Effect of zoledronic acid on the doxycycline-induced decrease in tumour burden in a bone metastasis model of human breast cancer

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1 ritish Journl of Cncer (7) 96, ll rights reserved 7 9/7 $3. Effect of zoledronic cid on the doxycycline-induced decrese in tumour burden in bone metstsis model of humn brest cncer WCM Duivenvoorden 1,, S Vukmirović-Popović, M Klin 1, E Seidlitz 1 nd G Singh,1, 1 Jurvinski Cncer Centre, 699 Concession Street, Hmilton, Ontrio, Cnd L8V 5C; Deprtment of Pthology nd Moleculr Medicine, McMster University, 18 Min Street W, Hmilton, Ontrio, Cnd L8N 3Z5 one is one of the most frequent sites for metstsis in brest cncer ptients often resulting in significnt clinicl morbidity nd mortlity. isphosphontes re currently the stndrd of cre for brest cncer ptients with bone metstsis. We hve shown previously tht doxycycline, member of the tetrcycline fmily of ntibiotics, reduces totl tumour burden in n experimentl bone metstsis mouse model of humn brest cncer. In this study, we combined doxycycline tretment together with zoledronic cid, the most potent bisphosphonte. Drug dministrtion strted 3 dys before the injection of the MD-M-31 cells. When mice were dministered zoledronic cid lone, the totl tumour burden decresed by 43% compred to plcebo tretment. dministrtion of combintion of zoledronic cid nd doxycycline resulted in 74% decrese in totl tumour burden compred to untreted mice. In doxycycline- nd zoledronte-treted mice bone formtion ws significntly enhnced s determined by incresed numbers of osteoblsts, osteoid surfce nd volume, wheres decrese in bone resorption ws lso observed. Doxycycline gretly reduced tumour burden nd could lso compenste for the incresed bone resorption. The ddition of zoledronte to the regimen further decresed tumour burden, cused n extensive decrese in bone-ssocited soft tissue tumour burden (93%), nd sustined the bone volume, which could result in smller frcture risk. Tretment with zoledronic cid in combintion with doxycycline my be very beneficil for brest cncer ptients t risk for osteolytic bone metstsis. ritish Journl of Cncer (7) 96, doi:1.138/sj.bjc Published online 17 pril 7 Keywords: bone metstsis; brest cncer; tetrcyclines; bisphosphontes one is one of the most frequent sites for metstsis of brest nd prostte cncer. one metstses re often ssocited, especilly in brest cncer, with extensive osteolysis of minerlised collgenous bone mtrix (Knis, 1995) nd subsequent hyperclcemi resulting in significnt clinicl morbidity. Currently, there is no dequte curtive regimen for ptients with bone metstsis. The existing stndrd of cre for the tretment of brest cncer-relted bone metstsis is the bisphosphonte fmily of drugs tht effectively inhibit the cncer-ssocited resorption. They re lso used s therpeutic gents in clinicl disorders chrcterised by incresed osteolysis, such s osteoporosis (erenson et l, 1). Unfortuntely, tretment with bisphosphontes is not curtive, but rndomised control trils hve shown tht zoledronte, the most potent bisphosphonte, decreses the frequency of skeletl-relted events, delys the first occurrence of such n event, nd reduces pin in bone-metsttic brest cncer ptients (Rosen et l, 4; Kohno et l, 5). During the -yer dily dministrtion of orl Correspondence: Dr G Singh, Jurvinski Cncer Centre, 699 Concession Street, Hmilton, Ontrio, Cnd L8V 5C; E-mil: gurmit.singh@hrcc.on.c Revised 16 Mrch 7; ccepted 19 Mrch 7; published online 17 pril 7 clodronte, one of the erlier bisphosphontes, to ptients with operble brest cncer without evidence of metsttic disese the development of bone metstses is significntly reduced (Powles et l, ). Therefore, preclinicl evidence tht zoledronte my lso be used to prevent the onset of bone metstsis would be very worthwhile. We hve previously shown tht doxycycline reduces the tumour burden from brest cncer metstsis in nude mice substntilly (Duivenvoorden et l, ). Doxycycline hs severl properties relevnt for tretment of bone metstsis, s doxycycline cn inhibit tumour cell prolifertion (Vn den ogert et l, 1983; Fife nd Sledge, 1995; Duivenvoorden et l, 1997) nd MMP ctivity, nd ccumultes t high concentrtions in bone (Golub et l, 1991). We hve lso shown evidence to support the concept tht the reduction in tumour burden by doxycycline is likely due to its properties s n inhibitor of tumour cell prolifertion nd tht doxycycline significntly increses severl prmeters of bone formtion (Duivenvoorden et l, ). isphosphontes re stble pyrophosphte nlogues tht lso ccumulte in bone nd very effectively inhibit osteoclstmedited bone resorption. The most potent bisphosphonte is zoledronte, which induces poptosis of the osteoclst nd interferes with the opertion of the ruffled border (Green, 4). In the present study, we combined doxycycline together with zoledronic cid s possible modlity to prevent or reduce the

2 tumour burden from bone metstsis using the experimentl brest cncer MD-M-31 mouse model. MTERILS ND METHODS Cell line The humn brest denocrcinom MD-M-31 cell line obtined from the mericn Type Culture Collection (Mnsss, V, US) ws mintined in Dulbecco s miniml essentil medium supplemented with 1% fetl bovine serum nd ntibiotics (1 U ml 1 penicillin sodium, 1 mgml 1 streptomycin sulphte nd. 5 mgml 1 mphotericin ; Invitrogen Cnd Inc., urlington, Ontrio, Cnd). nimls ll protocols for niml studies were reviewed nd pproved by the niml Reserch Ethics ord of McMster University (Hmilton, Ontrio, Cnd). Per tretment group 1 femle inbred nude (lb/c nu/nu) mice (Chrles River, St. Constnt, Quebec, Cnd) 5 weeks of ge (15 g) were used. Control nimls, not injected with tumour cells, were lso included. Intrcrdic injections of MD-M-31 cells were done ccording to rguello et l (1988). Mice were nesthetised by isoflurne inhltion nd the cells (.1 ml of cell suspension contining cells) were injected into the left ventricle of the hert using 6-guge needle inserted percutneously ner the midline. Twenty-eight dys fter injection of the cells, high-resolution rdiogrphic scns of ll mice in the prone nd lterl position were tken under inhled isoflurne nesthesi with Fxitron X-ry system MX- (Fxitron X-ry Corportion, Wheeling, IL, US). The nimls were killed nd both tibie, femor, nd humeri, nd the spinl column were dissected, fixed in formlin, declcified, using Declcifier I (Surgipth, Winnipeg, Mnitob, Cnd) nd embedded in prffin. Tretment Doxycycline-contining pellets (1 mg per pellet with timedrelese of 1 dys; Innovtive Reserch of meric, Srsot, FL, US) were implnted subcutneously 3 dys before cncer cell injections. Plcebo pellets were used in control nimls. t the sme time, pellets contining.5 mg of 17b-estrdiol (1 dyrelese) were implnted in ech niml. On the sme dy, zoledronic cid (Novrtis, Dorvl, Quebec, Cnd) tretment ws strted, t dose of. mg per mouse of zoledronic cid (s mgml 1 PS), given s subcutneous injection. The zoledronic cid injections were repeted nine times, every dys. Histology nd histomorphometry Tumour burden in the long bones nd the spinl column of ech mouse were mesured using stereologicl technique s described before (Duivenvoorden et l, ). Longitudinl sections (thickness 4 mm) were cut through the middle prt of the bone nd stined with hemtoxylin nd eosin (H&E). The totl cumultive tumour re per niml ws clculted using point grid (point re of.9 mm ) nd expressed s bone tumour burden. Histomorphometric mesurements were performed on cncellous re of the left nd right femor strting 1 mm below the epiphysel growth plte. Mesurements were performed using digitl color cmer ttched to light microscope nd Northern Eclipse imge-nlysing softwre (Empix Imging Inc., Mississug, Ontrio, Cnd). The following prmeters were mesured: volume occupied by bone, nd tumour s frction of the totl tissue volume (V/TV nd TuV/TV (%), respectively), number of osteoclsts nd osteoblsts per length of trbeculr bone surfce (N.Oc/S nd N.Ob/S (mm 1 ), respectively), eroded bone surfce, ctive resorption surfce beneth or in contct with osteoclsts, nd osteoid surfce s frction of the trbeculr bone surfce (ES/S, Oc.S/S, nd OS/S (%), respectively). To detect trtrte-resistnt cid phosphtse (TRP) ctivity in the osteoclsts, nphthol S-I phosphte (Sigm-ldrich, Okville, Ontrio, Cnd) ws used ccording to vn de Wijngert nd urger (1986). Sttisticl nlysis Tumour burden dt were tested for differences between mens using Student s t-tests. Histomorphometric dt were tested for differences between medins using two-tiled Mnn Whitney test. Differences were considered significnt t Po.5. RESULTS Severl bisphosphontes hve been used in preclinicl models of brest cncer bone metstsis. Tretment of mice with ibndronte (Hirg et l, 1), risedronte (Sski et l, 1995) or zoledronte (Green et l, ) results in reduction of 35, 5 nd 8% in osteolytic lesion re s determined by X-ry utordiogrphy, respectively. Previously, we hve shown tht doxycycline lso significntly reduces tumour burden in the MD-M-31 brest cncer bone metstsis model (Duivenvoorden et l, ). In this study, we describe the effect of zoledronic cid on the skeletl tumour burden in mice concurrently treted with doxycycline. Doxycycline ws dministered to lb/c nu/nu mice s described previously (Duivenvoorden et l, ), resulting in delivered dose of pproximtely 15 mg kg 1 dy 1 strting 3 dys before MD-M-31 cell injections. Zoledronte ws dministered t. mg per mouse every dys strting t the sme time s the doxycycline tretment. We did not observe ny dverse effects of ny tretment protocol in the mice over the study period of 8 dys. Six long bones (both femor, tibie nd humeri), nd ech vertebrl body of the spinl column of ech mouse were screened for the microscopicl presence or bsence of tumour. Previously, it hs been shown tht the mjority of the tumour burden occurs in these bones (Sski et l, 1995; Duivenvoorden et l, ). Occsionlly, the mxille nd pelvic bone show presence of tumour. Doxycycline lone nd in combintion with zoledronte significntly reduced the tumour burden. Figure 1 shows the totl tumour burden s determined in the femor nd spine. These two bones contributed the most to the totl tumour burden per niml (Figure 1). In the plcebo group the totl cumultive tumour burden (for spine, femor, tibie nd humeri) per niml mounted to mm in tumour-bering nimls (n ¼ 15), wheres the totl tumour burden in the doxycycline group (n ¼ 1) ws mm, significnt reduction of 5%. In the group of nimls tht received zoledronte lone (n ¼ 1) the tumour burden ws mm, number tht decresed to mm when doxycycline ws dded to the tretment protocol (n ¼ 6). The tumour burden in the ltter group ws lso significntly decresed when compred to the plcebo-treted nimls. Interestingly, zoledronic cid effected primrily reduction in bone-ssocited soft tissue tumour burden, wheres doxycycline decresed both the bone- nd ssocited soft-tissue tumour burden. Zoledronte cused 93% reduction in soft-tissue tumour burden, irrespective of doxycycline tretment, wheres the totl tumour burden ws merely reduced by 74 nd 43%, in the presence or bsence of doxycycline, respectively. The combintion tretment decresed the tumour burden of both bone nd ssocited soft tissue to lrger extent thn either single tretment lone (Figure 1), lthough this trend did not rech sttisticl significnce. The numbers of mice per group ended up dissimilr, even though, per repeted experiment, five mice per group were 157 ritish Journl of Cncer (7) 96(1),

3 158 injected. The numbers lso excluded mice tht died very soon fter tumour cell injection nd ones tht did not develop tumours. Moreover, the control nd doxycycline-treted mice included mice from previous experiments (Duivenvoorden et l, ). one histomorphometry showed tht both doxycycline nd zoledronte significntly incresed severl prmeters of bone formtion in the femor, including osteoid volume (dt not shown), osteoid surfce nd the number of osteoblsts per bone surfce (see Tble 1, Figure nd D). When zoledronic cid lone Tumour burden (mm ) one tumour burden (mm ) one Plcebo Doxycycline b Zoledronte Combintion one-ssocited soft tissue Totl Femur Tibi Humerus Spine Figure 1 Effect on tumour burden in mouse bone metstsis model of humn brest cncer (MD-M-31). Mice received 1-dy timedrelese pellets contining 1 mg doxycycline 3 dys before intrcrdic MD-M-31 injections. Other groups of mice received either zoledronic cid (t. mg per mouse s.c.) every dys lone or in combintion with doxycycline. plcebo group ws lso included. Mice were killed 8 dys fter cell injection. Longitudinl sections of the spine nd femor of tumourbering nimls (n ¼ 15 in plcebo group, n ¼ 1 in doxycycline-group, n ¼ 1 in zoledronte-group, n ¼ 6 in combintion group,) were nlysed to determine () tumour burden in bone nd bone-ssocited soft tissue tumour burden nd () verge tumour bone burden per evluted bone (spine, humeri, femor nd tibie). Dt represent mens7s.e. of the combined vlues of ll nimls in ech of the groups in t lest two seprte experiments. Significntly different from corresponding plcebo group (Po.5). Significntly different from corresponding doxycycline-lone group (Po.5). b Significntly different from corresponding zoledronic cidlone group (Po.5). ws used, there ws drmtic nd significnt increse in bone volume in control nimls, which ws mintined in the tumourbering nimls (Tble 1). The ddition of doxycycline to the regimen of zoledronic cid sustined the increse in bone volume nd lso induced concomitnt decrese in tumour volume even though the difference did not rech significnce. The presence of brest cncer hs profound effect on bone. Skeletl MD-M-31 tumours showed extensive mrrow colonistion, significnt osteolysis, nd corticl perfortion. Wheres in control nimls, hrdly ny osteoclsts could be found, the number of osteoclsts nd the eroded bone surfce incresed mrkedly in tumour-bering bones (Figure nd C). In contrst, the structure of the trbeculr bone in mice treted with zoledronic cid lone nd in combintion with doxycycline (Figure 3D nd E) seemed better preserved, even in the presence of tumour cells. The mice treted with doxycycline in the presence or bsence of zoledronic cid both showed significnt increse in the osteoid surfce (Figure ); however, this effect could not be mintined in the presence of tumour. Hemtoxylin nd eosin sections of the femur (Figure 3) clerly show the osteolysis induced by the presence of tumour. Sections stined for TRP show the incresed presence of osteoclstic ctivity t the tumour bone interfce (Figure 4). In the presence of tumour, we observed noticeble osteolysis on H&E-stined sections nd incresed osteoclstic ctivity t the tumour bone interfce fter TRP stining. We lso noticed less TRP-positive osteoclsts present on the bone surfce in both zoledronic cid groups, in the presence or bsence of doxycycline (Figure C). mjor feture of brest cncer bone metstsis is the uncoupling of bone remodeling. We lso observed this phenomenon in tumour-bering bones nd show tht doxycycline nd zoledronic cid cn improve this to some level by incresing bone formtion. In doxycycline-treted tumour-bering mice, the tissue volume occupied by bone ws significntly incresed, 9.1% compred to.7% in plcebo (Tble 1). The bone volume further incresed to 3.8 nd 38.7% in nimls treted with zoledronic cid lone nd in combintion with doxycycline, respectively. These represented levels of bone volume tht were higher thn in control mice. This ws ccompnied by n increse in bone formtion prmeters (number of osteoblsts (Figure D) nd osteoid surfce (Figure ) nd volume (Tble 1)) nd concomitnt decrese in bone resorption prmeters, such s number of osteoclsts (Figure C) nd eroded bone surfce (Figure ). Interestingly, in control mice, zoledronic cid lone resulted in n increse in the number of both osteoclsts nd osteoblsts, suggesting n overll increse in bone remodeling, even in the bsence of tumour cells. Moreover, in utordiogrphs of mice treted with zoledronte, we lso observed dense bone nodules t the sites of rib rticultion (dt not shown). The berrnt bone formtion ws noted in zoledronte-treted nimls Tble 1 Effect of tretment with doxycycline or zoledronte lone or combined on bone nd tumour volume in mouse bone metstsis model of humn brest cncer (MD-M-31) s mesured by bone histomorphometry on longitudinl sections of the femor one volume V/TV (%) Plcebo Doxycycline Zoledronic cid Doxycycline nd zoledronic cid Control nimls Tumour-bering nimls Tumour volume TuV/TV (%) Tumour-bering nimls Dt represent mens7s.e. of the combined vlues of ll nimls in ech of the groups in t lest two seprte experiments. Significntly different from corresponding control (no tumour) group (Po.5). Significntly different from corresponding plcebo group (Po.5). Significntly different from corresponding doxycycline-lone group (Po.5). ritish Journl of Cncer (7) 96(1),

4 ES/S (%) 3 1 OS/S (%) Control Tumour o o 159 Plcebo Doxycycline Zoledronte Combintion Plcebo Doxycycline Zoledronte Combintion C NOc/S (mm 1 ) 1 Plcebo Doxycycline Zoledronte Combintion D NOb/S (mm 1 ) 6 4 Plcebo Doxycycline Zoledronte Combintion Figure Effect of doxycycline tretment on bone histomorphometric prmeters in mouse bone metstsis model of humn brest cncer (MD-M- 31). Longitudinl sections of the femor were nlysed to determine () eroded bone surfce s frction of the trbeculr bone surfce (%), nd () osteoid surfce s frction of the trbeculr bone surfce (%), (C) number of osteoclsts per mm trbeculr bone surfce, nd (D) number of osteoblsts per mm of trbeculr bone surfce. Dt represent mens7s.e. of the combined vlues of ll nimls in ech of the groups in t lest two seprte experiments (nx3). Significntly different from corresponding control group (no tumour cells injected) (Po.5). Significntly different from plcebo control nimls (no tumour cells injected) (Po.5). o Significntly different from plcebo tumour-bering nimls (Po.5). Significntly different from corresponding doxycycline-lone group (Po.5). b Significntly different from corresponding zoledronic cid-lone group (Po.5). C o b D E Figure 3 () Histologicl ppernce of the left femur of control mouse (untreted, no cell injections) stined with H&E. ( E) Histologicl ppernce of skeletl tumours in the left femor of mice injected with MD-M-31 cells fter H&E stining. Mice were treted with () plcebo, (C) doxycycline, (D) zoledronic cid, or (E) with both doxycycline nd zoledronic cid. (Originl mgnifiction 1, r ¼ 15 mm). o, bone; GP, growth plte; Tu, tumour. irrespective of doxycycline tretment or the presence of tumour cells. DISCUSSION Using the sme bone metstsis model, zoledronte hs been shown to mrkedly reduce the re of osteolytic lesions s detected by X-ry utordiogrphy, independent of time of dministrtion (concurrently with the tumour cell injections or fter the estblishment of bone metstsis) (Peyruchud et l, 1) nd of the dose dministered (., 1 nd 5 mg per mouse; Green et l, ). When GFP-lbelled MD-M-31 cells re used, the effect of zoledronte on the fluorescence s mesure of the mount of tumour cells is less pronounced thn on the osteolytic lesion re (Peyruchud et l, 1). Similrly, tretment with olpdronte, nother bisphosphonte, demonstrtes tht the tumour burden in the initil phses is more hevily impcted nd returns to vehicle- ritish Journl of Cncer (7) 96(1),

5 153 C D E Figure 4 () Histologicl ppernce of the left femur of control mouse (untreted, no cell injections) stined for TRP. No osteoclsts were observed. ( E) Histologicl ppernce of skeletl tumours in the left femor of mice injected with MD-M-31 cells showing TRP-positive osteoclsts (rrowheds). Mice were treted with () plcebo, (C) doxycycline, (D) zoledronic cid or (E) with both doxycycline nd zoledronic cid. (Originl mgnifiction 4, r ¼ mm). o, bone; Tu, tumour; rrowheds, osteoclsts. treted levels by the end of the 47-dy tretment. During the sme time frme, rdiogrphy shows sustined nd extensive decrese in osteolytic lesions (vn der Pluijm et l, 5), thus substntiting the primry mechnism of ction of bisphosphontes s nti-resorptive. We observed primrily lrge reduction in bonessocited soft tissue tumour burden (93%) fter dministrtion of zoledronte. This is lso supported by dt using 4T1/luc mouse brest cncer model showing tht zoledronic cid results in n inhibition of viscerl metstsis (Hirg et l, 4) nd by Corey et l (3), who provided evidence to suggest tht the inhibitory effects of zoledronte my not only be ssocited to its effect on osteolysis, but lso on cncer cell prolifertion nd poptosis. When compring both compounds, our results showed tht doxycycline induced lrger decrese in tumour burden, wheres zoledronte ws more cpble of sustining the bone volume, in spite of n increse in bone resorption. Similr results were reported using osteoblstic LuCP3.1 or osteolytic PC-3 prostte cncer cells injected into mouse tibie. Zoledronic cid tretment lso induces decrese in tumour volume nd concomitnt increse in bone volume (Corey et l, 3). When we dministered both drugs the result ws substntil decrese in tumour burden nd sustined bone volume. Future investigtions will focus on potentil effects on locl concentrtions of ech of the osteotropic drugs in the bone nd on the specific trgets of both drugs. The min trget of zoledronic cid ppers to be the induction of poptosis (Corey et l, 3; Quinn et l, 5), wheres doxycycline induces G1 cell cycle rrest (Vn den ogert et l, 1986). Hirg et l (1) describe substntil increse in poptotic MD-M-31 cells in the bones of mice treted with ibndronte, especilly t the bone tumour interfce. Severl other mechnisms of ction hve lso been suggested for both drugs. In vitro, zoledronte hs lso been reported to stimulte osteoprotegerin production in primry humn osteoblsts (Viereck et l, ), inhibit prostte cncer cell prolifertion (Corey et l, 3), MMP ctivity (oissier et l, ) nd dhesion to bone extrcellulr mtrix (oissier et l, 1997). Wheres the current study clerly demonstrtes the benefit of combined tretment when both drugs re dministered simultneously strting 3 dys before cell injections, future experiments will include sequentil dministrtion, nd strting severl weeks fter the cell injections. This will help to determine whether doxycycline could lso be beneficil for ptients with lredy estblished bone metstsis nd who re currently receiving zoledronic cid nd vice vers. In conclusion, doxycycline gretly reduced tumour burden nd could lso compenste for the incresed bone resorption frequently ssocited with bone metstsis from brest cncer. The ddition of zoledronte to the tretment regimen further decresed tumour burden nd effected n extensive decrese in bone-ssocited soft tissue tumour burden, independent of doxycycline tretment. Moreover, the combintion lso sustined bone volume, which could gretly reduce the risk of frcture. Tretment with zoledronic cid in combintion with doxycycline my be very beneficil for brest cncer ptients t risk for osteolytic bone metstsis. CKNOWLEDGEMENTS We thnk the Cndin rest Cncer Reserch Inititive nd Cndin Institutes of Helth Reserch for finncil support. We re indebted to Scott Su nd Dr S Lhoták for excellent technicl ssistnce. REFERENCES rguello F, ges R, Frntz CN (1988) murine model of experimentl metstsis in bone nd bone mrrow. Cncer Res 48: erenson JR, Rosen LS, Howell, Porter L, Colemn RE, Morley W, Dreicer R, Kuross S, Lipton, Semn JJ (1) Zoledronic cid reduces skeletl-relted events in ptients with osteolytic metstses. Cncer 91: oissier S, Ferrers M, Peyruchud O, Mgnetto S, Ebetino FH, Colombel M, Delms P, Delisse JM, Clezrdin P () isphosphontes inhibit ritish Journl of Cncer (7) 96(1),

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