from the
inconception,designandcoordinationoftheprojectandhelpeddraftthe
primary tumor at an early stage of the disease. Secondly, manuscript.CCG:participatedinconception,designandcoordinationofthe
the intracardiac injection model does not recapitulate projectandhelpeddraftthemanuscript.WJ:participatedinconception,
designandcoordinationoftheprojectandhelpeddraftthemanuscript.ST:
the requirement for cells to escape the primary site and
generatedstabletransfectedcells,performedopticalimaging,participatedin
so not all the steps of the metastatic process can be datatabulation,analyses,interpretationofdataanddraftedthemanuscript.
studied. Thirdly, the use of mouse models in itself has Allauthorsreadandapprovedthefinalmanuscript.
the limitation that ethical time points arrive much earl-
Acknowledgements
ier than for example in a rat model and it is uncertain
HK,CCGandSTacknowledgethesupportoftheDFGfundedinter-regional
whether a longer period of Sunitinib treatment may researchgroupSKELMET,theEuropeanRegionalDevelopmentFund(ERDF)
andtheZukunftsprogrammWirtschaft,Schleswig-Holstein.CSacknowledges
eventually hadaneffectonosteolyticsize.
thesupportofPfizerinmakingavailableSunitinibMalateandan
With an increasing number of targeted therapies
unrestrictedgrant.ThebreastcancercelllinewasprovidedbyProf.Dr
entering clinical trials, there is an urgent need to apply ToshiyukiYoneda(UniversityofTexasHealthScienceCenter).
these drugs for the prevention of fully developed meta-
Authordetails
static lesions arising from disseminated tumor cells. 1MolecularImagingNorthCompetenceCenter,DepartmentofDiagnostic
With advances in molecular biomarkers that refine and Radiology,UniversityHospitalSchleswig-Holstein,CampusKiel,Germany.
2DivisionofMolecularOncology,InstituteforExperimentalCancerResearch,
improve risk stratification in patients, those with a high
UniversityHospitalSchleswig-Holstein,CampusKiel,Germany.3Department
risk of developing bone metastases may benefit from
ofGynecology,UniversityHospitalSchleswig-Holstein,CampusKiel,
adjuvant or prophylactic treatment with targeted thera- Germany.4DepartmentofGynecology,UniversityHospitalAachen,Aachen,
Germany.
peutics, as long as the cytotoxicity is supportive of long
term chronic administration. The targeted therapies that
Received:1August2012Accepted:15January2013
may come into consideration are those that target genes Published:24January2013
involved in disseminated tumor cells infiltration,survival
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