[CANCER RESEARCH 39. 3861-3865.
October 1979]
0008-5472/79/0039-OOOOS02.00
Effect of Surgical Removal on the Growth and Kinetics of Residual
Tumor1
Nurten Gunduz, Bernard Fisher,2 and Elizabeth A. Saffer
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
ABSTRACT
Findings from this study using a transplantable C3H mam
mary tumor failed to indicate interaction relative to growth
parameters between two foci present in the same host. Whether
they were growing alone or in the presence of a second focus,
tumor growth rates were similar until the combined mass of
multiple tumors approached that which was incompatible with
survival. Only then was a difference in growth observed. Cy-
tokinetic parameters, i.e., labeling index, primer-dependent
DNA polymerase index or growth fraction, DMA synthesis time,
tumor doubling time, and cell cycle time, were also similar
whether tumors grew alone or in the presence of a second
focus. Following removal of a tumor, changes were observed
within 24 hr in the kinetics of the residual focus. There was an
increase in labeling index (duration =10 days) and primer-
dependent DNA polymerase index with a decrease in the tumor
doubling time. Minimal change was noted in DNA synthesis
time and cell cycle time. The kinetic changes observed were
reflected in a measurable increase in tumor size = a week
following tumor removal. Absence of an alteration in DNA
synthesis time and cell cycle time indicates that the increase in
tumor growth was probably due to a conversion of noncycling
cells in Go phase into proliferation. Relationship of the findings
to the use of adjuvant chemotherapy is considered.
INTRODUCTION
Numerous investigators have reported that the presence of
a primary tumor inhibits growth of métastases (1, 2, 4, 5, 8-
10, 18, 20) and that its removal accelerates their growth (7-9,
12, 15, 19). The degree of inhibition varied relative to the type
of tumor and its volume (6). Findings from those studies were
obtained by observation of gross tumor growth. None supplied
information relative to cellular change. With the advent of
methodology for ascertaining cytokinetic parameters, consid
erable information has accumulated regarding the growth char
acteristics of both primary and metastatic tumors. Few inves
tigations, however, have been carried out to determine the
effect of removal or manipulation of a primary tumor on the
kinetics of métastases. The reports by Simpson-Herren ef al.
(16, 17) describing the consequences of excision of primary
s.c. Lewis lung tumors on kinetics of lung métastasesprovide,
to our knowledge, the only information in that regard. They
observed that noncurative excision resulted in an increase in
the LI3 and growth rate with minimal changes in the Tc and Ts
Received March 20, 1979; accepted June 22, 1979.
1 Supported by USPHS Grants CA-14972 and CA-12102 and funds contrib
uted by Luis and Antoinette Nunez of Caracas, Venezuela.
2 To whom requests for reprints should be addressed, at Department
Surgery, University of Pittsburgh School of Medicine, 914 Scaife Hall. 3550
Terrace Street, Pittsburgh, Pa. 15261.
3 The abbreviations used are: LI, labeling index; Tc, cell cycle time; Ts, DNA
OCTOBER 1979
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15261
of the métastases.There was a consistent decrease in median
life span as a result of stimulation of growth of lung nodules.
Since much of the rationale for the use of postsurgical adjuvant
chemotherapy has arisen from those contributions, there is
clearly a need for further investigation utilizing a variety of
models to determine the universality of the findings. As a
consequence, the present studies using a C3H mammary tumor
have been carried out to determine the cell kinetics of multiple
tumor foci and to ascertain whether kinetic changes occur in
a residual tumor following removal of a distant focus.
MATERIALS AND METHODS
Animals. Eight- to 12-week-old female C3HeB/FeJ mice
from The Jackson Laboratory, Bar Harbor, Maine, were used.
All animals were housed in separate cages and fed standard
laboratory chow and water ad libitum.
Tumors. The spontaneous mammary adenocarcinoma orig
inating in a female C3H/HeJ mouse was passed by biweekly
s.c. transplant in C3HeB/FeJ mice. Tumor cell suspensions
were prepared by mincing tumor fragments with scissors on an
80-mesh nylon screen and washing the cells through the
screen with Medium 199. The cells were counted using trypan
blue exclusion as a test of viability. Viable tumor cells (2 x 105)
were inoculated s.c. in the left hind leg proximal to the popliteal
node to induce tumors arbitrarily designated as "large," and 3
x 10* tumor cells were injected into the right hind leg at the
same location to induce tumors designated as "small." In some
animals, large tumors were produced in both legs by inocula
tion of 2 x 10s cells.
Tumor Growth. Tumor growth was followed by caliper mea
surement of 2 perpendicular diameters. Tumor volumes were
calculated, and curves of mean volume versus time were plot
ted on semilog paper. Td was derived from the slope of the
curve.
Experimental Design. Eighty-six animals were given injec
tions of 2 x 105 and 3 x 104 tumor cells in the left and right
hind leg, respectively, at the same time from the same suspen
sion and divided into 6 groups consisting of 12 to 15 mice
each. The large tumors were removed from 3 of the groups at
14,21, or 28 days after inoculation of tumor cells. The remain
ing 3 groups served as controls for the tumor-amputated ani
mals. The left tumor-bearing leg was removed by amputation
under ether anesthesia. There was no operative mortality fol
lowing removal of 14- and 21-day tumors. Eighty % of animals
survived removal of 28-day tumors.
In Vitro Labeling Methods. Animals were sacrificed by cer
vical dislocation, and tumors were removed immediately. A
single-cell suspension was prepared by mincing the tumor in
of
synthesis time; Td, tumor doubling time; dThd. thymidine; POP. primer-dependent
DNA polymerase; PDPI, primer-available DNA polymerase index.
3861
N. Gunduz et al.
McCoy's medium with 20% fetal calf serum (Grand Island
Biological Co., Grand Island, N. Y.) at room temperature and
filtered through a nylon screen. The single-cell suspension (3
to 5 x 107 cells/ml) was incubated for 1 hr at 37° in fresh
medium with 2.5 /iCi [3H]dThd per ml (14 to 17 Ci/mmol) in
single-label studies and for an additional 30 min with 0.25 ^Ci
[MC]dThd per ml (54 mCi/mmol) for double labeling. Labeling
was terminated by inserting tubes containing cells into ice. The
cells were washed with cold Ca2+-Mg2+-free Eagle's minimal
essential medium (Grand Island Biological Co.). After washing,
the cells were digested in 0.25% Bacto-trypsin (Difco Labora
tories, Inc.) plus 0.1 mg DNase per ml (Sigma Chemical Co.,
St. Louis, Mo.) and incubated for 10 min at 37°. They were
centrifuged at 500 x g for 3 to 5 min and resuspended in fresh
medium. Viability tests were made by trypan blue exclusion.
Cell viability was in excess of 90% (100% viability was not
infrequent). The cells were centrifuged at 500 x g for 3 to 5
min, resuspended in cold 0.1 M citric acid for 2 min, and
centrifuged again at 500 x g for 3 to 5 min. They were
resuspended in Carnoy's fixative for 5 min at 4°,and "drop"
preparations were made.
PDF Assay. The growth fraction was estimated in vitro by
means of POP assay (13, 14). Tumor imprints were made from
the freshly cut surface of tumors on acid-cleaned slides. The
slides were air-dried, dipped in 0.25% agar solution, and dried
again. The slides were incubated for 45 min at 37°in 0.5 ml of
an incubating mixture containing dATP, dGTP, and dCTP; 5
mvi MgCI2; Ficoll (Sigma Chemical Co.); 5 jiCi [3H]TTP (17 to
54 Ci/mmol). The incubation mixture was buffered with Tris-
HCI, pH 7.4 at 25°. Following incubation, slides were fixed in
methanol, rinsed thoroughly in tap water and triple-distilled
water, and air-dried. The nuclei are provided with all the nec
essary components for DMA synthesis with the exception of
DMA polymerase and DMA capable of acting as primer tem
plate. When all the necessary elements are present, DMA
synthesis occurs. Incorporation of [3H]TTP was detected on
the slides by autoradiography. The fraction of PDP-positive
cells/total cells was determined. In this paper, the POP index
is referred to as the growth fraction.
IO O r
io
o O.I
CE
O GROWTH OF SMALL TUMOR
ALONE
IN PRESENCE OF
LARGE TUMOR
OOI
IO 20 30 IO
"I2 MICE/GROUP
Chart 1. Effect of the presence of a distant tumor focus on tumor growth.
3862
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Research.
Autoradiography. Autoradiography was carried out as pre
viously described in detail (3) using gold activation to intensify
the latent image in the photographic emulsion, thus shortening
the exposure time. With this technique, the exposure time for
single-labeled samples is reduced to one day and for POP
samples to 5 to 6 days. Double-emulsion autoradiography was
used to distinguish cells labeled with only [3H]dThd and those
labeled with [MC]dThd.
Counting Procedure. To determine LI, PDPI, and Ts, 700 to
1500 cells were counted for each tumor sample. Slides were
counted by either 2 or 3 observers, and the results, which were
in good agreement, were pooled to obtain a mean value. The
equations used in calculation of the kinetic parmeters are those
previously described by us (3).
RESULTS
Effect of the Presence of a Separate Tumor Focus on the
Growth and Kinetics of an Associated Tumor. Growth rates
were similar until the final stages of tumor growth, whether the
tumors were growing alone or in the presence of a second
focus (Chart 1). Only then were tumors in mice with a single
focus observed to grow at a greater rate than did their coun
terparts with 2 tumor foci. This occurred whether tumors arose
from large or small tumor cell inocula. The cytokinetic param
eters were similar whether the tumor grew alone or in the
presence of an additional focus (Chart 2). There was an equiv
alent decrease in LI during the period of observation, i.e., from
the second to fourth weeks of tumor growth. A similar decrease
in PDPI (growth fraction) occurred in both circumstances. A
similar increase in Ts and Tc occurred in the single growing
tumor and the one accompanied by a second tumor focus. The
kinetics during the 2 to 4 weeks of tumor growth when a small
growing tumor was used for comparison were similar to those
for a larger growing tumor and were not affected by the
presence of a second focus.
Effect of Tumor Removal on Growth and Kinetics of Cells
in a Residual Tumor Focus. Mice, each bearing a large and a
small tumor focus, were randomly assigned to one of 3 groups.
GROWTH OF LARGE TUMOR GROWTH OF LARGE TUMOR
o—o ALONE o—o ALONE
•—•
IN PRESENCE OF • •IN PRESENCE OF
SMALL TUMOR LARGE TUMOR
20 30 IO 20 30
DAYS OF TUMOR GROWTH
CANCER RESEARCH VOL. 39
 Growth of Residual Tumors after Surgery

LARGE TUMOR SMALL TUMOR

O ALONE O ALONE
•IN PRESENCE OF SMALL TUMOR •IN PRESENCE OF LARGE TUMOR
10 0
352515oÃ-50—
o*
e5*r i-Õr LU
to
•H

i•5tD?
?mÃ-iÃŽÃ--o-it:
40è*CL
o:
lfr O

30oÃ-IIii
<f;Ã-
0.1
1421 28 28 35
*
* TUMOR AGE (DAYS) AT REMOVAL
Chart 3. Effect of the removal of a distant tumor focus on tumor growth. •.
9X7tH
CC
°•
Ã- presence of distant focus; O, after removal of distant focus (12 to 15 mice/
group).

Table 1
I12.0
ÃŽ Effect of tumor removal on Td and Tc of a residual tumor focus
TdAge

of iso
17.0O
I3.3 I3.3 14.811.8 12.7 tumor after re iso metric tu
X--- 130 14.4 16.0-fi 11.6 12.6 removed moval tu metric tumor tu mor (con
(days)142128Time (hr)38±9.6"30±1.654±10.561±6.4131±63207±46217±5282±
mor (control)
(hr)34±3.131±1.662±16.899±11.5156±56.0217
(days)0137101421013710140137Residual mor
(hr)11.5±1.210.8±1.413.0±0.711.1
trol)
(hr)11.8±0.411.2±1.0
1 2 3 4 I 2 3 4 '

WEEKS OF TUMOR GROWTH

Chart 2. Effect of the presence of a distant tumor focus on tumor kinetics.
Points, mean ±S.E. of 6 mice.

In one of the groups, the large tumors were removed at 14

days of growth, in another group at 21 days, and in the third at
28 days of growth. Such removal resulted in acceleration of
the growth of the residual smaller tumor focus in all groups but
more prominently when 14- and 21-day tumors were ampu
tated (Chart 3). Following removal of a 14- or 21-day-old tumor,
a decrease in Td was evident for about a week when compared
to the Td of single tumors of the same size (isometric) (Table
1). When tumor removal was delayed for 28 days, no decrease
in Td was observed.
The cell kinetics of residual tumor foci were studied following
removal of large tumors after 14, 21, or 28 days of growth
(Chart 4). When compared with control values, i.e., those
values on the day of operation, the LI was found to have
increased. While there was an increase noted 1 day after
removal of tumors in all groups, the increase was greatest
when tumors were removed after 14 or 21 days of growth
(125% of controls in the former and 144% in the latter). The LI
remained greater than control values for =10 days after tumor
removal in those 2 groups. Removal of the large tumor at a
later stage of growth (28 days) also induced an increase in LI
(115% of control) but to a lesser extent than when 14- or 21- a Mean ±S.E. of 3 animals.

OCTOBER 1979 3863

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N. Gunduz et al.

rate of métastasesor on a second tumor transplant (1, 2, 4-

TUMOR AGE (DAYS)
AT REMOVAL 10, 16-18, 20). Despite their efforts, whether a primary tumor
40
14 influences the growth of métastases or wee versa remains
2l unclear because of the divergence of findings which are, in all
28 probability, related to differences in host, tumor type, size of
S? 30
inoculum used, immunological variation, and other factors.
Despite lack of evidence in this model to indicate interaction
20 relative to growth parameters between 2 separate tumor foci
present in the same host, removal of one was found to affect
O the other. Changes in the kinetics of the residual tumor were
60 observed within 24 hr. An increase in LI and PDPI (growth
fraction) together with a shortening of the Td indicated an
acceleration in growth of the residual tumor. Since there was
50 minimal change in Ts and Tc, the increase in growth following
removal of the "primary" tumor was probably not the result of
a more rapid proliferation of the dividing cells but was more
40 probably due to conversion of noncycling tumor cells in G0
l"1' ..-*•-.
phase into proliferation. Since the increase in the growth frac
tion observed at 1 and 3 days after tumor removal was no
30 longer evident by 7 days, it is possible that these stimulated
O cells returned to the nonproliferating population after a few cell
divisions. The increase in LI with minimum changes in Ts and
Tc in lung métastasesreported by Simpson-Herren ef al. (16)
following removal of a s.c. Lewis lung tumor is in accord with
our findings. While the kinetic changes observed were not
et
I immediately reflected in a measurable increase in tumor size,
the effect of removal of a tumor on the size of the residual
focus was evident after 1 week. The growth of that focus was
I 7 IO 14 2I then more rapid than if the second tumor had remained in
DAYS AFTER TUMOR REMOVAL place.
EACH POINT MEAN ± SE. 3 MICE If the rapid but transient change in the population kinetics of
the residual focus after removal of a 'primary" tumor, as
Chart 4. Effect of removal of a distant tumor focus on tumor kinetics.
described here and reported by others, is a consistent finding
day tumors were removed. The PDPI of residual tumors also under a variety of settings, it could have significant implication
increased to a maximum of 125, 121, and 108% of controls relative to the use of adjuvant chemotherapy. At present, it is
following removal of tumors at 14, 21, or 28 days, respectively. considered that certain factors negatively influence the suc
The Ts of residual tumors remained relatively constant following cess of chemotherapy given following removal of a primary
removal of large tumors (7.9 ± 0.12 (S.E.) hr). Following tumor. Nonproliferating (G0) cells which retain their potential
removal of a tumor after 14 days of growth, the Tc of the for proliferation, while sensitive to cell cycle nonspecific agents
residual tumor focus remained relatively unchanged throughout to some extent, are less responsive than those in the prolifer
the duration of the experiment (11.6 ±0.3 hr) (Table 1). Similar ating pool to cytostatic manipulation. Other factors which de
findings were noted when tumors 21 or 28 days of age were termine the responsiveness of a tumor population to chemo
removed. therapy are the variations of Tc and the degree of synchroni
zation of cell cycles. Although the present investigations failed
DISCUSSION to demonstrate that tumor removal affected Tc or Ts and
although they provided no information relative to synchroniza
The present investigation indicates that in the model used tion, they did indicate that noncycling cells became proliferative
there was little interaction between multiple tumor foci which and thus could become more vulnerable to cytostatic agents.
affected their growth rate. Only when the combined mass of The rapidity of the onset of the kinetic changes and their
multiple tumors approached that which was incompatible with relatively short duration provide a rationale for the use of
survival was a slower rate observed. Cytokinetic studies also adjuvant chemotherapy as soon as possible following primary
failed to indicate any interaction between the 2 tumor foci. In tumor removal. Confirmatory evidence from other host-tumor
contrast to our findings, DeWys (2) and Simpson-Herren ef al. systems is necessary to support or deny this hypothesis.
(16) have shown that in mice bearing 2 simultaneous s.c. tumor
implants, the total mass of the 2 tumors during their growth ACKNOWLEDGMENTS
was equivalent to that of a single s.c. implant. Our observations We acknowledge the technical aid of Judith Benson and Nancy Shuck in the
are more in keeping with those of Rockwell and Kallman (11) carrying out of these studies.
who reported that there was no significant difference in either
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3865
Effect of Surgical Removal on the Growth and Kinetics of
Residual Tumor
Nurten Gunduz, Bernard Fisher and Elizabeth A. Saffer

Cancer Res 1979;39:3861-3865.

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