effects of cytochalasin b in culture and in yivo on murine ......cytochalasin b and b16 and m109...

ICANCER RESEARCH 50. 1431-1439. March 1. 1990]

Effects of Cytochalasin B in Culture and in yivo on Murine Madison 109 LungCarcinoma and on B16 Melanoma1

Peter F. Bousquet, Lesa A. Paulsen, Christopher Fondy, Karen M. Lipski, Karen J. Loucy, and Thomas P. Fondy2

Department of Biology, Syracuse University, Syracuse, New York 13244

ABSTRACT

Cytochalasin B (CB), at 100 or at 10 mg/kg single dose s.c. incarboxymethyl-cellulose (2%)/Tween-20 (1%) 24 h after s.c. challengeof 1561)21i mice with trocar implants of B16F10 tumor s.c., delayed theappearance of measurable tumor nodules by 157 and 93%, respectively,and extended host survival by 65 and 26%. Tumor growth was alsodelayed when CB treatment was given 1 day after the appearance ofpalpable tumor nodules. By in vivo bioassay, in vitro cloning, and dyeexclusion measurements, solid tumor nodules treated in vivo with CB ateither 100 or 10 mg/kg showed the same viability and tumorigenicity asdid vehicle-treated nodules 4 and 6 days after drug treatment, at whichtime growth inhibition was still apparent. This indicates that growthinhibition by CB is not dependent on a gross cytotoxic effect. (1)21,mice challenged s.c. with Madison 109 lung carcinoma cells and treatedwith CB s.c. at 100 or 150 mg/kg 24 h later showed a 66% delay in themedian day of tumor nodule appearance. When administered under theseconditions or at the time of nodule appearance, CB markedly inhibitedthe rate of tumor growth, prevented tumor invasion at day 23, extendedlife span by 23%, and significantly inhibited spontaneous lung mÃ©tastasesmeasured 28 days after tumor challenge. Maximum tolerated doses ofCB administered i.p., s.c., or i.v. in suspension or in solution are defined.These results delineate the conditions under which CB can be tested forin vivo biological activities and establish that this microfilament-activenatural product in a single-agent protocol inhibits local tumor growthand extends survival in B16F10 melanoma and Madison 109 lung carcinoma, and, in the latter model, inhibits invasion and spontaneous lungmÃ©tastasesby mechanisms that do not appear to depend on cytotoxicity.This work on formulation, tolerated doses in vivo, and localized peritu-moral effects of CB now permits evaluation of systemic antitumor effectsof Cytochalasin B as a single agent. It also permits chemotherapy studiesusing CB as a potential amplifier of the activity of other antitumor agents

INTRODUCTION

Cytochalasins form a congeneric family of more than 24structurally related secondary metabolites produced by variousspecies of molds (1). These agents exert profound effects atmicromolar and submicromolar concentrations on the morphology and functions of a wide variety of cells and tissues inculture [for a detailed review see the preceding paper in thisseries (2) and two comprehensive review books (3, 4)]. Themorphological and functional effects produced by cytochalasinsappear to arise largely because of alteration in actin polymerization, in turn affecting the structure, function, and plasmamembrane interactions of microfilaments (5). Thus the cytochalasins are considered to be microfilament-directed agents inthe same sense that the Vinca alkaloids are microtubule directed.

A very large number of studies have been carried out on theeffects of cytochalasins on cell and tissue morphology andfunction in vitro, including comparisons among normal and

Received 2/15/89; revised 7/21/89. 11/13/89; accepted 11/27/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by the Stella Hardeman Memorial Grant for Cancer Research tothe American Cancer Society and by the Liposome Company. Princeton. NewJersey.

1To whom inquiries should be addressed.

neoplastic cells (6-26). Effects on cell motility, adherence,secretion, and drug efflux, among other effects, suggest to usthat the cytochalasins might produce significant responses inexperimental cancer chemotherapy model systems in vivo eitheras individual agents or, more likely, as agents amplifying responses produced by known antitumor drugs.

For reasons detailed earlier (2), very few in vivo studies havebeen carried out with the cytochalasins. In vivo toxicities ofvarious cytochalasins, Cytochalasin derivatives, and chaetoglo-bosins (27-29) have been investigated, but it does not appearthat CB3 itself has ever been evaluated in mice for in vivo

pharmacodynamic effects of any kind. Some direct attempts atcancer chemotherapy against LI210 leukemia. Ehrlich carcinoma, sarcoma 180, and some other neoplastic model systemsin rodents have been made using Cytochalasin D (27-30). However, these were tests designed to evaluate single-agent cytotoxicantitumor activity, a property unlikely to be important in cytochalasins, which are not especially cytotoxic. These earlychemotherapeutic studies did not reveal direct cytotoxic anti-tumor activity in vivo.

In experimental designs more relevant to the unusual properties of the cytochalasins, B16F10 murine melanoma cells (31)and TA3 murine mammary ascites carcinoma cells (32) weretreated with CB in vitro and transplanted in vivo, but, unfortunately, without the effect-sustaining direct administration ofCB in vivo. Intravenous challenge with B16F10 cells treatedwith CB under these conditions showed increased extrapulmo-nary mÃ©tastases(31). However, many of the important morphological and functional effects of cytochalasins in vitro areknown to be readily reversible (references detailed in Ref. 2).Thus, in vitro alteration of cells with cytochalasins followed byimplantation in vivo without the additional administration ofcytochalasins in vivo is not likely to reveal the full potential ofCytochalasin effects on cell functions in vivo.

A full evaluation of the in vivo utility of these agents willrequire that bioactive concentrations be achieved in target tissues and maintained for times sufficient to produce pharmacodynamic effects. Such in vivo evaluation necessarily requiresthat the agents be available in amounts that are generallyimpractical to obtain from commercial sources. It also requiresknowledge concerning formulation, administration, and dose-limiting host toxicities as a function of formulation, route ofadministration, and dose scheduling. Moreover, evaluation ofin vivo interactions of cytochalasins with other antitumor agentsnecessarily requires that the effects of the cytochalasins as singleagents be known. We have developed improved procedures forthe production of CB and have determined its distribution as afunction of time in 14 tissues following single dose bolus i.p.injection (2). In the present work we have determined themaximum tolerated doses of CB administrated by differentroutes as a bolus drug in a variety of formulations. The earlierCB production and tissue distribution work along with thetoxicity studies presented here permit us to examine the anti-

3The abbreviations used are: CB, Cytochalasin B: CM-cellulose. carboxyme-thycellulose; M109. Madison 109; DMSO. dimethyl sulfoxide; HPLC. highperformance liquid chromatography; TLC, thin layer chromatography.

1431

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CYTOCHALASIN B AND B16 AND M109 TUMORS /A' VIVO

tumor effects of cytochalasin B as a single agent in vivo inanticipation of later studies with in vivo drug combinations.

The present work details our results in culture, in vivo withthe B16F10 melanoma syngeneic in C57BL/6 mice, and in vivowith the Madison 109 murine lung carcinoma syngeneic inBALB/c hosts, a carcinoma which is invasive and metastaticfrom a s.c. implant. Using these systems we have determinedeffects on growth kinetics, localized invasion, spontaneous mÃ©tastases, and host survival.

MATERIALS AND METHODS

Animals and Tumors. The M109 lung carcinoma arose spontaneouslyin 1964 in the lung of an 18-month-old BALB/c mouse. The tumorwas characterized as an alveologenic carcinoma and has been highlyconsistent in its histology and growth characteristics (33, 34). Its valueas a chemotherapeutic model system has been examined using virtuallyevery known clinically active antineoplastic agent in a thorough analysisby Rose (35). The M109 lung carcinoma used in this work was obtainedfrom Dr. William Rose (Bristol Laboratories). All of our work withM109 was conducted with BALB/c and CD2F, (BALB/c female xDBA/2 male) mice. The M109 carcinoma was maintained by serial s.c.passage of 20% tumor brei (approximately 1 x IO6total cells) in BALB/

c mice every 12 days (35). Experimentation was done on CD2F, micewith M109 implanted either i.p., i.v. through the tail vein, or s.c.ventrally on the abdomen.

Tumor brei for passage was prepared by excising the tumor from thepassage mouse, removing any necrotic areas, weighing the tumor,mincing it aseptically, and suspending the fragments in 0.85% (w/v)sterile NaCI solution (l g tumor/ml) containing 0.4 units of penicillinand 0.4 n% of streptomycin/ml. The suspension was then diluted to20% with 0.85% sterile NaCI solution and 0.2 ml (2 x IO5trypan blue-

excluding cells) was implanted s.c. ventrally on the abdomen. For drugtesting, the 20% cell suspension was diluted to allow injection of 1 x10' trypan blue-excluding cells per mouse. Trypan blue exclusion was

determined using 0.2% dye solution in a 1:1 (v/v) ratio with cellsuspension. Viability of the tumor suspension by dye exclusion wasgenerally around 20%.

B16F10 murine melanoma selected by Fidler (36) for high lung-colonizing potential from i.v. challenge was received from the Divisionof Cancer Treatment, Tumor Depository. This subline was maintainedin culture in RPM1 1640 medium, containing 20 ITIM4-(2-hydroxy-ethyl)-l-piperazineethanesulfonic acid buffer, 100 ng/ml streptomycin,100 units/ml penicillin, 250 Mg/ml fungizone, 10% newborn calf serum(Grand Island Biological Company), and 2 g/liter sodium bicarbonate(hereafter called complete medium), and 5% carbon dioxide at 37Â°C

(pH 7.2). All media were filter-sterilized immediately prior to use. Cellswere harvested in log phase by detaching with 0.25% trypsin/0.02%EDTA for 1 min or less and then adding 10 ml of complete medium.The line was maintained by subculturing late log phase cells by 1:10dilution every 5-6 days. The original cell sample from the NationalCancer Institute was expanded and frozen in complete medium broughtup to 50% fetal bovine serum, 10% DMSO, v/v. After 10 passages,new stock was thawed, washed in complete medium, and maintainedin vitro. B16BL/6, a subline selected for bladder connective tissueinvasiveness (37), was also obtained from the Division of CancerTreatment, Tumor Depository, and maintained as above for the FIOsubline.

The FIO and BL6 sublines were used as challenge tumors either assuspensions prepared from cultured cells, or as trocar implants of solidtumor pieces prepared from s.c. tumors elicited by injection of suspended cultured tumor cells. Subcutaneous trocar implantation of asolid piece of tumor (0.5 mm') on the ventral surface of C57/BL6 micewas performed under Nembutal anesthesia with a 13-gauge tumorimplant needle (trocar). In experiments where in vivobioassay of treatedtumors was performed, single-cell suspensions were prepared by aseptically removing the treated tumors, mincing in sterile Â¡sotonicNaCIsolution, and passing the suspension through a sterile nylon filter toremove cell clumps and connective tissue.

Cytochalasin B. The compound was prepared in lots of 400 to 600mg from mold mattes of Drechslern dematioidea by the method ofLipski et al. (2), was purified by preparative TLC, and was analyticallypure at the 99% level as detailed in that work. Radioactive CB wasprepared as described previously (2).

In Vitro Growth Curves. B16F10 cells were routinely harvested fromflasks at late log phase, seeded into 24-well culture plates (Corning) ata concentration of 2.5 x 10' trypan blue excluding cells/cnr into wells

containing 2.0 ml complete medium, and allowed to attach overnight.After 24 h the medium was removed and replaced with 2.0 ml of freshcomplete medium. The wells were treated with various concentrationsof CB dissolved in DMSO. CB solutions were prepared from a stocksolution of 1 mg/ml, which was stored frozen and protected from light.CB was added in 5- to 20-^1 aliquots and all growth experimentsinvolved control wells, which received the equal volume of DMSO(0.25% to 1% final DMSO concentration). Identical wells were harvested in triplicate or quadruplicate, counted in a Coulter counter, andaveraged to determine absolute cell number for each CB concentration.Viability was determined by trypan blue exclusion.

Reversibility in Vitro. Cells were grown and monitored as in growthcurve experiments but were washed at various times. Washing involvedremoval of medium, addition of 2.0 ml fresh complete medium, gentlepipetting, removal of wash medium, and addition of fresh medium.This procedure was repeated two additional times.

Determination of CB in Treated Cells and Media. Plates were seededand treated as in growth curve studies. For each concentration threewells were harvested and pooled at the indicated times. Medium withresidual CB was separated from cells and each was analyzed separately.For analysis of CB in the medium. 5 ml of HPLC grade methanol wereadded to the 6 ml of pooled medium and centrifuged at 1000 x g for 5min and the supernatant was collected. HPLC grade water (15 ml) wasadded to give a 20% methanol final concentration. This was then passedthrough a CIS Sep-Pak (Waters) that had been washed with 4 mlmethanol followed by 6 ml HPLC grade water.

For analysis of CB in cells, the cells were harvested as in growthexperiments and 5 ml methanol were added. This mixture was homogenized with a Polytron and centrifuged and the supernatant was collected. This solution was made 20% methanol by adding water andpassed through a Sep-Pak cartridge as above. Extracts from cells ormedia were treated identically after this point. After drying overnightin a vacuum desicator, the samples were eluted from the CIS cartridgewith 2 ml HPLC grade ethyl acetate. The eluates were dried downunder nitrogen and redissolved in 70 ^1 methanol and 50 Â¿ilw-ereloadedonto an analytical Linear K silica gel TLC plate with a sample-concentrating pre-run zone. The plate was run in a solution of 15%methanol/85% toluene (v/v) or 3/1 benzene/acetone, dried, and stainedwith p-anisaldehyde as detailed previously (2). CB (1 and 5 ^g) wasadded to parallel cell and media samples as controls and to TLC platesdirectly as a marker.

In Vivo Chemotherapy Testing. When testing was done on animalschallenged with either B16F10 or M109 tumors passaged in vivo, theanimals were challenged either with a cell suspension (1 x 10' trypanblue-excluding cells for B16F10 or 1 x IO5trypan blue-excluding cellsfor M109) s.c. or Â¡.p.,or s.c. with an implant of tumor pieces (0.5-mmcubes) with a 13-gauge trocar on day 0. Tumor treatment was initiatedeither on day 1 or at the time of appearance of palpable tumor nodule.Animals were sedated for trocar implant with Nembutal at 40 mg/kg.One day prior to tumor challenge, hair in the region of trocar entryand at the site of tumor deposition was removed with electric clippersand chemical depilatory to permit sterile sealing of the entry woundand to facilitate caliper measurement of tumor dimensions.

CB Treatment. The CB was administered in solution in DMSO (s.c.)or suspended in 0.85% sterile NaCI solution (i.p. and s.c.) or in sterilepyrogen-free 2% CM-cellulose/1% Tween-20 (s.c., Â¡.p.,or i.v.) (a giftfrom Dr. Thomas Tice, Southern Research Institute. The CM-cellu-lose/Tween was found to be the most reliable vehicle for administrationof CB in suspension, since this vehicle minimized adherence of CB tothe syringe walls and permitted delivery of 80 to 90% of the nominaldose, as determined by TLC analysis of the residual CB in the syringes.For i.v. administration of CB in solution, the CB was dissolved in

1432



CYTOCHALASIN B AND B16 AND MI09 TUMORS IN VÃŒVO

ethanol and diluted to 48% ethanol using warm pyrogen-free NaClsolution immediately prior to tail-vein injection (0.1 ml total vehicle).Dilution to 33% ethanol also retained CB in solution but was not usedin this work.

Suspensions were achieved by weighing CB into a syringe, loadingthe vehicle into a second syringe, connecting the syringes with a double-hubbed emulsifying needle, forcing the suspension back and forthrepeatedly through the coupling tube until flow was impeded, and thenprogressively decreasing the bore diameter from 21 gauge to 23 gaugeand finally to 25 gauge. Syringes and couplers were washed withmethanol and the methanol wash was analyzed by TLC to determineresidual CB that was not actually delivered to the test animals.

CB Toxicity in Vivo. Maximum tolerated single bolus doses (LD5_10)as a function of vehicle and route of administration were estimated bycumulative experience with CB treatment of tumor-bearing animals(either M109 in CD2F or B16 melanoma in B6D2F, mice). Maximumtolerated doses estimated in tumor-bearing animals were confirmed innon-tumor-bearing CD2F,, B6D2F,, or C57B1/6 mice. 20 to 24 g, ofeither sex. The effects of subacute CB doses were monitored by determination of animal weights daily for 2 weeks. The vehicles employed[CM-cellulose/Tween or 1:1 (v/v) 95% ethanol/0.85% NaCl solution]were pyrogen free (Limulus amebocyte lysate assay; Whittaker M. A.Bioproducts, Walkersville, MD).

Effects on Primary Tumor and MÃ©tastases.Primary s.c. tumor grow thwas monitored by noting the day when tumor first became measurable.Growth rate was monitored by calculating tumor weight, usingmeasurement of two perpendicular dimensions and the equation(L x w2)/2 (38), and by host survival where animals were terminated

when tumors reached 25 mm in one dimension or when an animalappeared to be in distress. The validity of the tumor growth ratemeasurements was tested by comparing actual weights of tumors obtained at autopsy to values obtained prior to the terminal stages.Intraperitoneal and i.v. tumor growth was determined by host survival,which included animals terminated at the point where distress wasapparent and end stages predictable within 2-3 days. Invasion from s.c.implants of M109 tumor was readily apparent as breaking through theabdominal wall and into the peritoneum. MÃ©tastasesin the liver, spleen,lungs, and pancreas were determined either by necropsy of terminalstage animals or by necropsy of animals sacrificed at a constant timepoint, generally 23 to 30 days after tumor implant, as indicated. Organswere fixed in Bouin's solution (75 ml saturated picric acid, 25 ml

formalin, and 5 ml glacial acetic acid) and metastatic foci were countedunder a dissecting microscope. MÃ©tastasesare presented as the proportion of animals with one or more metastatic nodules and also as themean number of nodules per organ.

Measuring Viability of Tumor Cells in Vivo. Tumor cell viability wasdetermined by three methods: trypan blue exclusion, plating efficiency,and growth of tumor in secondary' recipients. In all cases, s.c. tumors

were removed from initial experimental mice, minced, and dissociatedto form a single-cell suspension in sterile isotonic saline. A 0.1-mlaliquot of suspension was added to 0.1 ml of 2% trypan blue. Totalcells and viable cell counts were made on a hemacytometer. Secondaryisogenic recipients were inoculated with 1 x IO6 viable cells s.c. and

monitored for tumor appearance and host survival.To determine plating efficiency, cells were plated in 6-well tissue

culture plates (Costar) at a density of 1 x 10' cells/cm2. Cell colonies

(50 or more cells clustered) were counted on day 4 of culture. Platingefficiency was defined as the ratio of the number of colonies to thenumber of cells plated, expressed as percentage.

Statistics. Significance was determined by Student's t test and by

Wilcoxon-Mann-Whitney two-sample test.

RESULTS

Effect of CB on B16F10 Growth and Morphology in Vitro.B16F10 cells were treated in vitro with concentrations of CBranging from 100 n\i to 4 UM 24 h after seeding in vitro, atwhich time the cells had a density of 3 x lO^/cm2. Fig. 1 showsthe dose-response effects of continuous exposure to CB for 3

CBFig. 1. Growth of B16F10 melanoma cells in vitro in the continuous presence

of cylochalasin B. Cells were seeded at 2.5 x IO4cells/cm2 at time 0. allowed 10

grow for 24 h. and then treated with CB in DMSO. DMSO concentrations rangedfrom 0.25 to 1.0% and did not affect cell proliferation significantly. Cell growthwas determined by Coulter counter after eell detachment.

and 4 days. A sharp cytostatic dose-response effect was observed, where cells treated by continuous exposure to 200 n\iCB grew to 70% of the density reached by DMSO-treatedcontrols after 4 days, while cells treated at l /IM grew to only10% of the control cell density. CB at 4 /Â¿Mwas completelycytostatic, but no evidence of cell destruction was seen evenafter 4 days of drug exposure in vitro. An IC5n value of 460 n\iwas determined after 3-day exposure and 370 HMafter 4 days.A departure from linearity was observed at the 1CÂ«?point whenexposure to CB was for 3 days. This does not affect comparisonsof ICso values but does suggest a concentration-dependent self-limiting effect, possibly cell cycle based, and has been observedby us with other neoplastic cell lines.4

Final DMSO concentrations in vitro ranged from 0.25 to1.0% and produced a maximum inhibition compared to untreated control cells of less than 30%. Cells exposed to CB atlevels above 100 nM showed alterations in morphology. Thecells became rounded, rather than exhibiting the fibroblasticspreading characteristic of DMSO-treated control cells or ofuntreated cells, and the CB-treated cells had additional pseu-dopodal appendages. These morphological changes appearedto be directly proportional to both the concentration and thetime of exposure (data not shown).

Reversibility of the Cytostatic Effect in Vitro. According to anumber of studies (cited in Ref. 2), most of the effects of CBon cells are rapidly reversible upon removal of the CB-contain-ing growth medium followed by washing and refeeding of thecells with fresh medium. In order to test the reversibility of thecytostatic effects produced by CB, cells were exposed to 1.0 or5.0 tiM CB for 7, 24, or 48 h prior to removal of the CB-containing medium, washing, and refeeding with fresh medium(Fig. 2). After a 24-h period to allow attachment and equilibration of the washed cells, growth rates for the various celltreatment groups were determined. The reversibility of theeffects of CB in this system was a function both of CB concentration and of duration of exposure to CB. Cells exposed to 1.0Â¿IMCB (Fig. 2/1) for either 7 or 24 h and then washed grewwithin 3 days after CB removal to a 4-fold greater density thancells continuously exposed to 1.0 /KMCB (unwashed controls).Under the same conditions, the cells which were exposed to 1.0n\i CB for 48 h prior to removal of CB took 6 days for thewashed cells to achieve a 4-fold increase in density comparedto cells continuously exposed to CB at l UM. A drug exposure

4J. M. Mitchell and T. P. Fondy. unpublished observations.

1433



CYTOCHALASIN B AND B16 AND M109 TUMORS IN VIVO

400Table 1 Cytochalasin B: bolus dose-limiting acute toxicities in mice

2 3456Days Post Washing

Fig. 2. Reversibility of CB effects in vitro. B16F10 cells were grown andtreated as for Fig. 1 but were rendered drug-free by repeated washing with freshmedium at the times indicated. Cell growth was determined daily by Coultercounter and compared to washed and unwashed control wells and to unwasheddrug-treated wells. A, 1 pM CB; B, 5 H.MCB.

of only 7 h at 5.0 ^M CB (Fig. 2B) was sufficient to producethe same level of growth inhibition as was produced by 1.0 //MCB after 48 h of exposure. When cells were treated with 5.0/iMCB for 48 h prior to washing, there was virtually no regrowtheven 6 days after reseeding in the absence of CB.

TLC Analysis of Washed and Unwashed Cultures. To determine the effectiveness of the washing procedure, cell cultureswere treated with CB and washed. CB levels in the originaltreated medium, in the combined washings, and in the washedcells were determined by TLC analysis (see "Materials andMethods"). CB was detectable only in the original drug-treated

medium, and the combined washings. It was below the limit ofdetection, that is less than 1 ^g of CB/106 cells, in the methanol

extracts of the washed cells.The completeness of CB removal by washing was also con

firmed using tritium-labeled CB. More than 99% of the addedradioactivity was recovered in the medium and washings whencells were treated with labeled CB and washed with completemedium (data not shown).

Maximum Tolerated Doses, Routes, and Vehicles. The maximum tolerated doses of CB by i.p., s.c., or i.v. bolus injectionwere established for various drug formulations as suspensionsor solutions. The vehicles tested for drug suspensions were 2%CM-cellulose/1% Tween 20, 0.85% NaCl solution, and 10%Intralipid (Cutter Laboratories). All vehicles were pyrogen-free.CB solutions were prepared in DMSO for i.p. or s.c. testingand, for i.v. injection, in 1/1 (v/v) ethanol/sterile pyrogen-free

0.85% NaCl solution, after initial solution in 95% alcohol.Suspensions in NaCl solution or in Intralipid proved to beunsatisfactory, since variable proportions of the suspended drugup to 80% on occasion remained adsorbed to the deliverysystem. More than 80% of the drug was consistently deliveredto test animals with the CM-cellulose/Tween vehicle, as determined by quantitative TLC analysis of drug that remainedtrapped in the delivery system.

Table 1 presents the detailed cumulative results obtained inour study of the in vivo toxicity of CB. Where deaths occurredat a given dose level in the first two mice tested, no additionaltesting was done at that dose. Maximum weight loss in groupsof animals where no deaths occurred generally was observed 2

CB treatment(mg/kg/day)0CB

administered i.p.5050'

10025

50CB

administered i.v.10204050510

2010

2040CB

administered s.c.'

100ISO'100

100*

150VehicleCMC/Tw*

CMC/TwCMC/TwDMSO

DMSOCMC/Tw

CMC/TwCMC/TwCMC/TwEtOH/NaCI'

EtOH/NaClEtOH/NaClEtOH/NaCl

EtOH/NaClEtOH/NaClCMC/Tw

CMC/TwDMSO

DMSODMSODrug

deathsfraction2/34c'"

0/24C'"5/9f'"'Â£6/jf0/5*

0/5"2/2"2/2"0/29*

0/4f3/4f0/5"

0/7"2/2"0/31'-"

4/28c5/1

6C0/1 Ã³"6/10'

" Day 1 only unless noted otherwise.* CM-cellulose, 2%/Tween-20, 1%, tested pyrogen-free.CCD2F|. normal or M109 tumor-bearing." B6D2F,. normal or B16F10 tumor-bearing.' Days 1 and 7 or days 1, 4. and 7./C57BL/6.* 1/1 (v/v) 95% ethanol/0.85% NaCl solution, pyrogen-free.' Drug administered over ventral wall of peritoneum except as noted.' Day 1 or days 1 and 4.* Drug administered over the lateral wall of the peritoneum.

or 3 days after drug treatment and was less than 5%. Drugsuspension in CM-cellulose/Tween administered at 50 mg/kgi.p. to either normal or tumor-bearing mice was the maximumconsistently tolerated i.p. dose (<LD,0). The presence or absence of tumor did not appear to affect drug toxicity, but theredid appear to be slightly greater sensitivity to the drug in CD2F,compared to B6D2F, mice. Additional bolus i.p. injectionsspaced 3 days apart were well tolerated and, in fact, the laterinjections appeared to cause less weight loss and less apparenthost distress than did the initial injection (data not shown).When CB was administered as a solution in DMSO it wasapproximately twice as toxic as was CB administered in suspension in CM-cellulose/Tween. Only 25 mg/kg CB was tolerated by i.p. injection in DMSO solution. The data shown inTable 1 for the toxicity of CB in DMSO were obtained inC57BL/6 mice, but the same higher toxicity was also seen inB6D2F, and in CD2Fi mice. CB was tested by i.v. administration either as a suspension in CM-cellulose/Tween or as asolution in 1/1 (v/v) 95% ethanol/0.85% NaCl solution. CM-cellulose/Tween was nontoxic when as much as 0.3 ml wasinjected i.v. The 48% ethanol vehicle given up to a volume of0.1 ml i.v. produced transient ethanol intoxication, but this wasdissipated within a few minutes, and there were no apparentdeleterious effects. The drug suspension in CM-cellulose/Tween contained microcrystals of CB after sonication that wereapparent at x 100 magnification. The physical presence ofmicrocrystals did not appear to contribute to host toxicity, sincethe same maximum tolerated dose was observed for suspensionsand for solutions of CB. The maximum-tolerated dose inB6D2F, mice was 20 mg/kg i.v. in either vehicle. The maximumtolerated dose in CD2Fi mice was 10 mg/kg. Even at high i.v.

1434



CYTOCHALASIN B AND BI6 AND M109 TUMORS /A' VIVO

100(X)

L

X60

40

Z 20

V 100 Mg/kg

D 10 M9/k9

X Uel-iiole

10 15 20Day

25 30 35

Fig. 3. Effect of CB s.c. on the time of B16F10 tumor nodule appearance.Animals under nembuta! anesthesia were implanted with a solid piece of B16F10tumor s.c. using a 13-gauge trocar. Treatment with CB in 2cc CM-cellulose/lÃ®r

Tween 20 at 10 or 100 nig/kg or with vehicle alone was given s.c. peritumorally1 day after tumor implant. Animals were considered positive for tumor noduleappearance when a definite mass could be palpated. Either treatment was significantly different from vehicle controls (P < 0.005. n = 6).

IODI?

IO 15 OftV 25 30 48

Fig. 4. Effect of CB on host survival with B16FIO tumor s.c. Animals werechallenged and treated as in Fig. 3. Animals were terminated when tumorsexceeded 2.5 cm in any one dimension or when animals appeared to be in distressor moribund. Median survival was significantly increased in the 100 mg/kg group(percentage of median life span treated/control = 165; P < 0.005).

doses, weight loss was minimal in animals surviving the acuteinitial response to CB. Multiple daily doses of CB were administered i.v. with no apparent cumulative toxicity (data notshown). Since there is a very sharp dose-response effect for thetoxicity of CB administered i.v., the drug was tested for in vivoeffects on tumor growth at 5 mg/kg, which is one half themaximum tolerated dose in CD2Fi mice.

CB in CM-cellulose/Tween suspension at 150 mg/kg administered s.c. ventrally over the peritoneal wall was approximatelythe LD,5 dose, and this dose could be used for in vivo testing,but 100 mg/kg was more reliably tolerated. CB dissolved inDMSO and administered s.c. was also tolerated at 100 mg/kg,but only if injected laterally on the peritoneal wall. Injection inDMSO s.c. at 100 mg/kg ventrally over the peritoneum wasrelatively more toxic, presumably due to rapid penetration intothe peritoneal cavity by the dissolved drug.

Effects of CB on B16F10 Implanted s.c. CB administered asa single bolus dose at either 100 or 10 mg/kg s.c. under thetumor challenge site, 24 h after trocar implant of B16F10 solidtumor s.c., had marked effects on tumor latency, tumor growthrate, and host survival. Fig. 3 shows the delay in the appearanceof measurable tumor nodules in the treated groups comparedto vehicle (CM-cellulose/Tween 20)-treated controls. The median of tumor appearance in the controls was 7, whereas treatment with CB at 10 mg/kg delayed the median appearance dayto 13.5, and at 100 mg/kg to day 18 (P < 0.005). Once thetumor nodules began to grow, their growth rates were com

parable in either of the treated groups, compared to controls(data not shown), but there was a marked increase in theduration of host survival in both of the treated groups (Fig. 4).Animals receiving 100 mg/kg CB on day 1 exhibited a mediansurvival of 28 days, compared to vehicle control survival of 17days (165% survival treated/control). At 10 mg/kg CB, survivalwas 126% (treated/control) (P < 0.005).

In a separate experiment where animals received 150 mg/kgCB s.c. and were sacrificed 12 days after tumor implant topermit direct measurement of tumor burden, there was also asubstantial inhibition of tumor growth. The treated group hadtumors with mean weights of 159 mg, compared to vehiclecontrols which had mean tumor weights of 1.25 g.

Cytochalasin B Effects on M109 Lung Carcinoma. Since neither the B16F10 nor the B16BL6 lines exhibited spontaneousinvasion and mÃ©tastasesfrom s.c. implants, we investigated theeffects of CB in vivo on invasion and spontaneous metastasisusing the M109 lung carcinoma model. The pattern of growthand mÃ©tastasesof the Madison 109 lung carcinoma is dependent upon the route of tumor injection. Table 2 presents themedian survival times and the patterns of metastatic spreadobserved for the different routes of tumor challenge. Intraperi-toneal tumor spread more extensively and killed more rapidlythan either i.v. or s.c. tumor challenge. The i.p. tumor spread

Table 2 Madison 109 lung carcinoma survival and mÃ©tastases

Tumorroute"i.p.

i.v.s.c.Median

survival(days)22

3532Number

of animals47

1958%

Animals with one or moremÃ©tastasesLiver89

4210Pancreas96

016Spleen6

00Lung159552

Â°I x IO5viable M109 cells injected.

4 1

3

>- CMC/TVEEN,DI

>- CB 150,01

(10)

uo

I-

14 15 24 25

DAYFig. 5. Growth of s.c. M109 tumors in animals treated s.c. with vehicle (2%

CM-cellulose/lco Tween-20) compared to animals treated s.c. with CB at 150mg/kg single bolus dose 1 day after tumor challenge (1 x IO5viable cells) (A) or

at 100 mg/kg single bolus dose administered when tumors first became detectable(B). (TAD. tumor appearance day; 4 to 6 days after tumor implantation), n.number of animals per group. Growth rates of treated versus vehicle controls aresignificantly different, P< 0.001 in A: P< 0.01 in B (Student's t test).

1435



CYTOCHALASINB AND BI6 AND M109 TUMORS /A' VIVO

Table 3 Cylochalasin B s.c. versus Madison 109 lung carcinoma s.c. (1 x 10* viable cells)

CBtreatment"mg/kgVehicle

150Vehicle

ISOVehicle

ISOVehicle

100Vehicle

100Day1

1111

11

TAD7TAD

TADNecropsy

(day)23

Z3282822-36''

36-38''28

2829-36''

28-50''Median

Lifespansurvival T/C(%)30

1003T12334

10040 118Tumor

weight atdeath(g)2.6

Â±0.5*0.6 Â±0.2e4.9

Â±1.14.4 Â±2.26.4

Â±0.35.7 Â±0.34.9

Â±1.13.8 Â±1.06.8

Â±1.03.7 Â±1.3Fraction

withinvasion9/10

1/10Â°4/5

1/33/5

2/34/5

4/52/42/6Tumor-free

survivors,day500/5

0/30/42/6

" Vehicle. CM-cellulose, 1%/Tween 20, 2%.* Mean Â±SE.' P = 0.001 compared to vehicle controls.''Terminal.' P < 0.05 compared to vehicle controls.7TAD. tumor appearance day.

primarily to peritoneal sites, whereas the i.v. injection producedtumor colonies (artificial mÃ©tastases)in the lungs of all challenged animals and in the livers of some of them. The s.c.challenge metastasized mainly to the lungs. These results areconsistent with the findings of other investigators (33, 39, 40).

When the s.c. M109 tumor was treated s.c. with CB suspended in CM-cellulose/Tween, following procedures used forthe B16F10 tumor models, tumor appearance was delayed by4 days (appearing by day 6 in the vehicle controls and inuntreated controls compared to day 10 in the CB-treated animals). As shown in Fig. 5, CB treatment produced a significantdecrease in tumor growth rate when animals were treated withCB either on day 1 or on the day when a measurable tumornodule was detected (generally around day 6). Increased mediansurvival time was seen in CB-treated animals, either thosetreated on day 1 or those treated when a tumor nodule wasdetected (Table 3). Of the animals treated in this latter group,33% (2 of 6) were tumor-free on day 50. In all experiments alower tumor weight at death was consistently observed in all ofthe treated groups. The difference was significant when tumorsexcised from treated and control animals sacrificed on day 23after tumor challenge were compared. The difference in tumorweights between CB-treated compared to control groups became less marked at later stages of tumor growth. Most importantly, invasion of the tumor into and through the peritonealwall when determined by necropsy at day 23 was stronglyinhibited by CB treatment in comparison with vehicle controls(Table 3). This effect of CB on invasion persisted but becameless marked at later stages of tumor growth.

Fig. 6 presents the effect of cytochalasin B s.c. on the formation of spontaneous pulmonary mÃ©tastasesfrom a s.c. tumor.A decrease in the proportion of animals exhibiting lung tumornodules determined either at day 28 or at the terminal stageswas seen in groups of animals treated with 100 mg/kg CB atthe time of primary tumor detection, when compared to vehiclecontrols. A marked decrease in the mean number of lung tumornodules was seen at day 28 (32 Â±15 in the controls, 3 Â±2 inthe treated; P < 0.05). This difference appeared to persist atthe terminal stages (43 Â±19 in the controls, 10 Â±9 in thetreated), even though one would expect tumor spread in treatedanimals to catch up to that in controls at their respectiveterminal stages. However this difference at the terminal stageswas no longer statistically significant (P< 0.2). Slight increasesin pancreatic mÃ©tastaseswere seen both at day 28 and at the

100

-i ^ 80

CD LUZ </i 60

r- +< Z 40

<

Z.

20

04-

VEHICLE,DAY 1 or TAD

CB: 100, TAD

S.E.M.7/9

5/5

DAY 28 TERMINAL

Fig. 6. Spontaneous pulmonary metastatic nodules in animals shown in Fig.5B. determined by necropsy either at day 28 after tumor challenge or at theterminal stage (days 29-36 for vehicle controls; 28-50 for CB-treated). Thedifference in the number of nodules determined by simultaneous necropsy oftreated animals versus vehicle controls on day 28 is significant (P < 0.05). Thefraction above the bars represents the number of animals bearing pulmonarytumors over the number of animals in the group.

terminal stage when animals were treated with CB at 150 mg/kg (data not shown). It should be noted that 150 mg/kg s.c.was an LD40 in this experiment, so the effects on lung andpancreatic mÃ©tastasesmay be secondary to the toxic effects ofCB in the animals that survived drug treatment. Spleen andliver were also examined for metastatic nodules in all groups,and no effect of CB administered s.c., either decreasing orenhancing metastasis to these organs, was observed (data notshown).

The effects of peritumoral CB administered s.c. in the regionof a s.c. M109 tumor challenge or growing tumor nodule havenow been confirmed with an intradermal tumor model, whereCB administered s.c. does not directly contact the growingtumor nodule. Single or multiple bolus doses (days 1, 3, and 5after tumor nodule appearance) retard tumor growth, prolongsurvival, and produce a significant level of tumor regressions.5

Effects of CB on Tumor Cell Viability in Vivo. The cytostaticrather than cytotoxic effects of CB in vitro suggest that in vivoeffects on tumor growth do not arise from cytotoxic effects.However, localized very high nominal concentrations of CB are

5J. M. Mitchell. C. M. Corsette, and T. P. Fondy, work in progress.

1436



CYTOCHALASIN B AND B16 AND MI09 TUMORS IN VIVO

Table 4 In vivocytotoxicity of cytochalasin B against BlfiFlO melanomaEstablished tumors (8 days after trocar implant s.c.) were treated s.c. with 10

or 100 mg/kg CB or vehicle on day 8 post tumor challenge. Tumors were removedon either day 12 or day 14 (4 or 6 days after CB treatment), at which times therewas a significant inhibition of growth of tumor in the CB-treated group. Tumorsfrom treated and control groups were analyzed individually for viability. Secondary recipients received 1 x 10* cells s.c. Three tumors were analyzed per group.

None of the comparisons are significantly different at the 0.05 level.

GroupVehicle

CB, 10mg/kg

CB, 100mg/kgAnalysis

day12

1412141214Cell

viability(%)Â°TBE23

1720131515PE18

1817151416Second

TAD*7

8

9Recipient

survival(days)'17

16

18Â°TBE, Trypan blue exclusion; PE, plating efficiency.* TAD, median tumor appearance day of secondary recipient.c Survival, median survival (days).

conceivable following s.c. treatment under the tumor site, so itwas necessary to determine more directly whether CB couldfunction as a localized cytotoxic agent in vivo. Animals werechallenged with B16F10 tumor s.c. and the tumors were allowedto grow to measurable size (8 days), at which time they weretreated with CB at either 100 or 10 mg/kg s.c. under the tumorsite or with CM-cellulose/Tween 20 vehicle. Four or 6 dayssubsequent to drug treatment, at which time growth inhibitionwas apparent, tumors were excised and the viability of the cellswas determined by plating efficiency, dye exclusion, and in vivobioassay in recipient mice. Table 4 presents the results of thisstudy. It is apparent that cell viability is not compromised byCB treatment, in comparison with vehicle control-treated cells.In vivo bioassay of the cells recovered from treated tumorsshows that the latency period prior to tumor nodule appearancein the recipients is not significantly affected in the CB-treateddonor tumors, nor is the survival of the recipient mice significant different when they received tumor from groups treatedeither with high or low dose CB, as compared to those challenged with vehicle-treated control tumors (P > 0.05).

The absence of cytotoxic effect of CB in vivo is furthersupported by experiments utilizing multiple bolus doses of CBadministered i.p. at 50 mg/kg to animals bearing s.c. B16F10tumors.6 Systemic CB administered on days 1, 4, and 7 after

s.c. tumor challenge produces a small but significant inhibitionof tumor growth rate compared to controls, suggesting thathigh, localized, potentially cytotoxic concentrations of CB arenot necessary for inhibition of growth of s.c. tumor nodules.

Effects of CB i.p. on B16F10 or M109 i.p. The absence ofdirect cytotoxicity by CB on B16F10 melanoma cells in vivo isalso apparent when CB at the maximum tolerated i.p. dose ofSO mg/kg is administered to animals bearing an i.p. tumorchallenge (Table 5). With CB treatment given either as a singlebolus dose 1 day after tumor challenge or as two bolus doseson days 1 and 7, increase in survival compared to vehiclecontrols was less than 20%. CB under these conditions alsoproduced a marked enhancement of metastasis to the spleen atthe terminal stage of the disease and appeared to accelerate theseeding of mÃ©tastasesto the liver and pancreas as well as to thespleen when necropsy was performed on day 12 after tumorchallenge.

As shown in Table 5, CB administered i.p. against a M109i.p. challenge produced a marked increase in the proportion ofanimals exhibiting splenic mÃ©tastasesand a significant increasein the mean number of nodules in the spleens, when compared

both to vehicle controls and to untreated controls. A significantincrease in the mean number of nodules in the liver was observed in the animals treated with a single dose of CB on day1, when compared to the controls, but in animals given CBtreatment i.p. on days 1,4, and 7 the increase in liver mÃ©tastasesversus vehicle controls was not statistically significant. Thismultiple-dose group showed a slight increase in survival (17%)compared to vehicle controls. These effects of Â¡.p.CB at 50 mg/kg are consistent with transient immunosuppression producedunder these conditions7 and are not indicative of cytotoxicity

against the tumor challenge.Effect of CB i.v. on M109 i.v. When CB was administered i.v.

at 5 mg/kg in 48% ethanol either 1 hour prior to i.v. tumorchallenge (1 x IO5viable cells) or 24 h subsequent to i.v. tumorchallenge, there was no increase in lifespan compared to vehicle-treated controls (7 or 8 animals/group; data not shown). Allanimals had multiple lung nodules when examined either atday 20 or at the terminal stage of the disease, and there was nosignificant difference in the number of nodules in the treatedcompared to the control groups. No mÃ©tastaseswere visible inthe spleen, liver, or pancreas in any of the mice, indicating thatCB administered i.v. does not enhance splenic mÃ©tastases.

DISCUSSION

More than 3000 papers dealing with the effects of CB in vitrohave been published, and some work has appeared on the invivo toxicity of other cytochalasin congeners (27-29). However,the work presented here is the first detailed in vivo evaluationof the toxicity of CB administered i.p., s.c., or i.v. and is thefirst in vivo evaluation of CB against a tumor model system. Itis apparent that against a s.c. tumor challenge a single dose ofCB administered s.c. at 100 mg/kg, which is below its LDi0, isable to delay the time of tumor appearance, inhibit tumorgrowth rate, and prolong host survival in B16F10 melanomaand M109 lung carcinoma. The growth inhibition and extendedsurvival is true whether drug treatment is given 1 day aftertumor challenge or treatment is withheld until palpable tumornodules have appeared. In the M109 model system a singledose of CB also inhibits tumor invasion and spontaneous lungmÃ©tastases.The effects on the B16F10 model persist at a 10-fold lower dose, and at either dose it does not appear that effectsare due to measurable gross cytotoxicity. As shown in Table 4,clonogenicity, tumorigenicity, and vital dye exclusion are notsignificantly altered by CB treatment in vivo, at times (4 and 6days after CB treatment) when tumor growth is significantlyinhibited in the treated animals. Evidence on CB clearance invivo (2)J indicates that CB would be cleared from the injection

site and from the animals long before the 4- to 6-day post-injection point, especially at the 10 mg/kg low dose treatment.Work with M109 lung carcinoma intradermal tumors treatedwith CB administered s.c. or i.v.* confirms this delayed effect

of CB on tumor growth, suggesting a noncytotoxic basis for theaction of CB.

The mechanistic basis for the inhibition of growth and metastasis remains to be established, but the effects of CB oncytoskeletal structure and function suggest that alterations ofeither tumor or host cell movement could lead to measurableeffects on local tumor growth, invasion, and metastasis. CB andother cytochalasin congeners have been shown to be noncytotoxic against a variety of cell types in culture at concentrationsup to 100 Mg/ml (200 MM)for short term exposures in the range

* McQuiggan and Fondy, unpublished observations.7 D. Bogyo and T. P. Fondy. unpublished observations.8J. M. Mitchell and T. P. Fondy, unpublished observations.

1437



CYTOCHALASIN B AND BI6 AND M109 TUMORS IN VIVO

Table 5 Effect of cytochalasin B i.p. on BI6FIO melanoma and on M109 lung carcinoma i.p.

CBdosemg/kgMI09,

1 x 10*viable cellsi.p.UntreatedVehicle505(1BI6F10,

1 x 10' viable cellsi.p.Vehicle5050Day1;

1,411.4,71.71,71Median

survival(days)20202224161614Fraction

with terminal mÃ©tastases(nodules)Liver5/6

(10 Â±3)Â°5/6

(10 Â±2)5/6(53Â±17)6/6(21

Â±5)3/5

(2Â±1)4/4(100 Â±0)4/4(87 Â±7.5)Pancreas5/6

(835/6(885/6(836/6

(83Â±17)Â±13)Â±17)Â±11)5/5

(3 Â±0.5)4/4(154/4(16Â±2.2)Â±

3.8)Spleen1/6

(0.2Â±2)1/6(1Â±0.6)5/6

(12 Â±5)6/6(12 Â±2)0/54/4(6.5

Â±1.8)4/4(4.5 Â±0.9)Lung0/60/60/62/6

(2Â±1/5(0.41/4(0.32/4(1.81)Â±0.4)Â±0.3)Â±D

' Mean Â±SE.* Vehicle. 2c.i carboxymethyl cellulose/1% Tween 20 in 0.85% NaCl solution.

of 2 h (41). The profound pleotropic effects produced by CB oncells in culture depend on continued presence of the drug andare in many cases reversible upon removal of the CB (fordetailed references see Ref. 2). The work presented here showsthat CB can produce irreversible effects on cell proliferativeability with prolonged exposure in vitro. However, in additionto the viability studies on B16F10 cells recovered from treatedmice (Table 4), other lines of evidence argue against generalizedcytotoxicity by CB in vivo. CB given i.p. as a single bolus doseat 50 mg/kg shows no effect on the life span of mice bearingan i.p. M109 tumor challenge, indicating the absence of significant cytotoxicity. When injected i.v. either immediately priorto i.v. tumor challenge or 24 h subsequent to challenge, CBagain shows no evidence of being able to measurably affect thegeneral overall viability of the tumor cell challenge. Sincesustained localized high concentrations of CB may be able toproduce irreversible effects, it is possible that the inhibition ofinvasion and possibly the growth inhibition itself are due toeffects on the viability of localized regions of tumor cells or ofhost cells necessary to support tumor growth and extension.Our methods would not be able to detect such localized cytotoxicity. Studies on the effects of systemically administered CBon the growth, invasion, and metastasis of localized tumorchallenges are underway to address this point.

In addition to the potentially beneficial antitumor effectsobtained with CB, it should also be noted that CB administeredi.p. enhanced splenic mÃ©tastasesfrom an Â¡.p.tumor challengein both the M109 and B16F10 systems. This effect couldconceivably arise if actin depolymerization and microfilamentalteration increased deformability (42, 43) to a greater extentthan it inhibited locomotion (44), potentially facilitating tumorcell invasion. Alternatively or additionally, deleterious effectsproduced by CB on host leucocyte-based antitumor responses(45) could lead to increased splenic metastasis. Enhancementof artificial M109 lung mÃ©tastaseswith agents affecting naturalkiller cell or macrophage activity has been observed (46).

The results should be of value in the study of the effects ofcytochalasin B and other cytochalasin congeners on tumormodel systems in vivo, particularly in determining the potentialutility of cytochalasins as chemotherapeutic amplifiers in combination chemotherapy in vivo. In vitro studies indicate to usthat cytochalasins may exert exploitable effects on inherent andinduced drug resistance in tumor models and might alter mo-tility-dependent functions such as tumor invasion, spontaneousmetastasis, and angiogenesis. If any such in vitro effects can beextended to in vivo models, the results could be of considerablevalue, particularly in view of the fact that more than 24 naturaland semisynthetic congeners are available for structure-activityevaluation and for the optimization of cytochalasin-based effects. Beyond these 24 congeners, a large family of additional

semisynthetic modifications of natural congeners is readilyaccessible.

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1439



1990;50:1431-1439. Cancer Res Peter F. Bousquet, Lesa A. Paulsen, Christopher Fondy, et al. Madison 109 Lung Carcinoma and on B16 Melanoma

on Murinein VivoEffects of Cytochalasin B in Culture and

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effects of cytochalasin b in culture and in yivo on murine ......cytochalasin b and b16 and m109...

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