[CANCER RESEARCH 37, 1864-1867, June 1977]
Survival Differences Exhibited by Normal and Transformed Rat
Liver Epithelial Cell Lines in the Aggregate Form1
Anton F. Steuer, Patricia M. Hentosh, Lella Diamond, and Robert C. Ting
Biotech ResearchLaboratories,Rockville, Maryland20852(A. F. S., P. M. H., R. C. T.J,and WistarInstitute of Anatomyand Biology, Philadelphia,
Pennsylvania19104(L. 0.1
SUMMARY
MATERIALS AND METHODS
Normal and transformed rat liver epithelial cell lines ex
hibited differences in the ability to survive in the aggregate
form. Normal nat liver epithebial cells in the aggregate form
underwent a rapid decline in the number of viable cells,
while counterpart transformed epithelial cells exhibited an
ability to survive and proliferate in the aggregate form. This
survival ability was found to correlate with colony formation
Cells. K16, a normal epitheliabrat liver (Sprague-Dawley)
cell line, and W8, an epithelial chemically transformed
in soft agam and tumonigenicity
in nude
mice.
Cell survival
in
the aggregate form could possibly serve as a criterion for in
vitro transformation of epithelial cells derived from matliven.
INTRODUCTION
A variety of criteria have been used to assess in vitro cell
transformation (2, 5, 7, 9, 11, 12, 14); these have been
reviewed by Steuer and Ting (18). Most of these assays have
been used to assess the transformation of fibrobbastic cells.
Morphological criteria used to detect fibroblastic transfor
mation have been reported to be not applicable to epithelial
cells (21). However, colony formation in soft agar has been
reported
to be a reliable
indicator
of epithebial
cell transfor
mation (21, 22). Since carcinomas anise from epithebiab
cells, it is desirable to establish additional criteria form vitro
epithelial cell carcinogenesis that correlate with tumoni
genicity.
We have previously reported that transformed fibroblastic
(rodent) cells in the aggregate form exhibited larger and
more numerous aggregates than counterpart normal cell
aggregate populations (17). It was suggested that these
aggregation differences were due to cell survival differ
ences in the aggregate form. Subsequent studies (15, 16,
18) showed that, over a 4-day period, normal cells in the
aggregate form underwent a rapid decline in the number of
viable cells, while counterpart transformed cells in the ag
gregate form exhibited an ability to survive and in many
cases to proliferate. This cell survival was found to correlate
with colony formation in soft agan and tumomigenicity (15,
16). This study demonstrates
that epithelial
cell populations
derived from rat liver are amenable to this aggregation
method for assessing transformation.
counterpart of Ki6, were kindly supplied by Dr. I. B. Wein
stein; these cell lines have been previously described (22).
Rat liver clone No. 5 (RLC15) is a normal epitheliab cell line
established in our laboratory from a newborn Fischer rat.
WIRL-3 cell lines were derived from the liver of a Wistar mat
(1). WIRL-3C is an untransformed epitheliab cell line, WIRL/
Sv is an SV4O-transfonmed
epithelialWIRL-3cell line, and
R72/3 is an epithelial cell line derived from a tumor pro
duced by a spontaneously transformed WIRL-3 subline. All
cell lines were grown as monolayers in RPMI2 Medium 1640
(Grand Island Biological Co., Grand Island, N. V.) supple
mented with 10% fetal bovine serum (Grand Island Biobogi
cal Co.) and antibiotics (penicillin, 100 units/mI; and strep
tomycin, 100 j.@g/mI).Cell cultures were maintained in an
atmosphere of 5% CO2:95% air at 37°.
Aggregation Method. Cell lines were harvested with
0.025% tnypsin:0.0i% EDTA solution (1:250) for a period of
time not greaten than 10 mm. Tryptic activity was terminated
by the addition of growth medium containing 10% FBS.
After centnifugation (540 x g for 3 mm), the cell pellet was
suspended in liquid growth medium at a concentration of 1
x i0@ viable
cells
pen
ml.
Cell
viability
was
measured
by
trypan blue exclusion. Two x 10@viable cells were seeded
per Falcon 35-mm plastic dish containing a 2-mb agan base
(0.9% Difco agan in McCoy's Medium 5A on RPMI Medium
1640 supplemented with 20% FBS) on a 2-mI agarose (Grand
Island Biological Co.) base (1% agamose electnophonetic
grade, in RPMI Medium 1640 supplemented with 10% FBS).
Dishes were incubated in a humidified atmosphere of 5%
CO2:95% air at 37°.Cell survival in the aggregate form was
assessed over a period of 4 consecutive days in the follow
ing manner. Each viability assessment for a particular cell
line was performed on triplicate dishes for each day. After
collection and centnifugation of cell aggregate populations
from each dish, the pellet from each dish was resuspended
in a solution of 0.05% trypsin:0.02% EDTA (Grand Island
Biological Co.). Trypsmnization was carried out at 37°for a
sufficient amount of time (5 to 15 mm) to cause dispersion
of aggregates into single cells. Growth medium containing
10% FBS was added to stop tryptic
activity.
This trypsin
dispersed single-cell suspension was pelleted and sus
I These
studies
were
supported
by Contract
N01-CP-55640,
cer Institute.
Received October 28, 1976; accepted March 17, 1977.
1864
National
Can
2 The
abbreviations
used
are:
RPMI,
Roswell
Park
Memorial
Institute;
FBS,
fetal bovine serum.
CANCER RESEARCH VOL. 37
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Epithelial Cell Survival in the Aggregate Form
pended in phosphate-buffered saline and trypan blue (0.1%
final dye concentration). Viable (trypan blue-excluding)
cells were counted with the use of a hemocytometer.
RESULTS
a
When rat liver epithelial cells were suspended in liquid
growth medium above an agamon agarose base which pre
vents cell attachment, cell aggregates formed sponta
neously within 24 hr. Figs. 1 to 5 show the morphobogies of
the aggregates 3 days after seeding under the described
conditions. Each cell type had its own characteristic aggre
gate morphology. When these aggregates were collected
and trypsinized in order to disperse the aggregates into
single cells, cell viability was easily assessed by enumema
ting the number of trypan blue-excluding cells. Cell survival
in the aggregate form was determined on 4 consecutive
days. From the survival profiles (Charts 1 and 2), it is appar
ent that the normal rat liver cell lines Ki6, WIRL-3C, and
RLCi5,
underwent
a rapid decline
in the number of viable
cells in the aggregate form, whereas the transformed cell
lines, W8, WIRL/SV, and R72/3, exhibited an ability to sum
vive and to proliferate in the aggregate form. The viability of
the aggregate could also be assessed by its microscopic
appearance. In general, the normal cell aggregates were
small and dank; they became necrotic so that, with time,
there was a buildup of cell debris in the dishes. The trans
formed cell lines formed more numerous cell aggregates
(diffuse or compact) which transmitted light readily. The
ability of cells to survive in the aggregate form correlated
with the ability to produce colonies in soft agar and tumors
in nude mice (Table 1).
uJ
-J
DAYS
Chart 1. Survival profiles of K16, RLCI5, and W8 cells in the aggregate
form. The number of viable (trypan blue-excluding) cells from trypsinized
aggregates was counted on 4 consecutive days. Points, mean count of
triplicate dishes. See “Materials
and Methods―for procedure. •,K16; A,
R1C15; C, W8.
0
(I)
0
0
DISCUSSION
a,
Ca
>
This study demonstrates that normal and transformed mat
liver epitheliab cell lines differ in their ability to survive in the
aggregate form. Normal epitheliab cells in the aggregate
form displayed a marked decline in the number of viable
cells over a 4-day period, while transformed counterparts
survived and proliferated in the aggregate form. This phe
nomenon has been previously described by us (15, 16, 18)
for fibroblastic cell populations. The bases for these sun
vival differences are currently unknown. Various factors
such as P02' pCO2, pH, nutrient availability, and cell prod
ucts may possibly influence the viability of cells in the 3dimensional aggregate form. Leighton and Tchao (8) have
described the effect of oxygen tension on cell viability in 3dimensional matrix culture of tumor explants. Folkman et
a!. (3) have reported the size limitation of cell sphenoids
grown in soft agamdue to the effects on diffusion rates of
nutrients and cell wastes. Under the aggregation conditions
reported here, normal cells may possibly be more sensitive
than transformed cells to metabolic gradients existing
throughout a cell aggregate. Another possible explanation
could be the importance of cell shape in affecting cell
survival. Different spatial tensions may exist between cells
in the 3-dimensional aggregate form, compared with those
occurring with cells interacting with. a culture support.
0
a
z
Days
Chart 2. Survival profiles of WIRL-3C, WIRL/Sv, and R72/3 cells in the
aggregate form. The number of viable (trypan blue-excluding) cells from
trypsinized aggregates was counted on 4 consecutive days. Points mean
count of triplicate dishes. See “Materials
and Methods―for procedure. •,
WIRL-3C; @,
WIRLJSv; A, R72/3.
Changes in the cell shape could possibly affect membrane
transport of essential nutrients. Normal cells in the aggre
gate form may be more sensitive to membrane changes
than transformed cells. Since normal cells require more
growth factors than transformed cells (6), limitation of es
sential growth factors by alteration in membrane transport
due to changes in cell shape could eventually lead to cell
death. The actual mechanisms for cell survival differences
in the aggregate form remain to be elucidated.
In vitro indices of cell transformation should correlate
with the neoplastic state. Stilesetal. (19) have reported that
JUNE 1977
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1865
A. F. Steuer et a!.
and transformed epithelial cell lines derived from other
species and other tissue sources exhibit cell survival differ
ences in the aggregate form.
Table 1
Correlationformationin
of cell survival in aggregate form with colony
soft miceCell
agar and tumorigenicity
survival in
nudeCell
miceRLC15
type
@
in nude
Plating effi-
the aggregate
form―
ciency in soft
aganb (%)
0/3K16
0/3WIRL-3C
0/3W8
—
—
—
3/3WIRL/SV
+
4
1/3R72/3
+
6
+
37
a
, cell line exhibits
in the aggregate
ity in
to remain
REFERENCES
3/3
viable
—, cell line exhibits
and to proliferate
a rapid decline
in the
number of viable cells in the aggregateform.
b Expressed
as
percentage
plating
efficiency
(number
of
cob
nies pen number of cells seeded x 100). The soft agar procedure
has previously been described (15). Colonies 0.1 to 0.125 mm and
larger were counted (averageof 3 dishes) after 2 to 3 weeks of
growth.
C Expressed
as
number
of
nude
mice
per number of nude mice inoculated.
cells/nude mouse s.c.
with
Animals
progressive
received
tumors
5 x 10@
SV4O-transfommedhuman fibnoblastic cell lines were nontu
monigenic in athymic nude mice. These cell lines were
considered transformed due to changes in morphology and
in vitro growth
behavior.
In contrast,
Shin et a!. (13), work
ing with virus-transformed rodent fibmoblastic cell lines,
reported that the only cell property that correlated with
tumorigenicity was anchorage-independent growth in vitro.
Other investigations have also indicated that colony forma
tion in a semi-solid medium without anchorage correlates
very well with tumonigenicity (4, 18). Colony formation in
soft agan has been reported to be a reliable index of in vitro
epithelial cell transformation (21, 22).
The aggregation method described here has the desirable
quality of approaching the 3-dimensional in vivo environ
ment of tumor formation. The formation of cell aggregates
has been reported to be the 1St stage of tumor formation in
vivo (3). Multicell sphenoids or aggregates have been pro
posed as an in vitro model for nodular carcinomas by Suth
emlandet a!. (20). In contrast to growth in soft agam,where
cells are not initially provided with a substratum for anchor
age, cells in the aggregation system initially adhere to one
another, providing a cellular substratum, which would seem
to favor cell survival. However, normal cell populations in
the aggregate form undergo a rapid decline in number of
viable cells.
Namba and Sato (10) have previously described aggrega
tion differences for normal and chemical-transformed rat
liver epithebial cells. Their studies indicated that chemical
transformed rat liven cells formed larger aggregates in a
rotation culture system over a 24- to 28-hr period than
counterpart normal rat liven cell lines. The results reported
here may explain these earlier observations. Since normal
epithelial cells in the aggregate form undergo a rapid de
dine in the number of viable cells, the resultant aggregates
would be smaller than counterpart transformed cell aggre
gate populations.
Assessments of cell survival in the aggregate form as
described here may provide a meaningful reflection of the
transformed status of matliven epithelial cell populations.
Investigations
1866
are in progress
ACKNOWLEDGMENTS
The authors wish to thank Clifton Perkins, Jr. , and Robert K. Bennington
for their excellent technical assistance.
0
0
0
an ability
form;
Tumonigenic
to determine
whether
normal
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CANCERRESEARCHVOL. 37
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Fig. 1. Three-day-old
photographed. x 120.
Fig. 2. Three-day-old
graphed. x 120.
Fig. 3. Three-day-old
dish was photographed.
Fig. 4. Three-day-old
‘
.@
.@-
@1
@.
4A@;@.
@,%,
‘&@@\
c@°•@i@@1r
aggregates of normal K16 cells. The aggregates are diffuse in form and few in number. Representative area of the dish was
aggregates of transformed W8 cells. The aggregates are diffuse and interconnective. Representative area of the dish was photo
aggregates of normal WIRL-3C cells. Aggregates are very small, compared with transformed counterparts. Representative area of the
x 120.
aggregated
of transformed
WIRL/SV
cells. Aggregates
are numerous
and compact
in form. Representative
area of the dish was
photographed. x 120.
Fig. 5. Three-day-old aggregates of transformed R72/3 cells. Aggregates are numerous and compact in form. Representative area of the dish was
photographed. x 120.
JUNE 1977
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.
1 867
Survival Differences Exhibited by Normal and Transformed Rat
Liver Epithelial Cell Lines in the Aggregate Form
Anton F. Steuer, Patricia M. Hentosh, Leila Diamond, et al.
Cancer Res 1977;37:1864-1867.
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