[CANCER RESEARCH 42. 72-78,
0008-5472/82/0042-OOOOS02.00
January 1982]
Isolation of Quiescent Cells from Multicellular Tumor Spheroids Using
Centrifugal Elutriation1
Kenneth D. Bauer,2 Peter C. Keng, and Robert M. Sutherland
Department of Radiation Biology and Biophysics [P. C. K., R. M. S.J and Cancer Center. Experimental
University of Rochester. Rochester. New York 14642
ABSTRACT
A quiescent (nonproliferating) subpopulation was identified
by flow cytometric analysis using two-step acridine orange
staining in the EMT6/Rochester,
N. Y. subline multicellular
tumor spheroid, an in vitro culture system which provides a
cellular microenvironment which mimics that of many in vivo
tumors. To isolate a viable quiescent cell subpopulation, cen
trifugal elutriation which allows for cell separation mainly on
the basis of size was used. This technique provided single cells
of relatively homogeneous cell volume which varied over a wide
range (approximately 100 to 5000 cu jum). Though the relatively
small cell volume fractions were the most enriched (82%) in
quiescent cells, such cells were also observed in significant
numbers (=20%) even in the largest cell fractions. The cell
clonogenicity of the various elutriation fractions was also as
sessed and shown to be lowest (plating efficiency =20%) in
the small spheroid cells but relatively constant in fractions
containing intermediate and large cells (plating efficiency =
50%). Continuous [3H]thymidine labeling indicated a slower
rate of accumulation of labeled cells in the small spheroid cells,
which may result from the transition of proliferating spheroid
cells to the quiescent compartment during the course of label
ing. These findings indicate the utility of centrifugal elutriation
for quiescent cell characterization in in vitro tumor systems.
INTRODUCTION
Q3-cells, i.e., nonproliferating
cells, have been shown previ
ously to be of relevance in cancer therapy. A preferential
sparing of Q-tumor cells and thus a relatively increased sensi
tivity of P-cells to many chemotherapeutic agents have been
documented (6). In addition, recent studies (12, 19) suggest a
relative radioresistance of Q-tumor cells and indicate that they
may play an important role in tumor regrowth after radiation
therapy (18).
Due to the documented inherent resistance of Q-tumor cells
to current therapeutic modalities, their biochemical and bio
physical characterization appears to be an important precedent
to the development of more effective treatment regimens. To
date, most characterization of Q-cells has come about from
1This research was supported by NIH grants CA 11198, CAO9363, and CA
11051 and Contract DE-ACO2-76EV03490,
United States Department of En
ergy. Part of the research was supported by the Cell Separation Facility of the
University of Rochester Cancer Center. Flow cytometry was performed at the
University of Rochester Cell Sorting Facility under NIH Grant GM-23088. Pre
sented in part at the Eighth Conference on Analytical Cytology and Cytometry,
Wentworth-by-the-Sea.
N. H., May 1981.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: Q, quiescent; P. proliferating; dThd, thymidine;
Li, labeling index; FCM. flow cytometry; AO, acridine orange; BME, Eagle's basal
medium; PBS, phosphate-buffered
saline [NaCI (8.0 g/liter):Na2HPC>4 (1.15 g/
liter):KCI (0.2 g/liter):KH2PO4 (0.2 g/liter)].
Received April 24, 1981 ; accepted October 5, 1981.
72
Therapeutics Division [K. D. B., P. C. K., R. M. S.],
cultures which were starved by serum or amino acid deprivation
and, thereby, highly enriched in Q-cells (2). This approach may
suffer from the limitation that such observations on Q-cells
simply reflect extreme cell starvation, a condition which may
be distinct from that of most naturally occurring Q-tumor cells,
which occur together with P-cells in complex microenvironments in which additional factors such as hypoxia, pH, and
osmolarity may be important.
One in vitro tumor model system which appears useful for Qcell characterization is the multicellular tumor spheroid. Al
though spheroid cultures are fed daily to provide nutrient
renewal, they nevertheless contain a mixture of P- and Q-cells
(10, 26). In addition, spheroids exhibit cellular subpopulations
which are resistant to chemotherapeutic agents (25, 32) and
radiation (24); in these and other ways, they show a striking
resemblance to small nodular carcinomas (24, 26).
Characterization of Q-cells from spheroid cultures (and from
in vivo tumors) until recently has been unsuccessful due to
difficulties both in the identification and isolation of the Q-cell
population previous to further investigation.
Recently, centrifugal elutriation has been utilized to separate
and synchronize in vitro (13) and in vivo (15, 16) tumor cell
suspensions. This technique allows for the separation of cells
primarily on the basis of cell size and has the advantage of
providing cells of high viability. Previous investigations (9, 20)
have presented evidence that centrifugal elutriation or sedi
mentation velocity methods might be useful in separating Pand Q-tumor cells on the basis of cell size. However, these
investigations suffered from the inability to clearly distinguish
these cell subpopulations, and the conclusions were based on
differences in the [3H]dThd LI of various fractions. Such mea
surements are complicated by the known wide range of cell
cycle transit times in many tumor systems (23) and therefore
are difficult to interpret in the absence of a "marker" for the Qcells.
Darzynkiewicz et al. (7) and Tráganos étal.(27) have devel
oped an FCM method which appears to discriminate the Qand P-cell subpopulations. The method utilizes AO staining and
allows the simultaneous assessment of cellular DNA (green
fluorescence) and RNA (red fluorescence) contents on a percell basis, thereby identifying a discrete Q-cell subpopulation
by a relatively lower RNA and DNA content (27). This method
appears useful for Q-cell identification in lymphocytes (27) and
leukemias (28), although it was unsuccessful in identifying a
discrete Q-cell population in plateau-phase HeLa-S3 (human
cervical carcinoma);'« vitro suspension culture (4). In a previous
report (30), evidence was provided that suggested that this
method appeared useful in the identification of Q-cells in EMT6
monolayer cultures, and this has been confirmed in the EMT6/
Rochester, N. Y. subline (EMT6/Ro) multicellular spheroid (5).
In this report, centrifugal elutriation has been utilized to purify
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1982 American Association for Cancer Research.
VOL. 42
Isolation of Quiescent Spheroid Cells
the Q-cell population as identified by AO staining and FCM
analysis using EMT6/Ro multicellular spheroids in an effort to
better characterize these therapeutically refractory cells.
MATERIALS
AND METHODS
Spheroid Culture. EMT6/RO murine mammary tumor cells were
grown in monolayer cultures in antibiotic-free BME supplemented with
50
15% fetal bovine serum, as reported earlier (11). Multicellular sphe
roids were initiated from asynchronously growing monolayer cultures
(doubling time, approximately 12 to 13 hr) by inoculating 5X10"
cells
ISO
250
RED FLUORESCENCE
(Channel Number)
in 5 ml of medium into microbiological Retri dishes. After maintaining
these cells for 4 days in a 37° humidified incubator equilibrated with
3% CO2, small spheroids were removed from the Retri dishes and
added to suspension flasks containing 75 ml of growth medium. Two
days later, the spheroids of homogeneous size were sorted out and
placed in suspension flasks at a final concentration of approximately
500 spheroids/200
ml of medium. The old medium was removed by
suction and replaced by fresh medium plus serum daily thereafter as
described previously in detail (11 ). The spheroids utilized in the present
investigation were approximately 850 /¿min diameter (15 to 17 days of
growth), the diameter representing the geometric mean of 2 orthogonal
diameter measurements of spheroids in complete medium on Retri
dishes, as estimated under a phase-contrast inverted microscope.
0
into single cells. After this time,
(10 min at 400 x g), after which
resulting cell pellet was dispersed
cold serum-free BME and DNase
the cell suspension was centrifugea
the supernatant was poured off. The
by agitating lightly, and 20 ml of iceI (103 Kunitz units/ml; Sigma Chem
ical Co., St. Louis, Mo.) were added prior to centrifugal elutriation.
Centrifugal Elutriation. The procedure for obtaining relatively ho
mogenous cell populations from EMT6 spheroid cells using centrifugal
elutriation was a modification of the long collection method which has
been detailed previously (13-15). Briefly, single-cell suspensions from
multiceli spheroids were elutriated in ice-cold serum-free BME. The
elutriator system had been sterilized previously (using 70% ethanol),
and the elutriator run was performed at 4°using a constantly main
tained flow rate of 35 ml/min in these experiments. After loading the
spheroid cells, the rotor speed was decreased in a stepwise fashion
with varying numbers of 40-ml cell fractions collected at each step.
The cells in each fraction were counted, and their volume distributions
were assessed with an electronic particle counter and channelyzer
system (Models ZB1 and C1000, respectively; Coulter Electronics,
Hialeah, Fla.). The median cell volume of each fraction was estimated
from the median channel number of the cell volume distribution with a
calibration constant determined previously by relating microscopically
measured EMT6/Ro cell size to electronic particle counter settings.
Two-Step AO Staining. The 2-step AO staining was performed by
a modification of the method of Darzynkiewicz ef al. (7) and Tráganos
ef al. (27) as described previously (3). A cell suspension was obtained
from dissociated spheroids at a final concentration of approximately
106 cells/ml in PBS + glucose (1 g/liter). Approximately 0.3 ml of the
cell suspension was mixed with 0.45 ml of 0.1% Triton X-100 in 0.08
N MCI + 0.15 M NaCI (pH 2.2) for 1 min on ice. After this time, the cells
were stained by the addition of 1.8 ml of chromatographically
purified
AO (12 /ig/ml; Polysciences, Inc., Warrington, Pa.) in 0.2 M NajHPO«:
0.1 M citric acid (pH 6.0) and 1 HIM sodium EDTA, resulting in a final
AO concentration of 2.8 x 10~5 M. The calculated ratio of /¿molDMA:
phosphate to /«molAO was approximately 8.6, which at the AO con
centration utilized offers good spectral discrimination between nuclear
DMA and cytoplasmic RNA (17).
ISO
250
IO
7.5
5
Spheroid Dissociation. EMT6/Ro spheroids were dissociated by
removing approximately 750 spheroids from suspension flasks, placing
them into a sterile tube, and rinsing them once in 15 ml of ice-cold
serum-free BME. After rinsing, 15 ml of trypsin (73.8 units/ml; Worthington Biochemical Corp., Freehold, N. J.) in PBS were added to the
spheroids. This mixture was agitated on a mechanical rotator for
approximately 20 min at 37°,a time sufficient to disperse the spheroids
50
RELATIVE RED FLUORESCENCE
(Channel Number)
2.5
"0
50
RELATIVE
ISO
250
GREEN FLUORESCENCE
(Channel Number)
Chart 1. Representative 3-dimensional fluorescence contour map (fop), red
fluorescence histogram (middle), and green fluorescence histogram (bottom)
obtained from dissociated EMT6/RO spheroid cells after 2-step AO staining and
measuring by FCM. The relative red and green fluorescence intensities (channel
number) for individual cells and the relative cell number as indicated by the
contour levels are shown on the contour map. Iso-cell contour levels of 10, 25,
50, 100, and 250 cells are shown. Dors, locations with 5 or more cells accumu
lated. For fluorescence histograms, the relative cell number (ordinate) is plotted
against cellular red or green fluorescence (channel number).
FCM. The fluorescence of AO-stained cells was monitored on a
EPICS IV flow cytometer (Coulter Electronics, Inc.) interfaced to a PDP11 /03 minicomputer (Digital Equipment Corp., Maynard, Mass.). The
cells were stirred and maintained at approximately 4° during FCM
analysis.
Laser excitation
(Model 164-05;
Spectra
Physics, Boston,
Mass.) was at 488 nm (300 milliwatts). A 510 nm interference barrier
filter was inserted in front of the right angle collection lens. A 560 nm
dichroic mirror was used to split the fluorescence signal. Green fluo
rescence (530 to 560 nm) was observed with the addition of a 530 nm
long-pass filter, while red fluorescence was simultaneously monitored
utilizing a 630 nm long-pass filter. A minimum of 1.5 x 10* spheroid
cells was analyzed for each FCM histogram (relative red or green
fluorescence intensity versus relative cell numbers) and 3-dimensional
contour map (cellular red and green fluorescence intensity and cell
number).
The proportion of Q-cells observed after AO staining was estimated
from 3-dimensional contour analysis using a computer program devel
oped by Salzman et al. (22) compatible with the interfaced PDP-11703
minicomputer. Briefly, the method involved plotting contours of suc
cessively larger numbers of cells per red and green fluorescence
channel number combination, which provided an estimate of the bound
ary between the P-like and Q-like cells. Next, the boundary of the
region of interest could be graphically "windowed,"
and the cell
number in the window of interest could be quantitated and compared
with the total cell number. For example, in Chart 1 (fop), the Q-like
population was estimated from the cell number occurring between
Green Fluorescence Channels 61 and 94 and Red Fluorescence
Channels 18 to 46.
Cell Clonogenicity. The cell clonogenicity of EMT6/RO cells was
JANUARY 1982
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1982 American Association for Cancer Research.
73
K. D. Bauer et al.
assayed by the method of Puck and Marcus (21). Approximately 75
and 150 cells from control or elutriated spheroid cell suspensions in 5
ml of BME + 15% fetal bovine serum were plated on 60-mm Retri
dishes with 5 replicate dishes plated per dilution. The cells were then
maintained in a 37°incubator for 10 to 12 days, i.e., un'il macroscopic
IO •
TOTAL EMT6/RO
SPHEROID CELLS
8-
colonies appeared. The colonies were then stained with crystal violet
and scored when consisting of 50 or more cells.
[3H]dThd Labeling and Autoradiography. For LI determinations,
[3H]dThd (specific activity = 20 Ci/mmol; New England Nuclear,
Boston, Mass.) was added to monolayer or multiceli spheroid cultures
at a final activity of approximately 0.025 /iCi/ml. The continuous LI for
the multiceli spheroids was determined by collecting 30 spheroids from
suspension flasks after various periods of [3H]dThd incubation at 37°,
trypsinizing
them to single-cell
suspensions,
centrifuging
the cells for
10 min at 200 x g, and fixing the resuspended cells in 1 ml methanol:
glacial acetic acid (3:1). For the monolayer cultures, individual 100mm plates were incubated with [3H]dThd for appropriate periods of
time, trypsinized. and fixed as for the spheroids.
After fixation, the cells were washed twice in methanohglacial acetic
acid, and the washed cells were dropped onto prewashed microscope
slides. The slides were dipped in NTB3 photographic emulsion (East
man Kodak Co., Rochester, N. Y.), stored at 4°for various lengths of
I
Si
SEPARATED EMT6/RO
SPHEROID CELLS
time, developed, and stained with hematoxylin and eosin. The slides
were developed between 2 and 10 days and microscopically scored
for labeled cells. The background grain counts were <1 grain/cell with
a labeling index threshold of 2 grains/cell. The reported continuous LI
was determined from the plateau region of temporal LI curves in all
cases. The pulse [3H]dThd LI was determined similarly after a [3H]dThd
exposure period of 30 min.
RESULTS
Chart 1 (top) shows a representative 3-dimensional contour
map of a 2-step AO-stained cell suspension derived from 850
cu firn EMT6/RO spheroids in which cellular red and green
fluorescence/cell
was assessed simultaneously. The corre
sponding histograms for green fluorescence (DMA content) and
red fluorescence (RNA content) are displayed in Chart 1 (mid
dle and bottom, respectively). On the contour map and the red
fluorescence histogram, a discrete cellular subpopulation with
a relatively low red fluorescence (mean channel number, ap
proximately Channel 23) is clearly identifiable. From the con
tour map, this population is further observed to possess a low
(i.e., Gì)DNA content (green fluorescence). This subpopulation
occurs only at very low frequency (<2.5%, data not shown) in
I
2
CELL VOLUME
(cu u,m xlO"
Chart 2. Representative cell volume profiles of dissociated EMT6/Ro sphe
roid cells. A, relative cell number versus cell volume obtained from nonelutriated
cells (total spheroids). B. result from 4 different fractions (Groups Q1, Q2, Q3,
and Q4/P) obtained after centrifugal elutriation which have median volumes of
approximately 800, 1250, 1650, and 2250 cu ¡im,respectively.
The cells in Chart 2 ß,Group Q1, are from an early elutriation
K. D. Bauer et al.
the relation between the relative clonogenicity of elutriated
cells and the estimated proportion of Q-like cells which was
assessed on the remaining cells using AO staining and FCM
analysis. Linear regression analysis of the raw data indicates
a significant (p < 0.05) correlation coefficient, r = -0.77,
suggesting an inverse relationship between cell viability (i.e.,
clonogenicity) and the proportion of Q-like cells. Since expo
nential EMT6/RO cells, which contain approximately 97.5% Plike cells, have a clonogenicity of approximately only 70% and
since the mean proportion of Q-like cells in each of the elutria
tion fractions shown in Chart 5 is higher than the mean pro
Ö 50
ISO
250
portion of nonclonogenic cells, it is suggested that a proportion
RED FLUORESCENCE
of the Q-like population is composed of clonogenic cells. Also,
(Channel Number)
it is apparent that the fractions with the smallest proportions of
Q-like cells (cells of largest size) have a somewhat lower
clonogenicity than do fractions with intermediate numbers of
2.0
such cells. These fractions composed of very large cells include
giant cells which have been shown previously (15, 16) to have
I.5
a lower clonogenicity.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1982 American Association for Cancer Research.
In classical cell cycle kinetic analyses, variations in the rate
Isolation of Quiescent Spheroid Cells
(Chart 3, Group Q1 ) is clearly enriched in the proportion of Qlike cells, with >99% of the cells having G,-phase DNA content
(Chart 3, lower). Chart 3, Groups Q2 and Q3, indicate that
successively larger cells also with predominantly G t-phase
DNA content show proportionately fewer Q-like cells. Despite
the similar DNA contents in the different groups, both the Plike and Q-like cells of Chart 3, Group Q3, appear to have a
somewhat higher red fluorescence (RNA content) than do those
of Groups Q1 and Q2.
The median cell volume, proportion of trypan blue-excluding
cells, calculated proportions of Q-like cells from various elutriation fractions, and proportion of the total spheroid cells which
the fractions represent are displayed on Table 1. This table
illustrates that centrifugal elutriation allows for the separation
of cells which vary widely in the proportion of Q-cells. The
earliest elutriation fractions isolated (those with median vol
umes <800 cm firn) were composed mainly of cell debris and
dead cells (approximately only 21 % of these cells were capable
of trypan blue exclusion). Most of these cells showed a green
fluorescence intensity (DNA content) and red fluorescence
intensity (RNA content) less than that of Gi-phase cells with
very few (approximately 1%) Q-like cells. Relative to total
spheroids, which contain approximately 21% Q-like cells, it
can be seen that, with the exception of the smallest fractions
(median volumes, <800 cu urn), the smaller volume fractions
appear the most enriched in the Q-like cells, although some
enrichment is evident in cell fractions as large as 1600 eu /¿m,
a median cell volume approximately that of the total spheroid
cells. Despite the marked enrichment of Q-like cells in the
small-volume fractions, elutriated cells of larger median volume
still contain significant numbers (=20%) of Q-like cells.
This finding is further exemplified in Chart 4 in which a 3dimensional contour map (top), red fluorescence histogram
(middle), and green fluorescence histogram (bottom) of elu
triated cells with a large median cell volume (corresponding to
that of Chart 28, Group Q4/P) are displayed. In this case,
Table 1
Enrichment of Q-like cells and trypan blue dye exclusion of elutriated
EMT6/RO spheroid cells
The estimated proportion of Q-like cells observed after AO staining and FCM
was derived from 3-dimensional contour analysis (cellular red and green fluores
cence intensities and cell number). See text for details.
Median cell volume8
% of Q-like cells6
% of trypan
blue dye-exeluding cells
2182.3±0.1°
}
<800800-900900-10001000-12001200-14001400-16001600-18001
8158.0±5.3
)
6.448.6
±
5.135.3
±
2.727.1
±
3.019.4
±
Proportion
of total
spheroid
cells (%)
^70
3.120.1
±
800-20002000-25002500-3000>30000.9
1.924.2
±
3.225.1
±
1.221.2
±
±5.890QO9A94831
Derived from the median channel number of calibrated Coulter channelyzer
measurements.
6 Total (nonelutriated) spheroids have a median cell volume of approximately
1600 cm ¡itn.
c Mean ±S.D.; n a 3 determinations.
200-150-100-50-0-QlÄKSr*---"r*.•0
200-,I50-IOO-50-nQ2^S=OD„
200
-,150-100-50-o.Q3Oè£=3^Ì&-•fi
"
250n§•4-3-2-1
50
150
0
50
150
0 50
250
RED FLUORESCENCE (Channel Number)
5-,
432
150
250
03
•n-QlL!L..J
"rTri*-w^.
5
IO-
Ql
7.5
5
2.5-1
O
0 50
150
0
250
O 50
150
250
O 50
RELATIVE RED FLUORESCENCE (Channel Number)
107.552.5-
Q2
10-
150
250
150
250
Q3
7.5
52.5-
0
0
150
150
250
0 50
250
0 50
RELATIVE GREEN FLUORESCENCE (Channel Number)
Chart 3. Three-dimensional contour map (top), red fluorescence histogram (middle), and green fluorescence histogram (bottom) from elutriated spheroid cells
obtained after AO staining and measuring by FCM. Q1, Q2. and Q3 illustrate these parameters in cells from fractions with increasing volume, which correspond
approximately to those of Cell Groups Q1, Q2, and Q3, respectively, of Chart 2B. The iso-cell contour levels and dot representations are as in Chart 1.
JANUARY
1982
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1982 American Association for Cancer Research.
75
K. D. Bauer et al.
'0
50
150
the relation between the relative clonogenicity of elutriated
cells and the estimated proportion of Q-like cells which was
assessed on the remaining cells using AO staining and FCM
analysis. Linear regression analysis of the raw data indicates
a significant (p < 0.05) correlation coefficient, r = —0.77,
suggesting an inverse relationship between cell viability (i.e.,
clonogenicity) and the proportion of Q-like cells. Since expo
nential EMT6/RO cells, which contain approximately 97.5% Plike cells, have a clonogenicity of approximately only 70% and
since the mean proportion of Q-like cells in each of the elutria
tion fractions shown in Chart 5 is higher than the mean pro
portion of nonclonogenic cells, it is suggested that a proportion
of the Q-like population is composed of clonogenic cells. Also,
250
RED FLUORESCENCE
(Channel Number)
it is apparent that the fractions with the smallest proportions of
Q-like cells (cells of largest size) have a somewhat lower
clonogenicity than do fractions with intermediate numbers of
such cells. These fractions composed of very large cells include
giant cells which have been shown previously (15, 16) to have
a lower clonogenicity.
In classical cell cycle kinetic analyses, variations in the rate
of increase in LI after continuous [3H]dThd labeling have been
2.0
1.5
1.0
interpreted to indicate variable rates of cell cycle transit (29).
To further characterize the elutriated spheroid cells, pulse- and
continuous [3H]dThd-labeling studies were performed prior to
0.5-
50
150
250
RELATIVE RED FLUORESCENCE
(Channel Number)
centrifugal elutriation. The results of this investigation are
shown on Chart 6. Chart 6A indicates the result from small
cells (median volume, approximately 800 eu /¿m)thus corre
sponding to those shown in Chart 2B, Group Q1, while Chart
6, ßand C, indicates the result in larger cells (median volume,
approximately 1000 and 1GOOcu firn, respectively). For com
parison, the result from total spheroids is shown in Chart 6D.
The smallest cells (Chart 6A, most enriched in Q-like cells)
show a significant but somewhat slower rate of increase in LI
(1.6%/hr)
relative to that observed in cells somewhat less
enriched in Q-cells (Chart 66, 1.9%/hr). Both of these groups
appear to increase the LI at a slower rate than do cells in Chart
6C (2.8%/hr) which contained nearly the same proportion of
„ EXPONENTIAL
70J_-"""~"MONOLAYER
CELLS
0
50
RELATIVE
150
250
GREEN FLUORESCENCE
(Channel Number)
Chart 4. Three-dimensional contour map (top), red fluorescence histogram
(middle), and green fluorescence histogram (bottom) from elutriated spheroid
cells of relatively large median cell volume (approximately 2250 cu /un), corre
sponding to Cell Group Q4/P of Chart 26. The iso-cell contour levels and dot
representations are as in Chart 1.
although a substantial enrichment of S- and G2-M-phase cells
was achieved relative to the total spheroid cells, a substantial
number of Q-like cells remain. Although Q-like cells with Giphase DMA content are again observed, other cells, possibly
corresponding to Q-like cells with S- and G2-phase DMA con
tents and somewhat higher RNA content, are also observed.
One advantage of centrifugal elutriation is that it is a mild
separation procedure which allows for the isolation of viable
cells. Trypan blue dye exclusion measurements performed
both on nonelutriated spheroid cell suspensions and on a
composite of all elutriated fractions suggested that centrifugal
elutriation did not induce gross cell killing at the cell membrane
level (i.e., 83% dye-excluding cells in nonelutriated spheroids
and 87% dye-excluding cells after elutriation). Chart 5 shows
76
60
IO
S
e>
50
4O
TOTAL
SPHEROID CELLS
2O
20
30
40
PROPORTION
50
OF Q-LIKE
60
70
80
90
CELLS
Chart 5. Relationship between cell clonogenicity and the proportion of Q-like
cells obtained by 2-step AO staining and FCM for elutriated EMT6/RO spheroid
cells.The plotted proportion of Q-cells represents the mean of at least 3 separate
determinations. Points, corresponding clonogenicity expressed as the group
mean; oars, S.D.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1982 American Association for Cancer Research.
VOL.
42
Isolation of Quiescent Spheroid Cells
documentation of heterogeneous Q-cells in solid tumors (8).
The present finding (Chart 4) that Gi-phase Q-cells appear to
reduce the effectiveness of obtaining synchronous populations
of S- and G2M-phase cells by elutriation may also be important
in the context of previous data which have indicated substantial
contamination of Gt-phase cells in S- and G2-M-enriched frac
tions in the EMT6/RO (1 5) and other (1 6) solid-tumor systems.
The relationship between clonogenicity and the P- and Qtumor cell compartments has long been speculated upon by
many investigators and remains somewhat controversial. The
current findings (Chart 5) suggest that the identified Q-cells in
EMT6/RO spheroids include both clonogenic and nonclonogenic cells, further emphasizing the heterogeneity of the Qcompartment. The additional observation in Chart 5 of an
apparent inverse relationship between the proportion of Q-cells
10
20
30
TIME (hr)
Chart 6. Continuous
40
[3H]dThd
SO
20
JO
TIME (hr)
40
LI (%) as a function
of time in EMT6/Ro
spheroid cells. A to C, determinations from elutriated spheroid cells of increasing
median volume, i.e., approximately 800, 1000. and 1600 cu ,um, respectively; D,
the same determination from total spheroids.
Q-like cells and rate of increase in L! as cells from total
spheroids, Chart 6D (22 and 2.6%/hr, respectively). These
data thus could be interpreted as indicating a relatively slower
rate of cell cycle transit in the groups (i.e., Chart 6, A and 8)
most enriched in Q-cells, relative to the total spheroid (Chart
6D).
DISCUSSION
These studies demonstrate that centrifugal elutriation can be
successfully used to separate cells from the multicellular sphe
roid tumor model into subpopulations which are quite homo
geneous in size. Small cell volume fractions were obtained
which consisted almost exclusively of G,-phase cells, indicating
the successful synchronization of G,-phase spheroid cells. By
combining centrifugal elutriation with AO staining and FCM,
which allows for the identification of Q-like cells on the basis of
relatively lower RNA and DNA contents than the P-cell coun
terparts, the Q-like cells have been markedly enriched (i.e., 80
to 85% Q-cells versus approximately 21 % in the total spheroid)
(Chart 3, Table 1) in elutriation fractions of small (i.e., 800 cu
jum) cell volume.
Centrifugal elutriation in the present investigation provided
approximately 87% trypan blue dye-excluding cells as com
pared with 83% dye-excluding nonelutriated spheroid cells.
Analysis of the composite of data from all elutriation fractions
(Table 1) revealed approximately 25% total Q-like spheroid
cells, as compared with approximately 21 ±3% (S.D.) Q-like
cells from nonelutriated EMT6 spheroids. Thus, the method
provided a benign method for spheroid cell separation, al
though we cannot rule out the loss (lysis) of a small proportion
of labile (presumably dead) cells by this procedure.
Despite success in enriching for Q-like cells in small-volume
spheroid cell fractions, such cells were also observed in sub
stantial numbers in fractions containing cells of much larger
median volume. Such size heterogeneity of Q-cells and the
possible appearance of Q-cells with varying DNA and RNA
contents (Chart 4) suggest different modes of arrest of P-cells
or different physiological states of Q-cells in the multicellular
spheroid cultures, a finding in agreement with the previous
and clonogenicity provides further support for the hypothesis
that Q-cells in in vitro tumor systems represent an intermediate
compartment between P-cells and dead cells. Finally, the pres
ent data corroborate previous in vitro studies (11, 25), which
suggested a decreased clonogenicity in Q- relative to P-spheroid cells but contrast with the previous conclusion of an equal
clonogenicity in these cell compartments in a rat rhabdomyosarcoma (1 ).
The multicellular spheroid is a dynamic cell system charac
terized by a varying growth fraction [i.e., P/P + Q(10, 31)], Pto Q-compartment transition (10, 11 ), cell loss (1 0), and vari
ations in the proportion of Q-cells as a function of the distance
between the center and periphery of the spheroid (1 1). Our
finding of marked increases in the continuous LI even in frac
tions substantially enriched in Q-cells (Chart 6, A and 8), which
has been related previously to cell cycle transit rate (29),
indicates that such an interpretation must be considered with
great caution, due to the probability that such LI increases
mainly reflect P- to Q-transition during the course of the labeling
studies (4). Since many tumors are also characterized low and
varying growth fractions (8) and substantial cell loss (23), the
present results argue that the use of continuous [3H]dThd
labeling for estimating the rate of cell cycle transit in such
tumor systems may not be warranted in the absence of Q-cell
identification. Furthermore, the Q-cells identified using this
assay probably represent only a subpopulation of the Q-cell
compartment.
ACKNOWLEDGMENTS
The authors wish to express their sincere appreciation to Dr. P. Horan for
helpful consultation and reviewing of this manuscript and to P. Alden for expert
technical assistance.
REFERENCES
1. Barendsen, G. W., Roelse. H., Hermens, A. F., Madhuizen, H. T., Van
Peperzel, H. A., and Rutgers, D. H. Clonogenic capacity of proliferating and
nonproliferating cells of a transplantable rat rhabdomyosarcoma in relation
to its radiation sensitivity. J. Nati. Cancer Inst., 57: 1521-1526,
1973.
2. Baserga, R. Resting cells and the G, phase of the cell cycle. J. Cell. Physiol.,
95:371-386,
1978.
3. Bauer, K. D., and Dethlefsen, L. A. Total cellular RNA content: correlation
between flow cytometry and ultraviolet spectroscopy. J. Histochem. Cytochem., 28:493-498,
1980.
4. Bauer, K. D., and Dethlefsen, L. A. Control of cellular proliferation in HeLaS3 suspension cultures: characterization of culture utilizing acridine orange
staining procedures. J. Cell. Physiol., 708. 99-112, 1981.
5. Bauer, K. D., and Sutherland, R. M. Identification of a quiescent cellular
subpopulation in EMT6 Ro multicellular spheroids using acridine orange
staining and flow cytometry. In: Proceedings of the First International Con
ference on Spheroids in Cancer Research, p. 50. Rochester, N. Y., June 16
to 20, 1980.
JANUARY 1982
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1982 American Association for Cancer Research.
77
K. D. Bauer et al.
6. Clarkson. B. D. The survival value of the dormant state in neoplastia and
normal cell populations. In: B. D. Clarkson and R. Baserga (eds.). Control of
Proliferation in Animal Cells, pp. 945-972. Cold Spring Harbor, N. Y.: Cold
Spring Harbor Laboratory. 1974.
7. Darzynkiewicz, 7... Tráganos. R., and Melamed. M. R. New cell cycle
compartments identified by multiparameter flow cytometry. Cytometry, '
98-108, 1981.
8. Dethlefsen, L. A. In quest of the quaint quiescent cells. In: R. E. Meyn and
H. R. Withers (eds.), Radiation Biology in Cancer Research, pp. 415-435.
New York: Raven Press, 1979.
9. Durand, R. E. Isolation of cell subpopulations from in vitro tumor models
according to sedimentation velocity. Cancer Res., 35. 1295-1300,
1975.
10. Durand, R. E. Cell cycle kinetics in an in vitro tumor model. Cell. Tissue
Kinet., 9. 403-412. 1976.
11. Freyer. J. P., and Sutherland, R. M. Selective dissociation and characteri
zation of different regions of multiceli tumor spheroids. Cancer Res., 40:
3956-3965.
1980.
12. Kallman, R. F., Combs. C. A.. Franks. A. J., ef al. Evidence for the recruit
ment of noncycling clonogemc tumor cells. In R. E. Meyn and H. R. Withers
(eds.). Radiation Biology in Cancer Research, pp. 397-414.
New York:
Raven Press. 1979.
13. Keng, P. C.. Li. C. K. N., and Wheeler. K. T. Synchronization of 9L rat brain
tumor cells by centrifugal elutriation. Cell Biophys.. 2. 191-206, 1980.
14. Keng. P. C.. U. C. K. N., and Wheeler, K. T. Characterization of the
separation properties of the Beckman elutriator system. Cell Biophys.. 3.
41-56, 1981.
15. Keng, P. C.. Wheeler. K. T., Siemann, D. W., and Lord. E. M. Synchronization
of cells from solid tumors by centifugal elutriation. Exp. Cell Res., 134:1522, 1981.
16. Meistnch. M. L., Grdina, D. J., and Barlogie, B. Separation of cells from
mouse solid tumors by centrifugal elutriation. Cancer Res., 37:4291 -4296,
1977.
17. Nicolini. C. M., Belmont, A., Pavadi, S . Lessin, S , and Abraham, S. Mass
action and acridine orange staining: static and flow cytofluorometry.
J.
Histochem. Cytochem., 27: 102-113, 1979.
18. Potmesil. M., and Goldfeder. A. Cell kinetics of irradiated experimental
tumors: cell transition from the nonproliferating to the proliferating pool. Cell
Tissue Kinet.. »5.563-570, 1980.
19. Potmesil. M.. Ludwig, D., and Goldfeder. A. Cell kinetics of irradiated
experimental tumors: relationship between the proliferating and nonprolifer
78
ating pool. Cell Tissue Kinet., 8. 369-385, 1975.
20. Preisler, H., Walczak. I., Renick, J., and Rustum, Y. M. Separation of
leukemic cells into proliferative and quiescent subpopulations by centrifugal
elutriation. Cancer Res., 37: 3876-3880,
1977.
21. Puck. T. T., and Marcus, P. I. A rapid method for viable cell titration and
clone production with HeLa cells in tissue culture: the use of X-irradiated
cells to supply conditioning factors. Proc. Nati. Acad. Sei. U. S. A., 41:432437, 1955.
22. Salzman, G. C., Hiebert, R. D., and Crowed, J. M. Data acquisition and
display for a high-speed cell sorter. Comput. Biomed. Res., il: 77-88,
1978.
23. Steel, G. G . Adams, K., and Barrett, J. C. Analysis of the cell population
kinetics of transplanted tumors of widely differing growth rate. Br. J. Cancer,
20. 784-800. 1966.
24. Sutherland, R. M., and Durand, R. E. Radiation response of multiceli sphe
roids—an in vitro tumor model. Curr. Top. Radiât.Res.. 11: 87-139, 1976.
25. Sutherland, R. M.. Eddy. H. A.. Bareham, B.. Reich. K.. and Vanantwerp, D.
Resistance to Adriamycin in multicellular spheroids. Int. J. Radiât. Oncol
Biol. Phys.. 5. 1225-1230.
1979.
26. Sutherland, R. M., McCredie, J. A., and Inch. W. R. Growth of multiceli
spheroids in tissue culture as a model of nodular carcinomas. J. Nat). Cancer
Inst.. 46: 113-120, 1971.
27. Tráganos. F., Darzynkiewicz. Z., Sharpless, T.. and Melamed, M. R. Simul
taneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells
using acridine orange in a flow cytofluorometric
system. J. Histochem.
Cytochem.. 25. 46-56, 1977.
28. Tráganos. F., Darzynkiewicz, Z., Sharpless, T., and Melamed, M. R. Erythroid differentiation of Friend leukemia cells as studied by acridine orange
staining and flow cytometry. J. Histochem. Cytochem., 27: 382-389, 1979.
29. Watanabe, I., and Okada, S. Stationary phase of mammalian cultured cells
(L5178Y). J. Cell Biol.. 35. 285-294. 1974.
30. Watson. J. V., and Chambers, S. H. Fluorescence discrimination between
diploid cells on their RNA content: a possible distinction between clonogenic
and nonclonogenic cells. Br. J. Cancer, 36. 592-600. 1977.
31. Yuhas. J. M.. and Li, A. P. Growth fraction as the major determinant of
multicellular tumor spheroid growth rates. Cancer Res., 38 1528-1532,
1978.
32. Yuhas, J. M., Tarleton, A. E., and Harman, J. G. /" vitro analysis of the
response of multicellular tumor spheroids exposed to chemotherapeutic
agents in vitro or in vivo. Cancer Res.. 38. 3595-3598.
1978.
CANCER
RESEARCH
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1982 American Association for Cancer Research.
VOL.
42
Isolation of Quiescent Cells from Multicellular Tumor Spheroids
Using Centrifugal Elutriation
Kenneth D. Bauer, Peter C. Keng and Robert M. Sutherland
Cancer Res 1982;42:72-78.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/42/1/72
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1982 American Association for Cancer Research.