(CANCER RESEARCH 49, 5148-5152. September 15, 1989]

A Human Tumor Lung Metastasis Model in Athymic Nude RatsInge Kj0nniksen,' Ritsa Storeng,1 Alexander Pihl, Theodore L. McLemore, and Oystein Fodstad2

Departments of Tumor Biology ¡I.K., 0. F.] and Biochemistry [R. S., A. P.], Institute for Cancer Research, The Norwegian Radium Hospital, Montebello 0310 Oslo 3,Norway; and the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda Maryland 20892 [T. L. M.]

ABSTRACT

Experimental lung métastases regularly developed in athymic\\M\-.rnulrnu Rowett rats after i.v. injection of LOX human malignantmelanoma cells. When 5 x 10*tumor cells were injected into 4-week-old

rats, 89% of the animals died of lung tumors, with a mean survival timeof 18 days. With 5- and 6-week-old rats, however, the fraction of animalsthat died decreased to 80 and 46%, with mean survival times of 35 and38 days, respectively. The number of detectable lung colonies in eachanimal was about 35 in 5- and 6-week-old animals, compared to nearly300 in 4-week-old rats. In the latter, a correlation was found between thenumber of tumor cells injected and the number of detectable lung colonies.The capacity of the LOX tumor to grow s.c. and to form experimentallung métastaseswas, by and large, similar in young nude rats and in nudemice, and no significant difference in morphology between the differenttumors in the two species was seen. A high-resolution radiographiemethod was used to visualize lung colonies in the nude rats, and singletumors with diameters as small as 2-4 mm could be detected. By thismethod, for the first time, the effect of chemotherapy on a human tumorgrowing in a visceral organ of a rodent host could be followed by repeatX-ray examinations, mimicking a situation commonly faced in the clinic.This procedure may prove particularly useful for experimental chemotherapy studies, and may be extended to other human tumors thatfrequently metastasize to the lungs. Indications were obtained that somehost-specific differences in tissue-preferenced growth might exist, apossibility that will be further explored.

INTRODUCTION

Progress in metastasis research depends to a large extent onthe use of experimental animal models that, as closely aspossible, mimic the situation in humans. Previously, most metastasis studies were performed in syngeneic murine and rattumor systems (1-4). The more recent development of humantumor metastasis models in athymic, nude mice (5-9) hasprovided improved possibilities for elucidating factors involvedin the metastatic process.

For some types of experiments, i.e., those involving technically difficult procedures such as various types of cannulationsand intracardial/intraarterial injections, it would be advantageous if larger animals could be used. One possibility is theathymic, nude rat, a mutant first described in 1978 (10). Theeffects of the nude mutation seem to be quite similar in ratsand mice (11-13), but the nude rats are known to be morerobust than the mice and can be kept free of disease also inconventional animal facilities (10). Despite this advantage, nuderats have been used only to a limited extent for experimentsinvolving human cancers, and to our knowledge no detailedstudy on human tumor metastasis formation in nude rats hasbeen reported.

Here we report on the establishment and usefulness of anexperimental metastasis model in nude rats in which the animals die within 3-5 weeks after i.v. injection of the humanmelanoma cell line LOX. It was found that the survival time is

Received 2/2/89; revised 5/30/89; accepted 6/8/89.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.

' Fellow of the Norwegian Cancer Society.2To whom requests for reprints should be addressed, at Department of Tumor

Biology. Institute for Cancer Research, Montebello 0310, Oslo 3, Norway.

dependent on the age of the rats at the time of tumor cellinjection. We demonstrate also that the change in size of thelung tumors can be followed, in individual animals, by employing a radiographie method.

MATERIALS AND METHODS

Animals. Congenitally athymic, nude rats (Han:rnu/rnu Rowett) werepurchased germ free and gnotobiotic from Zentralinstitut fürVersuchstiere, Hannover, West Germany, or bred in our own nude rodentfacility. Animals of both sexes were used in the experiments, and afterweaning at 3 weeks the rats were kept in positive pressure rooms withfiltered and humidified air. All animals proved to be disease freethroughout the experimental period.

Cell Line. The malignant amelanotic melanoma cell line LOX originates from a patient's lymph node metastasis (6). The cells werecultivated in monolayerin a 1:1 mixture of Dulbecco's modified Eagle'smedium and Hani's F12 medium supplemented with 10% fetal calfserum at 37°Cin a humidified mixture of 5% O2 and 5% CO2 in

nitrogen. The cells were subcultured twice weekly after detachmentwith trypsin-EDTA and were found to be free of mycoplasma infections.

Morphological Studies. Tumor tissue was fixed in buffered formaldehyde, dehydrated, and embedded in paraffin. Sections were stainedwith H & E, and the morphology of s.c. and intrapulmonar^ tumorsfrom rats was compared with that of corresponding tumors from miceand from the patient's lymph node metastasis.

Subcutaneous Tumor Growth. LOX tumor tissue cubes (2x2 mm)were prepared from s.c. growing xenografts and inoculated in the flanksof groups of rats of different ages (2-, 4-, or 6-week-old at the time oftransplantation). Two perpendicular diameters of the tumors weremeasured with calipers three times weekly. Tumor volumes were calculated according to the formula 0.5 x L x W2(14), and growth curves

were constructed.Experimental Metastasis Formation. Monolayer LOX cells were

grown to near confluence, washed twice with phosphate buffered saline,detached with EDTA (6) and resuspended in RPMI 1640. Nude ratswere anesthetized with halothane (Trofield Surgicals A.C., Zug, Switzerland) and N..(). and injected i.v., in a lateral tail vein, with differentnumbers of cells in 0.1-0.2 ml of RPMI. The rats were checked dailyfor up to 3 months. Animals with marked dyspnea were sacrificed by alethal dose of lialot hanc/N ..O, and the time from the day of tumor cellinjection was recorded. The lungs were inflated by intrabronchial injection of Bouin's solution, and the number of tumor colonies on the

surface of the lungs was counted by visual inspection.Radiographie Studies. The rats were anesthetized with a mixture of

2.5 mg/kg midazolam, 5 mg/kg fluanisone, and 0.1 mg/kg fentanyli.p., and X-ray films were taken using a Senographe 500T mammographdevice (Thomson-CGR Medical Corporation, Columbia, MD) employing molybdenum anode and filter, focus 0.3 mm, 25 kW exposure at16-20 mAs and a distance of 55 cm, essentially as previously described(15, 16). To achieve good immobilization, anterior-posterior compression was applied. The film, Ortho M (Kodak), was exposed in a KodakMinR-casette with Kodak MinR-screen.

Chemotherapy Experiments. Groups of 4-week-old rats carrying s.c.LOX xenografts were treated, when the tumors had reached a size ofapproximately 4x4 mm, with mitozolomide (15 mg/kg) or c/i-dich-lorodianimineplatinum(II) (4 mg/kg) i.v., or left as untreated controls.Assessment of antitumor effects (four tumors per group) was performedas previously described (6, 14).

In other experiments, 1 x IO6 LOX cells were injected i.v. into 4-and 5-week-old rats. Some animals were treated i.v. with mitozolomide(15 mg/kg), and repeat X-ray examinations were performed on the daysindicated in the text.

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RESULTS

Growth of s.c. LOX Xenografts. Tumor take was observed inalmost all 6-week-old rats in about 7 days after transplantationof LOX tissue obtained from s.c. xenografts in nude mice. Thetumors grew progressively and could thereafter be serially transplanted to other animals. The growth rate of the s.c. tumorswas similar to that observed in nude mice, with a doubling timeduring exponential growth of approximately 1.8 days (data notshown).

Due to the variable success of previous investigators in growing human tumors in nude rats, and since young nude mice arereported to support xenograft growth better than adult animals(17), we studied whether the age of the rats at the time oftransplantation would affect the growth of the s.c. LOX tumor.No clear differences in the time before tumor growth wasobserved, or in the growth rate of the established tumors, werefound between animals transplanted at 2,4, and 6 weeks of age.

Lung Colony Formation. In experiments in nude mice we havepreviously found (6) that after i.v. injection of 1 x IO6 LOX

cells from monolayer cultures the animals regularly die ofprogressively growing lung tumors in about 35 days. Using thesame number of cells, the survival time of 5-week-old nude ratswas also 35 days. Lungs removed from rats and mice killedwhen they started to become dyspneic, contained tumor colonies that seemed to have similar patterns of distribution, aswell as size relative to the size of the host lung (Fig. 1, A andQ.

As was found in mice (6), the number of rat lung coloniesdiffered from animal to animal, and in lungs with larger tumors,the colonies tended to become confluent. Light microscopicstudies of the rat tumors showed closely the same morphological picture as those of mouse lung colonies, s.c. xenografts inboth species, and of the original patients' tumor (not shown).

When LOX cells were injected i.v. into animals of differentages, it was found that the survival time and lung colonyformation differed with the age of the rats (Table 1), in contrastto the situation when the LOX tumor was grown s.c. The life-span of 4-week-old rats after injection of 5 x IO5 cells was 19±2 days, compared to 35 ±10 and 38 ±10 days for 5- and 6-week-old rats, respectively. It is also seen that after injection of1 x IO6LOX cells, 19/20 (95%) of 4-week-old rats died of lungcolonies, whereas only 72% (13/18) of 5-week-old and 47% (9/19) of 6-week-old animals died. Similar results were obtainedwhen 5 x IO5 cells were injected, and with 1 x IO5 cells thepercentage of animals that died was 100% in 4-week-old rats

Fig. 1. Tumor colony formation in the lungs of nude animals injected i.v. with1x10* LOX cells. To visualize the colonies, the lungs of animals sacrificed whenthey became dyspneic were inflated by intrabronchial injection of Bouin's Solu

tion. The picture shows the lungs of rats injected at 5 weeks of age (A) and at 4weeks of age (B), and of a nude mouse injected at 6 weeks of age (C).

and only 13% in 5-week-old animals. Also, the life-span of therats was clearly shorter for animals injected at 4 weeks of agecompared to those in other groups (Table 1).

In agreement with these data, the number of lung colonies inanimals that died of their métastaseswas much higher in youngrats, where a correlation was found between the number ofcolonies and the number of tumor cells injected. The effect ofage is also evident from the pictures demonstrated in Fig. 1. Itis seen that the lungs of a rat injected at 4 weeks of age (Fig.IB) contained numerous small, partly confluent colonies, mostof them less than 2-3 mm in diameter, whereas the lungs of a5-week-old rat (Fig. \A) had much fewer, but larger colonies.Altogether these results show a strong effect of animal age onlung colony formation, suggesting that some host defense mechanisms against tumor cells may be less well developed in theyoung rats.

Extrapulmonary métastaseswere observed in a few cases.Thus, in one case a tumor was found on the back of the animal,and some animals developed hind leg paralysis (6/39 of 5-week-old, and 5/40 of 6-week-old rats). These rats were killed andnot included in the survival studies. Autopsy and preliminaryhistológica! examination have revealed that these animals allhad lung colonies, and in addition métastasesclosely associatedwith the spine were detected in the lumbar region of the rats.This finding may explain the paralyses (18), and the phenomenon is now being studied in more detail. Importantly, similarmétastaseshave never been observed in nude mice. Of the ratsthat survived to the end of the 3-month observation period,approximately 1/3 had lung colonies.

Radiography of Lung Tumor Colonies. In attempts to simulatethe situation in patients with lung métastases,we used a technique employing the equipment used for clinical mammographyto monitor the growth of LOX lung colonies in rats. After i.v.injection of 1 x !()'' tumor cells into 5-week-old animals, lung

tumors became visible on X-ray films taken about 17 days later.In Fig. 2 pictures of films taken 1 week (left) and 3 weeks (right)after tumor cell injection are shown. Whereas in the first caseno lung tumors could be detected, after 3 weeks massive tumorinvolvement of the lungs was visible. The minimum size oftumors that could be detected by this method was dependenton the localization of the tumor in the lung, as well as on thetotal number of tumor colonies. In most cases, single colonieswith a diameter down to 2-4 mm were detectable, and asrevealed on autopsy, tumors larger than 7 mm were nevermissed. A higher resolution was not obtained, partly because ofthe breathing of the anesthetized animals.

Chemotherapy Experiments. We have previously shown (6,19) that s.c. LOX xenografts in nude mice are extremely sensitive to the investigational drug mitozolomide, and marginallysensitive to a'j-dichlorodiammineplatinum(II). In comparable

experiments in rats, the s.c. xenografts showed a similar response pattern (Fig. 3), indicating that the pharmacology of thedrugs used and the local tumor environment are comparable inthe two different hosts.

To see whether consecutive X-ray examinations could beused for assessing the effect of chemotherapy in our rat lungcolony model, we monitored the lungs of three 4-week-old ratsinjected with 1 x IO6 LOX cells. On Day 13, the examination

gave pictures that seemed to represent a high number of small,partly distinguishable tumors in the lungs (not shown). Threedays later, when the mitozolomide treatment was started, thecolonies had become more manifest (Fig. 4). A second dose ofchemotherapy was given on Day 23, and the effect of thetreatment is illustrated by a representative picture taken on Day

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Table 1 Lung colony formation in nude rats injected /.»'.with LOX cells. Importance of animal age and number of tumor cells injected

No. of cellsinjected1

x 10'5 x 10!1 x 10sDeaths/totalLife-span

(days)"Age

(weeks)*419/20

16/1810/10513/1812/152/1569/19

7/16NDf418±2

19 ±229 ±6Age

(weeks)*535

±1235 ±10

48628

±538 ±10

NDrNo4409

±97272 ±101

52 ±29.

of lungcolonies"Age

(weeks)*556

±2635 ±10

96121

±4435 ±24

NDC

°Mean ±SD.ftAge at time of tumor cell injection.' ND, not determined.

Fig. 2. X-ray pictures of a nude rat injected at 5 weeks of age. taken 1 week(left) and 3 weeks (right) after i.v. injection of 1 x 10' LOX cells.

28, where no tumors could be detected in the lung. Whereasuntreated rats died after a median time of 18 days, the treatedanimals survived for 3 months with no sign of disease.

In Fig. 5 the tumor response to chemotherapy in an animalwith more advanced disease is shown. In this case, one 5-week-old nude rat was injected with 1 x IO6 LOX cells and treated

twice (on Days 28 and 35) with mitozolomide. With the use ofthe mammography equipment, X-ray pictures were taken onDay 28, when the animal had clear signs of respiratory distress,and again on Day 50, when the rat showed no sign of lungdisease. As seen in Fig. 5, advanced tumor involvement of thelung was present before chemotherapy. In contrast, 22 daysafter the first treatment no tumors could be detected in theright lung, but some tissue infiltration seemed still to be presentabove the heart in the left lung.

The data show that the radiographie method can be used tofollow the course of the malignant disease in the rat lungs, aswell as to evaluate the effect of experimental therapy.

DISCUSSION

In the present study, it was found that the LOX humanmelanoma formed experimental lung metastasis after i.v. injection into athymic rats, in a similar way as previously observed(6) in nude mice. Moreover, we demonstrate that repeat X-rayexamination can be used in rats for monitoring the size of thelung tumors. This method may be particularly useful in therapyexperiments. Importantly, the number of lung colonies and thelife-span of the rats were influenced by the age of the animalsat the time of injection of the LOX cells, a situation not seenin mice.

7000-

6000-

5000-

0 3000

2000-

1000-

Control

CDDP

2 4 6 8 10

Days after start of treatment12 14

Fig. 3. Subcutaneous tumor growth of LOX xenografts in groups of nude ratstreated with mitozolomide. «.v-dichlorodiammincplatinum(Il) (CDDP) or left asuntreated controls. The drugs were given i.v. as a single dose when the tumorshad reached a size of approximately 4x4 mm. The tumor diameters weremeasured three times weekly with calipers, and tumor volume (mean ±SD) wascalculated.

For several purposes the size of the nude rat makes it anattractive host for tumor xenograft studies. However, a reducedmalignancy of xenografted tumors in nude rats was indicatedby reports that murine tumors, which are highly metastaticfrom s.c. sites in syngeneic mice, grew noninvasively and didnot produce spontaneous métastasesin nude rats (20, 21).Moreover, some authors have reported poor take rates of human cancers transplanted s.c. in nude rats, and other authorshave observed spontaneous regression of such tumors (13, 22-24). In contrast, Basiert et al. (25) and Salomon et al. (21)reported similar and high take rates for several human cancersin nude rats and in nude mice.

The apparent inconsistency in the published results of workwith xenografts in nude rats may have several explanations:The success in obtaining tumor take may differ with the ratstrain used, as it is possible that the effect of the nude mutationto some extent may differ with the genetic background of theanimals; the health status of the rats may influence the results;and the limited number of experiments performed, each involving relatively few animals, may not give a representative picture

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Fig. 4. X-ray pictures of a nude rat injected at 4 weeks of age with 1 x 10*LOX cells and treated on Days 16 and 23 with mitozolomide (15 mg/kg). Thepictures were taken at the start of treatment on Day 16 (left) and on Day 28(right).

Fig. 5. X-ray pictures of a nude rat injected at 5 weeks of age with 1 x IO6

LOX cells and treated on Days 28 and 35 with mitozolomide (15 mg/kg). Thepictures were taken at the start of treatment Day 28 (left) and on Day 50 (right).

of the usefulness of the nude rat for such research. In addition,it is well known (26-28) that the take rate of human cancers innude mice differ with tumor type, as well as with individualcharacteristics of the tumor.

In the present study, the take rate and the volume doublingtime of s.c. LOX tumors were the same in rats as those previously found in nude mice. The tumors could be passaged seriallywith no change in important characteristics. The morphologicalpictures of the s.c. and lung tumors in rats and mice wereclosely similar, and the mean life-span of 5-6-week-old ratsinjected i.v. with 5 x 105-1 x 10" cells varied between 28 and

35 days, similar to what was previously found in mice (6). Thedata suggest, therefore, that healthy Rowett nude rats as usedhere, have a promising potential for use in studies involvinghuman tumors. Together with the findings in mice (6), thepresent data show that the LOX cells have a preference forgrowth in the lungs of the host, in agreement with the seed andsoil theory originally proposed by Paget (29).

A clear relationship between colonization of the lungs andthe age of the animals was found. Thus, in 4-week-old rats thenumber of lung colonies was higher and the life-span was

shorter than in the older animals receiving the same number ofcells. In the young rats, a correlation was found between thenumber of cells injected and the number of detectable lungcolonies, whereas this relationship was less clear in older animals.

The pronounced differences between 4- and 5-6-week-oldrats in lung colony formation are surprising. However, otherinvestigators have observed variation in s.c. tumor growth withage of nude rats. Thus, Maruo et al. (30) and Partridge et al.(31) showed that human tumors implanted in 4-week-old ratsgrew more rapidly, and consequently higher tumor weights wereobtained within the observation time, than in 6-10-week-oldanimals. It is noteworthy that in the present study the s.c.growth of LOX xenografts did not differ with the age of theanimals.

In nude mice, age-dependent variation both in s.c. growthand metastasis formation of human cancers has been reported.Sordat et al. (32) obtained higher take rates in newborn nudemice than in adult animals, and Hanna and Fidler (17) observedincreased metastatic potential of several tumor lines in 3-week-old compared to 6-week-old mice. These findings may be explained by immunological factors, as it is known (17, 33) thatvery young mice are less immunologically mature than adultanimals. However, in a previous study in our laboratory wecould not demonstrate any difference in the life-span of youngand adult nude mice injected i.v. with B16F10 and LOX cells(33).

The present data obtained with LOX cells in rats suggestthat in this species immunological differences between 4- and5-6-week-old animals exist that may explain the higher takerate of i.v. injected cells in the younger animals. This notionwas further supported by the finding that a few 2-week-old ratsinjected successfully i.v. with 1 x IO6 LOX cells, died evenearlier (after 13-16 days) than 4-week-old animals (data notshown). The effector cells conceivably responsible for the observed age-dependent differences may include NK cells (30) andcytotoxic macrophages (31, 34), as well as B-lymphocytes andeven T-cell-like cells. Because of the complexity involved, elucidation of this issue may require rather comprehensive immunological studies.

The fact that athymic rats seem to be less susceptible toinfections than nude mice may indicate that the nude mutationhas a somewhat less pronounced effect on the immune systemin rats than in mice. The difference between the two species insusceptibility to growth of human tumor cells would then beexpected to increase during the first weeks after birth, reflectingthe maturation of the immune effector mechanisms presumablytaking place in this period. Altogether, the results obtained withthe LOX cells in nude mice and rats seem to be consistent withthis view, which, if correct, also would suggest that youngathymic rats may be used to obtain metastatic models involvingother human cancers.

Human tumor xenograft models in mice have proved particularly useful for experimental chemo- and radiotherapy (19, 26,27), and it is clear that the transplants by and large retain thesensitivity of the parent tumors (26, 27). It has been arguedthat chemotherapy studies involving s.c. xenografts may not berelevant for most human cancers which usually grow at othersites, implying that the local growth conditions may differ fromthose in the patients, and that the pharmacology of the drugstested may also be different (27). With the LOX melanoma,tested in rats (as here), or in mice (6, 19), the response to thecompounds examined did not differ whether the tumor wasgrowing s.c. or in the lungs. It seems clear, however, that for

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several purposes a lung colony model may be more clinicallyrelevant than studies on s.c. xenografts.

The objective with all models for chemosensitivity testing isthat the model shall mimic, as closely as possible, the situationin the patient, in whom change in tumor size during treatmentis most commonly assessed by repeat X-ray examinations.Moreover, it is important to avoid experiments in which theanimals suffer from advanced tumor involvement of any majororgan system, particularly experiments which entail survival asthe end point. The radiographie method described meets bothof these requirements, and here we have demonstrated for thefirst time how the effect of chemotherapy on human tumors ina visceral organ can be followed by the use of X-ray examinations. Since many human cancers frequently metastasize to thelungs, this method may prove particularly useful for therapystudies in those of such tumors that can be grown in the lungsof nude rats. Although the sensitivity of the method is stilllimited, the approach may be used to rapidly screen for significant drug effects.

The use of radiography to assess the growth of xenograftedhuman tumors was first studied in nude mice injected intra-bronchially or intrathoracically with human lung carcinomacells (15, 16). The advantage of the present model is that thecells are not deposited directly into the lung tissue and that thetumors develop more randomly in both lungs. Moreover, thesize of the rats may be advantageous for monitoring tumorgrowth, particularly for the purpose of evaluating the effect oftherapy.

Since the formation of manifest tumor métastasesis the resultof an interaction between the tumor cells and the normal hostcells, comparison of the metastatic abilities of human tumorsin nude mice and rats may provide a new means for elucidatingfactors involved in this interaction. The data obtained here withthe LOX cells demonstrated a similar pattern of metastasisformation in the two species. However, preliminary experiments where we have compared the metastatic patterns ofvarious human tumors, suggest that in some cases the tissuepreferences may indeed be different in the two species, asindicated here by the hind leg paralysis observed in rats but notin mice. This may open new possibilities for identifying factorsin the different tissues that somehow influence whether or notmicrodeposits of tumor cells will develop to manifest metastasis.

ACKNOWLEDGMENTS

The authors want to thank Carin van der Lelie and MarianneMellembakken for excellent technical assistance, and Frances Jaquesfor secretarial assistance.

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1989;49:5148-5152. Cancer Res   Inge Kjønniksen, Ritsa Storeng, Alexander Pihl, et al.   A Human Tumor Lung Metastasis Model in Athymic Nude Rats

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