Rates of Cell Division of Transplantable
Malignant Rat Tumors*
FELIXD. BERTALANFFY
ANDCHOSENLAU
(Department of Anatomy, Faculties of Medicine and Dentistry, Unirersity of Manitoba, Winnipeg, Manitoba, Canada)
SUMMARY
The mitotic rates of transplantable
Walker carcinosarcoma
256 and fibrosarcoma
1F16F were investigated in rats by the colchicine method. The mitotic rates of these
tumors were apparently not influenced by the time of day. In female rats the estrous
cycle did not seem to have appreciable effects on the mitotic rates of fibrosarcoma.
During the period of active growth of the tumors a constant increase in the number
of cells occurred each day until the onset of necrosis. During the 5th-10th day after
transplantation
about 60 per cent of the cells divided daily in Walker carcinosarcoma.
During the growth period of the fibrosarcoma—i.e., from the 15th to the 32d day after
transplantation—about
40 per cent of newly formed cells were daily added to this
tumor. These figures imply that the cell population of Walker tumor doubles about
every 1.7 days, of the fibrosarcoma every 2.5 days. The mitotic rates of these malig
nant tumors exceed those of most normal tissues and are surpassed only by those of
the epithelium (crypts) in the small intestine.
Tumor growth has been quantitatively
esti
mated by a variety of methods. Radioactive trac
ers, such as tritiated thymidine (e.g., 11), spectrophotometric determination
(e.g., 22, 23) of DNA
synthesis and content of malignant tissues have
been employed. Other methods entailed measure
ment of the size of tumors. Various formulas have
been proposed to calculate, particularly,
the vol
ume of tumors. Gaylord and Clowes (13) calcu
lated the volume of mass of tumors by the formula
V = (7T/6)¿i¿22;
d\ and ¿2represent two diameters
of the tumor. Woglom (21) used the formula V =
(4/3) irr3; r represents the average radius. Cham
bers and Scott (9) considered the volume to be a
function of (\X06)3; a and b represent two diam
eters of the tumor. Schrek (18) estimated the vol
ume of Walker tumors with the formula V =
0.5236d3; d represents the geometric mean of three
diameters of the tumor (the cube root of product
of the three dimensions). Tumor growth has been
estimated by graphically plotting the calculated
volume against time. The slope of the graph would
represent the rate of growth of the particular tu
mor.
Volume measurements of tumor growth are ap* This work was supported by a research grant from the
National Cancer Institute of Canada, Toronto.
Received for publication December 11, 1961.
plicable only to solid tumors. Disadvantages
were
that malignant tumors rarely are exactly spheri
cal, cylindrical, or conical, as is requisite for the
application of the above formulas. Moreover, for
example, the Walker carcinosarcoma
256 is not
necessarily solid throughout.
When applying the
formulas, particularly at later stages after trans
plantation, necrotic debris and fluid in the center
of the tumors, the result of degenerative processes,
would be included as viable parts of the tumor.
A more direct method for the determination
of
tumor growth is thus desirable. The colchicine
technic enables one to overcome to a large extent
the above-mentioned
difficulties. Size and shape of
the tumor, necrosis, and other factors do not
hamper measurement
of the mitotic rate by the
colchicine technic. Such factors as cell debris and
necrotic fluid do not present difficulties. Moreover,
growth of métastases, as well as the mitotic rates
of any type of animal tumor can be determined.
Colchicine, when injected into rats in a dose of
0.10 mg/100 gm of body weight, arrests in metaphase those cells that have entered division during
the time interval between injection and sacrifice
of the animal (12). Enumeration
of resting cells
and "colchicine metaphases" permits calculation of
the percentage of cells that divide in a tissue dur
ing a certain period of time (15, 16). It is thus
possible to estimate the percentage
increase of
627
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628
Cancer Research
newly formed cells in a tumor during 1 day (the
daily mitotic rate) and, further, the time required
for the addition of 100 per cent of tumor cells.
In some animal tissues, the mitotic activity ex
hibits appreciable diurnal fluctuations during the
24-hour period, whereas other tissues do not dis
play any daily rhythm. It has been reported (8,14)
that experimentally produced epidermoid carci
nomas in mice did not display diurnal variations
of mitotic rate, the latter remaining at an almost
constant level throughout day and night. A similar
finding was made on human carcinoma of the large
intestine (10).
In the present investigation the mitotic rates of
the Walker 256 carcinosarcoma and fibrosarcoma
1F16F in the rat were determined, from the time
sufficient tissues could be obtained after trans
plantation until the onset of extensive necrosis,
which automatically terminated the experiment,
often by killing the animals. Moreover, we investi
gated whether these tumors underwent a daily
rhythm of mitotic activity or whether they be
haved similarly in this regard to the above-men
tioned epidermoid and intestinal carcinomas.
MATERIALS AND METHODS
Walker carcinosarcoma.—Adult,male albino rats
of the Sprague-Dawley strain (Holtzmann Rat
Co., Madison, Wis.), ranging from 7 to 12 months,
were given transplants of Walker rat tumor tissue
(Carcinosarcoma 256). The tumor tissue was finely
minced and a saline suspension prepared. Of this
tumor cell suspension, 0.25 cc. was injected into
both thighs (hindlegs) of each rat. Each day, from
the 5th to the 10th day after the tumor transplan
tation, four rats were given injections at 10:00 A.M.
of 0.10 mg colchicine/100 gm of body weight; they
were sacrificed exactly 6 hours later.
To ascertain whether diurnal fluctuations of
mitotic rate occurred in the Walker tumor, sixteen
rats, on the 7th day after transplantation, were di
vided into four groups of four animals each. The
animals of each of these groups were given injec
tions of colchicine at a different time of the day—
i.e., at 10:00 A.M.,4:00 P.M., 10:00 P.M.,and 4:00
A.M.They were sacrificed 6 hours after injection.
Fibrosarcoma.—Adult female rats, of similar
strain, ranging from 7 to 12 months, were trans
planted with fibrosarcoma 1F16F—54FM tumor
(20)—tissue. Non-necrotic fibrosarcoma tissue was
removed from animals and cut in saline into small
pieces, about 2 mm. in diameter. These were in
serted into anesthetized rats through a small inci
sion over the xiphoid process beneath the epider
mis into the subcutaneous tissue. The incision was
subsequently closed with a metal clip; the latter
was removed after a week. A small tumor mass was
Vol. 22, June 1962
palpable 15 days after transplantation. Therefore,
beginning with the 15th day, four animals were
given injections of colchicine at 10:00 A.M. and
sacrificed 6 hours after injection. The colchicine
experiments were carried out on the 15th, 18th,
22d, 26th, 28th, and 32d day after transplantation.
Beyond this the tumors had become extensively
necrotic, and most became unsuitable for mitotic
counts. To ascertain whether a diurnal pattern of
mitotic rate was apparent in this tumor, sixteen
animals were given injections of colchicine at 4
different times of the day (as above), 32 days after
transplantation.
Following sacrifice, tissue samples from nonnecrotic areas were collected from each tumor mass
and immediately fixed in sublimate formalin. Par
affin sections, cut at 7 p., were stained with hematoxylin and eosin. With the aid of a hand tal
ly counter, more than 2000 resting nuclei were
counted (with medium-high magnifications) in tu
mor sections of each animal; the numbers of col
chicine metaphases that occurred in the counted
fields were recorded separately. The percentage of
cells that divided during the 6-hour period of col
chicine action was calculated for each animal. In
the two 24-hour experiments, each composed of
four groups of four rats (and each group covering
a different 6-hour period of the day), summation of
the four 6-hour percentages yielded the percentage
of cells that divided during 1 day—i.e., the daily
mitotic rate of the particular tumor. Knowing the
percentages of cells that divided in the tissues dur
ing 1 day, one can readily calculate the number of
days required for 100 per cent addition of tumor
cells.
RESULTS
With Walker carcinosarcoma no demonstrable
tumor mass could be observed prior to the 5th day
after transplantation; thus, not sufficient tissue
was available for mitotic counts at earlier stages.
With the tumor suspension used, few of the ani
mals survived longer than 14 days. Actively grow
ing cancerous tissue was present throughout the
tumor mass up to the 7th day after transplanta
tion. Subsequently, the central portion of the tu
mor underwent progressive necrosis, became lique
fied, and contained degenerated cell debris. After
the 9th day, most of the tumor consisted of necrot
ic debris, and the growing tumor tissue was con
fined to a peripheral zone. In deeper regions, the
mitotic rates varied greatly, depending on the de
gree of necrosis in the samples of tissue. Therefore,
determination of the mitotic rate was confined to
the periphery of the tumor, which was best pre
served. Subsequent to the 10th day after trans
plantation, mitotic counts became impossible be-
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BERTALANFFY
ANDLAU—CellDivision in Rat Tumors
cause enough well preserved tumor specimens could
not be obtained.
With fibrosarcoma, sufficient tumor tissue be
came available only 15 days after transplantation
of the tumor samples. From this time the tumors
grew steadily and rather rapidly until onset of ne
crosis occurring during the 4th or 5th week after
transplantation. Subsequently, necrosis progressed
rapidly, often resulting in extensive ulcération.It
was not possible to obtain suitable specimens later
than 5 weeks after transplantation. Accordingly,
the mitotic rates were estimated within the period
from 15 days until 32 days after transplantation.
To ascertain whether the mitotic rates of these
tumors underwent variations throughout the day,
the rates were determined during one complete 24hour period; for each tumor, four groups of four
animals each were given injections of colchicine
and sacrificed at four different time intervals of the
day. The results of the mitotic counts are tabu
lated in Table 1. Each one of the four percentages
shown for each tumor represents the average of the
individual percentages of the four animals in each
group for each 6-hour period. The variation in mi
totic rate of the animals in each group was ex
pressed as the standard deviation. Analysis of vari
ance revealed no statistically significant difference
among the four means of either tumor (variance
between means < variance within groups) and
thus no diurnal variations. The average mitotic
rate of the Walker tumor was estimated to be 15.8
per cent for any 6-hour period of the day, the total
standard deviation being 2.37 per cent (Table 1).
The average mitotic rate of fibrosarcoma for any
6-hour period of the day was estimated to be 10.7
per cent (Table 1). The daily mitotic rate (the per
centage of cells formed during one 24-hour period),
obtained by summation of the four 6-hour per
centages of each tumor, was 63 per cent for Walker
tumor, and 42.8 per cent for fibrosarcoma (Ta
ble 1).
Table 2 shows the results of the mitotic counts
of each tumor on different days after transplanta
tion. The percentage of the 6-hour period of each
day represents the average of the individual per
centages of the four animals composing each group;
TABLE 1
MITOTICRATESOFWALKERCARCIXOSARCOMA
ANDRATFIBROSARCOMA
DURINGA24-HouR PERIOD
Each 6-hour group consisted
NUCLEIWalker
of four animals
METAPHASESWalker
METAPHASESWalker
OF
DAT10
TIIIE or
«56977296979903102841F16F9066917589819112No.
25614121536150218221F16F10121030861988Daily
P.M.4
A.M.- 4
P.M.10
P.M.-10
AJl.4
PMA.M.-10
256
Mean±
dev.14.4±2.5S15.8
at.
Mean±jt.
dev.11.2±0.2011.2+0.289.6
2.3215.2
+
1.6917.7±2.4263.115.8±2.371F16F
+
1.8710.8+2.7342.8
+
4
A.M.No.
mitotic rate
Over -all average of 6-hour periodsPERCENTAGE
10.7±1.70
TABLE 2
MITOTICRATESOFWALKERCARCINOSARCOMA
FROMTHESTHTOIOTHDAYANDOFRAT
FIBROSARCOMA
FROMTHE ISTHTOTHE320 DAYAFTERTRANSPLANTATION
Each 6-hour group consisted
AFTERTRANSPLANTATIONWalker
DATS
NUCLEIWalker
of four animals
UETAPHASESWalker
UETAPBASESWalker
PERCENTAGE OF
256
Mean±
dev.15.7±2.2215.6±4.3614.
St.
Mean±»t.
dev.10.1±0.0710.8±0.069.8
25656789101F16F151822262882NO.
25610082929297729188898988661FI6F948390728932914466959066No.
256158614461412118913391289IF16F9599778779476371012Av.:6-HOCR
5512.9±1.7014.9
4 ±2.
0.1410.4
+
0.129.5
+
2.3914.5±2.5014.7±2.861F16F
+
1.8411.2
+
0.2010.3
+
+ 0.11
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630
Cancer Research
the variation in mitotic rate of each group is indi
cated as the standard deviation. With neither tu
mor did analysis of variance detect any significant
differences among the means of the six days (vari
ance between means < variance within groups).
The average mitotic rate of Walker carcinosarcoma over any 6-hour period of any day after
transplantation was 14.7 per cent, the total stand
ard deviation being 2.36 per cent (Table 2) ; this is
not significantly different from the over-all average
of 15.8 per cent obtained in the 24-hour experiment
(Table 1). The average mitotic rate of fibrosar
coma over any 6-hour period of any day was 10.3
per cent, the standard deviation being 0.11 per
cent (Table 2) ; this is nearly similar to the over-all
average of a 24-hour period of 10.7 per cent (Ta
ble 1).
With the fibrosarcoma, female rats in all stages
of the estrous cycle were used. The minimal differ
ences of mitotic rates of the animals would imply
that the rate of mitosis of this tumor remained un
changed during the estrous cycle.
DISCUSSION
Growth of a transplanted tumor can be divided
into three periods: (a) a latent period when the tu
mor adapts itself to the new host; (b) a period of
growth when the constituent cells actively pro
liferate and the newly formed cells outnumber by
far those lost by degeneration ; and (c) a period of
degeneration when the tumor undergoes extensive
necrosis and liquefaction. Death of the animal may
shortly follow this latter period. With the Walker
carcinosarcoma, the latent period was 4 days after
transplantation, with fibrosarcoma about 2 weeks.
Active growth of the Walker tumor proceeded be
tween the 5th and 10th day; it continued up to 4
weeks after transplantation with the fibrosarcoma.
Following the growth period was onset of necrosis;
most animals in the group with Walker tumor had
died 2 weeks after transplantation. Rats with fibro
sarcoma rarely survived 7-8 weeks.
The mitotic rates of Walker carcinosarcoma 256
and of fibrosarcoma 1F16F were determined on
various days after transplantation during the ac
tive growth period. In neither tumor were fluctua
tions in mitotic rate apparent during a 24-hour
Vol. 22, June 1962
period; tumor growth apparently continued at a
steady rate, unaffected by day and night, or by
sleeping and waking of the animals. A major ob
servation was that there was no significant differ
ence apparent between the mitotic rates of any
day during the growth period of either tumor. This
indicated that both tumors grew at a steady rate
from the time the tumor had adapted itself to the
host until the mitotic rate fell off with advanced
necrosis. During active growth, the daily increase
in the number of cells in the tumors remained con
stant and did not fluctuate appreciably. This ob
servation of constant tumor growth is in agreement
with a similar finding previously reported by
Schrek (18) on the growth of Walker carcinosar
coma and Flexner-Jobling rat carcinoma.
The average mitotic rate of Walker carcinosar
coma for any 6-hour period during the 5th-10th
day after transplantation was 14.7 per cent (Table
2). This indicated a daily cell addition to the tumor
of close to 60 per cent, or a 100 per cent cell in
crease every 1.7 days. In fibrosarcoma, the average
mitotic rate of any 6-hour period during the 15th32d day after transplantation was 10.3 per cent
(Table 2), indicating a 40 per cent addition of new
ly formed cells daily, or a cell increase of 100 per
cent every 2.5 days. These calculations are based
on the assumptions that all cells in the growth
areas counted were capable of cell division and
that their interphase was of similar duration. If
this is the case, the figures of 1.7 and 2.5 days, re
spectively, also denote the typical duration be
tween two divisions. Even though about 15 and 10
per cent of cells are added to Walker tumor and
fibrosarcoma, respectively, during each 6-hour pe
riod, it is assumed that most of the newly formed
cells would not divide during the next 24 hours
(i.e., the duration of a complete colchicine experi
ment—Table 1).
Comparison of the daily mitotic rates of Walker
carcinosarcoma and fibrosarcoma (60 and 40 per
cent, respectively) with those of normal rat tissues
reveals that their rates of cell formation exceed
those of most organs. The daily mitotic rates of the
tumors are considerably higher than those of the
urinary bladder—1.6 per cent (17) and ureter—3
per cent (4), bronchus and trachea—2-4 per cent
FIG. 1.—Portion of Walker carcinosarcoma 256 from a rat
given injection of colchicine. The cells varied greatly in shape
and size, and so did their nuclei. Some nuclei contained coarse
chromatin particles and large distinct nucleoli. Several "colchi
cine metaphases" are visible (e.g., in right lower corner), with
clumped, irregularly shaped black chromosomal material. H.
& E., X480.
FIG. 2.—Portion of fibrosarcoma 1F16F from a rat given in
jection of colchicine. The cells were large, and the nuclei dis
played marked anisokaryosis. Scattered among the resting cells
were several "colchicine metaphases" with darkly staining
clumped chromatin material. H. & E., X420.
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BERTALANFFY
ANDLAU—CellDivision in Rat Tumors
(3), epidermis—3-5 per cent (1, 19), colon—10per
cent (2), esophagus—11 per cent (2), sebaceous
glands—13 per cent (1), pulmonary alveolar cells
—15per cent (3, 5, 6), lymphocytes in bronchial
lymph nodes—15per cent (5), and rectum—16 per
cent (2). The daily mito tic rate of Walker tumor
is more than twice as high as those of buccal mu
cosa—24 per cent (2), and of anal epidermis—23
per cent (2). The daily rate of new formation of
cells in fibrosarcoma is exceeded by that in the
pyloric mucosa—54 per cent (16), and the daily
mitotic rates of both tumors are surpassed only by
those in the epithelium (crypts) of the small intes
tine—64-79 per cent, both of the rat (2, 15) and
man (7).
It may be of interest to compare and contrast
the significance of mitotic activity in normal and
malignant tissues. The mitotic rates of the malig
nant tumors exceed those of most normal tissues;
however, they are not higher than some mitotic
rates that may normally occur. During the period
of growth apparently most malignant cells retain
the ability to divide. In normal epithelia, chiefly
less differentiated cells (such as basal cells) undergo
mitosis, and the majority of newly formed cells dif
ferentiate, for example, into superficial squamous
cells, columnar secretory and absorptive cells, etc.,
which, after having reached maturity, divide rare
ly or not at all. In both malignant tumors a con
stant number of cells divide daily. Similarly, in
many adult normal tissues the numbers of cells
that each day undergo division are constant. How
ever, in the latter mitosis serves cell renewal,
and for each newly formed cell another, usu
ally more differentiated cell, becomes extruded;
the numbers of cells thus remain constant—or in a
steady state—in spite of an often considerable mi
totic activity. In contrast, in malignant tumors
mitosis results in cell addition (or accumulation)—
i.e., in growth of the tumor; thus, about 60 per
cent of new cells are added each day to Walker
carcinosarcoma, about 40 per cent to fibrosarcoma.
Cell loss may occur also from malignant tumors, as
by necrosis or cell desquamation. Cells are shed
particularly from epithelial tumors, and this is also
the basis of exfoliative cytology. However, unlike
normal tissues, cell formation and cell loss are not
balanced in an orderly fashion in malignant tu
mors, and during the period of growth cell loss is
usually exceeded by cell addition.
ACKNOWLEDGMENTS
The authors are grateful to Dr. A. C. Wallace, Cancer Re
search Laboratory, University of Western Ontario, London,
Ontario, for his kind advice and assistance; Dr. Wallace also
made available the tumor material for the transplants. We are
indebted also to Prof. I. Maclaren Thompson, Chairman of
this department, for his kind assistance with the preparation
of the manuscript and with the statistical evaluation of the
631
results. Technical assistance by Miss C. McAskill and Miss
R. Mueller is acknowledged.
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Rates of Cell Division of Transplantable Malignant Rat Tumors
Felix D. Bertalanffy and Chosen Lau
Cancer Res 1962;22:627-631.
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