[CANCER RESEARCH 37, 145-151, January 1977]

In a recent report, van Putten et al. (20) found a significant enhancement of lung colony formation by pretreatment of mice with various cytotoxic drugs, particularlywith cyclophosphamide. This report prompted us to studythe effect of various cytotoxic drugs on lung colony formation using the syngeneic KHT sarcoma in C3H mice. Thistumor tends to metastasize to the lungs under normal conditions and thus provides a model comparable to manycurrent clinical situations in which patients with varioussolid tumors receive prophylactic and/or therapeutic chemotherapy during and following the treatment of the primarytumor by surgery and/on irradiation (18, 21).

MATERIALS AND METHODS

Mice and Tumors.In all but2 experiments,femaleC3H/Km mice, 14 to 18 weeks old , weighing 28 to 42 g were used.Twelve mice were preassigned to each treatment groupaccording to weight so that each group had the same meanweight.

The tumor used with C3H mice was the KHT sarcoma, atumor that arose spontaneously at the base of the ear of aC3H/Km mouse in 1962 and that has since been maintainedby serial s.c. passage into syngeneic mice. The tumor in thishost is at most only weakly antigenic since repeated immunization with cells sterilized with a dose of 10,000 rads failsto modify the number of cells needed to produce tumors in50% of injected animals (9). In 1 experiment, EMT6 tumor

cells were injected into 3- to 4-month-old BALB/cKa mice.This tumor has been adapted for growth both in vivo and invitro and is demonstrably immunogenic in this host (14).The details of the propagation of the tumors, the preparation of single cell suspensions, and the counting of lungcolonies have been described previously (2, 14). Briefly,single-cell suspensions of KHT tumor cells were obtainedfrom s.c. tumors and injected via the tail vein in a volume of0.2 ml into the recipient mice. The animals were sacrificed17 to 20 days later, and the lungs were extracted and preserved so as to allow the counting of all surface lung cobnies. The mean number of countable colonies was found tobe proportional to the number of cells injected in a givenexperiment, but varied somewhat from experiment to expenimentfora givennumber of cellsinjected.Therefore,each experiment was performed with its own internal control groups. The number of KHT tumor cells injected permouse varied in different experiments, depending on theexperimental conditions. The range was from 4 x 10@to 5 x10@cells/mouse, the exact amount being based on a goal of

145JANUARY 1977

The Effect of Cyclophosphamide and Other Drugs on theIncidence of Pulmonary Metastases in Mice1

Richard J. Carmel2 and J. Martin Brown3

Divisionof RadiobiologyResearch,Departmentof Radiology,StanfordUniversitySchoolof Medicine,Stanford,California94305

SUMMARY

A study was made of the effect of various cytotoxic drugson the ability of i.v.-injected KHT sarcoma cells to form lungcolonies in syngeneic C3H mice. Some enhancement of thenumber of lung colonies following an i.v. injection was seenfollowing pretreatment of the mice with actinomycin D andmithramycin, while pretreatment with vinblastine, bleomycm, methotrexate, cytosine arabinoside, on 5-fluonounacilhad little or no effect on lung colony formation. Pretreatment of the mice with cyclophosphamide, however, greatlyincreased lung colony formation (by a factor of approximately 100). This enhancement in lung colony formationwas maximal when the drug was given 24 hr prior to theinjection of tumor cells, but was seen as early as 2 hr anc@persisted as long as 8 weeks prior to the tumor cell injection.

The degree of enhancement of lung colony formation wasrelated to the dose of cyclophosphamide and was present inweanling as well as adult mice. This enhancement was notsignificantly reversed by anticoagulation with either aspirinon wanfamin. Immunosuppression by whole-body irradiationdid not affect the number of lung colonies seen in cycbophosphamide-treated mice.

The mechanism by which cyclophosphamide enhancesmetastatic tumor growth within the lung is not known. Themajor effect, however, does not appear to be mediatedeither by specific immunological on clotting factors.

INTRODUCTION

Several recent investigations (3, 17, 22) have demonstrated that if the lungs of mice are irradiated prior to theanimals receiving an i.v. injection of tumor cells, the numben of pulmonary nodules so produced is greatly increased.It has also been shown that prophylactic lung irradiationincreases the incidence of pulmonary metastases from aspontaneous osteosarcoma in dogs (12). The clinical nelevance of these studies lies in the possibility that lung irmadiation might enhance pulmonary metastasis in patientswhose primary tumor has not been completely eliminated(13).

I This investigation was supported by USPHS Research Grants CA-i 5201

and CA-10372 from the National Cancer Institute.2 Present address: Division of Radiation Therapy, Department of Radio

biology, Stanford University School of Medicine, Stanford, Calmf.94305.3 To whom requests for reprints should be addressed.Received July 23, 1976; accepted October 8, 1976.

Research. on January 5, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

Effect on lung colony formation of cytotoxic drugs given 1day prior totumor cellinjectionNo.

of cellsNo. of lung cobDrugDose(mg/kg)injectednies0.9%

NaCIsolution6.0 x 1O@2.1 ± 1.6@Cycbophosphamide2006.0x 1O@169.9 ±14.40.9%

NaCIsolution8.5 x 10@2.2 ±0.4Adriamycin108.5x 1O@2.4 ±1.0O.9%NaClsolution1O@3.6±

1.4ActinomycinD0.271O@23.7 ±9.5Methotnexate501O@2.1

±0.65-Fluorouracil1401O@4.5±1.5Cytosine

arabinoside250010@1 .8 ±0.6Mithnamycin11O@12.8±4.1Bleomycin6

(units/kg)1O@5.2 ± 2.1

A. J. Carmel and J. M. Brown

obtaining approximately 50 colonies/mouse and a requirement not to exceed 200 colonies/mouse.

In the case of the EMT6 tumor, cells were obtained fromtissue culture passage, diluted appropriately, and injectedi.v. in a volume of 0.2 ml as before. The mice were sacrificed17 to 20 days after injection and counted as for the KHTtumor.

Drug Treatment. Drugs for screening (Table 1) were selected on the basis that they were in current common clinical usage as anticancer agents. All the drugs were injectedi.p. on a mg/kg basis using from 50 to 90% of the LD50,,0based on package insert data and the published data ofSkipper (16). The cyclophosphamide used was Cytoxan(Mead Johnson & Company, Evansville, Ind.), and it wasdissolved in sterile water immediately prior to use.

Anticoagulation. In the experiments involving anticoagulation by either warfanin or aspirin, the anticoagulationagent was administered in the drinking water for 4 daysprior to and for 3 days after the tumor cell injection. Aspirinwas dissolved in the drinking water in a concentration of625 mg/liter, while the wanfanin concentration was monitoned and regulated so as to prolong the mean 1-stageprothrombin time by 2 to 4 times the normal value for mice(2).

Irradiation.Thedetailsfor localthoracicandwhole-bodyirradiation have been previously described (3, 19). Briefly,for local thoracic irradiation, anesthetized mice were positioned in a lead box with only the thoracic region exposed.For whole-body irradiation, the mice were not anesthetized.In both cases, irradiation conditions were: 250 kVp X-rays;15 ma; focus skin distance, 44 cm; half-value layer, 1.3 mmCu ; and a total dose of 400 rads at a dose rate of approximately 100 rads/min.

RESULTS

Effect of CytotoxicDrugs on Lung Colony Formation.Various cytotoxic drugs found in common clinical usagewere injected i.p. into mice 1 day pmiorto the i.v. injection ofa known number of KHT tumor cells (Table 1). There was aslight enhancement of pulmonary metastases (lung colony

formation) by actinomycin D and mithramycin, but therewas fan greater enhancement with cycbophosphamide. Thiscapacity of cycbophosphamide for greatly increasing thenumber of lung colonies formed was not limited to the KHTcell system, but was also seen when BALB/cKa mice werepretreated with cyclophosphamide 1 day prior to the injection of EMT6 tumor cells (Table 2).

In order to check that the negative results obtained withsome drugs were not merely a question of different kineticsof development of the effect, adniamycin (10 mg/kg) wasinjected at various times from 7 days to 3 hr prior to injection of KHT tumor cells. No effect on the number of lungcolonies was found in any of the groups.

Effect of Dose of Cyclophosphamideon Lung ColonyFormatIon.To determinethe dosedependenceof the enhancing effect of cyclophosphamide on lung colony formation, mice were pretreated with different drug doses 1 dayprior to the injection of the tumor cells (Charts 1 and 2). It isapparent that the dose-response curve is highly sigmoidalin nature with a possible threshold up to almost 100 mg/kg(Chart 1). However, when the data are plotted in a semiboganithmic form (Closed circles, Chart 2), they fit closely to anexponential relationship (i.e. , the number of lung coloniesis proportional to the exponent of the drug dose). Alsoshown in Chart 2 are data from other experiments in whichvarying doses of cyclophosphamide were injected 1 dayprior to injection of KHT tumor cells. None of the linesthrough the data differ significantly in slope. Doses greaterthan 200 mg/kg caused some drug-related deaths and werenot evaluated for their effect on lung colony formation.

Effect of the Number of Tumor Cells Injected. In thisexperiment, groups of 0.9% NaCI solution- or cyclophosphamide- (200 mg/kg) treated mice received injections 1day later of varying numbers of KHT tumor cells. The resultsshow (Chart 3) that, within the range of number of cellsinjected, the relationship between number of colonies andthe number of cells injected is approximately linear. Howeven, there are definite departures from linearity near theorigin:for0.9% NaCI solution-injectedmice,the curveisconcave upward (or has a threshold) below 1O@cells,whereas, for cyclophosphamide-injected mice, the curve isapparently steeper or convex upward below 4 x 1O@injected

Table 1

a Mean ± S.E.

CANCER RESEARCH VOL. 37146

Research. on January 5, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

Comparison of the effect of cyclophosphamide (200 mg/kg) onlung colony formation by KHTcells in C3H/Km mice and EMT6 cells

in BALB/cKa miceNo.

oflungcoloniesNo.

of cells CycbophosphaTumor injected ControlmideKHT

6 x 10' 2.1 ±1.6a 169.9 ±14.4EMT6 1O@ 12.3 ±1.8 110.0 ±14.1a

Mean ± S.E.

Enhancement of Metastases by Cyclophosphamide

2 doses of cyclophosphamide, 100 mg/kg, is seen. The 2nddose of 100 mg/kg was always given 24 hr before the tumorcells were injected (as was the single dose of 100 mg/kg).The reduction in the number of colonies in mice that meceived 2 doses, as the interval between these doses increases, reflects 2 processes: 1st, a decay in the interactionbetween the 2 doses; and 2nd , a decay in the effect of the1st dose of cycbophosphamide, 100 mg/kg, as the intervalbetween its delivery and the injection of the tumor cellsincreases. The 2nd effect can be factored out by referring toChart 6, bottom (the data of which were obtained as part ofthe same experiment). It can be seen that, up to 5 daysbetween injection of cyclophosphamide, 100 mg/kg, andinjection of the tumor cells, there is no loss of the colony

Table 2

I I I ILUU,0IU,LU

z0

0U(3z

U.

0

LU

I

z

LUU)D0IU,LU

z0

0U(32

U.

0

LU

I

z

LUU,0IU,LU

20

0U(3z..aU.0

LU

I

z

175

125 -

75 -

25 —

—.---.---.—---—@•@ I I.@- 50 100 150 200

DOSEOF CYCLOPHOSPHAMIDE(mg/kg)

Chart 1. The number of lung colonies per mouse as a function of the doseof cycbophosphamide injected 1 day prior to the injection of i0@KHT tumorcells.

cells. This implies that the relative difference between thenumber of colonies in the cycbophosphamide-treated andcontrol animals (on the colony enhancement ratio) increases with decreasing numbers of tumor cells in thebloodstream.

Kinetics of Development and Decay of Colony Enhancement. Chart4 showsthe resultsof an experimentin whichcycbophosphamide (200 mg/kg) was given at intervals from2 hr to 7 days prior to the injection of 6000 KHT tumor cells.The enhancing effect is cleanly present at 2 hr after cyclophosphamide injection, seems to be at a maximum at 1 dayafter injection, and is still elevated 7 days later.

Chart 5 shows the results of an experiment in which KHTcells were injected up to 8 weeks after a single i .p. dose ofcycbophosphamide (175 mg/kg). It is apparent that, afterthis high dose of cyclophosphamide, much of the damagecausing the colony-enhancing effect is repaired 2 to 3weeks after injection, but there is still a residual effect 8weeks after the cyclophosphamide.

Effectof MultipleDosesof Cycbophosphamide.Becauseof the cleanly sigmoidal nature of the dose-response curve(Chart 1), it was of interest to evaluate the effect of divideddoses of cycbophosphamide. An experiment was penformed, therefore, to answenthe question of how much timewas needed between 2 doses of 100 mg/kg for the 2 dosesto act independently rather than as a single dose of 200 mg/kg. The results of this experiment are shown in Chart 6. InChart 6, top, the effect of varying the time interval between

90

DOSEOF CYCLOPHOSPHAMIDE(mg/kg)

Chart 2. The number of lung colonies per mouse as a function of the doseof cycbophosphamide injected 1 day prior to the tumor cell injection.

(x10@) (x1O@)NUMBEROF CELLS INJECTED

Chart 3. The number of lung colonies per mouse as a function of thenumber of KHT cells injected in both 0.9% NaCl solution- and cyclophosphamide-(200 mg/kg) pretreated mice, the pretreatment being given I day priorto the tumor cell injection.

JANUARY1977 147

Research. on January 5, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

A. J. Carmel and J. M. Brown

tion. In order to test whether the colony enhancement bycyclophosphamide was due to a generalized immunosuppressive effect, groups of animals were pretreated 1 dayprior to the tumor cell injection with various combinationsof radiation and cycbophosphamide. The results (Table 5)show that the enhancing effect of 400 nads local thoracic orwhole-body irradiation [a partially immunosuppressive dose(11)J on lung colony formation is small compared to theeffect of a high dose (200 mg/kg) of cyclophosphamide.Furthermore, the colony enhancement by cyclophosphamide is just as great in immunosuppressed mice ( 400 madswhole-body irradiation) as in control mice.

Effect of Anticoagulation on Lung Colony Formation.The effects of aspirin and wamfaninon lung.cobony formationwere studied by administering these drugs in the drinkingwater for 4 days prior to, and 3 days after, the injection ofKHT tumor cells. Half of the animals also received pretreatment with cycbophosphamide (200 mg/kg) (Table 6). Asexpected, both aspirin and wamfanin cause a significantreduction in lung colony formation in control (0.9% NaCIsolution-injected) mice. However no reduction in the numben of lung colonies was seen in the animals treated withcyclophosphamide.

Effect of Cycbophosphamide on KHT Cells. To determine

2504,@

£ £

z 4 6

INTERVAL BETWEENCYCLOPHOSPHAMIDEAND TUMORCELL INJECTION(WEEKS)

Chart 5. The number of lung colonies per mouse as a function of the timeinterval between injection of cyclophosphamide (175 mg/kg) and injection ofKHTcells.

LUU,0I

LU0.U,LU20

0U(32...aU.0

LU

I

2

6

INTERVAL BETWEENCYCLOPHOSPHAMIDEAND TUMORCELL INJECTION(DAYS)

Chart 4. Lung cobonyformation following an injection of 6 x 1O@KHT cellsper mouse as a function of the time interval between cyclophosphamidetreatment (200 mg/kg) and cell injection.

@1 I I

0 CONTROL,3x1O@CELLS. CYCLOPHOSPHAMIOE,3x105CELLS£CYCLOPHOSPHAMIDE,1O@CELLSA CONTROL.104CELLS

enhancement, whereas, in this same interval, virtually all ofthe interactive effect of the 2 doses of cycbophosphamide islost (Chart 6, top).

From this result, it could be predicted that weekly dosesof cycbophosphamide would merely add to each other intheir effect rather than interact to produce a large colonyenhancement. Table 3 shows the results of an experiment inwhich colony formation in mice pretreated with 4 weeklydoses of cycbophosphamide, each dose ranging from 50 to175 mg/kg, was compared with that in mice given only asingle dose. As expected, most of the enhancement in lungcolony formation results from the final dose of cycbophosphamide, since the mean number of lung colonies observedfor most dosage groups is only slightly higher for weeklycycbophosphamide, compared with single-dose treatment.

Effect of Mouse Age on LungColonyFormation.Lungcolony formation was studied in mice of ages 3, 7, 11, and15 weeks. One-half of the groups were pretreated withcyclophosphamide (200 mg/kg) 1 day prior to the tumor cellinjection (Table 4). The number of lung colonies is seen todecrease with increasing age of the mice. However, muchof this difference can be accounted for on the basis of bodyweight (and blood volume). The main point of the data isthat the colony enhancement by cycbophosphamide did notvary greatly as a function of age of the mice, although theredoes appear to be somewhat less enhancement in 7-weekold mice than at the 2 older ages. The experiments reportedin this investigation were done on mice 14 to 18 weeks ofage.

Effect of Immunosuppressionon Lung Colony Forma

200

LUU,D0IU,LU

20

0U(32D

U.0

@1O0

ID2

50

I I I I0 8

148 CANCERRESEARCHVOL. 37

Research. on January 5, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

Comparison of the effect of single dose versusweeklycyclophosphamideon lung colonyformationIn

each group, the final drug dose (or single dose) precededthetumorcell injection by 1day.Cycbophos-

No. of lung coloniesphamide dose No. of tumor

(mg/kg) cells injected Single dose Weeklydose50

3 x 1O@ 16.6 ± 2.@' 28.4 ±5.6753 x 10@ 21.0 ± 5.5 30,8 ±1.71003 x 1O@ 50.0 ± 5.8 64.0 ±6.41253 x 1O@ 63.5 ±14.8 106.4 ±19.7150

1O@ 41.8 ±10.3 61.4 ±6.61751O@ 80.0 ±11.4 66.3 ±12.8a

Mean ± S.E.Table

4Effectof mouse age on lung colonyformationNo.

of lungcoloniesCyclophosphamideAge

(wk) Control@'treated@'3

>188.0 ±20.0― >209.0 ±18.0790.5 ±15.8 67.3 ±12.91158.6 ± 8.4 83.9 ±9.81545.9 ± 7.0 81.3 ±10.3

Radiation and cycthe injection of 4 xlophosphamide

were both given 1 day prior to10@KHTcells.RadiationNo.

of lung colonies with cyclophosphamidedose0

mg/kg 100 mg/kg 200mg/kgNone

LTI, 400 radsWBI, 400 nads0.0

±0.0― 4.5 ±0.9 60.9 ± 6.70.4 ±0.1 11.7 ±3.8 77.3 ± 9.40.7 ±0.3 13.7 ±2.2 112.5 ±11.8

Enhancement of Metastases by Cyclophosphamide

limbs. Fourteen days following the limb amputation, theanimalsweresacrificedand thelungswereassayedforlungcolonies. Cycbophosphamide (175 mg/kg) was administeredto some of the animals 1 day prior to, or 16 days after,implantation of the tumors. The results are shown in Table7. In the control animals whose tumors were allowed togrow for only 12 days prior to amputation, no lung colonieswere observed. After 16 and 20 days, however, pulmonarymetastases were observed in all animals. In the animalstreated with cycbophosphamide 1 day pniorto tumor implantation, a few colonies were observed after 12 days, but foreach increasing interval prior to amputation, only a slightincrease in lung colony formation was seen relative to thatobserved in the untreated control animals.

However, when cyclophosphamide was given to animals16 days after tumor implantation and the tumor-bearing

Table3

LUU,0I

LU0.U,LU

20

0U(32

U.0

LU

I

2

5

TIME BETWEENCYCLOPHOSPHAMIDEANDTUMOR CELL INJECTION(DAYS)

Chart 6. Top, lung colony formation following 10@KHT cells as a functionof the interval between 2 doses of cycbophosphamide, 100 mg/kg. The 2nddose was given 1 day prior to the injection of the tumor cells. Bottom, lungcolony formation following 10―KHT cells as a function of the interval betweena single dose of cyclophosphamide, 100 mg/kg, and the tumor cell injection.

the sensitivity of KHT cells to the cytotoxic effects of cycbophosphamide, mice were treated with cyclophosphamide(175 mg/kg) at intervals of 1 hr to 9 days after the i.v.injection of KHT cells into C3H mice. The number of cobnies was reduced from approximately 70/mouse in the controls to less than 1/mouse at all the time intervals up to 9days. It is apparent that cyclophosphamide eliminates vimtually all lung colonies when it is given following the injection of the KHT cells, thus exposing these cells in vivo to thecytotoxic effect of the drug.

The Effect of Cyclophosphamideon SpontaneousPubmonary Metastases. In an attempt to provide a bettermodel for the clinical situation in which spontaneous pubmonary metastases are observed from primary tumors, 1-cumm fragments of KHT tumor were implanted in the hindlimbs of C3H mice. The tumors were allowed to grow forvarying periods prior to amputation of the tumor-bearing

a 2 x 10@ tumor cells were injected.

b Cycbophosphamide, 200 mg/kg, was given 1 day prior to injec

tion of 5 x 10@cells.CMean ±S.E.

Table 5Comparison of the effect of LTIGand WBlon lung colony formation

in cyclophosphamide-treated mice

a LTI, local thoracic irradiation; WBI, whole-body irradiation.

b Mean ± SE.

149JANUARY 1977

Research. on January 5, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

Effect of aspirin and warfaTin on lung colonyformationNo.

of lung coloniesinCyclophosphamide

No. of cells in 0.9% NaCI solution- injected (200mg/kg)TreatmentjectedinjectedmicemiceControl10@0.8

± 0.4@ 115.9 ±18.6Aspirin1O@100.9±18.8Control5

x 1O@130.2 ±15.5Aspirin5x 10@26.9 ±11.7Control3x 10@117.5 ±11.2Warfanin3x 10@62.1 ±10.3Control5x 1O@97.8 ±12.9Warfarin5x 10@96.0 ±18.1

Table7Spontaneouslung colony formation from KHTtumor inthehindlimb

as a function of the number of days fromtumorimplantationto hindlimbamputationDays

to Daystohindlimblung har- No. of lung cob

Treatment amputation vestniesControl

12 26 0 ±O.OaControl16 30 27.7 ±7.4Control20 34 60.2±10.8CYTb.e12 26 3.0±1.2CYTC16 30 34.0±9.0CYT―20 34 63.0±9.5CYTd

21 35 1.3± 0.4

A. J. Carmel and J. M. Brown

Table6

a Mean ± S.E.

cells injected 24 hr later, yet its effect on lung cloning willgreatly extend the range of cell survival oven which the lungcolony assay can be utilized.

The mechanism by which cycbophosphamide pretreatment enhances lung colony formation by injected tumorcells is uncertain. The short serum half-life of the drugprecludes any direct effect on the tumor cells. The effectrather must be on the host and, presumably, some changeoccurs within the pulmonary microvasculatune, where theinjected tumor cells must lodge in order to form a metastasis or lung colony. It is known from previous studies thatessentially all of the injected cells become lodged initiallywithin the lungs (2, 22). The trapped cells then may remainto form viable lung colonies. Most of the cells, however,either die while attached to the pulmonary capillary endothelium, on are dislodged to enter the systemic circulation.The initial attachment of the tumor cells to the endothelialwall is aided or maintained by the formation of a plateletfibmin thrombus around the cells (8). Those tumor cellsdestined to become metastases pass into the penivasculartissues where they establish lung colonies (8, 23). Anticoagulants such as wamfaninand aspirin are known to reduce thenumber of lung colonies formed by injected tumor cells,presumably by interfering with the formation of thrombiaround the tumor cells (2, 5). The enhancement in lungcolony formation by cyclophosphamide pretreatment wasnot affected, however, by anticoagulation with either aspimmon warfanin in our experimental system. van Putten et al.(20) also observed a very much smaller reduction in thenumber of lung colonies when warfanin was administered tocycbophosphamide-pretreated mice, compared with controlmice. We conclude from these results that the effect ofcyclophosphamide on lung colony formation is probablynot on tumor thrombus formation within the pulmonarymicrovasculature, although an effect on the stability of thethrombus cannot be eliminated.

Cycbophosphamide is known to suppress antibody production in mice, but only after prolonged weekly administration of 70 mg/kg i.p. (7). Our experiments show that inimmunodepressed mice (i.e. , those that received 400 nadswhole-body irradiation), the colony enhancement by cycbophosphamide is undiminished, compared with that in nonirradiated mice. Our data also support the previous finding

a Mean ± SE.

b CYT, cycbophosphamide, 175 mg/kg.

C One day prior to tumor implant.

d Sixteen days after tumor implant.

limbs amputated 5 days later, very few lung colonies penanimal were observed. This shows that not only did thecyclophosphamide injection eliminate those nodules present at 16 days, it also prevented further metastatic spread inthe following 5 days, presumably due to its highly cytotoxicaction on the primary tumor.

DISCUSSION

The preceding experiments show that pretreatment ofC3H mice with cycbophosphamide can greatly enhance lungcolony formation to a degree fan greater than that observedwith the other cytotoxic drugs tested . It is also greaten thanthe effect seen with local on whole-body irradiation (3, 13,17, 22). This finding agrees well with that ofvan Putten eta!.(20). This effect, i.e. , that pretreatment of mice with cycbophosphamide increases the cloning efficiency (number ofvisible lung colonies per number of viable tumor cells injected) by 100-fold or more, will undoubtedly prove useful inimproving the sensitivity of the lung colony assay for thesurvival Qf tumor cells in vivo to radiation or chemothemapeutic agents. Because of the short half-life (17 mm) of thecytotoxic effect of cycbophosphamide and its metabolites(10), the drug will not interfere with the viability of tumor

150 CANCERRESEARCHVOL. 37

Research. on January 5, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

Enhancement of Metastases by Cyclophosphamide

that the colony enhancement by radiation alone is due to alocal effect on lung tissue rather than to a generalizeddepression of the immune system (3, 22). This fact, togetherwith the great similarity between the kinetics of appearanceand decay of the enhancement of cloning efficiency byradiation (cf. Ref. 3) and by cycbophosphamide, arguesstrongly that these 2 agents act by the same mechanism.One such possible mechanism is that cycbophosphamideand radiation are effective in either destroying macrophagesor in preventing their killing the endothelium-trapped tumorcells. The data of van Putten et al. (20) suggest that, whileCorynebacterium parvum is highly effective in reducinglung metastases in control animals, it is far less so in cyclophosphamide-treated mice. Also, Bomford and Olivotto (1)have shown that radiation can inhibit the reduction in lungnodules produced by C. parvum injection. Although theseauthors did not establish the mechanism of action of C.parvum in reducing lung nodules, they did demonstrate thatit was not mediated through an immune response to tumorspecific antigens, and their data and that of others (24) isconsistent with C. parvum acting by stimulating macrophages or macmophage precursors.

The clinical significance of our findings of prolongedenhancement of lung colony formation by cycbophosphamide pretreatment is not clear. Cycbophosphamide eliminated virtually all lung colonies when the drug was givenafter the tumor cell injection, and it inhibited the development of spontaneous metastases from a thigh-implantedtumor. Clearly, in our mouse model, the cytotoxic effectoutweighs the potential enhancement of lung colony formation by cycbophosphamide. However, the cytotoxic effect ofcycbophosphamide on certain human solid tumors is probably less effective, since many patients with these tumors dodevelop pulmonary metastases despite treatment with cycbophosphamide and other cytotoxic agents. It is in thissituation that the clinician should weigh the theoreticallypossible harmful effects of cyclophosphamide against thepotential tumonicidal benefits.

The doses of cycbophosphamide used in this study arequoted in mg/kg. On this basis they appear roughly 10-foldhigher than the doses used in clinical practice. However, ithas been demonstrated that the proper basis for companison for drug toxicity studies is that of body surface area (4,6). For the mean mouse size used in this investigation, thelargest dose used, 200 mg/kg, converts to roughly 700 mg/sq m, a dose that is well within the range of that givenclinically in single i.v. injections either alone on in combina-,tion with other anticancer drugs (6, 15).

Finally, we feel that these data argue that it may be advantageous in some circumstances not to initiate chemotherapy with cyclophosphamide until after completion of treatment of the primary tumor, be this with surgery on radiothenapy. This would minimize the possibility that viable tumorcells will become lodged in pulmonary tissue made extremely receptive by cycbophosphamide to the formation ofblood-borne metastases.

JANUARY 1977 151

REFERENCES

1. Bomford, R., and Olivotto, M. The Mechanism of Inhibition by Coryne. bacterium parvum of the Growth of Lung Nodules from Intravenously

Injected Tumor Cells. Intern. J. Cancer, 14: 226-235, 1974.2. Brown,J. M. A Studyof the Mechanismby WhichAnticoagulationwith

Warfarin Inhibits Blood-Borne Metastases. Cancer Res., 33: 1217-1224,1973.

3. Brown, J. M. The Effect of Lung Irradiation on the Incidence of Pulmonary Metastases in Mice. Brit. J. Radiol., 46: 613-618, 1973.

4. Freireich, E. J., Gehan, E. A., RaIl, D. P. Schmidt, L. H., and Skipper, H.E. Quantitative Comparison of Toxicity of Anticancer Agents in theMouse, Rat, Hamster, Dog, Monkey, and Man. Cancer ChemotherapyRept., 50: 219-244, 1966.

5. Gasic, G. J., Gasic, T. B., and Murphy, S. Anti-Metastatic Effect ofAspirin. Lancet, 2: 932-933, 1972.

6. Goldsmith, M. A., Slavik, M., and Carter, S. K. Quantitative Prediction ofDrug Toxicity in Humans from Toxicity in Small and Large Animals.Cancer Res., 35: 1354—1364,1975.

7. Hoffsten, P. E., and Dixon, F. J. Effect of Irradiation and Cyclophosphamide on Anti-KLH Antibody Formation in Mice. J. Immunol., 112: 564-572, 1974.

8. Jones, D. S., Wallace, A. C., and Fraser, E. A. Sequence of Events inExperimental Metastases of Walker 256 Tumor: Light, Immunofluorescent, and Electron Microscopic Observations. J. NatI. Cancer Inst., 46:493-504, 1971.

9. Kallman, A. F., Silini, G., and van Putten, L. M. Factors Influencing theQuantitative Estimation of the In Vivo Survival of Cells from Solid Tumors. J. NatI. Cancer Inst., 39: 539—549,1967.

10. Kline, I., Gang, M., Tyrer, D. D., Mantel, N., Venditti, J. M., and Goldin,A. Duration of Drug Levels in Mice as Indicated by Residual AntileukemicEfficiency. Chemotherapy, 13: 28-41 , 1968.

11. Micklem, H. S., and Brown, J. A. H. Rejection of Skin Grafts and Production of Specific Isohaemagglutinins by Normal and X-lrradiated Mice.Immunology, 4: 318-328, 1961.

12. Owen, L. N., and Bostock, D. E. Prophylactic X-lrradiation of the Lung inCanine Tumours with Particular Reference to Osteosarcoma. EuropeanJ. Cancer, 9: 747-752, 1973.

13. Peters, L. J. The Potentiating Effect of Prior Local Irradiation of theLungs on the Development of Pulmonary Metastases. Brit. J. Radiol., 47:827-829, 1974.

14. Rockwell, S. C., Kallman, R. F., and Fajardo, L. F. Characteristics of aSerially Transplanted Mouse Mammary Tumor and Its Tissue-CultureAdapted Derivative. J. Natl. Cancer Inst., 49: 735-749, 1972.

15. Schein,P.,DeVita,V.,Canellos,G.,Chabner,B.,andYoung,R.ANewCombination Chemotherapy Program for Diffuse Histiocytic (DHL) andMixed (DML) Non-Hodgkin's Lymphoma: BACOP. Proc. Am. Assoc. CancerRes.,16:248,1975.

16. Skipper, H. E. Biochemical, Biological, Pharmacologic, Toxicologic,Kinetic and Clinical (Subhuman and Human) Relationships. Cancer, 21:600-610, 1968.

17. Van den Brenk, H. A. S., and Kelly, H. Potentiating Effect of Prior LocalIrradiation of the Lungs on Pulmonary Metastases. Brit. J. Radiol. , 47:332-336, 1974.

18. Van der Werf-Messing, B. The Treatment of Pulmonary Metastases ofMalignant Teratoma of the Testis. Clin. Radiol., 24: 121-123, 1973.

19. van Putten, L. M., and Kallman, R. F. Oxygenation Status of a Transplantable Tumor during Fractionated Radiation Therapy. J. NatI. CancerInst., 40: 441-451 , 1968.

20.van Putten,L.M.,Kram,L.K.J.,vanDierendonck,H.H.C.,Smink,T.,and Fuzy, M. Enhancement by Drugs of Metastatic Lung Nodule Formation after Intravenous Tumour Cell Injection. Intern. J. Cancer, 15: 588-595, 1975.

21. Wharam, M. D., Phillips, T. L., and Jacobs, E. M. Combination Chemotherapy and Whole Lung Irradiation for Pulmonary Metastases fromSarcomas and Germinal Cell Tumors of the Testis. Cancer, 34: 136-142,1974.

22. Withers, H. R., and Milas, L. Influence of Preirradiation of Lung onDevelopment of Artificial Pulmonary Metastases of Fibrosarcoma inMice. Cancer Res., 33: 1931-1@6, 1973.

23. Wood, 5., Jr., Holyoke, E. D., and Yardley, J. H. Mechanisms of Metastasis Production by Blood-Borne Cancer Cells. Can. Cancer Conf., 4:167-223,1961.

24. Woodruff, M. F. A, Ghaffar, A., and Whitehead, V. L. Modification of theEffect of C. Parvum on Macrophage Activity and Tumour Growth by xIrradiation. Intern. J. Cancer, 17: 652-658, 1976.

Research. on January 5, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from

1977;37:145-151. Cancer Res   Richard J. Carmel and J. Martin Brown  Incidence of Pulmonary Metastases in MiceThe Effect of Cyclophosphamide and Other Drugs on the

  Updated version

  http://cancerres.aacrjournals.org/content/37/1/145

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/37/1/145To request permission to re-use all or part of this article, use this link

Research. on January 5, 2019. © 1977 American Association for Cancercancerres.aacrjournals.org Downloaded from