(CANCER RESEARCH 49. 3747-3753, July 15. 1989]
Therapeutic Perturbation of the Tumor Ecosystem in Reconstructed Heterogeneous
Mouse Mammary Tumors1
Bonnie E. Miller,2 Fred R. Miller, and Gloria H. Heppner
E. Walter Albachten Department of Immunology Michigan Cancer Foundation, Detroit, Michigan 48201
ABSTRACT
We have measured the response to methotrexate in vivo of paired
mixtures of sister subpopulation lines from a mouse mammary tumor, as
a model of drug response of a heterogeneous tumor. The subpopulation
lines differed in intrinsic sensitivity to methotrexate. Response was
measured both as growth delay and as a shift in tumor cell population
distribution toward the more resistant cell line. We found differences
between two pairs of cell lines in growth delay: line 66 plus 4T07 mixtures
tended to be as responsive as was line 4107 (the more sensitive line)
alone, whereas line 168 plus 4T07 mixtures tended to be less responsive
than line 4107 alone. With both paired mixtures, the tumors arising after
treatment tended to contain more line 66 or line 168 than did untreated
tumors, but this shift was extremely variable among individual tumors.
Within most treatment groups, there was no correlation between the
growth rate of individual mixed tumors and the final tumor cell distri
bution. Likewise, between experiments, there was no correlation between
the amount of growth delay in mixed tumors and the final tumor cell
distribution. Thus, the cellular composition of treated tumors did not
directly reflect the response to therapy.
INTRODUCTION
General awareness of the existence within a single tumor of
multiple tumor cell subpopulations that differ in level of resist
ance to chemotherapeutic drugs (reviewed in Refs. 1, 2) has led
to the concept that therapy may selectively eliminate the sen
sitive cells, while allowing the remaining therapy-resistant cells
to grow. The result would be the "recurrence" of a progressively
growing tumor, no longer responsive to the original therapeutic
agent. This concept has been expressed in mathematical terms
by Goldie and associates (3). Although it may be correct in
general, it is important to recognize the many cellular interac
tions which exist in tumors and which are subject to disruption
by treatment. Thus, tumor subpopulation interactions have
been described that affect growth kinetics, immunogenicity,
ability to metastasize, and genetic stability, as well as sensitivity
to treatment itself. Our concept of a tumor as an ecosystem (4,
5) suggests that any perturbation, such as that induced by
therapeutic intervention, is likely to result in dynamic shifts in
tumor composition, in addition to depletion of the treatmentsensitive cells.
We have previously characterized several subpopulation lines
derived from a single mouse mammary tumor (6-13) and have
described interactions which occur among them which alter
growth properties (14, 15) and sensitivity to chemotherapeutic
agents (9, 10, 16). We have recently developed quantitative
methods to determine the subpopulation composition of tumors
formed by mixtures of variant subpopulations (17) and have
shown, using this methodology, that interactions between subpopulations can affect their growth properties in vivo (17-20).
Received 7/29/88; revised 3/13/89; accepted 4/17/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by USPHS Grant CA-27419 awarded by the National Cancer
Institute, Department of Health and Human Services. Presented in part at the
Conference on Prediction of Tumor Treatment Response, Banff, Alberta, Canada,
on April 21-24, 1987.
2To whom requests for reprints should be addressed, at Michigan Cancer
Foundation, 110 East Warren Avenue, Detroit, MI 48201.
We now describe experiments in which the effect of methotrex
ate treatment on tumor growth and subpopulation composition
were determined in tumors resulting from paired subpopulations that exhibit different growth interactions. We chose subpopulation pairs which differed in intrinsic response to meth
otrexate, and in which one of the lines contained an independ
ent, selectable marker so that, after noncurative therapy, we
could determine the proportions of each subpopulation in the
resulting tumors.
MATERIALS
AND METHODS
Cell Lines and Cell Culture. Cell lines 66, 168, and 410.4 were
isolated from a single, spontaneously arising mammary tumor of a
BALB/cfC3H mouse (6-8). Thioguanine-resistant, ouabain-resistant
cell line 44FTO was isolated from line 410.4 after mutagenesis with
ethyl methanesulfonate (10). Line 4T07 was isolated from line 44FTO
after seven in vivo passages through the lungs of syngeneic mice (17).
All cell lines were certified mycoplasma-free, using DNA fluorochrome
stain plus UV microscopy, by Bionique Laboratories (Saranac
Lake, NY). For routine cell culture, cells were grown in monolayer in
DMEMO medium, consisting of DME supplemented with mixed nonessential amino acids (1 HIM), 2 mivi 1-glutamine, 5% iron-supple
mented calf serum (low endotoxin serum, Hyclone Sterile Systems,
Inc., Logan, UT), and 5% fetal bovine serum (low endotoxin serum,
Grand Island Biological Co., Grand Island, NY). DME medium in
powder form and other culture supplements were from Grand Island
Biological Co. Cultured cells exhibit 90% inhibition of colony forma
tion at methotrexate concentrations of 90 and 23 n\i for lines 66 and
168, respectively (continuous exposure to drug) (20). Line 4T07 cells
are approximately as responsive to methotrexate as is their parental
cell line, 410.4, which exhibits 90% inhibition of colony formation at
14 nM methotrexate (20).
Mice. Male BALB/c mice, 8-13 weeks old, were produced in our
animal facility from a BALB/c breeding colony established by Cesarean
derivation of a litter of mice from BALB/cfC3H parents obtained from
the Cancer Research Laboratory, Berkeley, CA.
Methotrexate Treatment. On Day 1 after cell injection, mice were
treated with methotrexate (Lederle Laboratories, Pearl River, NY)
injected i.p. at 75 to 112 mg/kg in three divided doses, 3.5 h apart. We
have found that this injection schedule maximizes therapeutic index
with these tumors, both by maximizing response and minimizing toxicity. Methotrexate was diluted with Hanks' balanced salt solution and
injected at 0.01 ml/g mouse body weight. Control mice received Hanks'
solution alone. In Experiments 1-4, mice received one or two additional
sets of three methotrexate injections on Day 8 and/or Day 15 (see
Tables 1 and 2).
Tumors. Cells from monolayer culture were suspended in Hanks'
balanced salt solution and injected s.c. into mice in a volume of 0.1 ml.
Tumors were measured twice a week in two perpendicular dimensions
with Vernier calipers. Tumor size in mm3 was calculated by the formula
ab2/2, where b is the smaller and a the larger of the two tumor
dimensions. For each individual tumor, the tumor volume between
approximately 75 and 500 mm3 was fitted to an exponential growth
curve, using linear regression analysis of the logarithm transformation
of tumor volume. Values for time to reach 100 mm3 and tumor doubling
time were obtained from each fitted curve.
Tumor Cell Suspension. Mice were sacrificed and their tumors asep3 The abbreviations used are: DME, Dulbecco's modified Eagle's medium;
HAT, hypoxanthine-aminopterin-thymidine;
HGPRT, hypoxanthine-guaninephosphoribosyl transferase.
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THERAPY OF HETEROGENEOUS
tically removed for cell suspension when tumors reached a size of 4002300 mm1 (average size, 800 mm3 for 360 tumors harvested). Tumors
tumor cell lines that differ in intrinsic methotrexate sensitivity
were injected s.c. into mice, which were then treated with
methotrexate, in divided doses beginning on Day 1 after cell
injection. This early treatment mode was chosen to maximize
response. We have found that there is little or no detectable
response of these tumors to methotrexate if treatment is delayed
until tumors are palpable. Beginning treatment before vascularization is clearly not a good model system for studying treat
ment of solid tumors, but it may serve as a model for early
treatment of metastatic disease.
In two experiments we used mixtures of line 66, which is
relatively insensitive to methotrexate in vivo and in vitro (13,
18), mixed with line 4T07, which retains the relative sensitivity
to that drug of its parent lines, 410.4 and 44FTO (13, 18). We
injected various ratios of the two cell lines in order to vary their
proportions in the tumors which arose (see below). When
mixtures of this pair of cell lines were treated with methotrexate
early after cell injection, several of the groups of tumors which
arose were delayed in growth (time to reach 100-mm1 tumor
were randomly selected for harvest in groups of eight per day after they
reached the desired size range. Untreated line 66 tumors (11 in two
experiments) were harvested at an average size of 930 ±460 mm' (SD)
at 30 ±4 days after cell injection. Untreated line 4T07 tumors (32 in
five experiments) were harvested at an average size of 810 ±320 mm'
at 33 ±9 days after cell injection. Other groups of treated and untreated
tumors were harvested over a similar size range, except for line 168;
we tended to harvest line 168 tumors at a larger size due to their rapid
doubling time (see "Results"). Untreated 168 tumors (19 in three
experiments) were harvested at an average size of 1560 ±880 mm' in
29 ±4 days. Over the range we used, the size of the tumor did not
appear to affect the colony-forming efficiency of the harvested cells (see
"Results").
Tumors were cut into pieces with scalpels, digested for l h with 2
mg/ml collagenase, type 3 (Cooper Biomédical,Malvern, PA) plus 1
mg/ml hyaluronidase (Sigma Chemical Co., St. Louis, MO), as previ
ously described (17). Our previously published procedure was modified
in that the protease step was omitted. After collagenase digestion, cells
were rinsed, passed through a syringe, and suspended as previously
described (17). A portion of each cell suspension was diluted and
counted with Trypan blue to determine the percentage of live cells.
Identification of Tumor Subpopulations in Mixtures by Colony For
mation Assay. The description and validation of this method has been
published previously (17). Briefly, cells were diluted and plated at three
replicates each at 5000, 1000, and 200 live (Trypan blue-excluding)
cells per well in 6-well tissue culture plates containing an equal volume
of selective medium twofold concentrated in selective agent. Selective
media used were thioguanine medium (DME-10 containing 60 HIM6thioguanine) for line 4T07 and HAT medium (DME-10 containing 100
JIMhypoxanthine, 0.4 ^M aminopterin, and 16 UMthymidine) for lines
168 and 66. Thioguanine and HAT mixtures were obtained from Sigma
Chemical Co. (St. Louis, MO). Since line 4T07 is HGPRT negative, it
will grow in thioguanine medium, but not in HAT medium. Conversely,
since line 168 and 66 are HGPRT positive, they will grow in HAT but
not in thioguanine. Media used for growth of tumor cells were also
supplemented with penicillin (100 units/ml) plus streptomycin (100
Mg/ml). After 7-10-day incubation at 37°C,10% CO2/air atmosphere,
colonies were fixed in methanol:acetic acid (2:1), stained with crystal
violet, and counted with the aid of a dissecting microscope. In each
experiment, the percentage of each tumor line present in tumor cell
suspensions was calculated from the colony forming efficiencies of
suspensions of unmixed tumors in the two media as previously de
scribed (17). We assumed that within a given experiment, the colonyforming efficiencies of each of the paired cell lines in each mixed cell
tumor would be unknown, but their ratio would be constant over all
the tumors. This ratio was set equal to the ratio of the mean colonyforming efficiencies obtained from the unmixed tumors in the same
experiment (17).
Zonal Distribution of Subpopulations. For these experiments, 10"
thoroughly mixed cells (IO3 line 66 plus 9 x 10' line 4T07) were
injected in a 20 //I volume into the right inguinal mammary fat pads of
mice under pentobarbital anesthetic. When tumors were 440-860 mm3
(at 22-43 days after injection), they were aseptic-ally removed and cut
into six pieces each. From each sixth of tumor, one small piece of
approximately 2-8 mm3 was removed for separate preparations of
single cell suspensions and colony-forming assays. The remainder of
each tumor was repooled, and suspended and assayed as usual (the
pooled tumor pieces yielded 66-88% of the total tumor cells obtained
per tumor).
Statistical Analysis. The Wilcoxon two-sample procedure was used
to test for differences in the medians of the time to reach 100 mm3
volume, and of the tumor cell composition, between treated and un
treated groups.
RESULTS
Response of Reconstructed Heterogeneous Tumors to Met 1mtrexate in Vivo. The data presented here are part of a series of
experiments in which mixtures of two sister subpopulation
TUMORS
volume) just as much as were the groups of tumors which arose
from line 4T07 alone (Table 1). Overall, if all doses of metho
trexate are combined, line 66 tumors had median growth delays
of 0-4 days, line 4T07, 4-13 days, and the various mixtures,
4-21 days. The volume-doubling times calculated between 100
and 500 mm1 (not shown), which averaged 3.8 days for line 66,
and 6.3 days for line 4T07, respectively, were not affected by
these treatment regimens in either experiment. We have previ
ously reported similar results with a mixture of line 66 and line
44FTO (a cell line closely related to line 4T07), and with a
mixture of line 66cl4 (a thioguanine-, ouabain-resistant line
derived from line 66) and line 410.4 (18, 20).
In contrast, in three experiments using mixtures of subpopulation line 168 (intermediate in sensitivity to methotrexate in
vitro) and line 4T07, the tumors which arose tended to be less
sensitive to methotrexate (less growth delay) than were line
4T07 tumors (Table 2). Again, the volume-doubling times
(averaging 2.4 days for line 168) were not affected by treatment.
Median growth delay for the seven treatment groups of line
4T07 tumors (two groups at 210 mg/kg methotrexate) from
Tables 1 and 2 and the three treatment groups of line 66 tumors
(two groups at 210 mg/kg methotrexate) from Table 1 is plotted
versus total methotrexate dose in Fig. 1. Growth delay for each
individual treated tumor was estimated from its time to reach
100 mm1, by subtracting the median value of that parameter
obtained from the group of similar untreated tumors. Six of the
seven 4T07 treatment groups responded significantly to meth
otrexate (Tables 1 and 2), and thus had growth delays signifi
cantly higher than zero. However, if the set of all individual
data points for line 4T07 tumors is fitted to a single regression
line linking growth delay to methotrexate dose, the fit is ex
tremely poor (the correlation coefficient is 0.08). Although the
data are scattered, there appears to be a break in the doseresponse curve with increased response at or above 210 mg/kg,
corresponding to 2-day treatment at 105 mg/kg/day.
Also shown in Fig. 1 are the median growth delays of six
groups of tumors which arose from injected mixtures of line 66
plus line 4T07 cells. No attempt was made to fit them to a
single line since they arose from different cell ratios. Five of
the six groups responded better than did line 66 tumors; three
of the six groups had median growth delays at least as great as
line 4T07 tumors.
Fig. 2 shows the dose-response relationship between the four
treatment groups of line 168 tumors, the line 4T07 tumors, and
the seven 168-4T07 injected mixtures. Below 150 mg/kg, the
response of line 168 and 4T07 could not be distinguished; all
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THERAPY OF HETEROGENEOUS TUMORS
Table 1 Effect of methotrexate on growth of mixtures of lines 66 and 4T07
Cells were injected s.c. On the following day, methotrexate was administered i.p. in three divided doses, 3.5 h apart. Methotrexate treatment was repeated after 7
days.
Experiment
mouse1
to reach
100-mm3 vol
treatment
Significance'19(16-20)'19(19-20)12(10-16)20(16-34)11(7-12)32(18-41)13(11-25)20(16-25)17(1
ume (days)
Median growth delay (days)"
(mg/kg)0210"0210'0210''0210*0150«210*0ISO«210"0150«210"0150"210"Number
mice7101091413151288891010*121112*11911Time
of
Cells injected per
66(3
10')4T07(3
x
10')2
10')66:4T07,
x
1:29(3.3%)'(10"
2.9x10')66:4T07,
+
1:9(10%)'(3x
104 + 2.7 x
0.02P<
<
0.0001NS
0.09)P
(P <
66(3x
10')4T07(3
(18-23)21
(17-23)18(10-24)22(17-34)31
0.04P
<
0.04NSNS
<
10*)66:4T07,
x
(24-34)14(11-19)24(21-26)32(27-41)15(12-17)19(16-23)24
0.06)P<
(P <
1:5(17%)'(0.5
10')66:4T07,
x 10' + 2.5 x
f(1.5
0.001P<
0.001NSP
|:1 (50%)
x 10'+ 1.5 x 10')Methotrexate
(19-29)0821744413101849NS'P
" Difference in median time to reach 100-mm3 volume between tumors in treated and untreated groups.
* Comparison of treated and untreated tumors by Wilcoxon two-sample test.
c Median (25% and 75% quartiles).
'' Methotrexate treatment given as three divided doses of 35 mg/kg each on Day 1 after cell injection, repeated Day 8.
' Not significantly different than untreated tumors.
'in parentheses, the percentage of line 66 injected.
' Methotrexate treatment given in three divided doses of 25 mg/kg on Day 1 after cell injection, repeated Day 8.
* One mouse did not develop a tumor in 90 days. This mouse was assigned a value of >90 days for inclusion in statistical analysis.
< 0.002
Table 2 Effect of methotrexate on growth of mixtures of lines 168 and 4T07
Experiment
injected3
Cells
to reach
100-mm3 vol
treatment
(mg/kg)0224e0224'0224e0315*0315'0315'0315'075*105'075*IDS'075'105*075*105'Number
mice91310151514101010'10'1010910108'810816*9881078Time
of
(days)17(16-18)*19(19-24)12(11-15)26
ume
growth
(days)214551110443746325Significance*P
delay
168(3
10')4T07(3
x
10')4
IO4)5
0.01P<
<
10')168:4T07,
x
(50%)"(3 1:1
x 10' + 3 x
(22-30)12(11-13)17(15-18)21
168(3
10')4T07(3
x
(19-22)26
(24-30)15(11-18)26
0.004P
<
10s)168:4T07,
x
(91%)"(3 10:1
IO4)168:4T07,
x 10' + 3 x
(97%)"(3 30:1
x 10* +
(24-56)15(13-19)25
0.02/><0.01P
<
(22-28)22
(20-22)26
(24-30)16(15-18)20
0.005P
<
0.0001P
0.0002P
<
168(3
10')4T07(3
x
(18-25)19(19-23)15(9-16)22(17-27)19(16-41)13(12-17)19(13-25)16(12-23)12(9-21)14(12-17)1
0.04P
<
0.007P
<
10')I68:4T07,
x
0.007P<
<
0.001NSNSNSNS
(90%)"(2.7 9:1
IO4)168:4T07,
x 10s + 3 x
(97%)"(2.9 29:1
x 10*+ IO4)Methotrexate
" Comparison of treated and untreated tumors by Wilcoxon two-sample test.
4 Median (25% and 75% quartiles).
' Methotrexate treatment given as three divided doses of 37.5 mg/kg each on Day 1 after cell injection, repeated Day 15.
"in parentheses, the percentage of line 168 injected.
' Methotrexate treatment given as three divided doses of 35 mg/kg on Day 1, repeated Day 8 and Day 15.
'One additional mouse did not develop a tumor in 90 days; this mouse was assigned a value of >90 days for statistical analysis.
" Methotrexate treatment given as three divided doses on Day 1.
* Three additional mice did not develop tumors in 90 days; they were assigned values of >90 days for statistical analysis.
three of the mixtures treated at higher doses had median growth
delays less than line 4T07 tumors.
Composition of Heterogeneous Tumors after Treatment with
Methotrexate. In order to identify the proportions of each
subpopulation in tumors arising from injected mixtures, tumors
were removed when they reached a size of approximately 800mm3 tumor volume and made into single cell suspensions. Cell
yields for the three subpopulation lines averaged (11 ±6) x 10"
live (irypan blue-excluding) cells per gram tumor for 27 line 66
tumors (mean ±SD), (7 ±3) x IO7 live cells per gram for 50
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THERAPY OF HETEROGENEOUS TUMORS
25
20
(B
15
IO
o
(B
\
O -
100
Methotrexate
200
300
Dose (mg/kg)
Fig. I. Relationship between growth delay and total methotrexate dose in
tumors of line 4T07. 66, and their mixtures. Data were obtained from the
experiments of Tables 1 and 2. Growth delay for each individual treated tumor
was calculated from the time to reach 100-mm' tumor volume (T^). by subtracting
the median Tt from a similar group of untreated tumors. Points, medians; bars,
quartile range. O, line 4T07 tumors; O. line 66 tumors; •¿,
tumors arising from
mixtures of line 4T07 plus line 66.
25
20
15
10
o
O
100
Methotrexate
200
300
Dose (mg/kg)
Fig. 2. Relationship between growth delay and total methotrexate dose in
tumors of lines 4T07, 168. and their mixtures. Data analysis was as described for
Fig. 1. D, line 4T07 tumors: O. line 168 tumors: •¿.
tumors arising from mixtures
of linc4T07 plus line 168.
line 4T07 tumors, and (15 ±6) x IO7live cells per gram for 19
line 168 tumors. The mean percentages of live cells for these
tumors were 30, 23, and 64% for lines 66, 4T07, and 168,
respectively. Yields for the various mixtures fell within these
ranges.
The suspended cells were plated for colony formation in
HAT medium and in medium containing 60 \¡\\thioguanine.
As we have previously described ( 17), this assay can be used to
distinguish these cell lines: lines 66 and 168 will form colonies
in HAT, whereas line 4T07 will form colonies in thioguanine.
For cell suspensions from tumors arising from a single cell line,
three replicates each were plated at 1000 and 200 live cells per
dish in the appropriate selective medium, and at 5000 live cells
per dish in the other medium to check for mutants, revenants,
and/or fusion products. No colonies were ever formed in thio
guanine from 168 or 66 tumor cell suspensions; very rarely, 1
or 2 colonies per suspension were formed in HAT from 4T07
tumor cell suspensions. The overall colony-forming efficiency
(the number of colonies as a percentage of the number of live
cells plated) of line 66 tumor cell suspensions was 23 ±7%
(SD) for 29 tumors in two experiments; similar value for 168
tumors and 4T07 tumors were 12 ±7% (35 tumors in three
experiments) and 11 ±6% (73 tumors in five experiments),
respectively. For suspensions from mixed tumors, three repli
cates each were plated at 5000, 1000, and 200 live cells per well
in each of the two selective media, allowing us to detect cells
with colony-forming efficiencies greater than 0.007%. At this
maximum plating density of 500 cells/cnr, the presence of both
cell lines will not interfere with the detection and quantitation
of the subpopulations (17). Approximately 1 in 50 tumors had
low colony-forming efficiencies in both media (sum of colonyforming efficiencies <1%). Data from these tumors were dis
carded. Cell suspensions from the remainder of tumors which
arose from the injection of mixtures of cells had summed
colony-forming efficiencies similar in mean and range to those
of the unmixed cell lines.
The results of the colony-formation assays are shown in Table
3. Although in all five experiments, a higher percentage of
tumor cells tended to be identified as the more methotrexate
resistant cell line (line 66 or line 168) in at least one of the
treated groups, the shift towards the resistant cell line was not
always statistically significant. Three of the four mixtures with
lines 66 plus 4T07 treated with 210 mg/kg methotrexate were
significantly affected, but neither of the two mixtures treated
with 150 mg/kg were. Only two of the seven 168-4T07 mixtures
were significantly affected; two other groups were borderline,
with P values between 0.05 and 0.1. Both the significantly
affected groups were mixtures injected with a high ratio of 168
to 4T07 (97%).
As noted in "Materials and Methods," we were not able to
harvest tumors at a uniform size. Therefore, we examined the
relationship between the size of the tumor at harvest and the
day of harvest to the colony forming efficiency of the harvested
cells, by regression analysis. In none of the three cell lines was
there a significant correlation between either the tumor size or
day of harvest (over the range used) and the colony-forming
efficiency. This result is in accordance with previous findings
for lines 4T07 and 168 (17). Thus, we did not correct for tumor
size or day of harvest in calculating the proportions of each cell
line found in mixed-cell tumors. Similarly, there was never a
significant difference (by t test or Wilcoxon two-sample test) in
colony-forming efficiencies between treated and control tumors
in any of the three cell lines, so we pooled the colony-forming
efficiencies in those groups for use in calculating the mean
colony-forming efficiency of each cell line in each experiment.
This quantity was used in calculating the cell content of indi
vidual mixed-cell tumors, both treated and controls.
As we have previously described (17,19), in untreated tumors
arising from 168/4T07 mixtures, the composition at the time
of harvest is strongly enriched in line 4T07. Injection of a high
ratio of 168 to 4T07 cells is required to recover any 168 cells
from most mixed-cell tumors, even though 168 tumors have a
faster doubling time when grown in the absence of line 4T07.
The tendency of line 4T07 to dominate the mixed-cell tumors
appears to be established early after cell injection; even very
small tumors show the effect, and the ratio of cells recovered
does not change with tumor growth (19). Likewise, in the
experiments shown in Table 3, in untreated tumors the propor
tion of line 168 harvested was greatly decreased from the
proportion injected. In treated tumors, the proportion of line
168 increased, but still never reached the proportion injected
in any group. Regression analysis revealed no correlation be
tween the day of tumor harvest or the tumor size and the
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THERAPY OF HETEROGENEOUS
TUMORS
Table 3 Tumor cell content of mixed tumors after methotrexate treatment experiments correspond to those in Tables I and 2
Mcthotrexatetreatment"
of
rcsistant cell line
Experiment
mixture1
Cell
3.3%
6610%
662
(mg/kg)0210021001502100150210022403150315075105075105Number
tumors11131411U1010II89141410109IO9771066c,'f
tumors0.01
in
l)f21 (0-5.
(0.9-98)0.4
(0-5.8)51
(3.4-90)1.7(0.2-7.3)14(3.3-47)16(0.2-39)26(10-61)34
6650%
17%
663
1684
(9.4-68)94
(38-97)0.0(0-10)1.0(0-20)1.6(0.3-4.6)44
50%
0.06)NS(/><0.06)<0.01NSNSNSP
(P <
16897%
91%
(0.4-82)0.5(0.1-12.3)75
1685
(64-95)0.2
(0.02-29)34
(0-92)O.I
(0-36)0.0(0-1.4)0.034(4-61)Significance*<0.04<0.04NSrfNSNS<0.05NS
16897%
90%
168Methotrexate
< 0.04
a Total dose; doses were divided as described in Tables 1 and 2.
'Comparison of treated and untreated tumors by Wilcoxon two-sample test.
c Median (25% and 75% quartiles).
d NS, not significant.
proportion of line 168 in the harvested tumors in most of the
12 mixed-cell groups of tumors. Only in two groups were these
parameters significantly correlated: in Experiment 4, in 97%
untreated mixtures, the tumors harvested at a larger size had
significantly higher proportions of line 168 (correlation coeffi
cient of 0.76, P = 0.02). This correlation was not found in the
97% untreated mixtures of Experiment 5, however. In Experi
ment 5, in the group of 90% mixtures treated with low metho
trexate, the tumors harvested at later times had significantly
lower proportions of line 168 (correlation coefficient of—0.87,
P =0.01).
It is apparent from the data of Table 3 that in untreated line
66-4T07 mixed cell tumors, the proportion of line 66 harvested
was also less than the proportion injected, although this tend
ency was not as pronounced as in the 168-4T07 mixtures. For
example, whereas untreated tumors which arose from injection
of 50% 168 cells mixed with 4T07 cells yielded a mean of 7%
168 cells at harvest, untreated tumors which arose from 50%
66 cells with 4T07 cells yielded a mean of 35% 66 cells at
harvest. The amount of line 66 recovered from tumors arising
from mixtures appeared to be proportional to the number
injected: injection of 3.3,10, 20, and 50% line 66 yielded tumors
from which the median percentages of line 66 recovered were
0.01, 0.4, 1.7, and 26%, respectively. In all of the three groups
of 66-4T07 mixtures significantly affected by treatment, the
treated tumors yielded a higher percentage of line 66 than that
injected. As with most 168/4T07 mixtures, regression analysis
revealed no significant correlations between the tumor size and
the proportion of line 66 in the harvested tumors (here in 10 of
10 groups). However, there was some tendency for the tumors
harvested later to have a higher proportion of line 66 cells. This
was significant in two groups (untreated 10% 66 tumors, P =
0.01, correlation coefficient = 0.66; and treated 3.3% 66 tu
mors, P = 0.02, correlation coefficient = 0.64), and borderline
(P < 0.1) in two other groups.
Within each treatment group in each experiment, we deter
mined whether the final percentage composition of each mixedcell tumor was significantly correlated with either the time to
reach 100-mnr' tumor volume or the doubling time, by regres
sion analysis. There was no significant correlation between final
tumor composition and tumor doubling time in any of the 22
groups of tumors arising from either paired mixture. Neither
was there any significant correlation between final tumor com
position and time to reach 100 mm1 in any of the 12 groups of
tumors arising from mixtures of lines 168 and 4T07. In four of
10 groups of tumors arising from mixtures of lines 66 and
4T07, the final composition was significantly correlated with
the time to reach 100-mm' tumor volume. In Experiment 1, in
both the 10% 66 mixture without methotrexate, and the 3.3%
66 mixture with methotrexate, the tumors slower to reach 100
mm' contained a higher percentage of line 66 (P < 0.01 and P
< 0.003, respectively). In Experiment 2, in the 17% mixture
with 210 mg/kg methotrexate, the tumors slow to reach 100
mm1 contained a higher percentage of line 66 (P < 0.04),
whereas in the 50% 66 mixture with 150 mg/kg methotrexate,
the tumors slower to reach 100 mm' contained a lower per
centage of line 66 (P < 0.005). The significance of these
relationships is unclear. One might expect that among treated
tumors arising from mixtures, slower early growth/longer la
tency of individual tumors, indicating more growth delay, might
be correlated with higher 66 or 168 content. With line 66/4T07
mixtures, in some groups, the tumors which were slower in
early growth (up to 100 mm'), and thus were harvested later,
contained more line 66 at the time of harvest. This did not
appear to be true of line 168/4T07 mixtures.
It is of interest to compare the in vivo response to methotrex
ate (growth delay. Tables 1 and 2) with the consequences of
treatment in terms of tumor composition (Table 3), between
experiments. In seven of the 13 cell/drug combinations, meth
otrexate significantly delayed growth in mixtures; in three of
the seven the growth delay was equal to or better than that in
line 4T07 alone. In 11 of these 13 combinations, the median
proportion of tumor cells in the resulting tumors had shifted
towards a tumor cell ratio favoring the more methotrexateresistant line as a consequence of treatment; however, the shift
was statistically significant in only five of the 13 groups. These
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THERAPY OF HETEROGENEOUS
five consisted of three tumor groups in which significant growth
delay was seen, and two tumor groups without significant
growth delay. The eight cell/drug combinations which did not
result in significant shift toward the more methotrexate-resistant line also consisted of tumor groups which did respond to
methotrexate with growth delay (four groups) and those which
did not (four groups).
In Experiments 2 and 4, each tumor cell suspension was also
plated in three replicates of 5000 cells per dish in medium
containing HAT plus ouabain, in order to identify any fusion
hybrids between line 4T07 (thioguanine resistant, ouabain re
sistant) and line 168 or 66 (wild type). Only one colony was
found (colony-forming efficiency <0.01%) in 98 tumors ana
lyzed. Thus, cell fusion appeared to be a rare event, unlikely to
influence the outcome of these experiments.
Distribution of Tumor Subpopulations within Reconstructed
Heterogeneous Tumors. In the five experiments described thus
far, the whole of each individual tumor was minced, and the
entire minced suspension of each tumor was digested and
suspended in order to minimize sampling error due to any zonal
heterogeneity of cellular content within each tumor. In order
to assess the degree of zonal heterogeneity within individual
tumors arising from injection of mixtures of lines 66 and 4T07,
we separately analyzed six individual small (approximately 28 mm') pieces taken from different areas of each of 13 tumors.
Because these fairly large pieces were the smallest size we could
separately analyze, we used tumors which arose from injection
of a small number of cells (IO4 per mouse) injected intra-fat
pad in a small volume, in order to try to maximize the size of
any zone which might arise. These tumors reached 100 mm' at
TUMORS
20). We show here that several combinations of the related cell
line 4T07 with line 66 also are delayed in growth after metho
trexate treatment, as much or more than line 4T07 alone. In
contrast, line 168-4T07 combinations appear to be delayed in
growth less than line 4T07, even though line 66 is more resistant
to methotrexate than is line 168. Thus, the therapeutic respon
siveness of heterogeneous tumors varies depending upon factors
specific to the component subpopulations other than relative
drug sensitivity. What these factors are requires further study.
Moreover, in neither paired mixture is there a good correlation
between the immediate therapeutic response (growth delay),
and the ultimate consequence of this noncurative therapy, i.e.,
shift towards a more resistant cell line. This suggests that the
cellular composition of treated tumors may not directly reflect
response to therapy and implies that tumors which recur after
a partial response to a particular drug may not contain a higher
proportion of resistant cells than they did before therapy, so
they may respond as well to a second treatment regimen of the
same drug. We have seen an example of this in Experiment 2
in the tumors which arose from 17% 66 injected mixtures,
which responded strongly in growth delay without shifting
towards more line 66.
The 168/4T07 and 66/4T07 combinations were chosen for
comparison because of previous observations that showed dif
ferent types of subpopulation interaction between these two
mixtures. Line 4T07 tends to "dominate" line 168, even though
by itself line 168 has a much faster doubling time (17,19). The
mixture of 66/4T07 does not show this strong dominance of
4T07 over 66, as we show here. In fact, mixtures of 66 and
4T07's parent line, 44FTO, appear to form a stable mixture
a median time of 16 days for untreated, and 28 days for treated
tumors. The results of our analysis are shown in Table 4. In
this group of tumors, as in the tumors of Experiment 1, also
injected with 10% line 66 (Table 3), many had relatively few
line 66 cells at the time of harvest: in four tumors, <0.1 % line
66 (>99.9% line 4T07) was found in all six pieces plus the
pooled remainder. In the remaining nine tumors, it is clear that
line 66 was distributed unevenly among the six pieces. Thus
zonal heterogeneity can be detected by this method.
(21).
In two of three experiments with 168/4T07 mixtures, we
injected excess numbers of 168 cells, in order to achieve signif
icant, albeit in the minority, numbers of line 168 in the final
tumor composition (Table 3). To compensate with 66/4T07
mixtures, we injected most groups with excess 4T07, in order
to achieve a final tumor composition similarly favoring line
4T07 (Table 3). What ratio of viable cells is present at the time
of methotrexate treatment in each case is unknown. However,
even if we compare only experiments in which a 50% ratio of
each pair was injected (Experiments 2 and 3), the line 66/4T07
DISCUSSION
mixture still responded more strongly in growth delay (9 versus
5 days) than did the 168/4T07 mixture at similar methotrexate
We have previously shown that combinations of the cell lines
doses. Thus, the ability of 4T07 to dominate over the growth
66 and 44FTO and of 66cl4 and 410.4 were delayed in growth
of 168 does not extend to sensitivity to methotrexate treatment.
after methotrexate treatment as much or more than was the
We have previously described three possible outcomes when
more sensitive line in the mixture, line 44FTO or 410.4 (18,
two cell populations are mixed and injected, depending on
which cell populations are chosen: the development of a stable
Table 4 Zonal distribution of lines 66 and 4T07 in tumors arising from injection
of their mixture
mixture (21), the tendency to form homogeneous tumors of
Tumors were injected with 1:9 66:4T07 cell mixture as described in "Materials
either cell line (21), and the dominance of one cell line over
and Methods." From each tumor, six 2-8-mm1 pieces plus the remaining pooled
another (17). Other authors have described similar phenomena:
tumor were analyzed. Of 13 tumors analyzed, four untreated tumors contained
Leith et al. have described the development of stable mixtures
<0.1% line 66 in all six pieces plus the remaining pooled tumor (not shown).
between human colon carcinoma subpopulation lines grown in
66Metho
Percent of tumor cells identified as line
nude mice (22), and Kerbel et al. have described clonal domi
pieces2
tumor
trexateTumor
nance developing in mixtures of subpopulations of mouse mam
treated"ABCDEF
1_»0.43.32123— 3—
mary tumors in syngeneic hosts (23). In all these experiments,
—¿_
the composition of the tumors which arose from injected cell
—¿â€”
—¿â€”
subpopulation mixtures was not predictable, based on the
—¿â€”
growth rates of the individual subpopulations. This strongly
—¿â€”
suggests that the cells interact by some mechanism or mecha
—¿â€”
—¿â€”
+G
—¿1.538Sampled
—¿â€”
nisms, whether directly or through the host, in order to affect
+H
—¿0.74————1.5——0.20.85—1.0—905.5——0.62566.71.91299440.20.426100
+I
the balance between them. The experimental results described
+Pooledtumor
here suggest that interactions between tumor subpopulations
°-, no drug; +, 105 mg/kg in three divided doses on Day 1, repeated on Day
can affect drug sensitivity, and may be modified by drug treat
*-, <0.1% line 66.
ment. Thus, a second implication of our work is that /// vitro
3752
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THERAPY OF HETEROGENEOUS
assays for drug sensitivity which minimize the possibility of
interactions between tumor cells (such as clonogenic assays) are
unlikely to reflect what may happen in response to drug treat
ment in vivo. For example, the interaction between lines 168
and 4T07 causing the inhibition of growth of line 168 in vivo
can be seen in vitro as well, but only under conditions in which
cells can remain in contact; when cell mixtures are plated at
low density or when the two cell lines share medium without
cell-cell contact, inhibition does not occur (19). In vitro drug
sensitivity assay systems such as spheroid cultures (24-27) or
collagen gel assays (10, 13) may be better predictors of in vivo
drug response because they allow maximal contact between
heterogeneous populations, as well as more closely mimicking
in vivo conditions of cell cycle distribution, cell shape, and
microenvironment. We are currently testing this hypothesis
with mixtures of our mammary tumor sister subpopulation
lines.
Our method of identifying the composition of mixed tumors
with selective media assumes that the formation of hybrid cells
between thioguanine-resistant and wild-type cells is a rare event.
By utilizing a subpopulation with both thioguanine resistance
and ouabain resistance markers as one member of paired tumor
cell lines (line 4T07 in these studies), we confirmed this as
sumption; i.e., only very rarely were colonies found with the
expected characteristics of fusion products. The low rates of
fusion we observed with both pairs of cell lines indicates that
the difference between the pairs in response to methotrexate is
not related to the formation of fusion products.
One of the first experimental demonstrations of tumor het
erogeneity in regard to drug sensitivity was that of Hakansson
and Trope who showed differential sensitivities among pieces
from single neoplasms (28), suggesting not only that overall
tumor subpopulation composition affects drug sensitivity but
also that the geographic distribution of these subpopulations
may be important. Other workers (21, 29) have also described
"zoning" of tumor subpopulations within heterogeneous tu
mors. Interestingly, even when great care is taken to mix cell
populations prior to injection, reconstructed heterogeneous tu
mors also can be shown to contain such zones. How such
regional heterogeneity is regulated, how it interacts with other
heterogeneously distributed microenvironmental factors (vasculature, pH, nutrients, oxygen, host cells, etc.), and how it
contributes to overall drug response remain areas for further
research.
The idea that tumor heterogeneity is an important determi
nant of treatment response has become a guiding concept in
cancer therapy. Our data, and those of others, warn against an
overly simplistic acceptance ofthat idea. More testing in model
systems is necessary in order to learn the underlying rules that
order tumor ecosystems and that impose limits on our ability
to use the concepts of tumor heterogeneity in developing strat
egies for more effective treatment.
ACKNOWLEDGMENTS
The authors thank Todd Machemer, David Wilburn, and Diane
Jackson for technical assistance, Margaret Peterson for manuscript
preparation, and Phyllis Gimotty, of Michigan Cancer Foundation
Biostatistics Unit, for assistance with statistical analysis.
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Therapeutic Perturbation of the Tumor Ecosystem in
Reconstructed Heterogeneous Mouse Mammary Tumors
Bonnie E. Miller, Fred R. Miller and Gloria H. Heppner
Cancer Res 1989;49:3747-3753.
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