[CANCER RESEARCH 40, 3977-3981,
0008-5472 /80/0040-OOOOS02.00
November 1980]
Growth Interaction in Vivo between Tumor Subpopulations Derived from a
Single Mouse Mammary Tumor1
Bonnie E. Miller,2 Fred R. Miller, John Leith, and Gloria H. Heppner
Department of Immunology, Michigan Cancer Foundation, Detroit, Michigan 48201 [B. E. M., F. R. M.. G. H. H.]. and Department
Biology and Medicine, Brown University, Providence, Rhode Island 02912 [J. L.J
ABSTRACT
Our laboratory has previously isolated several tumor cell
populations from a single, spontaneously arising mammary
tumor of a BALB/cfC3H mouse and established them in tissue
culture as independent sublines. These subpopulations differ
according to many criteria including growth parameters and
expression of tumor-associated antigens. We have tested the
interaction in vivo of several of these subpopulations by inject
ing cell suspensions of the same or different sublines into
opposite flanks of BALB/cfC3H or BALB/c mice. The growth
characteristics of certain subpopulations were altered by the
presence of a different subpopulation on the opposite side. In
order to understand the mechanism of interaction, we chose
two subpopulations (410 and 168) for further study. In BALB/
cfCSH mice, the presence of line 410 tumors on one flank
inhibited both 410 and 168 tumors on the other flank. Line 168
tumors did not inhibit either 410 or 168 tumors. The inhibitory
effect of line 410 appeared to be immunological, since (a) it
was increased by injecting line 410 several weeks before line
168, (b) it was abrogated in mice subjected to 400-rad Xirradiation 2 days prior to tumor cell injection, (c) mice could
be made resistant to both line 410 and line 168 tumors by
implantation followed by surgical removal of line 410 but not of
line 168, and (d) resistance could be adaptively transferred
with lymph node cells from line 410-sensitized mice in Winn
assays. Thus, immunity to tumor-associated antigens may be
one way by which cells of a heterogeneous tumor can interact.
INTRODUCTION
Mouse mammary tumors are noted for the heterogeneity
which exists not only between tumors but within a single tumor.
There can be marked differences in histology, growth param
eters, and expression of differentiated function within a single
tumor cell population (9). In order to analyze the effect of this
heterogeneity on tumor behavior and response to therapy, our
laboratory has isolated a series of tumor subpopulations which
are ajl derived from the same spontaneously arising BALB/
cfCSH mouse mammary tumor (8, 19). We have shown these
lines to differ by many criteria including morphology and growth
characteristics in vitro; karyotype; expression of tumor asso
ciated antigens; and transplantation characteristics in vivo such
as the number of cells required to produce a tumor, length of
the latency period, and the growth rate (8). The immunogenicity
of these subpopulations also differs markedly (29), as does
1 This work was supported by USPHS Grant CA 27419 and by a grant from
the Concern Foundation.
2 To whom requests for reprints
should be addressed,
at Department
of
Immunology, Michigan Cancer Foundation, 110 E. Warren Ave., Detroit, Mich.
48201.
Received May, 5, 1980; accepted July 24, 1980.
NOVEMBER
1980
oÃ-Radiobiology.
Division ol
their sensitivity to standard chemotherapeutic agents (19). Our
results are similar to those of many other workers using differ
ent tumors (1, 10, 11, 13, 16, 17, 23-27, 31, 32, 35, 39, 4042) including human cancers (2-5, 36, 38).
Cancer is frequently defined as "relatively autonomous
growth," but if the component subpopulations of a tumor were
themselves autonomous, the slowly replicating subpopulations
should be rapidly overgrown. Since we have been able to
isolate cells of such widely differing tumorigenicity from a single
tumor, we hypothesized that intratumor homeostatic mecha
nisms exist which allow the fast- and slow-growing subpopu
lations to coexist in the parental tumor. The purpose of this
study was to determine whether intratumor growth interactions
could be demonstrated in vivo and, if so, what their mechanisms
might be.
MATERIALS
AND METHODS
Mice. Male BALB/cfC3H and BALB/c mice, 3 to 5 months
old, were purchased from the Cancer Research Laboratory,
University of California at Berkeley, Berkeley, Calif.
Tumors. All tumor lines used for these experiments were
derived from the same single, spontaneously arising mammary
tumor of a BALB/cfC3H mouse. Lines 68H, 168, 66, and 67
were derived in vitro from the original tumor (8). Line 168 was
further cloned in soft agar. Line 410 was derived from a
metastatic nodule in the lung of a BALB/cfC3H mouse carrying
the tenth s.c. in vivo passage of the parent tumor (19). The
tumor lines used for these experiments had been continuously
in monolayer culture for over 1 year. They were grown in
Waymouth's medium supplemented with 15% fetal calf serum,
2 HIM glutamine, penicillin (100 units/ml), and streptomycin
(100 /¿g/ml).Cells for injection into mice were harvested with
0.125% trypsin in 0.05% EDTA, washed once, and resuspended in 0.9% NaCI solution. Tumor cells were injected s.c.
in 0.1 ml of 0.9% NaCI solution into the flanks on either side of
mice, ventrally, in the area of the inguinal mammary gland.
Measurement of Tumor Growth. Mice were examined 2 or
3 times per week for palpable tumors. The latency period was
defined as the number of days between injection and the
appearance of a palpable, progressively growing tumor. The
mean latency period includes data only for those animals in
which tumors became palpable within 90 days after injection
(termination date for all experiments). The number of tumorfree days for each animal developing a tumor was defined as
the same number of days as the latency period for that tumor
and was set at 90 days for those animals not developing a
tumor in that period. The parameter "mean tumor-free days"
includes data for all animals given injections, thus combining
latency period and incidence into a single number. Tumors
were measured with vernier calipers in 2 dimensions. The
growth rate (tumor area in sq mm/day) of each mouse's tumor
3977
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B. E. Miller et al.
was calculated using linear regression from the data obtained
from approximately 2 weeks after first palpation until death.
(Tumors grew more slowly immediately after palpation, reach
ing linear growth after 10 days to 2 weeks.)
X-irradiation. BALB/cfC3H mice were immunosuppressed
2 days prior to cell injection with 400-rad whole-body X-irra
diation with a Philips X-ray machine (Philips Electronics, Mahwah, N. J.) operating at 280 kV, 20 ma, and half-value layer of
1.5 mm Cu.
RESULTS
Growth Interactions between Tumors on Opposite Sides
of Mice. In order to see whether the presence of one tumor
subpopulation could affect the growth of another, mice were
simultaneously given injections in opposite flanks with the same
or different subpopulations. The results of one large experiment
testing the interaction of tumor subpopulations 66 and 67 are
shown in Table 1. The contralateral presence of Tumor 67
increased the latency period of Tumor 66 by 2 weeks (p <
0.01), whereas Tumor 66 reduced the incidence of Tumor 67
from 63% to 36% (p < 0.001).
Six similar experiments (using fewer mice) were also carried
out with subpopulations 168 and 410. The pooled incidence
data for Tumor 168 are shown in Table 2. In these experiments,
it appeared that Tumor 410 was the "controlling"
tumor. The
incidence of 168 was reduced from 96% to 82% (p < 0.005)
in those animals simultaneously given injections of line 410 in
the opposite flank. No significant differences in the latency
periods (mean, 19 days) or growth rate (mean, 19 sq mm/day)
of line 168 in the 2 groups were seen. No effects on the growth
parameters of line 168 by contralateral injection of a second
line 168 inoculum were observed (data not shown). The growth
parameters of Tumor 410 were not affected by simultaneous
Table 1
Growth interactions between Tumors 66 and 67
Tumor cells (5 x 104 cells of line 66 or 2 x 10s cells of line 67) were
simultaneously injected into opposite flanks of BALB/c mice. Line 66 is more
tumorigenic than is line 67; hence, the inoculum of line 66 was smaller than was
that of line 67.
tumorIncidence264/272
of Flank 2
GroupABCDtumor66676766tumor66666767Growth
(97)a183/199
(days)36
1b50±2d57
+
(92)200/316
(63)Z1/1990
(36)Latency
±262
±2
Numbers in parentheses, percentages.
Mean ±S.E.
c Significantly (p < 0.001) smaller tumor incidence than in Group C by x2
analysis.
Significantly
t analysis.
(p < 0.01) longer latency period than in Group A by Student s
Table 2
Effect of line 4 W on the incidence of line 168 tumors
Tumor cells (105) were injected s.c. into opposite flanks of BALB/cfC3H
Group
Flank 1 tumor
Flank 2 tumor
A
168
168
B
410
168
Mechanism of Growth Interaction. We chose to look more
closely at the interaction between Tumors 168 and 410 by
examining the effect of the time of injection of the contralateral
tumor. We found that the effect of line 410 in altering the
growth of either a line 168 tumor or a second line 410 tumor
was heightened when the "controlling"
tumor was injected
some weeks before the tumor for which growth was being
measured. As shown in Table 4, Experiment 1, prior injection
of line 410 tumor cells strongly repressed the growth of a
second inoculum of line 410 tumor cells injected contralaterally. Shown in Table 4, Experiment 2, is the effect of line 410
on line 168. The inhibitory effect of line 410 was greater when
it was injected 3 to 6 weeks prior to, rather than simultaneously
with, line 168. Under this circumstance, not only was the
incidence of 168 reduced but the latency period was increased
as well. As mentioned above, the latter parameter was unaf
fected by simultaneous injection of line 410 cells.
We have further examined the mechanism of interaction
between Tumors 168 and 410 by determining their growth in
mice immunosuppressed by 400 rads of X-irradiation 2 days
before tumor injection. This experiment is shown in Table 5.
Irradiation enhanced the growth of both subpopulations (de
creased mean tumor-free days) but abolished the interaction
between the 2. The presence of a line 410 tumor in an irradiated
animal had no effect on the growth of either a second line 410
or a line 168 tumor on the contralateral flank.
The observations that pretreatment with 410 enhanced and
X-irradiation abolished the interaction with line 168 suggest an
immunological mechanism. To directly test this possibility, the
immunological relationships between the 2 subpopulations
were assessed. Two techniques were used. The first was the
induction of transplantation immunity. BALB/cfC3H mice were
Table 3
Effect of 410 on the growth of 410 tumors
Tumor cells (105) were injected s.c. into opposite flanks of BALB/cfC3H
tumorGroupABFlank
mice.
Incidence of Flank 2
tumor
49/60
(82)b
Computed as described in "Materials
mice.
Growth of Flank 2
1
2
tumor410Flanktumor410410Incidence8/918/20Latency
(days)16
±3"26
110/114(96)"
Numbers in parentheses, percentages.
1Smaller than incidence in Group A by if analysis; p < 0.005.
3978
injection of line 168 cells into the opposite flank (data not
shown). However, mice given injections of line 410 had more
tumor-free days (p < 0.05) when line 410 was injected in the
opposite side than they had when no second tumor was present
(Table 3). Thus, the simultaneous injection of line 410 tumor
cells in one side resulted in poorer growth of both line 168 and
line 410 tumors on the other.
An additional experiment was performed with subpopulations
68H and 168. The results were similar to those found with line
410 and line 168 in that the contralateral presence of line 68H
significantly ( p < 0.01 ) increased by 25% the number of tumorfree days of mice given injections of line 168 cells. Unlike the
situation with line 410, the growth of line 68H was influenced
by the presence of line 168; the incidence of line 68H tumors
was increased from 10% (2 of 20) to 50% (5 of 10; p < 0.05)
at an inoculum of 105 cells.
tumorfreedays32432C
±2Mean
and Methods," using 90 days as the
experiment termination date.
" Mean ±S.E.
c Significantly (p < 0.05) greater than in Group A by Mann-Whitney
CANCER
RESEARCH
U test.
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Tumor Subpopulation
Interactions in Vivo
Table 4
Effect of time of injection on the interaction between 410 and 168 tumors
Flank 1 tumor cells were injected either at the same time as were Flank 2 cells (Day 0) or 3 or 6 weeks
previously. All tumors were injected at a dose of 105 cells s.c. into BALB/cfC3H mice.
2Experiment12GroupABCDEFlank
Growth of Flank
tumor-free
1tumor410410410Time
of in
2tumor410410168168168Incidence9/94/1010/107/106/10Latency(days)8±1b^8
days0861°1747d50d
jection
(wk)-3-3-6Flank
217±
Computed as described
in "Materials
and Methods,"
128 +
523 ±
±3tumorMean
using 90 days as the experiment termination
date.
Mean ±S.E.
c Greater than in Group A; p < 0.001 by Mann-Whitney U test.
d Greater than in Group C; p < 0.01 by Mann-Whitney U test.
Table 5
Effect of immunosuppression on growth interactions
BALB/cfC3H mice were irradiated with 400 rads of whole-body X-irradiation 2 days prior to s.c. injection
of 105 tumor cells.
tumorGroup
IrradiationABC
Growth of Flank 2
tu
mor-freedays"8043°19"14"2465919"17"Growth
1tumor410168410168168410168410Flank
2tumor410410410410168168168168Incidence3/168/99/109/920/205/920/209/9Latency(days)39
rate(sq
/day)1.7
mm
±4"37
0.32.6±
211 ±
0.63.7±
+D
+EFG
+H
+Flank
Computed as described
0.5e4.8
±
114 ±
in "Materials
and Methods,"
124 ±
0.821 ±
245 ±
819 ±
±221
119 ±
117 ±
± 2Mean
124
using 90 days as the experiment
±
±1
termination
date.
Mean ±S.E.
c Less than in Group A; p < 0.01 by Mann-Whitney
U test.
Less than in Group A; p < 0.001 by Mann-Whitney U test.
" Faster than Group A; p < 0.05 by Mann-Whitney U test.
Faster than Group A; p < 0.01 by Mann-Whitney U test.
9 Greater than in Group E; p < 0.001 by Mann-Whitney U test.
Less than in Group E; p < 0.05 by Mann-Whitney U test.
given injections of line 410 cells, line 168 cells, or a mixture of
the 2. After 11 days, the nascent tumors were removed; 7 days
later, the mice were given injections of either line 168 or line
410 in the opposite flank. As shown in Table 6, growth of both
line 410 and line 168 tumors was suppressed in mice which
had been immunized with line 410; line 168 tumors did not
immunize against either themselves or line 410. Mixing line
168 cells with line 410 cells did not affect the immunogenicity
of the latter.
The second technique used to evaluate the immunogenicity
of line 168 and line 410 tumors was the Winn assay. BALB/
cfCSH mice were immunized by injection of either line 168 or
line 410 cells. The tumors were allowed to become palpable
and then were surgically removed. Two weeks later, lymph
node cells were taken from the immunized or normal mice,
mixed together with either line 168 or line 410 tumor cells
(ratio of lymphoid to tumor cells, 100:1), and injected s.c. into
syngeneic mice. Lymphocytes from mice immunized against
line 410 reduced the outgrowth incidence of line 410 tumors
from 100% to 0% (p < 0.01) and of line 168 tumors from
NOVEMBER
100% to 40% (p < 0.01). Lymphocytes from mice immunized
against line 168 had no effect on either tumor. Thus, line 410
cells not only induced immunity to themselves, but they were
also able to induce immunity against line 168 even though the
line 168 cells could not immunize against either subpopulation.
DISCUSSION
The problem of tumor heterogeneity has recently begun to
receive renewed attention by experimental oncologists (for
review, see Ref. 22). Many tumors are mixed populations of
cells that differ markedly in fundamental properties including
karyotype (36, 38), growth parameters (13), behavioral char
acteristics (10, 25, 35, 40, 41), expression of differentiated
cell products (13, 23, 26, 39), ¡mmunogenicity (4,11, 24, 31,
32), and sensitivity to therapeutic agents (1-3, 5, 16, 17, 27,
36, 38, 42). Our laboratory has demonstrated such heteroge
neity within a single BALB/cfC3H mammary carcinoma (8, 19,
29). We have also presented evidence indicating that hetero
geneity remains a feature of mammary tumors through at least
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3979
8. E. Miller et al.
Immuno/ogical cross-reactivity
Table 6
between lines 410 and 168: transplantation
assay
The immunizing tumor cells were injected s.c.; any palpable tumor plus the mammary fat pad and inguinal
lymph node underlying the injection site were surgically removed on Day 11. Challenge tumors were injected
on Day 18. The immunizing dose of line 410 and line 168 was 105 cells; the mixed dose consisted of 5 X
10" cells of each cell type (total 105 cells).
tumorABCDEFGH,JKLImmunizing
Growth of challenge
tumorfree
days"1539C2434C1646d1963"4282'3764
dose22225555105105105105x(days)152415251625192722281737+±+±++±±±±¿Ia42415134
tumor410168410 tumor168168168168168168168168410410410410Challenge
168410168410
+
10"x
10"x
10"x
10"x
168410168410
+
+ 168Challenge
' Computed as described
date
b
c
"
"
in "Materials
10"x
10"x
10"x
10"Incidence10/107/97/86/710/106/99/93/77/101/83/43/6Latency
and Methods."
termination
Mean ±S.E.
Significantly greater (p < 0.05; Mann-Whitney U test) than for unimmunized mice.
Significantly greater (p < 001 ; Mann-Whitney U test) than for unimmunized mice.
Significantly greater (p < 0.001 ; Mann-Whitney U test) than for unimmunized mice.
10 in vivo passage generations (15). The coexistence of tumor
cell subpopulations for prolonged periods suggests the exist
ence of mechanisms to maintain heterogeneity and to counter
selective forces acting on differential growth potentials. Several
such mechanisms can be envisioned. For example, our subpopulation 68H is capable, under certain conditions, of giving
rise to new variants that differ among themselves in growth
capacity (18). Thus, heterogeneity may be reintroduced into a
tumor population through the process of "stem cell" differen
tiation (30) or mutation. We also have preliminary evidence of
an inhibitory factor, detectable in vitro, that is produced by
certain of our subpopulations and that inhibits growth of some
mammary tumor cells (21). The experiments presented here
provide evidence for yet another mechanism by which tumor
cell subpopulations interact and possibly maintain heteroge
neity.
Tumor cell subpopulations were injected bilaterally, on op
posite sides of mice. The growth (incidence, latency period, or
growth rate) of the subpopulations was found to be influenced
by the identity of the contralateral tumor. Sublines 66 and 67
grew less well when paired with each other than they did when
paired with themselves. Subline 168 grew less well when paired
with line 68H than it did when injected opposite itself or when
injected only on one side. On the other hand, the incidence of
the very slow-growing (8) line 68H was increased when it was
paired with line 168 as compared to when it was paired with
itself or with no tumor. Injection of line 168 cells opposite the
slow-growing line 410 cells resulted in poorer growth of the
former with no effect on the latter. However, line 410 cells
grew better when injected alone than when injected opposite
a second line 410 inoculum. X-irradiation (2000 rads) of cells
prior to injection abrogated the effect of line 410 cells on the
growth of line 168 (21).
The mechanism of the interaction between subpopulations
168 and 410 was further investigated. Three types of evidence
suggest an immunological
basis: (a) the effect can be
3980
using 90 days as the experiment
heightened by preinjection of line 410, i.e., by immunization;
(b) the effect is abrogated by 400-rad X-irradiation of the mice
prior to tumor cell injection, i.e., by immunosuppression; and
(c) the direction of the effect is compatible with the immunolog
ical relationships between the subpopulations as revealed by
standard ¡mmunological assays. Thus, line 410 cells induce
strong immunity against themselves and against line 168 cells,
whereas line 168 cells do not immunize against either subpopulation. Mixing line 168 cells with line 410 cells does not affect
the immunogenicity of the latter. This "one-way cross-reactiv
ity" of line 410 with line 168 seems a likely explanation for our
results. Although we did not directly test the mechanism of line
66-line 67 and line 68H-line 168 interactions, the immunolog
ical relationships between these subpopulations (29) suggest
a similar effect.
The role of the immune response in neoplasia remains an
enigmatic and controversial subject. Evidence for immune in
hibition, enhancement, and indifference are abundantly avail
able in the cancer immunology literature. These divergent
results are in part due to the heterogeneity in the antigenicity
and immunogenicity of many tumors (4, 11, 24, 31, 32) includ
ing mouse mammary adenocarcinoma (29). Paradoxically, it
may be that the heterogeneity in immunity to tumors is involved
in countering forces that would tend to select against the more
slowly growing immunogenic subpopulations. In this view, ad
mittedly speculative, the immune response is considered as a
partner in the development of neoplastic complexity rather than
as a component of host defense.
The fact that subpopulations from the same tumor can influ
ence each other's growth adds a new dimension to the concept
of tumor heterogeneity: the behavior of heterogeneous cancers
is not necessarily predictable from knowledge of the behavior
of their component parts. Observations on growth interactions
between primary tumors and métastasesor other primaries (6,
7, 14, 28, 34,37, 43) also suggest this to be the case.
Intratumor interactions further complicate efforts to develop
CANCER
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Tumor Subpopulation
predictive assays for chemotherapy, since the drug sensitivity
of individual subpopulations, selected by their ability to grow in
soft agar (33) or as xenografts (12), may not be an accurate
guide to their sensitivity when growing in the presence of their
fellow subpopulations. Development of rational therapeutic pro
tocols may require the ability to anticipate how treatment of
certain subpopulations may affect the residual populations. It
may be that removal of the controlling influence of sensitive
cells would be a growth stimulus to insensitive populations.
Furthermore, if the growth of an otherwise sensitive subpopu
lation was slowed by the presence of another population,
sensitivity to cycle-dependent drugs might be lessened. It may
also be that treatment directed against sensitive subpopulations may "spill over" to cells which by themselves would be
unaffected.
We have preliminary evidence (20) for enhancement of the
drug sensitivity of one subpopulation by the presence of an
other. One might also predict that immunotherapy might be
beneficial against even nonimmunogenic cells if it could stim
ulate the type of one-way cross-reactivity exhibited between
line 410 and line 168. In any event, it would seem that a true
appreciation of the influence of heterogeneity on tumor behav
ior and response to therapy will require tumor "reconstruction"
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3981
Growth Interaction in Vivo between Tumor Subpopulations
Derived from a Single Mouse Mammary Tumor
Bonnie E. Miller, Fred R. Miller, John Leith, et al.
Cancer Res 1980;40:3977-3981.
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