characterization of metastatic clones derived from a metastatic … · that b16 melanoma cells...

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[CANCER RESEARCH 43, 5437-5442, November 1983] Characterization of Metastatic Clones Derived from a Metastatic Variant of Mouse Colon Adenocarcinoma 261 Takashi Tsuruo,2 Takao Yamori, Kuniko Naganuma, Shigeru Tsukagoshi, and Yoshio Sakurai Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo, 170, Japan ABSTRACT A spontaneously metastatic variant (P-26-select) was selected from the murine colon adenocarcinoma 26 after repeated (26 times) cyclic in vivo passage of tumor cells from the lungs of mice bearing s.c. tumors. Clones were established from the cultured P-26-select cell line. These clones, the parent [P-no- select (culture cell line of colon 26 without in vivo selection)] and the metastatic variant (P-26-select) were then studied using three different assays to determine their metastatic potential. These assays included experimental metastasis, spontaneous metas tasis from a nonresected s.c. growing tumor, and spontaneous metastasis following the resection of a primary footpad tumor. In general, there was an agreement among the results of these three assays of métastases,i.e., if a variant was spontaneously metastatic, it was also metastatic in the other assays of métas tases. However, some experimentally metastatic clones did not necessarily show a high spontaneous metastatic potential to the lung. These results might indicate that the metastatic variant (P- 26-select) is populated by spontaneously metastatic clones and also by clones which possess a propensity to metastasize ex perimentally. The morphology and growth properties of the clones were compared also. INTRODUCTION Recent studies on the pathogenesis of cancer metastasis indicate that metastasis is produced by the proliferation of a minor subpopulation of cells that preexist within the parent neoplasm (11, 16, 27). Only a few tumor cells survive the multistep process to produce metastasis (7, 13, 37), and the process is now believed to be a rather nonrandom selection of certain preexisting subpopulations with specific hereditary intrin sic properties (8,27,28,30,31 ). A growing body of data supports this pathogenesis and, at the present time, it is generally ac cepted that individual tumors contain populations of cells that are heterogeneous with regard to metastatic potential (1, 8,10- 12, 17, 20, 22, 24, 30, 33, 36). Recently, it has been reported that B16 melanoma cells contain 2 distinct metastatic popula tions (30). Some metastatic cell lines produce a lung colony after i.v. injection but rarely metastasize after s.c. injection; others are spontaneously metastatic to the lung but produce few lung colonies after i.v. injection. The former is a lung-colonizing (ex perimentally metastatic) population, and the latter is a lung- metastasizing (spontaneously metastatic) population of B16 melanoma cells (30). Many of the highly metastatic lines were isolated from mouse 'This work was supported by Grants-in-Aid (57010027 and 57010051) for Cancer Research from the Ministry of Education, Science, and Culture, Japan. 2To whom request for reprints should be addressed. Received February 7,1983; accepted August 9,1983. tumors. However, these lines were established mainly from soft tissue tumors and sarcomas and, to our knowledge, the isolation of metastatic variants from mouse carcinomas is rather limited (23). Mouse colon adenocarcinoma 26 is a chemically induced undifferentiated carcinoma (3, 4). We started the isolation of metastatic variants from mouse carcinoma, expecting that such variants would aid in the study of cancer metastasis. Colon 26 is one of the signal tumors used for the screening of chemother- apeutic agents by the National Cancer Institute, NIH (14). Estab lishment of a metastatic system by using metastatic variants of colon 26 would also aid in the study of antimetastatic activity of the drugs as well as of the antitumor activity of the compounds. We isolated a metastatic variant from colon 26 by the repeated in vivo procedure and established several clones. Among the clones, some populations have both lung-metastasizing and lung-colonizing activities, while other populations possess a pro pensity to produce an experimental (lung-colonizing) metastasis. MATERIALS AND METHODS Mice and Tumor Female BALB/c mice were obtained from Charles River Japan, Inc., Tokyo, Japan. Colon 26 is a /v-nitroso-N-methylurethan-induced tumor and, by histopathology, an undifferentiated carcinoma (3, 4). The tumor was supplied by Simonsen Laboratories, Inc., Gilroy, Calif., under the auspices of the National Cancer Institute, NIH, Bethesda, Md. Tumor Cell Culture The cells from solid colon 26 tumor or metastatic nodules were established by disaggregation of the tumor by collagenase (35). Briefly, tumors were treated twice with calcium- and magnesium-free HBSS3 containing 0.14% (w/v) collagenase (type I; Sigma Chemical Co., St. Louis, Mo.) and 0.03% (w/v) DNase I (type I; Sigma). The disaggregated cells were collected by centrifugation, and the cells were grown on tissue culture plastic dishes (Corning Glass Works, Coming, N. Y.) in Eagle's minimal essential medium (Grand Island Biological Co., Grand Island, N. Y.) supplemented with 10% calf serum and kanamycin (100 ^g/ml; growth medium) (34). Cells were grown at 37°in a humidified atmosphere of 5% CO2. Establishment of a Highly Metastatic Line in Vivo Colon 26 was implanted s.c. into the flank of 5 BALB/c mice. Three to 4 weeks later, the lungs were removed from individual mice and were implanted s.c. into the abdominal flank of the recipient BALB/c mice. Approximately 2 weeks later, palpable tumor appeared at the inoculation site. Three to 4 weeks later, the lungs were removed and again implanted s.c. into the abdominal flank of the recipient mice. We selected abdominal flank as the site of s.c. inoculation, because the site is relatively avasciHar and, thereby, implanted tumor cells would have less opportunity to be introduced artificially into the blood vessels as suggested previously (30). 3The abbreviation used is: HBSS, Hanks' balanced salt solution. NOVEMBER 1983 5437 on July 22, 2021. © 1983 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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Page 1: Characterization of Metastatic Clones Derived from a Metastatic … · that B16 melanoma cells contain 2 distinct metastatic popula tions (30). Some metastatic cell lines produce

[CANCER RESEARCH 43, 5437-5442, November 1983]

Characterization of Metastatic Clones Derived from a MetastaticVariant of Mouse Colon Adenocarcinoma 261

Takashi Tsuruo,2 Takao Yamori, Kuniko Naganuma, Shigeru Tsukagoshi, and Yoshio Sakurai

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo, 170, Japan

ABSTRACT

A spontaneously metastatic variant (P-26-select) was selected

from the murine colon adenocarcinoma 26 after repeated (26times) cyclic in vivo passage of tumor cells from the lungs ofmice bearing s.c. tumors. Clones were established from thecultured P-26-select cell line. These clones, the parent [P-no-

select (culture cell line of colon 26 without in vivo selection)] andthe metastatic variant (P-26-select) were then studied using three

different assays to determine their metastatic potential. Theseassays included experimental metastasis, spontaneous metastasis from a nonresected s.c. growing tumor, and spontaneousmetastasis following the resection of a primary footpad tumor.

In general, there was an agreement among the results of thesethree assays of métastases,i.e., if a variant was spontaneouslymetastatic, it was also metastatic in the other assays of métastases. However, some experimentally metastatic clones did notnecessarily show a high spontaneous metastatic potential to thelung. These results might indicate that the metastatic variant (P-26-select) is populated by spontaneously metastatic clones and

also by clones which possess a propensity to metastasize experimentally. The morphology and growth properties of theclones were compared also.

INTRODUCTION

Recent studies on the pathogenesis of cancer metastasisindicate that metastasis is produced by the proliferation of aminor subpopulation of cells that preexist within the parentneoplasm (11, 16, 27). Only a few tumor cells survive themultistep process to produce metastasis (7, 13, 37), and theprocess is now believed to be a rather nonrandom selection ofcertain preexisting subpopulations with specific hereditary intrinsic properties (8,27,28,30,31 ). A growing body of data supportsthis pathogenesis and, at the present time, it is generally accepted that individual tumors contain populations of cells thatare heterogeneous with regard to metastatic potential (1, 8,10-

12, 17, 20, 22, 24, 30, 33, 36). Recently, it has been reportedthat B16 melanoma cells contain 2 distinct metastatic populations (30). Some metastatic cell lines produce a lung colony afteri.v. injection but rarely metastasize after s.c. injection; others arespontaneously metastatic to the lung but produce few lungcolonies after i.v. injection. The former is a lung-colonizing (experimentally metastatic) population, and the latter is a lung-

metastasizing (spontaneously metastatic) population of B16melanoma cells (30).

Many of the highly metastatic lines were isolated from mouse

'This work was supported by Grants-in-Aid (57010027 and 57010051) for

Cancer Research from the Ministry of Education, Science, and Culture, Japan.2To whom request for reprints should be addressed.

Received February 7,1983; accepted August 9,1983.

tumors. However, these lines were established mainly from softtissue tumors and sarcomas and, to our knowledge, the isolationof metastatic variants from mouse carcinomas is rather limited(23). Mouse colon adenocarcinoma 26 is a chemically inducedundifferentiated carcinoma (3, 4). We started the isolation ofmetastatic variants from mouse carcinoma, expecting that suchvariants would aid in the study of cancer metastasis. Colon 26is one of the signal tumors used for the screening of chemother-

apeutic agents by the National Cancer Institute, NIH (14). Establishment of a metastatic system by using metastatic variants ofcolon 26 would also aid in the study of antimetastatic activity ofthe drugs as well as of the antitumor activity of the compounds.We isolated a metastatic variant from colon 26 by the repeatedin vivo procedure and established several clones. Among theclones, some populations have both lung-metastasizing andlung-colonizing activities, while other populations possess a propensity to produce an experimental (lung-colonizing) metastasis.

MATERIALS AND METHODS

Mice and Tumor

Female BALB/c mice were obtained from Charles River Japan, Inc.,Tokyo, Japan. Colon 26 is a /v-nitroso-N-methylurethan-induced tumor

and, by histopathology, an undifferentiated carcinoma (3, 4). The tumorwas supplied by Simonsen Laboratories, Inc., Gilroy, Calif., under theauspices of the National Cancer Institute, NIH, Bethesda, Md.

Tumor Cell Culture

The cells from solid colon 26 tumor or metastatic nodules wereestablished by disaggregation of the tumor by collagenase (35). Briefly,tumors were treated twice with calcium- and magnesium-free HBSS3

containing 0.14% (w/v) collagenase (type I; Sigma Chemical Co., St.Louis, Mo.) and 0.03% (w/v) DNase I (type I; Sigma). The disaggregatedcells were collected by centrifugation, and the cells were grown on tissueculture plastic dishes (Corning Glass Works, Coming, N. Y.) in Eagle's

minimal essential medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 10% calf serum and kanamycin (100 ^g/ml;growth medium) (34). Cells were grown at 37°in a humidified atmosphere

of 5% CO2.

Establishment of a Highly Metastatic Line in Vivo

Colon 26 was implanted s.c. into the flank of 5 BALB/c mice. Threeto 4 weeks later, the lungs were removed from individual mice and wereimplanted s.c. into the abdominal flank of the recipient BALB/c mice.Approximately 2 weeks later, palpable tumor appeared at the inoculationsite. Three to 4 weeks later, the lungs were removed and again implanteds.c. into the abdominal flank of the recipient mice. We selected abdominalflank as the site of s.c. inoculation, because the site is relatively avasciHarand, thereby, implanted tumor cells would have less opportunity to beintroduced artificially into the blood vessels as suggested previously (30).

3The abbreviation used is: HBSS, Hanks' balanced salt solution.

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T. Tsuruo et al.

This process was repeated 26 times. Metastasis was not usually grosslyevident before the 20th passage, although histopathological examinationrevealed some micrometastases in the lung. After approximately 20passages, metastatic nodules could be observed macroscopically. Afterthe 26th passage, all mice usually showed macroscopic lung noduleswhen examined 5 weeks after the implantation of the lung.

In Vitro Cloning

Several lung nodules, formed after the 26th passage in vivo, wereremoved and dissociated as described above. The tumor cells werecultivated in the growth medium and were maintained for 2 weeks in invitro culture. This in vivo selected (26 times) culture line was designatedas P-26-select. (The culture line from colon 26 tumor without in vivoselection was designated as P-no-select.) The cells were then cloned in

growth medium by the limiting dilution technique. Cells were diluted to 3cells per ml of the growth medium, and 0.15-ml aliquots were distributed

to each of 96 wells in Microtest II plates (Falcon Plastics, Cockeysville,Md.). After 1 week, wells containing a single colony were marked. Thosewells which infrequently contained 2 clones were discarded. The cloningefficiency was approximately 30%. Approximately 50 clones were isolated. After sufficient growth (5 to 7 x 107 cells), the cloned cells were

stored frozen until used in the experiment. The clones usually weremaintained in vitro for less than 2 months (10 to 12 passages), and freshcells were routinely recovered from the frozen stock.

Assay of Metastatic Ability

The clones were grown in growth medium to subconfluency (5 to 10x 104/sq cm) and were harvested by rinsing the monolayers with calcium-

and magnesium-free HBSS containing 0.14% collagenase (type I;

Sigma):0.03% DNase I (type I, Sigma). After 1 min, the collagenasesolution was removed by aspiration, and the monolayer was incubatedat 37°.After 15 to 20 min, 2 ml of calcium- and magnesium-free HBSS

containing 1% mouse (BALB/c) serum (HBSS-serum) were added, the

monolayer was dislodged by gentle pipeting, and the cells were washedwith 15 ml of HBSS-serum solution by centrifugation. The cells wereresuspended in HBSS-serum and kept in ice for 10 min before they were

counted and injected. Mouse serum was used in order to avoid cellaggregation. Only suspensions containing single cells of more than 95%viability as determined by trypan blue dye exclusion test were used forinjection.

Quantitative Lung Colony Assay

Inoculation i.v. into the Tail Vein. The mice were given injections of5 x 104 tumor cells in 0.2 ml of HBSS-serum into the tail vein and were

killed 23 days later. The lungs were removed, rinsed in distilled watercontaining heparin (1 unit/ml), and fixed overnight in Bouin's solution (9).

The number of tumor foci was determined by counting the surfacecolonies with the use of a dissecting microscope. In some experiments,the removed lung was rinsed in 0.9% NaCI solution containing heparin(1 unit/ml) and was trimmed from the bronchi. The lung was weighed bya microbalance to quantitate tumor metastasis.

Inoculation s.c. into the Flank. The mice were given injections of 5x 10s or 1 x 106 tumor cells in 0.2 ml of HBSS-serum into the flank and

were killed 25 or 29 days later, respectively. The lung colonies werecounted as above. The tumor size (cu cm) was determined at necropsyby: aoz/2 where a and b are the largest and smallest diameters, respec

tively (5).Inoculation s.c. into the Forefootpad. The mice were given injections

of 2.5 x 105 tumor cells in 0.05 ml of HBSS-serum into the right

forefootpad. On Day 17 after inoculation, the right forearms of the miceincluding original tumors were amputated, and the tumor size (thickness)was measured with microcaliper. On Day 40 after tumor inoculation, themice were killed, and the lung colonies were counted as above.

RESULTS

Clonal Heterogenicity of Metastatic Ability after i.v. Inoculation. Metastatic properties of 23 clones isolated from culturedP-26-select were examined after i.v. inoculation into BALB/cmice (Table 1). The in vivo selected parent cell line (P-26-select)showed greater lung-metastasizing potential when the cells wereinoculated i.v. Clones NL-17, NL-18, NL-22, and NL-33 also

produced a larger tumor burden in the lung. The rest of theclones showed much less lung-metastasizing potential. This

might suggest an instability of metastatic potential of cloned cells(1, 2, 21, 25). The metastatic properties of 5 metastatic ornonmetastatic clones from those listed in Table 1 were examinedmore precisely.

The cultured parent colon 26 line without in vivo selection (P-no-select) and the in vivo selected parent cell line (P-26-select)

formed a relatively large number of metastatic nodules in thelung after i.v. inoculation (Table 2). NL-18, NL-17, NL-33, andNL-44 showed a rather weak metastatic potential to the lung.Among the highly metastatic clones, NL-18 formed rather smallnodules in the lung, while NL-17 and especially NL-22 formedrather large nodules in the lung. NL-33 as well as P-no-selectand P-26-select formed medium-sized nodules.

Metastatic Potential of the Clones after s.c. Inoculation.The potential to produce spontaneous metastasis varies amongthe clones, as shown in Table 3. We observed more metastaticnodules in Experiment 2, in which the inoculum size was largerthan that in Experiment 1, and the necropsy was carried out 4days later than that done in Experiment 1. Although the potencyof NL-22 to produce experimental metastasis was not the highestamong the clones (Table 2), NL-22 formed an impressive numberof tumor foci in the lung after s.c. inoculation. NL-17, NL-33, and

Table 1

Metastatic potential of cloned cells to the lung after i.v. inoculationCloned cells (5 x 104) were injected i.v. (tail vein) into each of 10 mice/clone.

The mice were killed 23 days later, and the weight of the lung was measured. Theaverage weight of lungs from a group of mice was compared to that from control

CloneNL-7NL-8NL-9NL-10NL-11NL-14NL-16NL-17NL-18NL-19NL-20NL-21NL-22NL-23NL-26NL-27NL-31NL-32NL-33NL-35NL-38NL-40NL-44P-26-setectMetastaticpotential"+±±±±—±++++++±—+++±__±±++±±±—++Extrapulmonarymetastasis1/1

Oliver1/10

ovary1/1

Oliver2/10ovary1/1

Oliver, 1/10 ovary* Average weight of lung of 10 control mice was 260 mg. Metastatic potential

with regard to lung weight is defined as follows: -, <260 mg; ±,260 to 389 mg;+, 390 to 519 mg; ++, 520 to 649 mg; +++, 650 to 780 mg.

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Metastatic Clones of Colon 26

also parent P-26-select were metastatici however, their potencywas less than that observed for NL-22. These variants were alsometastatic after i.v. inoculation (Table 2). NL-18, NL-44, and P-no-select were rather nonmetastatic in this experiment, althoughthese clones were metastatic after i.v. inoculation. Tumor sizes(mean ±S.D. of 10 mice) of NL-22 and P-26-select at necropsywere 333 ±71 and 386 ±63 cu mm, respectively, in Experiment1, and 1030 ±108 and 1078 ±58 cu mm, respectively, inExperiment 2. These findings might suggest that the strongability of NL-22 to produce spontaneous metastasis is an intrinsiccharacteristic of the cell. Among the clones we have examinedso far, the NL-22 metastasized most efficiently to the lungspontaneously.

NL-22 also metastasized to the lung after inoculation into theforefootpad (Table 4). In this experiment, the original tumor wasremoved on Day 17. Approximately 20 nodules were counted inthe lung. Although NL-17, NL-33, P-26-select, and P-no-selectdid not form impressive metastasis in the lung after inoculations.c. into the flank (Table 3), these clones, especially NL-33,showed an impressive metastatic ability to the lung after s.c.inoculation into the footpad. The tumor sizes (thickness at theresection; mean of 10 mice) of NL-22 and NL-33 were 5.32 ±0.38 and 5.99 ±0.18 mm, respectively. These values are almostthe same as that of P-26-select (5.66 ±0.25 mm). Highlymetastatic properties of NL-22 and NL-33 after footpad inoculation seem to be an intrinsic characteristic of the clone. NL-44and NL-18, which showed no significant metastatic ability to the

Table 2

Metastatic potential of several clones isolated from in vivo selected colon 26 cellsafter i.v. inoculation

Cloned cells (5 x 104)were injected i.v. into the tail vein of BALB/c mice. The

mice were killed 23 days later, the lungs were removed, and the number of tumorfoci was counted.

No. of pulmonarymétastasesCloneNL-17

NL-18NL-22NL-33NL-44P-26-selectP-no-selectMedian53.5

>2003449.51251.597.5Range13->200

75->2000-720-1070-36

22-9116->200Inci

dence20/20

6/619/2019/2018/1910/1010/10PPre

select">0.05

<0.001<0.01>0.05<0.001>0.05P-no-

select6>0.05

>0.05<0.005<0.05<0.001>0.05

lung (Table 3), showed some potential to metastasize to the lungfrom the footpad. The relative vascularity of the footpad mightafford a better opportunity for a metastatic clone to enter intothe blood vessels.

DoublingTime, Size, and Morphologyof Cloned Cells. Thein vivo selected (26 times) parent cell line (P-26-select) wassignificantly smaller in size than the nonselected cell line (P-no-select) (Table 5). Morphologically, these 2 cell lines were fibro-blastic (spindle shaped) (Fig. 1) and grew rather rapidly in culture(Table 6). The nonmetastatic clone NL-44 was spindle shapedand rather small. NL-17 and NL-18, which preferentially metastasized to the lung after i.v. inoculation, were also spindle shaped(the shape of NL-18 is not shown Fig. 1). The NL-17 and NL-18clones were significantly larger than the P-26-select line. NL-17grew rapidly in culture. NL-33 and especially NL-22 were composed of spindle-shaped cells. NL-22 was highly metastatic tothe lung after s.c. inoculation into the flank and footpad, and NL-33 was highly metastatic to the lung after s.c. inoculation intothe footpad (Tables 3 and 4). These clones were larger thanthose of P-26-select and grew rather slowly in culture.

NL-22 and NL-33 grew slightly more rapidly than P-26-selectand P-no-select in mass doubling time in vivo, which was determined from the slope of the growth curve at 10 to 20 days afters.c. inoculation. NL-17 and NL-18 showed almost the samemass-doubling time as did the parent tumors. NL-44, which hadvery low metastatic potential, grew rather slowly in vivo. Although mass doubling time determined at 10 to 20 days afterinoculation does not necessarily reflect the exact tumor size at

Table 4Pulmonarymetastaticpotential ot the clones after s.c. inoculation into forefootpad

Cloned cells (2.5 x 105)were injected s.c. in the right forefootpad of BALB/cmice. The forearms including original tumor were amputated on Day 17 afterinoculation.The mice were killed on Day 40 after inoculation, lungs were removed,and the number of tumor foci was counted.

No. ofmétastasesCloneNL-17

NL-18NL-22NL-33NL-44P-26-selectP-no-selectMedian9.5

119.5

160.566Range0-15

0-226-362-360-60-170-13Incidence9/10

5/1010/1010/105/108/109/10PPre

select">0.05

>0.05<0.02<0.05<0.05>0.05P-no-

select6>0.05

>0.05<0.005<0.01<0.01>0.05

" Probability of no difference from P-26-select (Mann-WhitneyU test).6 Probability of no difference from P-no-select(Mann-WhitneyU test).

8Probabilityof no difference from P-26-select(Mann-WhitneyU test)."Probability of no difference from P-no-select(Mann-WhitneyU test).

Table 3Pulmonarymetastaticpotential of the clone after s.c. inoculation into the flank

Cloned cells (5 x 105in Experiment 1 and 10" in Experiment 2) were injected s.c. in the flank of BALB/c mice. The mice were killed 26 days (Experiment 1) or 29

days (Experiment 2) later, the lungs were removed, and the number of tumor foci was counted.

Experiment 1 Experiment 2

Clone

No. of métastases

Median Range Incidence

No. of métastases

Median Range Incidence P-26-select" P-no-setect6

NL-17NL-18NL-22NL-33NL-44P-26-selectP-no-select0.50330.5010-50-12-1410-40-10-25/102/1010/105/101/107/10>0.05<0.05<0.001>0.05<0.02246.54.5700-1711-1780-130-210-06/1010/109/109/100/10>0.05<0.001>0.05<0.005<0.025<0.001<0.005<0.005

"Probability of no difference from P-26-select(Mann-WhitneyU test)."Probability of no difference from P-no-select(Mann-WhitneyU test).

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7. Tsuruo et al.

Table 5Size ot isolated clones

Cell size was determined when the cells were in log-phasegrowth with a ModelZBI Coulter Counter equipped with a channelizer

CloneNL-17

NL-18NL-22NL-33NL-44P-26-selectP-no-selectSize

(cufirn)1666±161a'6

1516 ± 84"1360 ±132°1480 ±122"1270± 95°1230±102°1760 ±305"

aMean ±S.D. of triplicate determinations."Significantly different (p < 0.05) as compared to P-26-selectby f test.cSignificantly different (p < 0.05) as compared to P-no-setectby i test.

TableóCell and tumor-doubling times of the clones

Doubling time

CloneNL-17

NL-18NL-22NL-33NL-44P-26-selectP-no-setectIn

vitro(hrf21.2±0.2C

22.1 ±1.424.5 ±1.7*

25.3 ±2.822.6 ±2.221.4 ±0.520.7 ±2.0In

vivo(days)62.33

±0.15"

2.57 ±0.202.05 ±0.15'2.13 ±0.20'5.15 + 0.332.56 ±0.102.51 ±0.08

aCalculated from slope of a 96-hr growth curve of cells in log-phase.6Calculated from the slope of growth curve 10 to 20 days after s.c. inoculation

into the abdominal flank. Tumor size was calculated by:

where a and ùare the larger and smaller diameter (5).cMean ±S.D. of triplicate determinations."Mean ±S.D. of triplicate determinations. Eachdetermination was the average

of tumor size of 3 mice."Significant (p < 0.05) by i test as compared to the value obtained with pre

select.Significant (p < 0.05) by i test as compared to the value obtained with P-no-

select.

necropsy, this parameter could be one of the determinants oftumor metastasis.

DISCUSSION

Mouse Lewis lung carcinoma, B16 melanoma, and colon ad-enocarcinomas 26 and 38 were widely used as signal solidtumors in cancer chemotherapy (14). Among these solid tumors,Lewis lung carcinoma is widely used for evaluation of antimeta-static drugs (18). Several metastatic variants have been isolatedfrom the B16 melanoma by Fidler et al. (8,12,15,26). Metastaticpotential of several cultured colon tumors has been examined,and colon 36 cells have been reported to possess a high lung-metastasizing potential (29). However, no metastatic clone orline has been isolated from colon adenocarcinomas. We couldselect a highly metastatic variant from colon 26 by an in vivoprocedure as described. In this in vivo selection process, weinoculated whole lung into the recipient mice because, during theearly in vivo passage, we could not detect any metastatic nodulesmacroscopically in the lung. We needed 20 to 26 passages invivo to obtain reproducible, visible metastatic nodules in the lung.The reasons for this time-consuming selection are not known;however, it is possible that the metastatic population within colon26 tumor is rather limited and that the metastatic potential of theclones is unstable. More importantly, it is possible that wholelung inoculation (but not tumor cell inoculation) may retard the

establishment of a metastatic line because host cell infiltrationor inflammation could occur when the whole lung is inoculated.Furthermore, we must consider the host response to tumor cells,because chemically induced colon 26 could be highly antigenic.Recent reports indicate that macrophage content of neoplasmsis not related to metastatic potential (6, 19, 32). At the presenttime, however, we cannot neglect the influence of lymphocytesand other host cells upon the selection pressure of a metastaticvariant. In this regard, the immunological properties of metastaticvariants are interesting subjects for future studies.

NL-17 and NL-18 might possess a propensity to be classifiedas lung-colonizing clones. According to Stackpole (30), there are

2 distinct cell populations in B16 melanoma, lung colonizing andlung metastasizing. Three of 5 lung metastatic lines were bothmetastatic and colonizing, and the other 2 lines were potentiallymetastatic but at most only poorly colonizing. NL-33 and especially NL-22 possess a propensity to be classified as lung-

metastasizing clones, but these clones also possess enoughpotency to metastasize to the lung after i.v. inoculation. Fromthese findings, we can speculate that lung-metastasizing cloneshave some potency for lung colonizing, while lung-colonizing

clones are not, or are not necessarily, metastatic to the lung.The clone having no colonizing ability, NL-44, does not seem to

be spontaneously metastatic to the lung.The relationship between the metastatic potential and the

characteristics of the clones is not clear at the present time,because the number of clones we examined is rather limited.Some interesting observations are as follows: (a) An in vivoselected line (P-26-select) which has a more enhanced spontaneous metastasizing potency than P-no-select is rather small insize (Table 5); (to) high colonizing clones NL-17 and NL-18 arerather large in size (Tables 2 and 5); (c) the cells of lung-metastasizing clones NL-22 and NL-33 appeared to be roundmicroscopically (Fig. 1); and (d) noncolonizing and nonmetasta-sizing clone NL-44 grows rather slowly in vivo (Table 6). The

reason for this slow growth is not known; however, slow growingproperties in vivo could be one of the most important determinants for the poor formation of lung metastasis after i.v. inoculation. We intend to examine the mechanisms of metastasis ofcarcinoma more closely by using the isolated clones as well astheir potential for use in chemotherapy of metastasis.

ACKNOWLEDGMENTS

We thank Dr. Talmadge for his kind and valuable discussion on the manuscript.We also thank M. Shimizu and E. Sass for typing and editing the manuscript.

REFERENCES

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T. Tsuruo et al.

P-26-selectP-no-select

Fig. 1. Cellularmorphologies of cloned cells. Phase-contrastmicroscopy, x 100. Bar, 0.1 mm.

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1983;43:5437-5442. Cancer Res   Takashi Tsuruo, Takao Yamori, Kuniko Naganuma, et al.   Variant of Mouse Colon Adenocarcinoma 26Characterization of Metastatic Clones Derived from a Metastatic

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