(CANCER RESEARCH 48, 2432-2439, May I, 1988]
Role of Necrosis in Regulating the Growth Saturation of M uItice IInlar Spheroids1
James P. Freyer2
Cell Biology Group LS-4, Mail Stop M888. Los Alamos National Laboratory, Los Alamos, New Mexico 87545
ABSTRACT
Growth curves for nuiltici-Mularspheroids of 15 different tumor and
normal cell lines were characterized by doubling times which decreased
with increasing growth until a stable saturation was attained. In spite of
the identical and constant conditions during growth, the size at saturation
varied by factors of 67 in spheroid volume and 75 in cell content. These
saturation sizes showed no correlation with the monolayer doubling times
or clonogenic efficiencies, the initial spheroid growth rate or clonogenic
capacity at saturation, the cell packing density, or the species of origin
and type of cell line. There was a strong correlation between the maximal
spheroid size and the size at which necrosis initially developed, suggesting
control by necrosis. Crude extracts prepared from spheroids with exten
sive necrosis showed dose-dependent cytostatic and cytotoxic activities
against monolayer cultures, while similar extracts from spheroids without
necrosis had little effect. This activity was also detected in the culture
medium to which the large spheroids had been exposed prior to prepa
ration of extracts, suggesting that the responsible factor(s) can diffuse
through the spheroid. The extract from spheroids of one cell line inhibited
the growth and clonogenicity of four other cell lines, including human
diploid fibroblasts. DNA content profiles measured during exposure to
this extract showed that the cytostatic effect was not due to the arrest of
cells in a specific cell cycle phase. The cell volumes were increased during
culture in medium containing the extract from spheroids with extensive
necrosis. These data support the hypothesis that growth saturation in
spheroids is regulated by factors produced, released, or activated during
the process of necrosis and suggest that these toxic factors have potential
therapeutic use.
INTRODUCTION
Recently, considerable knowledge has been gained concern
ing the regulation of tumor cell growth and viability. Tumor
cells can respond to a wide range of growth stimulatory sub
stances, either generated externally or produced by the tumor
cells themselves ( 1-5). Cell viability can be affected by a variety
of environmental factors, usually those involved with energy
metabolism (6-8). Substances have been isolated from both
tumor and normal cell cultures which inhibit the proliferation
and viability of tumor cells (9-14); similar inhibitors have been
identified in several in vivo systems (15-17). Factors which
stimulate growth can be inhibitory for other cellular processes
(18). It has also been known for some time that inhibitory
substances can be produced or released in the necrotic regions
of tumors in vivo (19-22). There are several distinct substances
known to be produced by immunological cells in vivo which
specifically inhibit the viability of tumor cells (23-25). Taken
together, these findings suggest that the growth and viability of
cells in a tumor may be regulated as much by the microenvironmental conditions as by the intrinsic genetic or epigenetic
properties of the individual cells. Due to the complex nature of
the tumor microenvironment in vivo, the exact interactions
among stimulatory and inhibitory substances in tumors are
poorly understood, as is the relationship between the regulation
of cell proliferation and viability.
The multicellular tumor spheroid culture system is a useful
model to investigate these relationships in vitro (26). The onset
of necrosis in spheroids, used as an assay of the loss of cell
viability, has been shown to be regulated by the availability of
several compounds involved in cellular energy metabolism (2729). An advantage of this system is that the intraspheroid
concentrations of several of these substances can now be assayed
(30-31). Much less is known about the regulation of tumor cell
proliferation in this system. Several reports have demonstrated
a correlation between the external concentrations of low-mo
lecular-weight nutrients and spheroid growth (32-34). In other
reports, cell proliferation is considerably reduced at relatively
high metabolite concentrations (28, 31, 35). Models for the
growth of spheroids assume that the extent of the proliferating
cell layer is controlled by the limited inward penetration of
growth-stimulatory substances (36, 37), but the discovery that
many tumor cells produce such substances themselves casts
some doubt on the validity of this hypothesis.
One aspect of the growth of spheroids which may be exploited
to study the relationship between proliferation and viability is
the phenomenon of growth saturation, whereby the spheroid
growth rate decreases with increasing diameter until a stable
maximal size is attained (28, 38). A similar spheroid growth
retardation occurs in vivo if vascularization is inhibited (39).
Although growth saturation was originally attributed to surface
area limitations for nutrient supply, consumption/diffusion
models have shown that this is not the case. The spheroid
growth model proposed by Landry et al. (37) suggests that
growth saturation is due to the production of growth-inhibitory
substances. Recent studies by Freyer and Sutherland (28, 35)
demonstrate that spheroid growth saturation can be manipu
lated by altering the external growth conditions. A correlation
was found between the onset of central necrosis and the sphe
roid saturation size. This led to the development of a model in
which cellular proliferation and viability are regulated by inde
pendent mechanisms at small spheroid sizes; as the spheroid
grows, these cellular processes become connected through the
formation of toxic products during the process of necrosis. This
manuscript validates the generality of the relationship between
growth saturation and necrosis and demonstrates the cytostatic
and cytotoxic properties of extracts from spheroids with exten
sive necrotic centers.
MATERIALS
AND METHODS
Cell Lines. The EMT6/Ro mouse mammary carcinoma and V79
Chinese hamster fibroblast cell lines were originally obtained from Dr.
Robert Sutherland of the University of Rochester Cancer Center and
have been maintained in our laboratory for 5 yr. The human colon
adenocarcinoma, lung carcinoma, and melanoma cell lines were ob
tained from the American Type Culture Collection; the human fibro
sarcoma cell line was obtained from Dr. Robert Tobey of Los Alamos.
The
mouse adenocarcinoma, lung carcinoma, and fibrosarcoma cell
Received 9/15/87; revised 1/22/88; accepted 2/3/88.
lines were obtained from Dr. Nobuhiko Tokita at Los Alamos. The
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
mouse colon carcinoma cell line was obtained from Dr. Harry Crissman
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
at Los Alamos. The Chinese hamster lung fibroblast cells were obtained
1Supported by NIH through the National Cancer Institute (Grants CA-36535
from Dr. Jacques Pouyssegur of the University of Nice, France, while
and CA-2258S) and the Division of Research Resources (National Flow Cytomthe two Chinese hamster ovary cell lines were obtained from Dr. David
etry Resource Grant RR-01315), and by the United States Department of Energy.
1To whom requests for reprints should be addressed.
Chen of Los Alamos. The 9L rat brain gliosarcoma cells were supplied
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REGULATION
OF SPHEROID GROWTH SATURATION BY NECROSIS
by Dr. Peter Keng of the University of Rochester Cancer Center, while
the rat fibrosarcoma cell line was established in our laboratory from a
radiation-induced tumor isolated by Dr. Albert van der Kogel at Los
Alamos. The human skin fibroblasts were obtained from Andrew Ray
of Los Alamos.
Monolayer Growth Conditions. All cells were maintained in monolayer culture in a humidified incubator equilibrated with 5% CO2 in air
at 37*C using minimum essential medium, a-formulation (Grand Island
Biologicals) containing 10% (V:V) fetal bovine serum (Hyclone Labo
ratories), 100 units/ml potassium penicillin, and 100 Mg/ml strepto
mycin sulfate. Cultures were passaged twice weekly by treatment for 10
min with 0.25% trypsin (Sigma Chemical Co.) in Hanks' balanced salt
solution containing 25 HIM EDTA and 10 HIM4 (2~hydroxyethyl)-lpiperazineethane sulfonic acid (pH 7.4), washing by centrifugaron,
then placing 5 x 10" cells in 75-cnr culture flasks (Corning) containing
15 ml of complete medium. All cell lines were found to be free of
mycoplasmal contamination using a simple DNA staining technique
(40); the cells routinely carried at Los Alamos were also found to be
contamination free using a culture technique (41). The clonogenic
efficiencies of these different cell lines were assayed using standard
techniques (28).
Spheroid Culturing. Spheroids were initiated and cultured essentially
as described previously (28). The medium in the spheroid culture flasks
was changed on the second and fourth days and daily thereafter. The
spheroids were assayed for mean size and cell content as described
below, and spheroids were removed from the cultures during medium
change such that the total number of cells in the spinner flask never
exceeded 2 x IO5 per ml of medium. Previous studies with the EMT6/
Ro cell line have demonstrated that these procedures ensure fairly
constant medium conditions throughout the growth period, with the
oxygen and glucose concentrations and the pH never decreasing by
more than 5% of the values in fresh medium (28).
Spheroid Growth Curve Measurement. Spheroids were assayed for
growth at medium replenishment times by measuring the population
mean diameter and the number of cells per spheroid as described
previously (28). The spheroid cell number at saturation was also com
bined with the spheroid volume and the extent of necrosis to estimate
the mean cell packing density in terms of the number of cells per unit
of viable spheroid volume.
Histological Measurement of Necrosis. Spheroids were fixed,
mounted in paraffin, sectioned, and stained for histológica!observation
as described in detail elsewhere (28). Measurement of the thickness of
the viable cell layer was done using a calibrated reticle in a microscope;
measurements of the central sections of each of 25-35 spheroids 8001200 nm in diameter were made as described previously (28). This
previous work demonstrated that for spheroids cultured in normal
concentrations of oxygen and glucose there was no significant variation
in the viable rim size over this range of spheroid diameters.
Necrotic Extract Preparation. In order to test for the presence of any
toxic properties of necrosis in spheroids, extracts were prepared in the
following manner. Three hundred EMT6/Ro spheroids were cultured
to a size of -1500 ^m; at this size the thickness of the viable rim was
~250 urn, so that these spheroids were composed of ~30% necrotic
material by volume. The spheroids were removed from the spinner
flask and placed into a 15-ml centrifuge tube and allowed to settle, the
medium was removed, the spheroids were washed quickly with 10 ml
of PBS, then resuspended in 10 ml of double-distilled water. This
spheroid suspension was homogenized using a tissue homogenizer
(Sorval Instruments) for 5 min, then placed into a —20°C
freezer for
storage. Extracts from spheroids without central necrosis were prepared
in a similar manner using 1 x IO4 spheroids of -450 ¿andiameter,
which represents an equivalent volume of material as was used for the
spheroids with necrosis. After defrosting, various dilutions of these
extracts were added directly to complete culture medium and mixed for
1 h. After mixing, the pH of the medium/extract mixtures was adjusted
to 7.4 using l N NaOH, then they were sterilized by passage through
0.22-jum nylon membrane filters (Millipore).
In order to be able to quantitate the amount of extract being added
to the cultures, a second method of preparation was used. The spheroids
were grown, isolated, washed, and frozen in PBS3 as described above.
These were then placed into a freeze-drying apparatus, lyophilized, and
stored at -20°Cuntil use. The freeze-dried spheroids were then ground
to a fine powder and added to culture medium at a concentration of 1
mg/ml. After mixing for l h at room temperature, the medium with
extract was adjusted and filtered as described above.
Monolayer Growth Curve Measurement. In order to initiate monolayer growth curves, cells obtained from exponentially growing monolayer cultures were washed by centrifugal ion and 10s cells were inocu
lated into each of 30 60-mm culture dishes containing 5 ml of the
medium of interest which had been preequilibrated to the proper pH
and temperature. At various times after plating, three dishes were
removed from each experimental group, the medium was removed, the
dishes were washed with 5 ml of PBS without calcium or magnesium,
and 1 ml of standard trypsin solution was added to each dish. These
were incubated at 37*C for 10 min, then 2 ml of complete medium was
added, the cell suspensions were mixed with a Pasteur pipet 10 times,
and an aliquot of each dish was added to a counting vial containing
PBS. Three counts of the cell suspension from each dish were then
made using the Coulter counter and pulse-height analyzer. The pulseheight analyzer was also used to obtain an estimate of the mean cell
volume using computer analysis described elsewhere (28). The loga
rithms of the mean cell numbers per dish as a function of time of
growth were then fit to a linear equation using a least squares method
to obtain an estimate of the exponential growth rate. The remainder of
the cell suspensions from the three dishes in each experimental group
was then pooled and prepared for flow cytometric DNA content analysis
as described below. In some experiments, the cells were allowed to
attach in normal medium for 24 hours, then the medium was replaced
by either control or extract-containing medium.
Flow Cytometric DNA Content Analysis. Cells were analyzed for total
DNA content using a flow cytometric technique which has been detailed
previously (28). The cell suspensions were analyzed for total DNA
content on a Los Alamos flow cytometer (42) with the laser operating
at 457 nm and collecting wavelengths longer than 512 nm. Histograms
containing >5 x IO4cells were collected and analyzed to determine the
percentages of cells in each of the cell cycle phases using standard
techniques (43).
RESULTS
Spheroid Growth Curves. In order to study the generality of
the relationship between the saturation of growth and the onset
of central necrosis, spheroids of 15 different cell lines were
cultured under identical nutrient conditions until a stable max
imal size was attained. Representative growth curves for sphe
roid volume and total cell content are shown in Fig. 1. In every
case, spheroid growth was characterized by an initial exponen
tial phase, a second phase in which the growth rate was contin
ually slowing, and a final plateau in growth. These data were
fit to the Gompertz equation by a nonlinear least squares best
fit routine in order to arrive at objective estimates of the initial
spheroid growth rate and the spheroid size at plateau. The
estimated values of these parameters are shown in Table 1 for
all of the cell lines. As can be seen from these data and the
curves in Fig. 1, there was a considerable variation in the initial
growth rates and the saturation sizes among these different cell
lines. The initial doubling times varied by a factor of 4.4 when
measured by volume growth and 3.7 in terms of spheroid cell
content. The saturation sizes varied by factors of 67 and 75 for
volume and cell number growth, respectively. There was no
significant correlation between the initial growth rate and the
saturation size, either in terms of spheroid volume (r2 =
0.000095) or total cell number (r2 = 0.013). The means and
confidence limits for the maximal size attained by spheroids of
a given species showed that there was no significant difference
3The abbreviation used is: PBS, phosphate-buffered saline.
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REGULATION OF SPHEROID GROWTH SATURATION BY NECROSIS
however, that there was a 10-30% reduction in colony-forming
ability comparing cells from large spheroids to those from
monolayers. Calculations of the cell packing density in the
spheroids at saturation also showed no correlation with the
saturation size (r2 < 0.028). Comparison of any of these cellular
10",
icfi
10'i
IO8
parameters with the logarithm of the maximal sizes also re
vealed no significant correlations (r2 < 0.32).
to',
There was a significant correlation between the thickness of
the spheroid viable cell rim and the growth saturation size, both
in terms of spheroid volume (r2 = 0.55) and total cell content
(r2 = 0.56). Previous studies with the EMT6/Ro cell line grown
10'
id1
Iff
O
5
10 15 20 25 30
O 5
10 15 20 25 30
TIME OF GROWTH
(days)
Fig. 1. Growth of spheroids in terms of total spheroid volume and total
spheroid cell content as a function of time. Points, means of 50 spheroids measured
as described in "Materials and Methods;" lines, nonlinear least squares best fits
to the Gompertz equation, fop two panels, spheroid volume growth; bottom two
panels, total cell number growth of the same spheroids; left two panels, mouse
colon carcinoma cells; right panels, human colon carcinoma cells.
in growth saturation among the spheroids derived from these
different mammals. Similarly, spheroids of different cell types
in those instances in which more than one measurement was
available (colon and lung carcinomas, adenocarcinomas, fibrosarcomas, and fibroblasts) also showed no dependence of satu
ration size on cell type.
Several other cellular parameters were measured for these
different cell lines, as shown in Table 1. The monolayer dou
bling times were correlated with the initial spheroid growth
rates (r2 > 0.85), but there was no correlation between the
monolayer growth rate and the size at saturation (r2 < 0.14).
Measurements of both the monolayer clonogenic efficiency and
the spheroid cell clonogenic capacity at growth saturation also
showed no correlation with the initial spheroid doubling times
(r2 < 0.15) or the growth saturation sizes (r2 < 0.19). Note,
Table 1 Monolayer and
Comparison of the size of spheroids at growth
type, the monolayer doubling time and clonogenic
and cell packing density at saturation. There were
and the spheroid saturation size (r2 < 0.3).
under different culture conditions showed that the spheroid size
at which necrosis initially developed could be reliably estimated
over a wide range of values by doubling the measured thickness
of the viable cell rim (28). Fig. 2 shows a semilogarithmic plot
of the estimates of the spheroid size at the onset of necrosis
and the spheroid size at saturation. These parameters were
highly correlated, both in terms of spheroid volume (r2 = 0.85)
and total cell number (r2 = 0.72). There was no correlation
between the spheroid size at the onset of necrosis and the initial
spheroid growth rate (r2 < 0.21). Additionally, there was no
correlation between the size at which necrosis initially devel
oped and any of the cellular parameters detailed above (r2 <
0.21). Also shown in Fig. 2 are values obtained for the spheroid
diameter at the onset of necrosis and the saturation of growth
in the EMTo/Ro cell line grown under different glucose con
centrations (28). Pooling these data together with the measure
ments on the different cell lines in the same medium increases
the correlation between necrosis formation and growth satura
tion both for spheroid volume (r2 = 0.92) and total cell content
(r2 = 0.89).
Effects of Necrotic Extract. In order to directly test the toxic
properties of necrosis in spheroids, extracts were prepared from
large (—1500 urn diameter) and small (~400 urn diameter)
EMTo/Ro spheroids as detailed in "Materials and Methods."
EMTo/Ro monolayer cells were then exposed to complete
medium to which various dilutions of these extracts had been
spheroid growth, plating efficiency, saturation size, viable rim and cell packing parameters
saturation (in terms of spheroid volume and total cell number) with the species of origin of the cell line, the cell
efficiency, and the spheroid initial doubling time, thickness of the viable cell rim, clonogenic efficiency at saturation,
no statistically significant correlations between any of the monolayer or spheroid numerical parameters shown here
MonolayerSpecies/
cellInitial volumeSpheroid
Maximal
Initial
Maximal
time
efficiency(%)527880676385675872637152637169Spheroid
doubling time
size
doubling time
size
(cells)1924201422162224212818641620161.0
(h)
(urn3)
(h)
lineMouseMAC2MCC26EMT6/ROMLC2PINChinese
cell
typeAdenocarcinomaColon (h)12139.5172010131415181218IS1110Plating
10'3.2
x
10'°1.2
x
carcinomaMammary
10'°1.9
x
carcinomaLung
10'°5.6
x
carcinomaFibrosarcomaFibroblastFibroblastFibroblastFibroblastGliosarcomaFibrosarcomaColon
10'7.5
x
hamsterV79CHO-K1CHO-XRSCHL-O23Rat9LRF1HumanHCC1HLC1HT1080MEL28Cell
10'4.8
x
10'5.6
x
10«4.2
x
10'2.5
x
10'°1.8
x
10'4.0
x
Cell
rim efficiency
packing(%)
(c/,im3)ND526251ND73NDNDNDND5
fam)1182572512132381809885198225139176104228
10'9.2
x
10*5.5
x
10sND1.5
x
IO-11.7
x
IO-18.6
X
10-'ND5.2
x
10*NDNDNDND4.7
x
10-'NDNDNDND5.7
x
10'4.7
x
IO'52.6
x
10'5.5
x
IO43.2
x
10"1.1
x
IO4ND3.1
x
10'°6.0
x
x 10'ND"262416ND19NDNDNDND226018ND19ND2.4
x IO5Viable
carcinomaLung
carcinomaFibrosarcomaMelanomaDoubling
IO"51.2
x
IO"1ND1.2
x
* ND, not done.
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x IO'4
REGULATION
OF SPHEROID GROWTH SATURATION BY NECROSIS
25-
I ICN
2015-
li i(fì
10_,
i l(f
ol
%
¡I
uì
li lcf
530252015-
icf
105200
400
600
001
ONSET Of NECROSIS
(diameter in f¿m)
Fig. 2. Relationship between spheroid saturation size and the size ot spheroid
at the onset of central necrosis. The size of spheroid at the onset of necrosis was
estimated from measurements of the thickness of the spheroid viable cell rim as
explained in the text. Maximal size values are individual determinations from
best fits to the Gompertz equation for data similar to that shown in Fig. 1. Lines,
linear least squares best fits to all data points. •,data for IS different cell lines
cultured in the same medium; O, comparable data for EMT6/Ro spheroids
cultured in medium containing four different glucose concentrations (28). Top,
spheroid saturation size in terms of the maximal spheroid volume attained;
bottom, this in terms of the maximal spheroid cell content.
10 7
10':
a^,
^•3
il
io5.
IO4
o
30
60
90
130 150 180
TIME OF EXPOSURE
(hours)
Fig. 3. Representative monolayer growth curves for control EMT6/Ro cul
tures (O) and identical cultures exposed to a 1:1 dilution of spheroid necrosis
extract (•)as explained in "Materials and Methods." Points, mean of three dishes
(SE within the size of the data points); lines, linear least squares best fits to all
data points.
added, and the growth rates of the cells were measured. Fig. 3
shows representative monolayer growth curves for cells exposed
to complete medium and to medium to which an extract from
spheroids with necrosis has been added in a 1:1 dilution. All of
the growth curves for the various dilutions of extracts from
spheroids with and without central necrosis were similar to that
shown in Fig. 3; i.e., the cell number increased exponentially
with time. These data were fit to the exponential growth equa
tion using a linear least squares technique in order to obtain
estimates of the cell doubling times. Fig. 4 shows the depend
0.1
DILUTION FACTOR
(extract/medium)
Fig. 4. Monolayer culture doubling times of EMT6/Ro cells as a function of
the dilution either of the spheroid extract (A) or of the medium from spheroid
cultures (B). O, extract/medium from spheroids without necrosis; •.data for
extract/medium from spheroids with extensive necrosis, as detailed in "Materials
and Methods." Values are slopes of linear least squares best fit lines to growth
curve data such as that shown in Fig. 3; bars, one SE of the best fit estimate
(within the size of the data point where not shown); lines, hand-drawn fits to the
data.
enee of the doubling time of EMT6/Ro cells in monolayer
culture on the dilution of extract present in the medium. In
addition, the culture medium in which these spheroids had been
incubated for the 24-h period immediately prior to preparing
the extracts was filtered and added in various dilutions to
complete medium; the effects of this spheroid-exposed medium
are also shown in Fig. 4. There was a slight effect of the extract
from spheroids without necrosis at the higher dilutions (dou
bling time increased by a factor of 1.3) but no such effect for
the medium from the small spheroids. After correcting for the
effect of the extract from spheroids without necrosis, the highest
concentration of the extract from spheroids with necrosis in
creased the cell doubling time by a factor of 2.3. This repre
sented a cell doubling time 3.2 times longer than the value in
complete medium with no extract added (8.7 h). Note that the
cell growth was slowed even at the lowest concentration, a 0.03
dilution, of the extract from spheroids with necrosis. The effect
of the medium from the spheroid cultures was somewhat less,
since the highest concentration of this medium increased the
cell doubling time by a factor of 2.0.
To obtain more detail about the mechanism of this growthinhibitory action, cells obtained from monolayers at various
times were assayed for cellular DNA content by flow cytometry.
Analysis of the DNA content histograms showed no significant
variation among the different culture conditions using a Stu
dent's t test (P > 0.25). Throughout the exponential growth
period, 35% of the cells were in the Ci-phase of the cell cycle,
with 47% in S-phase and 15% in the G2-phase. There was a
variation of 3-5% of these mean values among the different
sample times in one growth curve or among the mean values
for the different growth curves.
The effect of the spheroid extracts and the spheroid-exposed
media on the clonogenic efficiencies of EMT6/Ro monolayer
cells was also measured, as shown in Fig. 5. In this case, the
cells were plated at low density (200 per dish) and exposed to
the appropriate media throughout the 7-day period required to
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REGULATION OF SPHEROID GROWTH SATURATION BY NECROSIS
io7
100a
7550-
10",
le*
p
25-
31
G-5
r°
^ o
-3 I.
^^"
S
100-1 B
irf-
75-1
IO4
30
001
01
DIUL'TIONFACTOR
(extract medium)
60
1
90
120
150
TIME OF EXPOSURE
(hours)
Fig. 5. Clonogenic efficiency of EMT6/Ro cells as a function of the dilution
of extract/medium in which the cultures were incubated. Symbols represent
cultures as described in Fig. 4 and show the mean ±SE of 8-10 dishes; lines.
hand-drawn fits to the data.
grow colonies. Again, the extract or medium from spheroids
without central necrosis had no significant effect on the ability
of the cells to form colonies, even at the highest concentrations.
The extract and medium from spheroids with extensive central
necrosis showed a dose-dependent inhibition of colony-forming
ability. At the highest concentration (a 1:1 dilution), the cell
clonogenic efficiencies were reduced by factors of 2.9 and 1.8
for the extract and spheroid-exposed medium, respectively.
Again, note that the clonogenic efficiency of the cells was
slightly reduced even at the lowest concentration of the extract
from spheroids with extensive necrosis.
The data in Figs. 3-5 were all obtained with extracts or
medium from EMT6/R.O spheroids and tested against EMT6/
Ro monolayer cells. In order to test the generality of these
effects, monolayer cells of several different cell lines were
exposed to medium which had been mixed with a lyophilized
extract from EMT6/Ro spheroids with extensive necrosis, as
explained in "Materials and Methods." For these experiments,
the cells were inoculated at high (5 x 10" per dish) and low
(200 per dish) density in complete medium and incubated for
24-26 h. Then the medium on the dishes was removed and
replaced with control medium or medium mixed with 1 mg/ml
of the lyophilized extract, and the dishes were handled for.
growth curve analysis and clonogenic efficiency determination.
Fig. 6 shows the monolayer growth curves obtained for 9L rat
gliosarcoma cells. All of the monolayer growth curves had this
appearance, i.e., a reduced but still exponential growth rate in
the extract-containing medium. The magnitude of the growthinhibitory effect was cell-line dependent, as shown in Table 2.
For exposure to the identical extract-containing medium, the
monolayer doubling times were increased by factors of 1.2 to
5.6. Comparison of the doubling times in these two conditions
using a Student's r test shows that the monolayer growth rates
are significantly different for each cell line (P < 0.005). In every
case except the 9L cells in the extract-containing medium, the
cells were cultured until they reached a plateau. There were no
significant differences in the number of cells per dish at plateau
(P > 0.5). Table 2 also shows the clonogenic efficiencies of
Fig. 6. Growth of 9L rat gliosarcoma cells in monolayer culture; at 24 h, the
medium was replaced with either control medium (O) or medium containing a
lyophilized extract from EMT6/Ro spheroids with extensive central necrosis (•),
as described in "Materials and Methods." Points and lines, as described in Fig. 3.
Table 2 Monolayer growth and colonogenicity in control and
extract-treated medium
Comparison of the effect of an extract from EMT6/Ro spheroids with exten
sive necrosis on the growth and clonogenicity of monolayer cells of different cell
types. Doubling times are given as best fit values ±SE of the fit for growth curves
similar to those shown in Figs. S and 8 (n = 6-8). Control cultures were grown
in normal complete medium; experimental cultures were exposed to medium
containing an extract from spheroids as detailed in "Materials and Methods."
Doubling time (h)
Plating efficiency (%)
lineEMT6/RO
Cell
±0.055
±0.15
±2.2
±1.2
HT1080
10.2 ±0.072 12.2 ±0.11 76.0 ±2.1 24.7 ±1.5
MEL28
9.82 ±0.082 13.1 ±0.11 80.7 ±1.0 10.8 ±0.57
9L
17.6 ±0.15 99.2 ±6.0
<0.0005
58.3 ±1.9
HSFControl9.25
25.3 ±0.67Extract11.8
<0.0002
36.7 ±0.69Control83.3
25.2 ±3.2Extract32.9
these different cell lines in the two conditions. Again, in every
case there were significantly fewer colonies formed in the ex
tract-containing medium (P < 0.005). In the case of the 9L
cells, there were no colonies seen out of the total of 2000 cells
plated; for the HSE cells; there were no colonies seen after
plating 5000 cells.
During the monolayer growth curve measurements, the cells
removed from the dishes as a function of time were assayed to
obtain cell volume and DNA content distributions as detailed
in "Materials and Methods." The mean cell volumes were
increased by 16-35% in the cells cultured in the extract-exposed
medium; this difference was significant (P < 0.05) for the
EMT6/RO, HT1080, MEL28, and HSF cells but not for the
9L cells (P > 0.05). There were no significant differences (P >
0.10) in the cell cycle distributions of cells cultured in these two
conditions during the exponential phase of the growth curves,
despite the significant differences in growth rates (Table 2).
DISCUSSION
One of the important results of this study was that a large
number of basic cellular and spheroid parameters had no rela
tionship to the spheroid saturation size. There was no correla
tion between the spheroid saturation size and the species or cell
type from which the spheroids were cultured. The tumor cell
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REGULATION OF SPHEROID GROWTH SATURATION BY NECROSIS
lines studied did not consistently grow to a larger or smaller
final spheroid size than did the cells of normal tissue origin. As
one might expect, there was a positive correlation between the
monolayer and initial spheroid growth rates. There were no
correlations between the monolayer or spheroid growth rates,
or their respective clonogenic capacities, and the spheroid sat
uration sizes. There was no relationship between the cell pack
ing density at saturation and the saturation size (Table 1). The
cell lines studied covered a large range in all of these cellular
parameters, strongly suggesting that spheroid growth saturation
is not determined by the intrinsic growth properties of the
individual cells.
The only spheroid parameter measured which was positively
correlated with the saturation size was the thickness of the
viable cell rim. Despite the fact that all of the spheroids were
cultured in identical and constant conditions, there was a large
variation (85-257 urn) in this critical parameter. There is con
siderable evidence that the onset of necrosis is determined by
the availability of sufficient metabolites for the maintenance of
the energy requirements of the cells in the inner spheroid region
(27-29). Thus, the range in viable rim sizes observed in the
present study may be due to cell line differences in any of
several parameters, including metabolite consumption, energy
production and utilization, and the sensitivity to low metabolite
levels. In addition, several metabolites and catabolites may
interact in determining the viability of cells in spheroids (26).
The data in Fig. 2 demonstrate that the onset of necrosis is
closely coupled with the later development of growth saturation.
Calculations from the data in Table 1 show that, in spite of the
variation in rim sizes, spheroid growth saturation was attained
at a size at which central necrosis constituted 50-70% of the
total spheroid volume.
The observation that the only parameter which correlated
with the spheroid saturation size was the diameter at which
necrosis developed argues strongly against the hypothesis that
growth saturation is due to the limited surface area available
for supplying nutrients to the cell volume (38, 39). The surfaceto-volume ratios at growth saturation measured in the present
study varied by a factor of 4.2. It is difficult to invoke a nutrientsupply mechanism to explain spheroid growth saturation in
some cases at 970 ¿¿m
and in others at ~4000 urn in the same
medium. Mathematical models (36) have shown that as a
spheroid grows, the nutrient supply by diffusion from the
medium becomes similar to the situation in an infinite sheet of
a thickness equal to that of the viable cell rim. Under these
conditions, a spheroid should be able to grow indefinitely (37).
Therefore, it may be invalid to invoke the mechanism of limited
inward penetration and supply to explain spheroid growth
saturation.
Growth saturation in spheroids is more likely related to the
expansion of central necrosis through the formation, release,
or activation of cytostatic factors. One of the most striking
aspects of Fig. 2 is the similarity in the data for different cell
lines cultured in the same medium and for a single cell line
cultured in different media. This clearly suggests that the rela
tionship between the development of necrosis and growth sat
uration is a fundamental one. The nature of this relationship
was first detailed in a model developed by Landry et al. (37) in
which cellular proliferation in the outer rim of the spheroid is
reduced by the action of cytostatic factors diffusing out of the
necrotic core. As later expanded by Freyer and Sutherland (28),
the model proposes that spheroid cell proliferation and viability
are regulated independently at small spheroid sizes, but that
these processes become indirectly related when products gen
erated during the process of necrosis inhibit cell proliferation.
This may be an important consequence of the fact that cell loss
in spheroids (and primarily in tumors) occurs through the
process of necrosis, as opposed to the more orderly mechanism
of apoptosis (44). The data in this manuscript, especially the
finding that central necrosis is necessary in order to demon
strate a cytostatic effect in spheroid extracts (Fig. 4), strongly
support this model. The discovery of a cytostatic effect of the
medium exposed to spheroids with extensive central necrosis
(Fig. 4) also supports the critical requirement that the putative
cytostatic factors diffuse through (and out of) the spheroid. The
connection between the regulation of proliferation and viability
is an important contribution of the spheroid system to an
understanding of tumor cell biology and should have revelence
to the analogous situation in tumors in vivo.
The data presented allow some limited characterization of
the mechanism of action of the toxic extracts from spheroids.
The information in Fig. 6 and Table 2 demonstrate that the
extract from spheroids of one cell line is active against other
cell lines. This may indicate that the responsible factor(s) has a
general mechanism of action. Such a cross-reactivity of tumor
cell inhibitory factors has been found in other systems (13, 14);
interestingly, the spheroid necrosis extract also inhibited the
growth of human diploid fibroblasts. Preliminary data with
extracts from necrosis-containing spheroids of the HT 1080
human fibrosarcoma cell line demonstrate the presence of both
cytostatic and cytotoxic activities, suggesting that the toxic
effects of spheroid necrosis are also a general phenomenon. An
interesting finding of the present studies is the fact that the
spheroid necrosis extract does not induce the arrest of cells in
a particular cell cycle phase. Two reports of growth inhibitors
produced by normal cells show specific inhibition of the Gi/S
transition (10, 15), but at least one tumor cell growth inhibitor
found in normal human serum shows no cell cycle phase spec
ificity (16). The fact that the spheroid extract growth-inhibitory
activity slows traverse through all phases of the cell cycle may
have some relationship to the presence of nonproliferating Sand Gz-phase cells in several spheroid systems (45, 46).
There are two other findings in the present study which may
have some implications for the mechanism of action of the
growth inhibitory activity. The first is that the volumes of the
cells exposed to the extract were significantly increased. This
effect has not been reported previously for tumor cell growth
inhibitory factors and suggests some mechanism involving the
cell membrane or perhaps the general cellular metabolism. The
second interesting effect is the cytotoxic activity of the extract,
as measured by a clonogenicity assay. Cytotoxic activities of
tumor cell growth inhibitors have been reported in other sys
tems (14, 17). This could occur by the same mechanism which
causes the growth inhibition, or it may be an inhibition of cell
attachment as has been reported for extracts from necrosis in
tumors (21).
There are several possibilities to explain the origin of toxic
factors which are specifically present in spheroids with central
necrosis. The spheroid cell mass generates metabolic waste
products which can be found in the spheroid extracellular space
(26, 30), and such catabolites can inhibit growth (47). It is
unlikely that common catabolites are responsible for the ob
served growth and viability inhibition, however. There was little
effect of the extract from spheroids without central necrosis,
yet these spheroids carry out metabolism and presumably con
tain catabolites. Also, there was a significant effect of the extract
from spheroids with necrosis even at very high dilutions in
which the amount of any added catabolites would be quite low.
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REGULATION OF SPHEROID GROWTH SATURATION BY NECROSIS
We also have preliminary data showing that the cytostatic
activity in necrotic extracts is found in a high-molecular-weight
fraction. A second possible source of the toxic activity is the
release of materials from the cells during the process of necrosis,
for example, lysosomal enzymes (19, 22). Again, the finding of
activity only in extracts from spheroids with necrosis suggests
that such a mechanism would have to be specific to cell death
by necrosis. A third intriguing possibility is that substances
released or generated during necrosis could themselves generate
a toxic compound through enzymatic or chemical action. Such
a modification of growth-inhibitory activity by proteolytic ac
tion has been shown in several systems (15, 19, 48, 49). The
microenvironment in the necrotic region of spheroids is poorly
understood; it is plausible that toxic factors are released or
activated in this region and then alter the environment around
the viable cells.
Regardless of the mechanism, the presence of growth-inhib
itory factors in spheroid necrotic regions has some interesting
implications regarding the concept that growth saturation in
spheroids is analogous to the situation of a nonvascularized
tumor or metastatic nodule in vivo (39). The current results
suggest that the size to which a nodule can grow without
vascularization can vary greatly; there is no doubt a greater
variation in the complex in vivosituation than is reported here
for spheroids in vitro. Secondly, it is possible that the original
impetus for the vascularization of a tumor nodule is the for
mation and expansion of necrosis. Studies with the spheroid as
an immune response stimulus in vivosuggest that several types
of host cells are attracted by the necrotic region (50). Factors
released from the necrotic region of a small tumor nodule may
actually assist in the future expansion of the tumor by inhibiting
the further growth of the tumor cells in a reversible manner
while concurrently serving as a signal for the expansion of the
vascular system through angiogenesis. The spheroid system
provides a suitable model for investigating the initial events in
tumor nodule vascularization and the role of necrosis in this
process.
Another critical area that the present results have important
implications for is the use of spheroids as a therapeutic test
system. The spheroid growth model proposed by Freyer and
Sutherland (28), and supported by this manuscript, assumes
that growth regulation in spheroids is a competition between
stimulatory and inhibitory factors; the relative importance of
these factors changes as the spheroid grows. Thus, it may be
improper to interpret results obtained with therapeutic agents
at one spheroid size as applying in general, especially if the
agent used is dependent on cellular proliferation. This is an
important caution, as the majority of published reports using
therapeutic agents in spheroids have not investigated a range
of spheroid diameters. Another consideration is that many
therapeutic modalities can alter the spheroid microenvironment
and cell subpopulations, possibly altering the balance between
growth inhibition and stimulation. Again, there is considerable
work which could be done with the spheroid system to better
understand the changes in cell growth regulation which occur
after therapeutic treatment. This would be especially true for
treatments which themselves induce the formation of necrosis.
Finally, it should be productive to investigate the possibility of
enhancing the cytostatic and/or cytotoxic properties of necrosis
as a new therapeutic approach. Seen in this context, the nu
trient-deprived conditions (such as hypoxia) in spheroids and
tumors, which are generally seen as an advantage in surviving
treatment, could be made harmful to the tumor by manipulating
the toxic properties of the necrosis that develops in such re
gions. This approach would have the advantage of being specific
to tumor microregions, as these are generally the only locations
of necrosis in vivo.
ACKNOWLEDGMENTS
I wish to acknowledge the following persons: Patricia Schor for her
assistance with some of the spheroid growth measurements, the monolayer growth curves, and the flow cytometry; Kathy Thompson for
preparation of the histology sections; Mark Wilder for assistance with
the flow cytometry; and especially Dr. Mudundi Raju for his support
throughout this project. Drs. Raju, Art Saponara, and Kip Wharton
provided critical suggestions for improving the manuscript. I also thank
the Pfizer Chemical Company for their generous gift of mithramycin.
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Role of Necrosis in Regulating the Growth Saturation of
Multicellular Spheroids
James P. Freyer
Cancer Res 1988;48:2432-2439.
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