[CANCER RESEARCH 41, 438-444, February 1981 ]

Expression of Metastatic Potential of Allogenic and Xenogeneic Neoplasmsin Young Nude Mice1

Nabil Manna2 and Isaiah J. Fidler

Cancer Metastasis and Treatment Laboratory. NCI Frederick Cancer Research Center. Frederick. Maryland 21701

ABSTRACT

Several different mouse and rat tumors were injected s.c. ori.v. into specific-pathogen-free young (3-week-old) and adult(6-week-old) nude mice. All tumors grew s.c.; however, follow

ing i.v. injections, only metastatic neoplasms produced visiblepulmonary tumor foci in the 3-week-old (but not in the 6-week-

old) nude mice. The quantitative differences in metastatic potential among tumor lines and clones observed in syngeneichosts were maintained also when 3-week-old nude mice wereused. The enhanced survival of tumor cells in the lungs andthe large numbers of visible pulmonary métastasesobservedin the young nude recipients correlated closely with the lowlevels of natural killer cell activity detected in 3-week-old (butnot in 6-week-old) nude mice. In vivo activation of natural killercells by treatment of mice with polyinosinic-polycytidylic acidor Corynebacterium parvum, 24 hr prior to tumor challengerendered the 3-week-old nude mice resistant to development

of métastases.We conclude that specific-pathogen-free 3-week-old nude

mice, which lack functional T-lymphocytes and demonstrate

low natural killer cell activity, could serve as an in vivo modelto ascertain the metastatic potential of allogeneic and xeno-geneic tumors.

INTRODUCTION

The successful grafting of allogeneic and xenogeneic tumors(including human neoplasms) into athymic nude mice (6, 15,18) has provided an in vivo model for evaluating tumorigenicityof transformed cells of various origins (20, 24, 25). The usefulness of nude mice for studies of cancer metastasis, however,has been limited, inasmuch as most malignant neoplasmstested in this model rarely metastasized after being grafted intoadult nude mice (3, 15, 18, 21). However, métastaseswereobserved when newborn or X-irradiated adult nude mice wereused as recipients (16, 23). Recently, we have reported that 3-week-old syngeneic mice are highly susceptible to the devel

opment of experimental pulmonary metastasis (7). The enhancement of metastasis development in these young micecorrelated with low levels of activity of NK3 cells detected by in

vitro assays (7). These findings prompted us to investigatewhether the age dependence of NK cell activity in nude micealso correlates with successful experimental metastasis of allogeneic or xenogeneic tumors in young (3-week-old) but notadult nude mice. Furthermore, we examined the applicabilityof this in vivo model for detecting qualitative and quantitative

1 Research sponsored by the National Cancer Institute under Contract N01-

CO-75380 with Litton Bionetics, Inc.2 To whom requests for reprints should be addressed.3 The abbreviations used are: NK. natural killer; C3H , C3H/HeN(MTV ).

Received February 4, 1980; accepted October 16, 1980.

differences in the metastatic potential of well-defined tumor

lines.

MATERIALS AND METHODS

Animals

Specific-pathogen-free 3- and 6-week-old NIH-Swissathymic nude mice and 6- to 8-week-old C57BL/6 and C3H~

mice were supplied by the Animal Production Area, FrederickCancer Research Center. The mice were given injections oftumor cells immediately after they arrived in our laboratory.

Reagents

Corynebacterium parvum (Burroughs Wellcome and Co.,Research Triangle Park, N. C.) was administered i.p. at a doseof 175 /ig/mouse. Polyinosinic-polycytidylic acid (1.0 mg/ml;Sigma Chemical Co., St. Louis, Mo.), prepared in phosphate-buffered saline, was administered i.p. at a dose of 100 jug/mouse.

Tumor Cell Lines

Murine Tumors. The UV fibrosarcomas (1591, 1316, 1422,and 2237) were induced in C3H~ mice by chronic irradiation

with UV light (10). SF-19 is a fibrosarcoma that arose spontaneously in a C3H~ mouse (4), and MCA-3256 is a fibrosarcomathat was induced in a C3H~ mouse by s.c. injection of 1 mg

methylcholanthrene. K-1 735 melanoma is syngeneic to C3H

mice. The induction and properties of this tumor have beendescribed elsewhere (11). The in vitro production and characterization of a large number of cloned cell lines from the UV-2237 (13) and K-1 735 (11 ) tumors have been described previously. The B16-F1 (low lung colonization capacity), B16-F10(high lung colonization capacity) and B16-F10Ll (lymphocyte

resistant, low lung colonization capacity) cell lines, syngeneicto the C57BL/6 mouse, were described previously (2).

Rat Tumors. MADB-100 and MADB-200 are mammary ad-

enocarcinomas induced in different F344 rats given a singleP.O. dose of 20 mg of 9,10-dimethyl-1,2-benzanthracene(Sigma). MADB-105 is a selected variant cell line obtained from

a pulmonary metastasis produced by the i.v. injection of cellsof the MADB-100 tumor (22).

All tumor cultures were maintained in Eagle's minimal essen

tial medium supplemented with 5% fetal calf serum, vitaminsolution, sodium pyruvate, nonessential amino acids, and L-

glutamine. The components of this medium, designated complete minimal essential medium, were obtained from Flow Laboratories, Rockville, Md. All cultures were incubated at 37°in

a humidified atmosphere containing 5% CO2. All cultures werefree of Mycoplasma and the following pathogenic viruses:reovirus type 3, pneumonia virus of mice, K-virus, Theiler's

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Metastasis in Nude Mice

Table 1Experimental pulmonary métastasesproduced by murine tumor cells injected i.v. into 3- or 6-week-old nude mice

Mice were given i.v. injections of the designated dose of viable tumor cells and killed 4 weeks later. The number of lung tumor colonies wasdetermined with a dissecting microscope.

Pulmonary métastasesSource of tumor

6-wk-old syngeneic mice 3-wk-old nude mice 6-wk-old nude mice

StrainC57BL/6C3HTumorB16-F18B16-F10aBIG-FIO^SF-19"MCA-3256U

V-1591UV-1316UV-1422"UV-22373Cell

dose5X10"2.5

x10"5x10"2.5

X10'5X10"2.5x10"2

X10s1X10s1X1051X1051x1051x 10sIncidence8/1066/1010/1010/106/103/105/103/100/100/1010/1010/10Median82102481121008061Range0-360-1746-19021-890-20-20-60-7009-16714-138Incidence5/1062/1035/3510/101/101/104/102/50/100/1010/1020/20Median5080361052005790Range0-560-117-3417-960-10-10-100-4006-1213-428Incidence1/1968/201/90/100/50/100/50/100/10Median010000000Range0-10-190-1000000

" The number of métastasesproduced in 3-week-old mice differed significantly from that in 6-week-old nude mice (p < 0.001)."*Number of mice with metastases/total mice given injections.

encephalitis virus, Sendai virus, minute virus of mice, mouseadenovirus, mouse hepatitis virus, lymphocytic choriomenin-

gitis virus, ectromelia virus, and lactate dehydrogenase virus(Microbiological Associates, Bethesda, Md).

Assay for Experimental Pulmonary Metastasis

For in vivo studies, we harvested the tumor cells from sub-

confluent cultures in exponential growth phase by overlayingthe cells with a thin layer of 0.25% trypsin-0.02% EDTA for 1

min. The flask was tapped sharply to dislodge the cells, andcomplete minimal essential medium was added immediately.The cells were then washed and resuspended in Hanks' bal

anced salt solution. Tumor cell viability was about 95%, basedon the ability of the cells to exclude trypan blue. Only suspensions composed of single cells were used for i.v. inoculations.Unanesthetized mice were given injections either s.c. or i.v.The inoculum volume was always 0.2 ml/mouse; however, cellnumbers varied (see Tables 1 and 2). Tumor growths s.c. weremonitored biweekly. Mice were killed 3 to 5 weeks followingtumor cell injection. The s.c. tumors were fixed for histopath-ological examination. Lungs of mice given i.v. injections wereremoved and fixed in Bouin's solution to facilitate visualization

of tumor colonies (2). The number of tumor colonies wasdetermined by 2 independent observers with the aid of adissecting microscope. Tumor deposits were confirmed histo-

logically.

Quantitative Analysis of Tumor Cell Arrest and Survival inVivo

B16-F10 tumor cells prelabeled in vitro with ['"IJiododeox-yuridine (2) were harvested as described above, and 106 single

viable cells were injected i.v. into 3- and 6-week-old nude mice.

Triplicate samples were monitored for radioactivity to determine the amount incorporated per cell. Groups of 5 mice werekilled at intervals ranging from 10 min to 14 days after i.v.tumor cell inoculation. The lungs were excised and processedas described previously (2), and the number of viable labeledtumor cells present in the lungs at time of death was calculated.In addition, the number of visible lung tumor colonies at Day

Table 2Experimental pulmonary métastasesin 3-week-old nude mice given i. v.

injections of cells of metastatìcor nonmetastatic murine tumors

Ten mice per group were given i.v. injections of the designated dose of viabletumor cells and killed 4 weeks later. The number of lung tumor colonies wasdetermined with a dissecting microscope.

PulmonarymétastasesSource

oftumorB16-F10K-1735UV-2237UV-1316UV-1591Celldose1

0,0005,0002,50050.00025,00012,5006,25050.00025,00012,5006,250600,000400,000200,000100,000600.000400.000200.000100,000Median24327118443884000000000Range1-790-640-3015-2108-960-331-200-890-360-200-300000000

14 after tumor injection was determined using a dissectingmicroscope.

Assay for NK Cell-mediated Cytotoxicity

A modification of the 24-hr in vitro cytotoxicity assay described by Shiku ef al. (19) was used. Briefly, tumor targetcells prelabeled with [3H]proline (specific activity, 20 to 40 Ci/

mmol; New England Nuclear, Boston, Mass.) were dispersedinto flat-bottomed wells (10" cells/well) of a microtest culture

plate (Falcon Plastics, Oxnard, Calif.) 24 hr before testing.Effector spleen cells were added to the tumor target cellmonolayers in quadruplicate and incubated at 37°in a humid

ified atmosphere of 5% CO? for 24 hr. The cultures were

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N. Hanna and l. J. Fidler

washed thoroughly with warm medium, and the remainingviable adherent cells were lysed with 0.1 ml of 2% sodiumdodecyl sulfate. The contents of the wells were transferred toglass vials containing 10 ml scintillation fluid, and the amountof radioactivity was monitored in a ßscintillation counter. Thedegree of killing was calculated according to the formula:

% of cytotoxicity = 1 -

Statistical Analysis

cpm in test wells

cpm in media control wellsx 100

The results of in vivo experiments were evaluated by Mann-Whitney U test (2-tailed).

RESULTS

Metastasis Formation. Tumor cell lines and clones differingin their metastatic potential and/or immunogenicity (as determined in normal syngeneic hosts) were injected s.c. or i.v. into3- and 6-week-old nude mice. All tumors grew s.c. in mice of

both age groups. Differences in formation of grossly visiblemétastases(Fig. 1), however, were quite striking.

The data shown in Table 1 demonstrate that tumor cells thathave high metastatic potential in syngeneic recipients produceonly a few lung tumor colonies following i.v. injection into 6-week-old nude mice. In contrast, the same number of cellsinjected i.v. into 3-week-old nude mice produced a significantlyhigher number of pulmonary métastases.The B16-F10 mela

noma variant selected for its high lung colonization potential inC57BL/6 mice (2) produced significantly more tumor coloniesthan did the B16-F1 or B16-F10Lr variants with low lung colo

nization potential. The outcome of experimental metastasis inthe young nude mice paralleled the pattern observed when theB16 lines were injected i.v. into syngeneic C57BL/6 recipients(Table 1).

We also examined the metastatic behavior of several UV-induced fibrosarcomas (syngeneic to C3l-r mice) following i.v.injection into 3- and 6-week-old nude mice and 6- to 8-week-old C3H mice. UV-induced fibrosarcomas are known to differ

in their immunogenicity and metastatic potential (10, 13). Forexample, UV-1316 and UV-1591 are nonmetastatic, highly

immunogenic tumors that regress when transplanted into normal C3H~ mice. However, these tumors will grow progressively,

albeit locally at an injection site, in immunosuppressed recipients such as UV-irradiated syngeneic recipients. No sponta

neous or experimental metastasis can be produced by thesecells in any type of host (Tables 1 and 2) (4). Here again, theinjection of cells from UV-1316 or UV-1591 into either 3- or 6-week-old nude mice failed to produce pulmonary métastases.

As stated above, tumor growths did occur when cells wereinjected s.c. This finding and our previous results (4) indicatethat the failure of these tumors to metastasize is not necessarilyrelated to their immunogenicity. The i.v. injection of cells fromUV-1422 and UV-2237 tumors into 3- but not 6-week-old nude

mice resulted in the formation of large numbers of métastases.The low-metastatic SF-19 and MCA-3256 fibrosarcomas produced only a small number of lung tumor colonies in 3-week-old nude mice but produced none in 6-week-old nude mice.

Again, the incidence and extent of experimental metastasisfound in 3-week-old nude mice closely paralleled that observedin adult syngeneic recipients (Table 1).

In this and previous studies (4, 5), we classified tumors ashaving low to high metastatic potential. These are obviouslyrelative terms. Therefore, we next examined the formation ofpulmonary métastasesin 3-week-old nude mice following the

i.v. injection of graded doses of tumor cells. The data shown inTable 2 demonstrate that, for the metastatic tumors, B16-F10,K-1735, and UV-2237, even a low number of injected tumor

cells yielded some métastases.In contrast, the i.v. injection ofup to 6 x 106 cells of the nonmetastatic UV-1591 and UV1316

fibrosarcomas yielded no pulmonary métastases.The injectionof more than 6 x 105 cells into 3-week-old nude mice resulted

in immediate death from pulmonary embolism.Mouse neoplasms are known to be heterogeneous and to

contain subpopulations of cells with differing metastatic potential (2, 5, 13). We tested the metastatic behavior of severalcloned populations isolated from the parental tumors UV-2237(2, 13) and K-1735 melanoma (11) following i.v. injection into3-week-old nude mice (Table 3). The UV-2237 clones 38 and

46 are highly immunogenic and weakly metastatic when injected into normal C3H~ mice (2, 12, 13). Following i.v. injection into specifically immunosuppressed, UV-irradiated C3H~

mice or 3-week-old nude mice, cells of clone 46 (but not clone

38) produced a large number of pulmonary métastases(Table3). The failure of some UV-induced tumors O.e., UV-1316, UV-

1591, clone 38) to grow in lungs of nude mice is not caused byan inability of these cells to proliferate. These tumors growprogressively when injected s.c. into nude mice. We obtainedsimilar results with clones isolated from the K-1 735 melanoma

(Table 3). Clones 13,14, and 16 are of low metastatic potentialin normal syngeneic mice, whereas clone 13, similar to clone46 of UV-2237, readily metastasizes following injection intoyoung nude mice. The parent K-1735 line and clones 2 and 4

produced large numbers of lung colonies following injectioninto both normal syngeneic C3H~ and 3-week-old nude mice.

We next examined the metastatic potential of rat tumor cellsfollowing i.v. injection into 3- and 6-week-old nude mice (Table4). Injections of rat MADB-100 and 105 variant cell lines

produced a significantly larger number of lung tumor coloniesin 3-week-old than in 6-week-old mice. Moreover, in both agegroups, the MADB-105 variant, selected for high lung coloni

zation potential in syngeneic F344 rats, was more metastaticthan its MADB-100 counterpart. Cells from the MADB-200tumor formed few lung tumor colonies in both 3-week-old nude

mice and syngeneic rats. Thus, the biological behavior of therat tumors following injection into young nude mice paralleledthat observed in syngeneic normal recipients (Table 4).

Expression of Natural Cytotoxicity in Vitro against B16Melanoma, UV-2237 Fibrosarcoma, and K-1735 MelanomaTumor Cell Lines. NK cells play a significant role in thedestruction of circulating tumor emboli (7, 17). In this study,we tested whether there is a correlation between the levels ofNK cell activity in 3- and 6-week-old nude mice and the

outcome of experimental metastasis in these recipients. TheB16, UV-2237, and K-1 735 tumors were tested for sensitivity

to killing by normal spleen cells obtained from adult nude mice.All 3 cell lines were lysed by NK cells in a 24-hr in vitrocytotoxicity test. The UV-2237 and K-1735 tumor cells were

more susceptible to killing than the B16 melanoma (Table 5).Spleen cells obtained from specific-pathogen-free 3-week-old

nude mice exhibited low levels of NK cell activity (2 to 20%),whereas high levels (20 to 80%) consistently were found in 5-

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Metastasis in Nude Mice

Table 3

Experimental pulmonary métastasesproduced by murine tumor cells injected i. v. into syngeneic normal orUV-irradiated mice and into 3-week-old allogeneic nude mice

Mice were given i.v. injections of 105tumor cells and killed 3 to 4 weeks later. The number of lung tumor

colonies was determined with a dissecting microscope.

Pulmonary métastases

Normal C3HT UV-irradiated C3H 3-wk-old nude mice

Source oftumorUV-2237

parentClone38Clone46aK-1

735parent"Clone

16Clone14Clone13aClone

4Clone2Median500.513302.

0156123Range25-1740-120-50-1260-20-100-2101-19641-168Median148139Range96-2170-417-64Median902.5115226001845104Range3-2480-2769-30035-3710-201-8014-1329-284

The number of lung tumor colonies developed in 3-week-old nude mice or UV-irradiated recipientsdiffered significantly from that in normal C3H mice (p < 0.001).

Table 4Experimental pulmonary métastasesproduced by xenogeneic rat tumor cells injected i.v. into 3- and 6-week-old nude mice and F344 rats

Mice or F344 rats were given i.v. injections of 1 x 105viable tumor cells and killed 3 to 4 weeks later. The number of lung tumor colonies

was determined with a dissecting microscope.

MADB100F344

rat3-wk-oldnude mice

6-wk-old nude miceIncidence6/10a20/20

9/9Median1034

2Range0-406-820-6MADB

105Incidence10/10"

10/1010/10Median41

918Range6-21120-3202-49MADB

200Incidence4/10a

6/101/9Median4

80Range0-150-260-3

3 Number of animals with metastases/total animals given injections.0 The number of métastasesproduced in 3-week-old mice differed significantly from that in 6-week-old mice ( p < 0.001 ).

Table 5Levels of NK cell activity and experimental tumor métastasesin 3- and 6-week-old nude mice

3-wk-old mice with the followingtreatment36-wk-old

miceSource

oftumorB16-F100

UV-2237'K-1735%

of cyto-toxicity(range)20-45

45-8042-77Pulmonary

métastasesMedian1

00Range0-8

00-1%

of cyto-toxicity(range)02-5

5-187-20NonePolyinosinic-polycytidylic

acidPulmonary

métastasesMedian108

65112Range32-300"

11-135"28-300"%

of cyto-toxicity(range)622-45

55-8451-75Pulmonary

métastasesMedian12

57Range0-45e0-9e0-17eC.

parvum%

of cyto-toxicity(range)620-49

47-6843-72Pulmonary

métastasesMedian

Range231-68e

11 0-18"ND9

Mice were treated i.p. with 100 fig polyinosinic-polycytidylic acid or 175 jugC. parvum 24 hr before in vitro testing for NK cell activity or in vivo challenge withtumor cells.

b Range of killing percentage by normal spleen cells at 1.5 x 106cells/well obtained in 6 individual tests.0 50,000 cells/mouse." The number of lung tumor colonies in 3-week-old nude mice differed significantly from that in 6-week-old nude mice (p < 0.001).e The number of lung tumor colonies in 3-week-old nude mice pretreated with polyinosinic-polycytidylic acid or C. parvum differed significantly from that in

untreated 3-week-old control mice (p < 0.001).' 100,000 cells/mouse.3 ND, not determined.

to 10-week-old nude mice. A similar relationship between age

and NK cell activity has been reported previously (8). Thetreatment of 3-week-old nude mice with agents such as polyinosinic-polycytidylic acid and C. parvum that induce interferonboosted NK cell activity to levels comparable to those found in10-week-old mice (Table 5). Three-week-old nude mice weretreated with polyinosinic-polycytidylic acid or C. parvum 18 to24 hr prior to the i.v. injection of either B16-F10, UV-2237, orK-1 735 cells. As shown in Table 5, activation of NK cells by

such agents markedly inhibited the production of pulmonarymétastases. Effective prevention of metastasis developmentcould be achieved only when the treatment was initiated shortly

before but not after tumor cell inoculation. Administration ofeither drug 48 hr after i.v. challenge was ineffective.

Kinetics of Tumor Cell Arrest and Survival in 3- and 6-Week-Old Nude Mice. The high susceptibility of 3-week-old

nude mice to experimental metastasis could have been due toincreased efficiency of tumor cell arrest in the vascular bed ofthe lungs or to enhanced survival of the arrested tumor cells.To distinguish between these possibilities, we investigated thepattern of arrest and clearance of [125l]iododeoxyuridine-la-

beled B16-F10 tumor cells following i.v. injection into 3- and 6-week-old nude recipients.

As illustrated in Chart 1, the numbers of tumor cells arrested

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N. Hanna and l. J. Fidler

100000

lc 10000

1000

100

10

4 24

Time after tumor injection (ht)

96

Chart 1. Arrest and survival of 1 x 105 ['25l]iododeoxyuridine-labeled B16-

F10 tumor cells in lungs following i.v. injection into nude mice 3 (•)and 6 (•)weeks old. Each point represents the mean value of 5 mice. The variability withinthe groups did not exceed 10% The responses of the different age groupsobserved at 4. 24. and 96 hr after tumor challenge differed significantly (p <0.001)

initially in the pulmonary vascular bed were similar in both agegroups. However, the survival of tumor cells (4 to 96 hr afteri.v. injection) in lungs was significantly higher in 3-week-oldthan in 6-week-old recipients (Chart 1). The enhanced survivalof tumor cells in 3-week-old mice correlated with increased

numbers of visible métastasesdetected 14 days after tumorchallenge (>300 tumor colonies).

DISCUSSION

In the present study, we have shown that 3-week-old nudemice that lack functionally mature T-cells and exhibit low NKcell activity are susceptible to experimental metastasis of allo-

geneic and xenogeneic metastatic tumors, including those thatare highly immunogenic or sensitive to NK cell killing. Thevalidity and applicability of this in vivo model for determiningmetastatic potential of tumor cells were tested by comparingthe in vivo behavior of several tumor cell lines, variants, andclones with well-defined metastatic characteristics followinginjection into syngeneic normal and immunosuppressed hostsas well as into 3-week-old nude mice (Tables 1 and 2).

The metastatic patterns of allogeneic and xenogeneic tumorswere strikingly similar when injected into syngeneic hosts and/or young nude mice. All metastatic tumors produced lung tumorcolonies in young nude mice, whereas nonmetastatic neoplasms did not. Moreover, the quantitative differences in formation of experimental métastases among tumor lines andclones observed in syngeneic hosts were also found whenyoung nude mice served as recipients.

There are at least 2 possible explanations for the ability orinability of various tumor cells to metastasize. Tumor cells maypossess all properties required to complete the metastaticprocess but they also may be highly immunogenic. Such cellswill be rejected in a normal host but will metastasize in animmunoincompetent syngeneic animal (4) or in young nudemice (Table 3). Alternatively, the degree of immunogenicity ofthe tumor cells may not be pertinent because the cells mayalso lack one or another property necessary to complete the

complex metastatic process (e.g., detachment, invasion, extravasation, etc.). The use of immunosuppressed syngeneic hosts(4) or young nude mice (Tables 1 and 2) allowed us to distinguish between these; thus, metastatic tumor cells that arehighly immunogenic are able to successfully metastasize (Table3, clone 46 of UV-2237 fibrosarcoma). In contrast, tumors that

are nonmetastatic, irrespective of their immunogenicity, do notmetastasize in either normal or immunosuppressed hosts (Table 2, UV-1316 and UV-1591 fibrosarcomas).

Although i.v. injection of tumor cells bypasses the first fewsteps in the metastatic process (i.e., detachment from theprimary tumor mass and invasion of blood vessels), manysubsequent barriers must be overcome before lung métastasescan be established (5, 26, 27). In 3-week-old nude mice, theNK and T-cell immunological barriers are removed, thereby

enhancing tumor cell survival and formation of visible lungtumor colonies (Chart 1). Differences in initial tumor cell arrestin the lung could not account for the high susceptibility of theyoung nude recipients to metastasis development. Moreover,activation of NK cells by treatment with Interferon inducersbefore tumor challenge rendered the young nude mice resistantto metastasis (Table 5). Such treatment was found to enhancetumor cell death and clearance from the lungs without influencing initial tumor cell arrest (17). Further support for the role ofNK cells in prevention of experimental metastasis comes fromstudies in which partial depletion of NK cells by cyclophospha-

mide treatment led to a dramatic enhancement of tumor métastases in both syngeneic (7) and 10-week-old nude mice.4

Adoptive transfer of nylon wool-nonadherent, non-T spleen

lymphoid cells before tumor challenge abolishes the enhancingeffect of cyclophosphamide on metastasis formation (7). In thissystem, the reconstitution of drug-treated mice with spleencells obtained from adult 6- to 10-week-old nude donors was

more successful than that with spleen cells obtained fromyoung 3-week-old nude mice (7).

Although certain immune adjuvants activate NK cells andmacrophages, the difference in the time required for in vivoactivation of these cells rules out macrophage involvement inthe present system. For example, activation of NK cells by C.parvum takes place 16 to 24 hr after treatment (7), whereasthe same agent requires 5 to 7 days to activate macrophages.4

In our present study, the inhibitory effect of adjuvant treatmenton metastasis formation could be demonstrated only whenstimulants were given before tumor challenge. Treatment at orlater than 24 hr after i.v. tumor inoculation was ineffective. Thisindicates that the effector cells are functional during a shortperiod of time following tumor cell injection (12 to 24 hr) andthat the circulating tumor cell is the primary target for theeffectors generated. The effector cells active against the [3H]-

proline-labeled adherent tumor target cells used in the present

study appear to be similar, if not identical, to NK cells activeagainst lymphoma target cells in the short 4-hr 51Cr release

assay. They are cyclophosphamide sensitive, nylon wool non-

adherent, and present at low levels in beige mice; the expression of the activity is age and strain dependent. Moreover, theyare sensitive to treatment with anti-NKi ? antibodies and complement.5

It must be emphasized that the use of specific-pathogen-free

4 N. Hanna, unpublished observations.5 N Hanna, manuscript in preparation.

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Metastasis in Nude Mice

nude mice is crucial for the successful growth and metastasisof allogeneic and xenogeneic tumors. Recent reports clearlydemonstrate that nude mice infected with murine hepatitis virusor other pathogenic organisms exhibit increased NK cell activity (1) and enhanced resistance against transplanted xenogeneic human neoplasms (9, 14). The ability of allogeneic murineand xenogeneic rat tumor cells to establish lung tumor colonieswhen injected i.v. into specific-pathogen-free young nude micecould provide the basis for future investigations of the meta-

static potential of other xenogeneic tumors, including those ofhuman origin.

ACKNOWLEDGMENTS

We thank Zoa Barnes, Karen Cooper, and Mark Schneider for their technicalassistance.

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FEBRUARY 1981 443

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Fig. 1. Visible métastasesin cross-sections of lungs collected from young nude mice given i.v. injections of tumor cells 4 weeks earlier. A, B16 melanoma; B. UV-

2237 fibrosarcoma; C. K-1735 melanoma.

444CANCER RESEARCH VOL. 41

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1981;41:438-444. Cancer Res   Nabil Hanna and Isaiah J. Fidler  Neoplasms in Young Nude MiceExpression of Metastatic Potential of Allogenic and Xenogeneic

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