Download A Comparison of Three in Vivo Assays for Cell

[CANCER RESEARCH 34, 105-108, January 1974]
A Comparison of Three in Vivo Assays for Cell
Tumorigenicityl
John C. Petricciani, Roslyn E. Wallace, and Donald W. McCoy
Division of Pathology, Bureau of Biologies, Food and Drug Administration, Rockville, Maryland 20852 fJ. C. P./, and Lederle Laboratories, Pearl
River, New York 10965 [R. E. W., D. W. M./
cortisone-treated adult hamster with those in the ATS-treated
newborn hamster and the ATG-treated nonhuman primate.
SUMMARY
The cortisonized adult hamster, the antithymocyte-treated
newborn hamster, and the antithymocyte
globulin-treated
monkey were compared as in vivo test systems for the
tumorigenic potential of cells. The newborn hamster and
monkey systems were more consistent in allowing the
expression of tumors than was the cortisonized hamster when
each of three human tumor cell lines was assayed. Cells derived
from normal tissues failed to show evidence of tumorigenicity
in any of the three animal systems.
INTRODUCTION
Animal systems remain the most reliable approach to assess
the tumorigenic potential of cells because any in vitro test
such as growth in solid agar still requires validation by a
sensitive in vivo assay system. In 1956 it was demonstrated (2)
that cell lines derived from neoplastic tissues produced frankly
invasive tumors in the cheek pouches of hamsters treated with
cortisone. This in vivo system became the most widely used
test for potential malignancy of cells in culture, and it has
remained the traditional system against which all others are
compared. An alternative assay system using weanling mice
treated with ALS2 was described in 1968 (10), and the
usefulness of the ALS-treated mouse was quickly confirmed
(12). Newborn hamsters treated with ATS were also described
(16) as a simple, rapid, and more sensitive assay system than
was the standard cortisone-treated
adult hamster for the
purposes of testing heterotransplantability
of cells in culture.
More recently, nonhuman primates treated with ATG have
been shown to be sensitive hosts for progressive growth of
human neoplastic cells and virus-transformed cells (8, 9), and
the athymic or "nude" mouse has been found useful for
tumorigenicity studies (11). The question of whether a given
cell line has tumorigenic potential for man cannot be answered
unequivocally using any of the above systems; however, the
nonhuman primate assay offers the closest reasonable ap
proach to human experimentation. This study was directed at
comparing the results of tumorigenicity
assays in the
MATERIALS AND METHODS
ALS, ATS, and ATG. Hamster ATS was prepared as
described previously (16) by giving rabbits 3 injections of
adult hamster thymocytes in adjuvant and collecting the sera 1
week after the last injection. A 1:4 dilution of this serum
allowed 2.5 X 10s KB cells to grow to an average size of 14.2
cu cm in newborn hamsters at 4 weeks. Commercial hamster
ALS was obtained from Microbiological Associates, Inc.,
Bethesda, Md., and Grand Island Biological Co., Grand Island,
N. Y. A 1:2 dilution of these preparations resulted in average
KB tumors of 1.3 cu cm in 1 case, and the other was inactive
in the newborn hamster. Monkey ATG was also prepared in
rabbits as described previously (9) in a manner similar to that
used for the hamster ATS. This material prolonged skin
allograft survival by 9 to 12 days in rhesus monkeys. Rhesus
monkey ALS prepared from lymph node tissue was purchased
from Microbiological Associates.
Cells. Table 1 describes the origin, passage level, and
pertinent characteristics of each of the cell cultures used. The
KB cell line (1) was initially obtained from Dr. George Foley
(Children's Cancer Research Foundation, Boston, Mass.). Cell
line WI-38 (3), RMK, VMK, BSC-1 (4), and HEp-2 (13) cells
were all supplied by the Division of Virology, Bureau of
Biologies, Food and Drug Administration.
Cell lines
DBS-FRhL-2 (7, 18), Led-130 (14), DBS-FCL-1 (7, 17),
Led-T, (15), FRhL4 (17), and Led-WIDR (R. Wallace, R. W.
March, and D. W. McCoy. Production of Carcinoembryonic
Antigen by a Cell Line Established from Human Colon
Carcinoma, unpublished.) were established by Dr. R. E.
Wallace. LLC-MK-2 (5) was originally obtained from Flow
Laboratories, Rockville, Md. Cells were propagated in Eagle's
minimal essential medium or Waymouth's MAB 87/3 medium
supplemented with fetal bovine serum at 10% concentration.
Suspensions of each cell type for inoculation were made in
medium without serum and adjusted to provide 1 X IO6 to 7
X IO7 cells/test in monkeys and 2.5 X 10s to 5 X IO6
cells/test in hamsters.
Animals and Methods of Inoculation. Syrian hamsters
1This work was supported in part by Contracts NIH-69-100 and
(Mesocricetus auratus) approximately 24 hr old were inocu
NIH-69-2264 from Bureau of Biologies, Food and Drug Administration.
2The abbreviations used are: ALS, antilymphocyte serum; ATS, lated s.c. with 0.1-ml cell suspensions. The needle was inserted
antithymocyte serum; ATG, antithymocyte globulin; i.d., intrader- just above the base of the tail and directed under the skin
towards the neck to prevent leakage of the inoculum.
mally.
Received February 15, 1973¡accepted October 1, 1973.
Immediately after inoculation, 0.05 ml of ATS was injected
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1974
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105
John C. PetrÃ-cciani,Roslyn E. Wallace, and Donald W. McCoy
Table 1
Description of cells used in tumorigenicity tests
The cells described below were grown in Eagle's minimal essential medium or Waymouth's
MAB 87/3 medium supplemented with 10% fetal bovine serum. Cells were harvested at
confluency and suspended in medium without serum before inoculation into the various test
animals.
CellsNeoplastic
and
passage35-50°10-20°17-25102-110KaryologyAneuploidAneuploidAneuploidAneuploidOrigin
commentsHuman
originKBHEp-2Led-WIDRVirus
epidermoidcarcinomaHuman
laryngealcarcinomaHuman
colonadenocarcinomaHuman
transformedLed-T,Culture
lung fibroblasts transformed
with SV40 virus
Abnormal karyology
BSC-1
86-100
Aneuploid
I RhL-4
60-65
Aneuploid
LLC-MK-2
12-20°
Aneuploid
Normal primate cell lines
Wl-38
18-24
Diploid
Led-130
DBS-FRhL-2
28-30
13-47
Diploid
Diploid
DBS-FCL-1
13-42
Diploid
Primary
RMK
1-3
Diploid
VMK
1-3
Diploid
African green
monkey kidney
Rhesus monkey
fetal lung
Rhesus monkey
kidney pool
Human fetal lung;
substrate used in
virus vaccine
production
Human fetal lung
Rhesus monkey fetal
lung; candidate for
use in virus vaccine
production
African green monkey
fetal lung
Rhesus monkey
kidney
African green
monkey kidney;
substrate used
in virus vaccine
production
" Represents number of passages at Bureau of Biologies or Lederle Laboratories, Pearl River,
N. Y., not total culture passages.
i.p. In most tests a 2nd injection of ATS was given 7 days
later. Cell suspensions in 0.1 ml were also inoculated i.d. into
the cheek pouches of adult, 60- to 70-g Syrian hamsters by the
technique of Foley et al. (2). Adult hamsters were given s.c.
injections of 2.5 mg of cortisone in 0.05 ml once on the day
the cells were inoculated and thereafter twice a week. All
hamsters were observed once each week for at least 4 weeks.
Three diameters of the tumors were measured with a
graduated caliper and the tumor volume was expressed as 0.52
of the product of the diameters in cu mm. Selected animals in
each experiment were killed and their tissues were preserved
for histological examination.
Newborn, infant, and juvenile rhesus monkeys (Macaca
mulatta) were obtained from either the Primate Quarantine
Unit, Veterinary Resources Branch, NIH, Bethesda, Md.. or
Litton Bionetics, Inc., Kensington, Md.; African Green
monkeys (Cercopithecus aethiops) of the same age ranges were
106
from the NIH colony or from the colony maintained at Litton
Bionetics by the Bureau of Biologies, Food and Drug
Administration.
ATG was injected s.c. on the abdomen of test monkeys the
day before inoculation of the cells and on Days 1 and 3 after
cell inoculation. Newborn or infant monkeys received ATG,
50 mg/kg, at each injection, whereas the juveniles received
ATG, 7 to 13 mg/kg, at each injection. Cell suspensions (1.0
ml) were inoculated i.m. into the midbiceps or anterior thigh
on Day 0. KB cells served as the positive control for each test
animal and were inoculated into the right biceps, since it had
been shown previously that these cells grew progressively in
this system. Each cell type was inoculated into a minimum of
3 monkeys, and all monkeys were observed for evidence of
tumor formation; but an open biopsy of the inoculation site
was performed between Days 9 and 16 even without gross
evidence of tumor formation. Biopsy material was fixed in
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Comparison of Cell Tumorigenicity Assays
mercuric sublimate-formal (B-5) (6), dehydrated, cleared, and
embedded in paraffin. Histological sections were stained with
hematoxylin and eosin.
RESULTS
Table 2 summarizes the results of cell inoculations into the
3 animal systems studied in this report. Those cells that were
tumorigenic in the hamster cheek pouch (KB, HEp-2, Led-Ti,
and Led-WIDR) were also tumorigenic in the ATS-treated
newborn hamster and the ATG-treated monkey. However, the
tumorigenicity of these cell lines was more consistent in the
ATS-treated newborn hamster than in the adult hamster test
(p = 0.05). The ATG-treated monkey was a more consistent
host for KB and Led-Ti cells than was the adult hamster (p =
0.05). One commercial preparation of anti-hamster ALS was
of comparable potency to our ATS preparation as judged by
the high percentage of KB tumors, but the average tumor size
was less than 50% of that seen in ATS-treated animals. The
other commercial source of anti-hamster ALS showed no
immunosuppressive activity in this system. The one commer
cial preparation of anti-rhesus ALS that was tested showed no
activity in terms of allowing the development of typical KB
tumors.
DISCUSSION
The results of this study confirm and extend our previous
observations (16) that the newborn hamster treated with ATS
is more sensitive than the cortisone-treated
adult for the
purpose of testing heterotransplantability
of cultured cells.
The tumors produced by the same inoculum of KB cells were
significantly larger in hamsters treated with ATS; and the
latter system supported the growth of HEp-2, Led-WIDR, and
Led-T! cells more consistently than did the cortisone-treated
adult hamster. The ATG-treated monkey is equally as sensitive
an assay system for cell tumorigenicity as is the ATS-treated
hamster; and, even though relatively fewer monkeys could be
used for testing, they were more sensitive than the adult
hamster in the case of KB cells and Led-Ti cells.
Although the "nude" mouse appears to be a useful animal
model for tumorigenicity studies, the ALS- or ATS-treated
rodent offers several advantages. Pathogen-free environments
are not required, large numbers of animals are readily available
for testing, and the general condition of the animals does not
deteriorate even with repeated doses of ALS or ATS.
The variability in potency of ALS preparations was clearly
pointed out in tests using 3 commercial sources. Of particular
importance in this regard may be the source of lymphocytes
used in the preparation of a given lot of ALS. Our potent
anti-monkey ATG was made against thymocytes from young
monkeys, while the commercial ALS which failed to show
activity in monkeys was prepared against lymphocytes derived
from peripheral lymph nodes. We have also found that
multiple injections of thymocytes can raise the cytotoxic
antibody titer, but this results in the production of inferior
ATS with lowered immunosuppressive activity.
On the basis of the results reported here, the most
Table 2
Tumorigenicity of various cells in 3 in vivo assays
The cell types listed below were inoculated into: (a) cheek pouches of adult Syrian hamsters
treated with 2.5 mg cortisone on the day of inoculation and twice a week thereafter; (b)
1-day-old Syrian hamsters treated with ATS on the day of inoculation and again 1 week later;
and (c) arms and legs of monkeys treated with ATG the day before inoculation and I and 3
days following inoculation. From 1 X 10' to 7 x 10' cells/test were inoculated into the
monkeys, and 2.5 X 10s to 5 X 10* cells/test were used for the hamster studies. The larger cell
inocula were used for those cells that showed no evidence of tumorigenicity in the initial trials
with lower numbers of cells.
No. with tumor/no, inoculated
inoculatedNeoplastic
Cells
cheek
pouch27/328/1012/189/180/90/30/120/30/30/120/30/240/6ATS-treated
hamster100/106
newborn
monkey38/38
originKBHEp-2Led-WIDRVirus
22/24")42/4327/2850/540/250/70/410/60/80/520/100/810/22ATG-treated
(0/5°;
(0/3°)6/66/66/60/40/30/40/50/40/50/90/40/4
transformedLed-T,Abnormal
karyologyBSC-1FRhL-4LLC-MK-2Normal
primatecell
linesWI-38Led-130DBS-FRhL-2DBS-I;CL-1PrimaryRMKVMKHamster
a Commercial ALS.
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1974
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107
John C. Petricciani, Roslyn E. Wallace, and Donald W. McCoy
reasonable approach to determining the tumorigenic potential
of any given cells would seem to be to test them first in the
rapid, inexpensive, and easily available ATS-treated mouse or
newborn hamster system. If such tests are negative and if
further evaluation of the cells is indicated, additional assays in
the ATG-treated monkey should be considered. Any cells to
be used in the production of material for human use should
ultimately be tested in the ATG-treated nonhuman primate.
As we and others have stated previously (8, 9), none of the
currently available tests can give complete assurance that a cell
line is not tumorigenic. This study suggests that the 3 in vivo
tests examined are not equivalent in their sensitivity and that
the greatest level of confidence can be placed in the results of
tests in the ATS-treated newborn hamster and in nonhuman
primates treated with ATG. Further refinement of these tests
is in progress and may lead to an even greater degree of
sensitivity and reliability.
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CANCER RESEARCH VOL. 34
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A Comparison of Three in Vivo Assays for Cell Tumorigenicity
John C. Petricciani, Roslyn E. Wallace and Donald W. McCoy
Cancer Res 1974;34:105-108.
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