[CANCER RESEARCH 40, 2310-2315,
0008-5472/80/0040-OOOOS02.00
July 1980]
Loss of Malignancy during Serial Passage of Human Carcinoma in Culture
and Discordance between Malignancy and Transformation Parameters1
L. Ossowski2 and E. Reich
The Rockefeller University. New York. New York 10021
ABSTRACT
Human epidermoid carcinoma (HEp-3), a tumor which grows
on chorioallantoic membrane and metastasizes with high effi
ciency into the organs of chick embryo, was adapted to growth
in culture.
Upon serial passage in vitro, a progressive loss of metastatic
potential and of tumorigenicity was noted. The metastatic po
tential was completely lost within the first 2 to 10 weeks in
culture (4 to 14 passages), while tumorigenicity declined rap
idly during a period of 50 passages, as reflected in the pro
gressively larger inoculum required for development of detect
able tumors. Beyond this time, macroscopically visible tumors
were not observed during the standard 7-day growth period
even when the inoculum was increased 100-fold to 107 cells.
Identical results were obtained with six individual tumors
adapted to grow in vitro. Metastatic potential could be re
covered from cell populations that retained some degree of
tumorigenicity. This required at least two sequential passages
on the chorioallantoic membrane.
Loss of metastatic ability and tumorigenicity was accompa
nied by change in hormone responsiveness and by a distinct
improvement in growth efficiency in culture as manifested by
shortened doubling time and increased saturation density. Fur
ther, anchorage independence and serum independence, two
properties that are generally correlated with the transformed
phenotype, were inversely correlated with malignancy.
The malignant and the nontumorigenic cells were compared
with respect to production of plasminogen activator, an enzyme
associated with malignancy and transformation. Malignant cells
produced larger amounts of plasminogen activator, and en
zyme production was resistant to modulation by several hor
monal and nonhormonal effectors; in contrast, plasminogen
activator synthesis in nontumorigenic cells was stimulated by
cholera toxin and inhibited by glucocorticoids.
INTRODUCTION
The potential attractiveness of cell culture as an adjunct in
cancer research has been appreciated for decades, and many
studies of tumor biology have made use of cells that were
maintained in culture for long periods of time. Such cell strains
or cloned cell lines are obtained as slowly developing out
growths from neoplastic tissue that is mixed with and includes
a variable proportion of normal cells; it is therefore often
unclear whether the cell population that emerges in culture is
derived from the neoplastic component. In favorable situations,
e.g., melanomas, distinctive phenotypic properties such as
1 This study was supported
by American Cancer Society Grant ACS POT 1
and National Cancer Institute Grant CA 08290.
2 To whom requests for reprints should be addressed.
Received October 22. 1979; accepted April 8. 1980.
2310
melanin production may establish beyond doubt that cultured
cells and the original tumor belong to the same cell type,
although the neoplastic nature and origin of the culture remains
uncertain. However, even if it is assumed in such cases that
the culture population must have been derived from some
tumor cell(s), there is no way of verifying whether the minor
cell fraction that survives and grows in culture has retained the
important neoplastic characteristics of the original tumor. This
uncertainty applies especially to the fundamental criterion of
malignancy, namely, the ability to form métastases,a property
that can be assessed only in vivo. Indeed, the 2 growth envi
ronments being quite different, it is reasonable to suspect that
the phenotypes (and genotypes) which determine malignancy
in vivo are prone to disappear in vitro, since the characteristics
that confer selective advantage in culture are not necessarily
related to those required for malignancy.
Considerations such as these have stimulated many investi
gators to search for in vitro phenotypes, particularly those
associated with transformation in culture, that could be used
as markers for malignancy. Correlations between tumor growth
in vivo and several characteristics in vitro have been reported
by a number of laboratories (9, 14, 21) and questioned by
others (5, 12, 24). There are few reports of studies specifically
designed to explore either the retention of metastatic ability in
culture or in vitro correlates of this determinant of malignancy
(for review, see Ref. 22). Athymic mice have been used exten
sively as hosts to assay the neoplastic properties of cultured
human tumor cells and human tumor xenografts; when pro
gressive tumors develop in this system, they are rarely meta
static, suggesting either that this phenotype is rapidly extin
guished, is strongly selected against in culture and/or nude
mice, or is expressed only by a very small fraction of the tumor
cell population.
In the accompanying communication (17), we have de
scribed a quantitative experimental metastasis model, consist
ing of a transplantable human tumor strain, HEp-3 (26), that
grows and metastasizes in the chick embryo. This system is
convenient for testing the neoplastic properties of cultured
cells because the metastatic potential of small numbers of cells
can be assayed rapidly and easily. We have succeeded in
maintaining and growing HEp-3 in culture, and we present here
our initial observations. This work was undertaken with the
following aims in mind: (a) to determine whether, and to what
extent, tumorigenicity and metastatic ability were retained dur
ing serial passage in culture; (b) to assess the correlations, if
any, between the accepted criteria of transformation in culture
and tumorigenicity and malignancy in vivo; (c) to follow the
changes, if any, in the production and regulation of PA,3 the
synthesis of which is generally enhanced both in malignancy
3 The abbreviations
used are: CAM. chorioallantoic
membrane; PA. plasmin
ogen activator.
CANCER
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Loss of Malignancy during Culture in Vitro
and transformation (18, 19, 27) and in association with normal
tissue remodeling and cell migration (19, 20); (of) to define the
life cycle and population kinetics of metastatic cells and, if
possible, to explore how these may determine the cellular basis
of metastasis.
The last of these aims is still under investigation and will be
considered in a later paper.
MATERIALS
AND METHODS
All materials used were as described in accompanying paper
(17) except that cholera toxin was obtained from Schwarz/
Mann, Division of Becton, Dickinson and Co. (Orangeburg,
N. Y.), phorbol-12-myristate-13-acetate
was from P. Borchert
(University of Minnesota), and agar (purified for cell culture)
was from Difco. Chick embryo extract was prepared as de
scribed (3) except that incubation with hyaluronidase was
omitted and all centrifugations were at 27,000 x g.
125l-Fibrin plate assay, tissue extraction, PA determination,
preparation of single-cell suspensions of tumor and detection
of métastaseswere all performed as described in the accom
panying paper (1 7). Growth in semisolid agar was tested using
the method described by MacPherson and Montagnier (11 ).
Maintenance of Tumor Cells in Culture. Single-cell suspen
sions of tumor cells from HEp-3 tumors grown on the CAM
were obtained by mincing and collagenase treatment. Cells
were plated at a concentration of 4 to 6 x 106/100-mm Petri
dish in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum. Initially, the medium was replaced
every 3 days. The cells were detached with trypsin and pas
saged weekly for the first 4 to 6 weeks and twice weekly
thereafter. Care was taken to avoid prolonged trypsinization.
In many cultures, floating round cells could be seen (up to 10%
of the total population). These cells retained viability as deter
mined by trypan blue exclusion and by the ability to reattach to
plastic. The metastatic properties of freely floating cells were
found to be similar to those of cells that grew while remaining
attached to the dish. The cultures were regularly checked for
pleuropneumonia-like
organisms using biological methods of
detection and were always free of contamination.
Growth in Suspension. Petri dishes (60 mm) were first
coated with 5 ml of 1% agar in growth medium and serum and
then inoculated with single cells, obtained from collagenasedigested HEp-3 tumors and suspended in 4 ml of medium with
serum. Under these conditions, the cells grew in suspension
forming progressively larger clusters of fully viable cells. For
passaging, the clusters were disaggregated to single cells by
repeated pipetting.
RESULTS
Toolan and her colleagues, who adapted HEp-3 to grow in
immunosuppressed rats and in chick embryos (2, 26), were
able to maintain and propagate this tumor in culture using a
variety of complex media (15). We found that HEp-3 could also
be cultured successfully from cell suspensions, prepared by
collagenase treatment of CAM tumors, and inoculated into a
routine medium consisting of Dulbecco's modified Eagle's me
dium supplemented with 10% fetal bovine serum. There was
often a lag period lasting several days before a significant
increase in cell number was observed, but the procedure was
JULY 1980
entirely reproducible and primary cultures could be established
at will under these conditions. During the course of this work,
primary cultures were initiated independently and propagated
by serial passage for long periods; all gave qualitatively iden
tical results with respect to cellular growth properties in vitro
and in vivo and to PA production.
Change of Tumorigenicity and Metastatic Ability as a
Function of Growth in Culture. Tumor growth in vivo and
metastasis were evaluated as described in the companion
paper (17), 7 days after a defined inoculum of single cells was
applied to the CAM of 10-day embryos. It was of interest to
find that a cell suspension prepared directly from CAM tumor
yielded CAM tumors that were reproducibly somewhat smaller
than those produced by similar inocula from primary HEp-3
cultures grown in culture for 4 to 7 days (Table 1). The same
result was obtained in 5 independent experiments with different
tumors. We have not established the basis of this phenomenon,
but we suspect that abrupt loss of the in vivo proliferative ability
of CAM tumor cells may occur more readily than in cell cultures
owing to the more intense proteolysis that is required to prepare
single-cell suspensions from a solid tumor. More impressive
was the progressive loss, first of metastatic potential and then
of tumorigenicity, that accompanied serial passage in culture.
Tumorigenicity decreased gradually but progressively in a way
that could not be compensated, after numerous passages, by
increasing inoculum size; cells from passage 50 and beyond
did not form macroscopically visible tumors even when the
inoculum was increased 100-fold.
In Vitro Properties of Tumorigenic
(Early-Passage) and
Nontumorigenic (Late-Passage) HEp-3 Cells. The following
properties of HEp-3 cells were evaluated as a function of time
in culture: plating efficiency, growth rate, and saturation density
at different serum concentrations; anchorage dependence; and
PA production. High plating efficiency, rapid growth, high sat
iable 1
Change in tumorigenic and metastatic properties of tumor cells cultivated in
vitro for various periods of time
Tumor dissected from CAM was dissociated into single cells by collagenase
treatment, and 10s cells were inoculated onto the CAM of each of four 10-day
chick embryos. The remaining cells were plated in Dulbecco s medium with 10%
fetal bovine serum at 5 x I06/100-mm Petri dish. Cells were passaged serially,
and samples of cells were frozen in liquid nitrogen at intervals. To test their
growth properties in vivo, samples were thawed, cultured under standard con
ditions for 5 days, and inoculated onto the CAM'S of 10-day-old chicks In order
to obtain visible tumor with cells from passage 9 and beyond, the inoculum had
to be increased gradually up to 106 cells. Following 7 days of incubation, the
tumors were excised and weighed. Lungs from each embryo were dissected and
divided into 2 parts; one part was used for protein and PA determination, and the
second was inoculated again on CAM'S of 2 embryos and incubated for an
additional 7 days. The PA activity is expressed in Ploug milliunits of urokinase
per mg of protein (17).
lungsgrown
in
lungs(milliunits/mgprotein)2533028728181291211119PA
in
CAM(milliunits/mgprotein)NT"NT2618N
on
No. of pas
wt(mg)15037022016013080605015PA
5)1.01.01.02.05.010.010.010.010.0Tumor
10
sages
vitro01691217242948Control
in
ininoculum(x
of cells
1Control
2No.
NT, not tested
2311
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L. Ossowski and E. Reich
uration density, and low serum requirement are well-estab
lished parameters of transformation in vitro (7, 25). We com
pared, in all of these respects, primary HEp-3 cells after 5 days
in culture and a strain that had been maintained by serial
•passage
for approximately 5 months. Cells were plated at 1.5
x 105/35-mm Retri dish and incubated in medium containing
fetal bovine serum at 10%, 0.2% (Chart 1), or 2% (data not
shown). Both plating efficiency and growth rate at all 3 serum
concentrations were higher for nontumorigenic late-passage
cells; correspondingly, saturation density at 10% serum was
3.3 x 105/sq cm for late-passage cells compared with 0.9 x
106/sq cm, or nearly 4-fold lower for early-passage cells. The
respective doubling times, calculated from the exponential
portion of the growth curves at each serum concentration, are
given in the legend to Chart 1 and reveal the tumorigenic cells
to have a higher degree of serum dependence and slower
growth rate.
Loss of anchorage dependence, as determined by ability to
grow in sermisolid media, has been proposed as the most
reliable in vitro correlate of tumorigenicity, at least for transfor
mation of permanent cell lines (4). This correlation did not hold
in the HEp-3 system when we tested cell populations of known
tumorigenic and metastatic potential derived from cultures
maintained in vitro for different periods. The results summa
rized in Table 2 show that highly metastatic cells (passages 0
and 1) did not grow efficiently in soft agar and that anchorage
independence developed during serial propagation in culture.
Plasminogen Activator in "Early-" and "Late-Passage"
HEp-3 Cells. Tumorigenic, metastasizing populations and non
tumorigenic HEp-3 cultures were compared with respect to
intracellular and secreted PA. Both cell types were plated at 8
x 106/60-mm dish, and dishes from each group were assayed
at daily intervals. Intracellular PA was initially 3-fold higher in
tumorigenic cultures, but this difference disappeared during
further incubation as intracellular enzyme increased more rap
idly in late-passage cultures (results not shown). The major
difference between tumorigenic and nontumorigenic cultures
was observed in the levels of secreted PA (Chart 2), the
tumorigenic cells releasing 20- to 25-fold more enzyme
throughout the course of the experiment; this difference was
also reflected in the ratio of extra- to intracellular PA (Chart 2,
Table 2
Growth in semisolid agar of strains maintained in vitro for different numbers of
passages
Cells from exponentially growing cultures (except passage 0. which was from
a collagenase-dissociated
tumor) were plated in semisolid agar at 2 different
concentrations, each in quadruplicate. Cultures were scored for the presence of
macroscopically visible colonies 12 days after seeding. Data were obtained from
the concentrations giving the most conveniently readable colony counts.
No. of passages
vitro01111522275156No.
in
ofplated5
cells
formed
(%
total)<0.02a<0.005a<0.01a5.31.07.72
of
1032x10"1
x
1041
X
1045
x
1031x10"5
X
1031x10'5
X
X 103Colonies
Only very small colonies containing approximately 2 to 8 cells were present.
IO7
10
E
3
IO6
2
3
4
5
6
Days of incubation
Chart 1. Growth of tumorigenic cells and their nontumorigenic
5
7
T
"derivatives"
J.
in 10 and 0.2% fetal bovine serum. Cultures of HEp-3 cells grown in vitro for 51
passages during 5 months and tumor cells in culture for 5 days were trypsinized
and plated at 1.5 x 105 cells/35-mm dish in Dulbecco's medium supplemented
with either 0.2% or 10% fetal bovine serum. Two cultures in each experimental
group were trypsinized and counted daily. Medium was replaced daily in all
cultures kept beyond Day 4. •,passage 1, 0.2% fetal bovine serum; O, passage
1. 10% fetal bovine serum; A, passage 52. 0.2% fetal bovine serum; A. passage
52, 10% fetal bovine serum. Doubling times (hr) of tumorigenic and nontumori
genic cells at different concentrations of serum are:
Passage in vitro
1
52
10% fetal bovine
serum
2% fetal bovine
serum
0.2% fetal bo
vine serum
19.2
8.0
32.4
120.0
27.2
11.5
The doubling time for each sample was determined from the exponential part of
the growth curve.
2312
-ho5
2
3
Incubation (doy«]
Chart 2. PA production by early-passage (passage 1) tumorigenic and latepassage (passage 75) nontumorigenic cells. HEp-3 cells obtained from collagen
ase-dissociated tumor and grown in culture for 4 days (passage 1) and cells
maintained in culture for approximately 7 months (passage 75) were plated at 8
x 105 cells/60-mm
Petri dish in Dulbecco's medium with 10% fetal bovine
serum. Two cultures of each passage were washed daily with 3 changes of
medium and incubated for 24 hr in 5 ml of serum-free medium. The conditioned
medium was collected, and the cells were detached with 5 mw EDTA, counted,
and extracted with Triton X-100 (0.5%). Intra- and extracellular PA were deter
mined in the usual way. Each point represents the mean of duplicate cultures.
A
A, PA passage 75; A
A, cell number, passage 75; • •,PA
passage 1; •
•,cell number, passage 1.
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40
Loss oÃ-Malignancy during Culture in Vitro
inset). Since total secreted enzyme greatly exceeded the intracellular PA, it follows that PA synthesis by tumorigenic cells
was correspondingly higher than by nontumorigenic cultures
and hence that PA production decreased during prolonged
growth in culture. Jones ef al. (8) have also reported that
animal passage of a tumorigenic cultured cell line substantially
increased the rate of PA secretion.
Modulation of PA Production. PA production in many cell
types can be modulated by a large number of agents, including
hormones (1, 10, 16), tumor promoters (6, 13, 29-31), and
retinoids (13, 31), among others (28, 29), and the responses
of normal cells may differ from those of tumors that arise in the
same tissue (16). With these facts in mind, we compared the
responses of the 2 HEp-3 populations to a variety of effectors
that are known to modulate PA production in other cells.
PA production by nontumorigenic cells was stimulated 2- to
3-fold by chick embryo extract (Chart 3/4) and by cholera toxin
(Chart 3B). Simultaneous exposure to both agents gave an
additional increase in PA, although a synergistic increase,
which could not be reproduced, was observed on one occa
sion. Hydrocortisone (1CT6 M) strongly inhibited enzyme syn
thesis in these cells, whereas phorbol ester (2 x 10~7 M)
produced little effect (Table 3). Early-passage tumorigenic and
metastatic cells differed in response to all of the above agents;
PA production was essentially unaffected by chick embryo
300-
200-
I
E
100
-12
Embryo extract (%)
-II
-10
-9
Modulation of PA production
Table 3
in early-passage
(tumorigenic)
and late-passage
(nontumorigenic) HEp-3 cells
Hydrocortisone and phorbol ester were present in cultures for a total of 48 hr,
the initial 24 hr in the presence of 5% fetal bovine serum followed by 24 hr in
absence of serum. PA activity was measured both in culture medium and in cell
homogenates. At the end of the experiment, the cell number in cultures of
tumorigenic cells was in the range of 6 to 9 x 105; for cells passaged in vitro, the
cell number was 1.6 to 2.0 x 106. The lowest cell numbers were observed in
tumor cell cultures treated with phorbol ester. The control values (100%) for PA
activity were as follows. Early passage: intracellular PA, 2300 milliunits/mg
protein; extracellular PA. 2600 milliunits/ml of medium from 106 cells. Late
passage: intracellular PA, 1614 milliunits/mg
milliunits/ml of medium from 106 cells.
control)Treatment
protein;
extracellular
PA. 255
PA (% of
tumor cells
(passage
1)Intracellu-
tumor cells (pas
sage 54)Intracellu-
Concentration(M)Hydrocortisone
Secreted
lar82
1 x 10~6
Phorbol ester 2x10"'Early
34
Secreted
lar5
75
8Late
91
13
90
extract, cholera toxin, and hydrocortisone but was inhibited by
phorbol ester.
Comparison of Monolayer and Suspension Cultures. A
HEp-3 cell suspension was prepared by collagenase digestion
of a CAM tumor, and separate aliquots were propagated in
parallel as monolayer cultures on plastic or as suspension
cultures over agar. For passaging, these were subjected, re
spectively, to trypsin treatment or mechanical dissociation, and
each strain was tested for tumorigenicity and metastatic ability
at regular intervals. As seen in Chart 4, tumorigenicity de
creased at equal rates during early passages in both types of
culture. Thereafter, tumorigenicity in monolayers continued to
decrease progressively, but suspension cultures stabilized at
a reduced level of tumorigenicity. Metastatic ability was lost in
parallel in both types of culture, disappearing by passage 13.
As long as some degree of tumorigenicity was retained,
metastatic potential could be recovered by serial passage on
CAM. This is illustrated in Table 4 for a strain that had been
passaged 19 times as a monolayer on plastic. Although only
weakly tumorigenic at this stage, inocula of 2 x 106 cells
reproducibly formed small tumors on the CAM, but the lungs
showed no métastaseseither by assay of PA of human origin
or by secondary CAM culture (17). During serial subculture on
CAM, these tumors gave rise to metastasizing tumors.
Cholera toxin concentration (M)
Chart 3. PA production by tumorigenic and nontumorigenic cells: effects of
embryo extract and cholera toxin. HEp-3 cells were obtained from collagenasedigested tumor (passage 0) or from cell culture by trypsinization (passage 51)
and plated in Dulbecco's medium supplemented with 5% fetal bovine serum. The
number of cells plated was calculated to yield a cell density of approximately 5
x 105/60-mm dish at the beginning of the experiment. After 2 days of incubation,
the spent medium was removed, the cultures were washed 3 times with 5 ml of
Dulbecco's medium, and the medium was replaced with fresh serum-free medium
containing increasing concentrations of chicken embryo extract or with medium
containing 5% fetal bovine serum and increasing concentrations of cholera toxin.
The conditioned media were collected after 24 hr, at which time cells were
detached with 5 mw EDTA in phosphate-buffered saline, counted, and extracted
with 0.5% Triton X-100. PA content was determined as usual in conditioned
medium and in extracts. The PA content in control cultures of HEp-3 passage 0
in serum-free medium was 3680 milliunits/ml of conditioned medium taken from
106 cells. 2620 milliunits/ml for the same cells grown in medium with serum,
255 milliunits/ml from HEp-3 passage 51 without serum, and 94 milliunits/ml
from passage 51 with serum. Intracellular PA contents in passage 51 cells were
2100 and 1750 milliunits/mg protein, respectively, for cells in serum-supple
mented and serum-free medium. Intracellular PA of cells from passage 0 (results
not shown) was not affected by either embryo extract or cholera toxin. •PA in
medium, passage 51 ; O, intracellular PA; A, PA in medium, passage 1.
DISCUSSION
The results described in this paper establish 2 facts. The first
is that metastatic ability and tumorigenicity of HEp-3 cells
disappear during prolonged passage in culture. This loss oc
curs gradually and progressively over a period of weeks and is
accompanied by qualitative changes in hormone responses as
reflected in modulation of plasminogen activator production.
The simplest explanation of these observations is that HEp-3
tumors on the CAM, or more precisely, the cultures derived
from them contain at least 2 cell types: (a) tumorigenic, perhaps
metastasizing, and poorly adapted to growth in culture; and (to)
nontumorigenic, and capable of efficient growth in culture. If
that were the case, preferential selection and overgrowth of
nontumorigenic cells in culture could easily account for our
results (The nontumorigenic cells are of human origin, since
they are lysed by rabbit anti-human serum and complement;
JULY 1980
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2313
L. Ossowski and E. Reich
400,-
conclusion is not fully justified by the available data. All of the
results thus far available suggest that métastasesare formed
with high probability only after a threshold is reached in the
number of tumor cells on the CAM. If the concentration of
3OO
tumorigenic cells in the original inoculum were very low, failure
of efficient metastasis formation might occur because the nec
essary threshold was not achieved at a proper time in relation
to the limits set by the standard 7-day assay. The resolution of
f 200
ï
this uncertainty will also require further work. Whatever the
mechanisms that underlie the loss of tumorigenicity and meta
static ability in culture, they probably do not apply to HEp-3
100
alone. Hence, the in vivo relevance of results obtained with
human tumor cell lines or strains maintained in culture for
significant periods seems open to question.
Our second finding is that HEp-3 tumorigenicity and meta
4
8
12 16 20 24 28
static ability are not correlated with several of the phenotypes
Number of passages m vitro
ordinarily used to define transformation in culture. Indeed, in
Chart 4. Retention of tumorigenicity during growth in suspension culture.
vivo growth and metastasis were inversely correlated with
Cells obtained from collagenase-disaggregated
tumor were plated either in
serum and anchorage independence, high growth rate, and
ordinary plastic dishes or over an agar layer (0.5%) to prevent attachment. Both
types of cultures were incubated with Dulbecco's medium containing 10% fetal
high saturation density. While these criteria were established
bovine serum and passaged twice weekly. After 10 passages, the growth rate of
in studies of fibroblastic cells with a mesodermal origin that
cells growing on plastic was one-fourth to one-third more rapid than that of cells
probably differs from that of HEp-3, it is likely that the patterns
growing in suspension. At the indicated times, cells were tested for tumorigenicity
on 10-day chick embryo CAM'S by inoculating between 1.5 x 105 and 1.5 x
that we have observed will apply to tumors other than HEp-3.
106 cells, each cell concentration being tested in 2 to 4 embryos. Macroscopically
The usual criteria for transformation all reflect, in some way,
visible tumors formed by the smallest inoculum of each cell type were dissected
increased growth efficiency under a particular and arbitrary set
and weighed. The tumor weights shown here were obtained using inocula of 2
x 10s cells between passages 1 and 13 and 10s cells for passages 14 to 26.
of conditions imposed in culture. These conditions would be
O, cells passaged on ordinary plastic dishes; •,cells passaged over agar layer
expected to be selective for cells of the appropriate phenotype,
(in suspension).
and it is significant that comparable transformed phenotypes
Table 4
were acquired by HEp-3 populations as they were maintained
Recovery of metastasîzingcells by serial transplantation on CAM
and propagated in culture. Tumor formation in vivo is a much
HEp 3 cells maintained in culture for 19 passages were trypsinized. counted,
more complex process, and it occurs under conditions very
and inoculated onto the CAM of three 10-day embryos at a concentration of 2
x 10s cells/embryo. The 3 resulting tumors were weighed, pooled, minced, and
different from those prevailing in culture; hence, there are no
distributed onto the CAM of 4 embryos. These second-generation tumors were
reasons for assuming that the determinants of fitness for growth
again weighed, pooled, minced, and inoculated onto the CAM of a second group
in the 2 environments are related or that the phenotypes
of three 10-day embryos. All incubations were for 7 days. PA activity in the lungs
of each tumor-bearing embryo was determined at each passage. Conspicuously
selected for growth in each case would necessarily be corre
high lung PA was observed after the second and third passage.
lated in any particular way. These arguments apply even more
strongly to metastasis, a purely in vivo phenomenon with no
counterpart in culture. In the absence of selective value under
inlungs(milliunits/mg)3307030
inlungs(milliunits/mg)4751411543Tumor
inlungs(milliunits/mg)5252Tumor
conditions that promote rapid cell multiplication in culture, the
wt(mg)504030PA
wt(mg)30506060PA
Tumor
wt(mg)8080501PA
physiological mechanisms and phenotypes related to metas
tasis would also be lost. The work of Stanbridge and Wilkinson
(23) has already shown that transformation parameters and
neoplasia can be separated in cell hybrids.
With respect to PA and in contrast to the other transformation
parameters tested, high levels of enzyme production, resistant
no lysis is observed with rabbit anti-chicken serum and com
to modulation by all but one of the agents tested, were well
correlated with retention of tumorigenicity and metastasis. Like
plement.). This mechanism, if it is operating, involves a number
wise, PA levels of cultured nontumorigenic cell populations
of quantitative implications, which have yet to be tested, con
cerning the origin, frequency, and growth rate of nontumoriwere lower than those in CAM tumors, although they exceeded
genic cells, both in vivo and in vitro. Further, several other
the values usually found in normal tissue. These results are
cellular mechanisms that might be responsible for the loss of consistent with a large body of evidence documenting the
HEp-3 tumorigenicity and metastatic ability in culture remain to association of malignancy with high levels of PA. Nevertheless,
while high levels of PA are conceivably related to some phebe explored; it therefore appears preferable to postpone spec
notypic aspects of tumors in vivo, there is no known reason to
ulation about this point until additional data are at hand. An
swers to many if not all of these questions may emerge from a assume that the enzyme is required for growth in vitro and thus
clonal analysis of HEp-3 cell populations, which we are cur
no positive selection tending to maintain this property during
long-term culture.
rently attempting.
All of the systematic observations reported in this and in the
Because metastatic ability decays much more rapidly in
accompanying paper (17) concern a single tumor, HEp-3,
culture than does tumorigenicity, it might be concluded that
there are 2 tumorigenic populations, only one of which was growing in a single host, the chick embryo. Although there is
truly malignant, i.e., capable of giving rise to métastases.This
no reason to assume that the basic mechanism of tumor
2314
CANCER
RESEARCH
VOL. 40
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Loss of Malignancy during Culture in Vitro
metastasis will show interspecific differences, the general ap
plicability of our conclusions must await a thorough assessment
of HEp-3 behavior in other hosts such as immunosuppressed
rodents.
ACKNOWLEDGMENTS
We wish to thank Gloria Bertuzzi. Kristen Wiese, and Diane Biegel for expert
technical assistance.
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2315
Loss of Malignancy during Serial Passage of Human Carcinoma
in Culture and Discordance between Malignancy and
Transformation Parameters
L. Ossowski and E. Reich
Cancer Res 1980;40:2310-2315.
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