[CANCER RESEARCH 31, 185—190,February 1971]
Effects of Chemotherapeutic Agents on Normal Mouse Bone
Marrow Grown in Vitro
ClarenceH. Brown,Ill,' andPaulP. Carbone2
With the technical assistance of Eleanor Stashick
Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland 20014
SUMMARY
The effects of several antineoplastic chemotherapeutic drugs
on normal mouse bone marrow were studied with the use of
an enriched methylcellulose culture system in which marrow
forms discrete colonies of myeloid and mononuclear cells.
Twenty-four hr after incremental doses, cyclophosphamide,
nitrogen
mustard,
and
1,3-bis(2-chloroethyl)-1-nitrosourea
resulted in exponential decreases in the fraction of surviving in
vitro colony-forming
cells per femur, while vinblastine sulfate,
methotrexate,
and polyinosinic-polycytidylic
acid resulted in
dose-response curves which showed that despite increasing
doses a maximal kill of colony-forming cells was approached.
The repopulation
of bone marrow colony-forming cells
following
a single large dose of cyclophosphamide
been introduced by Pluznik and Sachs (1 1) and Bradley and
Metcalf (2). Their assay utilizes an agar medium for the
colonization of mouse granulocytes and mononuclear cells.
Worton et al. (18) have modified
the method
using an enriched
methylcellulose medium rather than agar. While the spleen
colony assay is a measure of the pleuripotential stem cell
compartment, the in vitro culture system most likely reflects
the proliferative
capacity of a committed
granulocyte
precursor(l,
18).
Using the methylcellulose
culture technique , we have
investigated the effects of several chemotherapeutic agents on
mouse
bone marrow
in order to establish
a laboratory
model
for future studies in man.
was also
studied. The in vitro culture technique provides another
system of assessing bone marrow effects following tumor
MATERIALS
therapy
marrow.
Animals.
Six- to 10-week-old
C57BL/6N
male mice
weighing 15 to 25 g were used for these studies.
Drugs. The test drugs were given in incremental
dosages.
INTRODUCTION
Each dose was administered as a single i.p. injection. The
appropriate diluent for each agent was injected in the same
volume into control animals. At least 3 mice were given
and may be adaptable
to investigation
of human bone
AND METHODS
Measurement of peripheral blood counts and histological
examination of the bone marrow are the most frequently used
means of assessing the suppressive effects of antitumor
chemotherapy on the marrow. Usually, these parameters are
maximally affected in a few days or as long as several weeks
after therapy and therefore reflect with an inherent time lag a
complexity of effects, including reduction in stem cell
injections
number, as well as the proliferation,
functional formed elements.
was submerged in 3 ml of McCoy's SA (modified) medium,
and the marrow plug was gently flushed out of the shaft with
repeated aspirations into the syringe. This resulted in the
pooling of 6 femoral marrow plugs from each group of mice in
maturation,
and release of
With the development of the mouse spleen colony assay
(15), a technique that measures the progenitor capacity of the
pleuripotential
hemopoietic
stem cell, the relatively
immediate
effects of chemotherapy on murine bone marrow (4, 5, 16,
17), both normal and malignant, can be assessed. This
technique has provided much fundamental information about
the kinetics of drug-induced effects on mouse bone marrow;
however, its obvious limitation to small laboratory animals has
necessitated the development of other assay systems that can
be used in man.
A method of growing hemopoietic precursors in vitro has
I Clinical
Associate,
Medicine
Branch,
National
Cancer
of each dosage and diluent.
The drugs, dosages, and
appropriate diluents are shown in Table 1.
Preparation
of Marrow
Suspension.
Twenty-four
hr after
injection, both femora were aseptically removed from 3 mice
in each dose group and the appropriate control group. The tips
of each femur shaft were removed, and a 2 1-gauge needle on a
l-ml syringe was inserted into one end of the bone. The bone
3 ml of collecting fluid. Counts of n.c.3 were then performed
on each poo1 with duplicate hemacytometers,
and the counts
were converted
to n.c. per femur. One ml of the counted
sample was then diluted to a final concentration of 750,000
n.c./ml.
Culture Medium and Plating Technique. The method of
Worton
et al. (1 8) with minor
variations
was used in these
experiments. Briefly, 0.5 ml of the marrow sample containing
750,000 n.c./ml was mixed in a Falcon plastic test tube with 5
ml of a suspension of methylcellulose, McCoy's medium, fetal
Institute,
NIH, Bethesda, Md. 20014.
2 Chief,
Medicine
Branch,
National
Cancer
Institute,
Md. 20014.
Received April 17, 1970; accepted October 15, 1970.
FEBRUARY
NIH,
Bethesda,
3The abbreviations used are: n.c., nucleated cells; IVCFC, in vitro
colony-forming
cells; BCNU, l,3-bis(2-chloroethyl)-1-nitrosourea;
LD, @,
, 10%lethaldose;PolyI-C,polyinosinic-polycytidylic
acid.
1971
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1971 American Association for Cancer Research.
I85
Clarence H. Brown, III, and PaulP.
Carbone
Table1
n.c. /femur, colonies/plate, and absolute number ofl VCFC/femur for each dose of
drug tested 24 hr after treatment
DrugDose
S.E.)Cyclophosphamide
Marrow nc. X 107/femur
(mg/kg)Colonies/plate
357NaCl
(0.85%01.75104
solution)―3
30
150
3002.05
71Nitrogen
±1.524,308
±
±2.1
± 557
90 ±3.2
22,707 ± 809
26 ±1.9
3,727 ± 283
4 ±0.921,840 424 ±
1.90
1.10
0.8580
±
5.737,605
mustard (0.85%02.45115
1,883NaC1
solution)5
25BCNU
±3.1
10
1.25
15
201.7@
0.85
0.6582
(0.85% NaCI solu
916tion
with 10% ethanol)1
16±L6
2±1.0
1 ±0.418,500
02.2082
10
44 ±
1.90
1.3088
±
±1,764
87 t 5.7
26,027 ±1,729
56 ±3.1
14,187 ± 753
23 ±2.228,748 3,943 ±
1.10
0.65
0.5540
±4.243,278
±
±3.4
± 570
36 ±1.6
5,289± 246
33 ±2.5
2,825 ± 220
25 ±1.66,8361,827±
1.70
1.30
1.25108
±3.924,667
±
±2.4
± 501
73 ±3.1
16,603± 701
78 ±3.2
13,433 ± 568
76 ±2.123,040
12,584 ±
2.25
1,477NaClsulfate (0.85%02.60125
solution)6.25
12.5
25
501.30
108Methotrexate
(2% sodium02.1093
1,058bicarbonate)5
50
250
5001.60
337Poly
±
± 672
2,714± 284
226± 103
±3.123,907
±5.4
50
1002.45
377Vinblastine
(mean ±SE.)IVCFC/femur
(mean ±
±5.928,160
I-C(0.15M02.2096
1,752phosphate-buffered
saline)0.2
±3.9
±
±1,043
19,500 ± 667
2
1.80
10
1.25
81 ±2.9
71±3.3
202.05
1.10110
86 ±3.829,999 12,577 ± 550
11,833± 540
aDiluents
foreach
drug
are
shown
inparentheses.
calf serum, bovine serum albumin, and colony-stimulating
factor,4 the latter known to be a necessary ingredient for the
in vitro system (2, 8). Four 1.l-ml aliquots, each with 75,000
n.c.,were
plated in separate 35- x 10-mm plastic Petri dishes
and incubated in a humidified chamber with 10% CO2 at 37
Colony Counting and Morphology Studies. After 7 to 10
days of incubation, the total number of colonies containing 50
or more cells (range, 50 to >1000) were counted on each plate
with the use of an inverted
microscope
at a magnification
of X
50. Morphological studies carried out to identify the cell type
present
consisted
of
myeloperoxidase
(Kaplow)
and
Wright's-Giemsa staining. For these microscopic studies, a
single colony was removed from the plate under a
magnification
of X 50 with a finely drawn Pasteur pipet. The
tip of the pipet containing the colony was then submerged in
10 to 15 drops of McCoy's medium plus 1 drop of 10% bovine
albumin in a cytocentrifuge receptacle (Shandon Scientific
Co., Inc., Sewickley, Pa.). After repeated aspirations of the
medium
4 A
single
into the pipet
source
of
to wash the cells into the fluid, the
supernatant
from
a
monolayer
(929) was used throughout as the stimulating factor.
186
of
mouse
L-cells
specimen
was spun at 500 rpm for 10 mm. The cellular button
was then stained and examined with light microscopy.
Calculations.
The number
of colonies
on each plate
represents
the number of IVCFC per plate. To obtain the
number of IVCFC per femur, the number of IVCFC per plate
was multiplied
by the n.c. count per femur of the tested
marrow
sample and then divided by the number of n.c.
inoculated on a single plate, which was 75,000 in all of these
experiments. To obtain the fraction of surviving IVCFC per
femur, the number of IVCFC per femur determined
for each
test sample was divided by the number of IVCFC per femur in
the respective control.
Repopulation
of Bone Marrow after a Large Dose of
Cyclophosphamide. In a separate experiment, a large group of
mice was given 200 mg/kg (approximately
4 mg/mouse)
of
cyclophosphamide,
the approximate
o dose. Every 1 to 3
days thereafter
for 25 days, the marrows from 3 mice were
pooled
and plated in the same manner
experiments.
Animals given injections
as for the 24-hr
of 0.85% NaC1 solution
were used for control purposes. Serial marrow n.c. counts,
colony counts, IVCFC per femur, and fraction of surviving
IVCFC per femur were determined.
CANCER
RESEARCH
VOL.
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31
Effects
ofChemotherapy
on Mouse Bone Marrow
RESULTS
Cyclophosphamide,
shows
the results
Nitrogen Mustard, and BCNU. Table 1
of marrow
n.c. counts,
colony
counts,
and
absolute number of IVCFC per femur 24 hr after incremental
doses of cyclophosphamide. Chart 1 shows the dose-response
curve plotting the log fraction of control surviving IVCFC per
femur on the ordinate and the dose of drug on a linear
abscissa.
The dose is also expressed
in terms of mg/mouse
U)
U-
and
LD10 . Each point represents a single plate and is based on the
mean control value. Charts 2 to 6 for other drugs tested are
plotted in a similar fashion. There was a progressive reduction
in marrow cellularity, colonies per plate, and IVCFC per femur
@
dose of cyclophosphamide.
@
C.)
with doses of cyclophosphamide above 30 mg/kg. Above this
dose, an exponential decrease in fraction of surviving IVCFC
per femur was observed. An approximate
o dose of 200
mg/kg would reduce the fraction to 0.07 on the curve plotted.
The results with nitrogen mustard are shown in Table 1 and
Chart 2. As with cyclophosphamide, there was an exponential
decline in the fraction of IVCFC surviving with incremental
doses. The approximate
o dose of 5 mg/kg reduced the
surviving fraction to 0.5, nearly 10 times less than the
BCNU also reduced
U-
C-)
oO.OI
IC.)
U-
the surviving
fraction exponentially (Table 1, Chart 3), with an
dose
of approximately 35 mg/kg reducing the fraction to 0.58.
Vinbiastine
Sulfate,
Methotrexate,
and Poly I-C. As seen in
Table 1, there was progressive diminution
0
5
I
I
I
0
0.1
02
03
0 4 mg/mouse
.7
3.4
5.2
6.9 in termsof LD10
c@i
of n.c. counts with
20mg/kg
5
S
-J
NITROGEN MUSTARD
.0
Chart 2.
Li
ULi
0.
Li
U-
U)
0.
C.)
>
>
z
0
C,)
C.)
C.,
C.,
I
0
II
C.)
0
I
I
I
I
1
1
I
I
0.02 0.2
II
I
toomg/kg
50
(
-J
I
003 0.3
2 mg/mouse
I
(
I
5
I
3lnlermsof LD10
BCNU
Chart 3.
increasing doses of vinblastine sulfate; however, the colony
counts
3
30
IIIIII
U__j
300mg/kg
50
0.06 0.6
I
I
IIIIII
3
u_ .J@.
_j___@I
0.01 0.13
0.65
CYCLOPHOSPHAMIDE
@
Chart 1.
FEBRUARY
-J
6 mg/mouse
-J
.3 in termsof LD@@
did not significantly
decrease
with doses above 6.25
mg/kg. Chart 4 shows that the fraction of IVCFC per femur
surviving a dose of 6.25 mg/kg was 0.15 of control. Increasing
the dose did not result in an exponential
kill of IVCFC,
but
rather in a curve that is asymptotic, indicating that a maximal
kill was approached despite increased doses. The
dose of
3 mg/kg would reduce the surviving fraction to 0.40 on this
1971
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1971 American Association for Cancer Research.
187
Clarence H. Brown, III, and PaulP.
curve.
All doses
reduced
of methotrexate
the number
Carbone
that
of marrow
were
tested
significantly
n.c. per femur; however,
the
reduction was no greater for 500 mg/kg than for 5 mg/kg.
Colony counts were slightly reduced with doses of 50 mg/kg
or more, but t-test analysis revealed that these counts were not
significantly lower than controls. Chart 5 shows that a
maximal
point
of depression
in the
fraction
0.
z
of surviving
C,)
C.)
I.0
C-,
>
z
0
C.)
U)
U-
.2
2
II
I
to
I
I
I
0.0040.04
U)
IL
0.
I
20 mg/kg
I
I
I
I
1
0.2
I
I_
I
I
0.01 0.1
I
I
I
I
0.5
POLYI—C
>
I
0.4mg/mouse
I
I in termsof LD10
Chart 6.
U)
C.)
C.)
Table2
n.c/femur of marrow pooled from both femora of 3 mice at intervals of
0
1 to
cyclophosphamideDay
3 days following
C-)
treatment
with
200 mg/kg
of
countsfrom
0 count was obtained before drug administration. Serial
U.
donesimultaneously
mice given injections of 0.85% NaCl solution were
significantlydifferentfrornDay
with drug-treated
determination.Days
0
groups and were not
after cyclophosphamide
/femur0 injection
6.25 2.5
25
2.351
50 mg/kg
0.652
0.244
0.606
1.658
2.1011
1.7013
1.6015
2.0018
2.3521
1.9525
II1I00i30.250.5IIII
I_I1I
I mg/mouse
IIII02.34.6
18.5in termsof LD10
9.3
n.c. X 10'
VINBLASTINE
Chart4.
I.0
2.30
I.
Li
U-
IVCFC was reached with 250 mg/kg. All points on the
curvefrom
Li
0.
thecontrol.
50 to 500 mg/kg
thiscurve.
C')
decreases
different
from
o dose of 75 mg/kg
the surviving fraction
to 0.65 on
6)produced
Incremental doses of Poly I-C (Table 1, Chart
methotrexate.The
results almost identical with those for
thesurviving
approximate
o dose of 20 mg/kg reduced
C.)
jo I
0.45.Marrow
fraction
Tablemg/mouse
250
550
500
mg/kg
@
0
I
It I115
I0.07
1
I
I
II—
0.73.5
days0.1
I
In terms of LD102
METHOTREXATE
188
were significantly
formethotrexate
The approximate
>
Chart 5.7
to
Repopulation following Cyclophosphamide.
shows serial marrow n.c. counts following a single injection
of 200 mg/kg of cyclophosphamide. In Chart 7 are shown the
corresponding
colony
counts.
The fraction
of surviving IVCFC
per femur, which is based on n.c. counts and colony counts, is
showninChart8.
Morphology Studies. Colonies consisted predominantly of
peroxidase-positive
granulocytes
ranging from promyelocytes
to rare mature neutrophiles
when examined from 3 to 5
CANCER
RESEARCH
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VOL. 31
Effects ofChemotherapy
400
surviving colony-forming
cells per femur
showed that mice treated with cortisone
380
both serum levels of colony-stimulating factor, as well as the
number of colony-forming cells. Dose-related suppression of
340
bone
320
Thus, the relative effects of standard toxic doses of several
marrow
agents
280
function
can be evaluated
can be compared.
For example,
with
this method.
the present
studies
showed that the
o dose of cyclophosphamide
was
significantly more toxic to the granulocytic precursor than the
N
0
260
dose
of
any
other
agent
studied,
most
of
which
reduced
the fraction of surviving colony-forming cells to between 0.45
and 0.65
of control,
as compared
to 0.07
for
240
_1
0.. 220
cyclophosphamide.
U)
colony-forming
0. 200
@
(3 , 6). Metcalf (9)
exhibited a fall in
360
300
@
on Mouse Bone Marrow
single-injection
80
With
cells
methotrexate,
were
killed
o dose, suggesting
35%
with
75
of
the
mg/kg,
the
that animals dying from
this dose do so from causes in addition to or other than
hemopoietic suppression.
With appropriate
experimental
planning, recovery of
8 60
40
colony-forming
ability
following
chemotherapy
can be studied
with the in vitro system. In this study, cyclophosphamide
caused a marked reduction in marrow cellularity and colony
numbers
within
24 hr after
treatment.
The
depression
in
marrow cell counts persisted for at least 4 days, while colony
counts
20
I
I
@@“!T—:--•@@'
1'@T@@@T1
I
II
rapidly
rose, indicating
that of the constant
marrow inoculum an increased proportion
75,000 cell
of cells capable of
in vitro proliferation
was present. However,
the absolute
number of IVCFC per femur, which is calculated from colony
_0
4
2
6
8
0
2
4
6
8
DAYSAFTER TREATMENT
20
22
24
26
Chart 7. Serial colony counts following 200 mg/kg of
cyclophosphamide. Each point represents a single plate. Shaded area,
control range.
after incubation.
number
of
From Day 6 through
colonies
contained
Day 10, an increasing
peroxidase-negative
mononuclear cells. These morphological results were seen
irrespective of the dose or type of chemotherapy used and are
not different
from those previously
reported
(10).
CD
DISCUSSION
@
U)
From these observations, it is apparent that quantitative
studies of mouse hemopoietic function with tissue culture
technique can add yet another parameter, in addition to
peripheral blood counts and marrow morphology, to evaluate
bone marrow status following chemotherapy.
Success in
U)
culturing
U-
marrow
cells with the use of this system is dependent
C.)
UC)
0
C.)
0.1
upon the use of highly enriched media supplemented with an
appropriate stimulating factor. In our experiments, mouse
L-cells provided the source of stimulating factor. The method
for preparation of this particular source has been reported
(18).
Because
provides
of its quantitative
a method
nature,
of evaluating
the in vitro assay
the effects
of chemotherapy
on a class of bone marrow cells that are apparently committed
to the formation of granulocytes. Several studies to this end
have been recently
reported.
L-Asparaginase
has been shown
to decrease the total number of marrow n.c. and fraction of
FEBRUARY
DAYSAFTERTREATMENT
Chart 8. Log fraction of surviving IVCFC/femur at intervals after
cyclophosphamide treatment. Each point represents a singleplate and is
based on the mean control value.
1971
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1971 American Association for Cancer Research.
189
Clarence H. Brown, III, and Paul P. Carbone
counts
and marrow
for evaluation
n.c. counts,
REFERENCES
is the most relevant parameter
of actual marrow function.
The fraction of
control IVCFC per femur in this recovery experiment
was
markedly reduced 24 hr after cyclophosphamide
treatment
( Chart
8).
After
nearly
complete
recovery,
a
secondary
depression occurred 2 to 5 days later. Although this delayed
fall is not entirely explicable, it is possible that cells initially
exposed to drug were capable of undergoing an additional
division before manifesting the damage sustained earlier. With
time-related
recovery
curves such as this, proper scheduling
of
sequential doses of antineoplastic therapy might be more
accurately determined.
With this culture system, the mode of action of
chemotherapeutic
agents
can
be
studied.
Information
concerning the differential effects of chemotherapy on normal
mouse hemopoiesis and mouse cancers has already been
provided
( 4,
5,
for
by several workers
16,
normal
17).
From
and
the
using the spleen colony technique
character
malignant
of the
spleen
dose-response
colony-forming
curves
cells,
a
possible mechanism of action of the drugs studied has been
inferred (5). Drugs that are generally considered not cell-cycle
specific, e.g., the alkylating agents, produce exponential
survival curves with the use of the spleen colony technique.
Similar dose responses were obtained with nitrogen mustard,
cyclophosphamide,
and BCNU in our study. On the other
hand, agents that probably kill cells in only 1 portion of the
generation
cycle decrease survival to a constant value when
studied by spleen colonization.
This type of response was
observed with vinblastine, methotrexate, and Poly I-C in this
study. By comparison of the cell survival curves of agents with
actions that are generally understood with survival curves
obtained with new agents, some basic assumptions can be
made regarding the mechanism of action of the latter.
Finally,
the
in
vitro
culture
system
may
afford
the
opportunity for study of the effects of chemotherapy on
human bone marrow, which the spleen colony technique does
not. Several investigators
are currently
noting success in
culturing human bone marrow in both agar (7, 12—14) and
methylcellulose (C. H. Brown and P. P.
data; J. Senn, N. Iscove, and E. A.
communication), and as soon as further
it is likely that studies similar to those
performed
Carbone, unpublished
McCulloch, personal
refinements are made
reported here will be
in humans.
ACKNOWLEDGMENTS
We are grateful to Dr. Joseph Gallelli of the Pharmaceutical
Development Service, Clinical Center, NIH, for assistance in the
preparation of the bovine serum albumin used in this study. To the
following pharmaceutical companies, we are appreciative for the drugs
used in this investigation: Mead Johnson and Co., Evansville,Ind., for
cyclophosphamide (Cytoxan); Merck Sharp and Dohme, West Point,
Pa., for nitrogen mustard (Mustargen HC1); Ben Venue Laboratories,
Inc., Bedford, Ohio, for BCNU; Eli Lilly and Co., Indianapolis, Ind, for
vinblastine sulfate (Velban); Lederle Laboratories, Pearl River, N. Y.,
for methotrexate; and Charles Pfizer and Co., Inc., New York, N. Y.,
for Poly I-C.
190
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CANCER
RESEARCH
VOL.
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31
Effects of Chemotherapeutic Agents on Normal Mouse Bone
Marrow Grown in Vitro
Clarence H. Brown III, Paul P. Carbone and Eleanor Stashick
Cancer Res 1971;31:185-190.
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