Download The Role of a Cytophilic Factor from Challenged

[CANCER RESEARCH 34, 3089—3094,November 1974]
The Role of a Cytophilic Factor from Challenged Immune Peritoneal
Lymphocytes in Specific Macrophage
1
Elisabeth
Pels and Willem Den Otter
Department ofPathology, State University, Pasteurstraat 2, Utrecht, The Netherlands
SUMMARY
A specific-macrophage-arming
factor is released into a
growth medium (Fischer's medium plus 10% heatinactivated
fetal bovine serum) when sensitized peritoneal lymphocytes
from C57BL mice immunized with the DBA/2 SL2 lymphoma
are cultured in vitro with these lymphoma cells. This factor
renders a monolayer of normal peritoneal C57BL macrophages
specifically cytotoxic
to the lymphoma cells. Trypsin
treatment
of
macrophages
coated
with
this
specific-macrophage-arming
factor
terminates
cytotoxic
activity. The factor also binds to lymphoma cells and is
present in the peritoneal cavity of immunized mice. The
demonstration of the presence of this cytophiic factor in the
peritoneal cavity provides more insight into the mechanism of
the inhibition of tumor growth by immune macrophages.
INTRODUCTION
There are many reports that show that peritoneal
macrophages from mice immunized i.p. against a tumor are
specifically cytotoxic to the target cells (6, 7, I 1—14,18, 21).
However, how these macrophages acquire cytotoxicity to the
target cells is far from being clear. The release of SMAF's2
from immune spleen lymphocytes incubated with specific
target cells have been reported (1 , 8, 9). SMAF renders
macrophages specifically cytotoxic. In this paper we will show
that SMAF is also released from immune peritoneal
lymphocytes that are incubated with specific target cells and
that this factor binds to peritoneal macrophages, rendering
them cytotoxic. The relationship between armed and immune
macrophages will be discussed.
MATERIALS AND METHODS
Animals and Immunization
Pure-bred C57BL and DBA/2 mice, 8 to 10 weeks old, were
used. The DBA/2 lymphoma, SL2, which arose spontaneously
as an ascitic tumor, and the C57BL lymphoma, TLX9, which
arose as a thymoma following X-irradiation, were maintained
by weekly i.p. passage. Both lymphomas were obtained from
Dr. P. Alexander, Sutton, England.
A single dose of i0@ SL2 cells was injected i.p. into
allogeneic C57BL mice. Unless stated otherwise, peritoneal
exudate cells from the immunized mice were harvested from
C57BL mice 21 days after immunization.
Cell Cultures
Lymphocyte
Cultures. Peritoneal exudate cells were
harvested from the cavity and seeded into 3.0-cm culture
dishes (Nunclon) in Fischer's medium supplemented with 10%
heat-inactivated
fetal bovine serum (growth medium).
Macrophages were allowed to adhere for 20 mm at 37°.
Lymphocytes and nonadhering macrophages were removed by
gentle washing with a Pasteur pipet and seeded again into a
culture dish for 20 min at 37°.After 3 seedings and washings,
the nonadhering cells were spun down, resuspended in growth
medium,
and
counted
in an
improved
Neubauer
hemocytometer.
These lymphocytes
had less than 2%
macrophage contamination.
Macrophage Cultures. Pentoneal exudate cells containing 2
x 106 macrophages
wereseededinto culturedishesas
described by Evans and Alexander (7). Macrophages were
allowed to adhere for 30 mm. Cultures were washed
thoroughly with jets of medium from a Pasteur pipet before
use in experiments to remove lymphocytes and other cell
types. The adhering cells were spread out forming confluent
monolayers as observed by a phase-contrast microscope.
Lymphoma Cell Cultures. The SL2 or TLX9 cells were
harvested from the peritoneal cavity of mice 7 to 10 days after
transplantation,
washed 3 times in Fischer's medium, and
finally suspended at a concentration of 1.5 X l0@ cells/ml of
growth medium. Both types grow in suspension with a
doubling time of 13 to 16 hr during the 1st days of
cultivation.
Cytotoxicity
1 This
study
was
financed
by
grants
from
the
Stichting
Koningin
Wilhelmina Fonds of the National Cancer League and by the Utrecht
University. This paper has appeared in preliminary form (20).
2The abbreviation used is: SMAF, specific macrophage-arming
factor.
Received
March 26, 1974; accepted
NOVEMBER
July 16, 1974.
Cytotoxicity was assessed by comparing growth of lym
phoma cells in cultures of normal, armed, and immune
macrophages, which were challenged with 2 X l0@ SL2 cells in
3 ml growth medium. The cells were counted in a hemocytom
1974
3089
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E. Pels and W. Den Otter
eter. Cytotoxicity
expressed as
was assessed 24 hr after challenge and
%GI=
(N - T)
XJOO
N
where % GI is the percentage of growth inhibition, N is the
number
of lymphoma
Table1
Demonstration of the production of the macrophage-arming factor by
challenged immune lymphocytes
cells in controls,
and Tis the number
SL2cells/cultureGrowth
No. of
inhibition24
hr afterat
24
hrCulturesachallenge(%)Normal5.4x
in
test system.
Twenty % growth inhibition is generally regarded as the
level of significance, since repeated cell counts on the same cell
population varied up to 10% (8). The results were expressed as
the mean values of at least 3 experiments performed in
duplicate.
l0@0Normal
supernatantfrom:Normal
exposed to
10'0Unchallenged
lymphocytesb5.4
x
immune4.5
2dlymphocytes―Challenged
x i0@17
±
normal5.1
2lymphocytesChallenged
x l0@5
±
X 10°58
±
immune2.2
6lymphocytesNomacrophages5.Ox
Demonstration
of the Factor
10°0
Two X 106 immune lymphocytes were challenged with 2 X
l0@ SL2 cells in 3 ml ofgrowth medium and incubated at 37°
a Monolayers
of
2 x
106
normal
peritoneal
macrophages
(C57BL)
for 24 hr. The cell-free supernatant was added to normal challenged with 2 x l0@ DBA/2 SL2 lymphoma cells in 3 ml of growth
macrophage monolayers for 24 hr. After several washes the medium.
b Two x 106 peritoneal
C57BL lymphocytes.
macrophage cultures were incubated with 2 X i0@ SL2 cells in
C Peritoneal
C57BL
lymphocytes
harvested
21
days
after
i.p.
3 ml of growth medium for 24 hr at 37°.Cell cultures were
immunization
with 10@ SL2 cells.
always incubated in an atmosphere of 5% CO2 in air.
d Mean ±S.E.
Cytotoxicity was assessed 24 hr after challenge of cultures.
in 3 ml of growth medium at 37°for various time intervals.
The cell-free supernatants were collected and added undiluted
Complement
to normal macrophage monolayers for 24 hr. Table 2 shows
that the factor is produced within 1 hr.
Guinea pig complement was obtained from Rijks Instituut
Titer of the Factor. Serial dilutions of the factor containing
voor de Volksgezondheid, Utrecht, The Netherlands. It was
supernatant were prepared, and the different dilutions were
absorbed with SL2 cells as well as with C57BL liver cells ( 1/ 1,
added to monolayers of normal macrophages for 24 hr. The
v/v) for 30 mm at 4°, centrifuged, and stored at —20°.
cultures were washed and challenged with 2 X I 0@SL2 cells in
Complement
was used to detect whether SMAF is a
3 ml of growth medium. Growth inhibition was assessed 24 hr
complement-dependent
cytotoxic antibody. The activity of
after challenge of the cultures. The factor-containing super
complement was shown by lysis of sheep RBC exposed to
natant was collected 6, 24, and 48 hr after challenge of the
specific rabbit anti-sheep RBC antibody in the presence of this
immune lymphocytes and the arming capacity of serial
complement. The antibody also was obtained from Rijks
dilutions of these supernatants was tested. There was no
Instituut Voor de Volksgezondheid.
difference between supernatant collected after 6 and 24 hr.
The 48-hr supernatant, however, had lost most of its arming
Trypsin
capacity (Table 3).
Trypsin, twice crystallized (Sigma Chemical Co., St. Louis,
Mo.), was dissolved in Fischer's medium and used as described
Specificity
of the Armed Macrophage.
Table 1 shows the normal growth of SL2 cells in growth
medium only and when placed on normal macrophage
monolayers, compared with the decrease in numbers when
placed on monolayers exposed to the supernatant from
challenged immune lymphocytes (armed macrophages). No
growth inhibition was obtained when normal macrophage
monolayers were exposed to supernatant from unchallenged
normal
lymphocytes,
unchallenged
immune
lymphocytes,
challenged normal lymphocytes.
Kinetics of the Production of the Factor.
tosupernatants
Macrophage cultures exposed
from immune lymphocyteGrowth
atculturesa
challenged with24
hr2
for(%)15mm48@8blhr65±43hr77±66hr70±424hr66±6Normal
x l0@ SL2 cells
a0supernatant
control
(not exposed
inhibition
to
and
Two X 106
immunelymphocyteswere incubatedwith 2 X l0@SL2 cells
3090
was tested
Table 2
Kinetics of the production
in the text.
RESULTS
Specificity
a Two
X
106
immune
C57BL
lymphocytes.
b Mean±S.E.
CANCER RESEARCH
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A Cytophiic
Table 3
Titer of the factor in cultures ofchallenged immune lymphocytes
inhibitionAt
Macrophage cultures exposed
hrcollected
to
supernatantaGrowth
afterDilution(%)—
24
6hr1
1/5
50±2
1/25
54±3
224hr1
1/125
1/62570@2b
35 ±8
33 ±
248hr1/5
1/5
1/25
1/125
1/62566±6
55±4
40±3
35±4
34 ±
30±2
21 ±8
9±2Normal
1/25
1/125
1/62514±5
control0
a Supernatant
from
2 X 10'
immune
C57BL
lymphocytes
chal
lenged with 2 X 10@DBA/2 SL2 cells.
0
Mean
± S.E.
by exposing armed macrophages to either SL2 cells or the
CS7BL lymphoma TLX9. Inhibition of SL2 cell growth was
found in the armed cultures, while TLX9 grew normally.
Nonimmune C57BL cultures supported growth of SL2 (Table
4).
Coating by the Factor. To show that the arming factor coats
macrophages
as
well
as lymphoma
cells,
either
normal
macrophage cultures (2 X 106 macrophages) or lymphoma
cells (2 X 106) were incubated with 2 ml of the undiluted
factor containing supernatant for 15 min, 1 hr, and 4 hr at
37°.After washing of the cells the armed macrophages were
incubated with 2 X i0@ 5L2 cells, while 2 X iO@ of the
supernatant-treated
lymphoma cells were added to 2 X 106
normal macrophage cultures. Growth was inhibited to the
same extent
in both
experiments,
but no growth
inhibition
was obtained when macrophages as well as lymphoma cells
were exposed to the factor for 4 hr (Table 5).
If we are dealing with a cytophilic factor on the alloimmune
Table 4
Specificity of the factor
Macrophage
culturesChallenge
lymphomaGrowth
inhibition
at 24 hr
(%)NormalSL2
0Armed―
±(versus
C57BL macrophagesSL266
SL2)TLX919
TLX90
±3
a Normal C57BL macrophages exposed for 24 hr to the factor
containing
supernatant;
the factor arose in C57BL mice immunized
with DBA/2 SL2 lymphoma cells.
b Mean ±S.E.
NOVEMBER
Factor from Immune Peritoneal Lymphocytes
macrophage cell membrane, trypsin should be able to suppress
the cytotoxic activity. Thus, monolayers of macrophages
exposed to the factor for 24 hr were washed and incubated
with 0.1% tiypsin in Fischer's medium for I hr. The
monolayers were washed and challenged with 2 X 10@cells in
3 ml of growth medium. The armed macrophages had
apparently lost cytotoxicity after incubation with trypsin.
These macrophages could, however, be rearmed by incubation
with the factor for 1 hr at 37 (Table 6).
Complement. To test whether the factor was a complement
dependent antibody, complement was added to SL2 cells
suspended in the factor containing supernatant and to SL2
cells coated by the factor. The results indicated that the factor
was not a complement-dependent cytotoxic antibody.
Cytotoxicity of Peritoneal Macrophages and the Arming
Potential of the Factor. At various times following im
munizationwe tested cytotoxicity of peritonealmacrophages
as well as the arming factor-producing capacity of immune
peritoneal lymphocytes. Chart I shows the decreasing cy
totoxicity of the macrophages from 21 to 173 days following
immunization.The supernatantwas collectedfromchallenged
immune and from challengednormal lymphocyte cultures.
Normal macrophages were incubated with the cell-free
supernatants for 24 hr and cytotoxicity of these armed
macrophages was tested. The production of the factor, as
measured by the arming capacity, decreases before the
cytotoxicity of the peritoneal macrophages.
The Factor in the Peritoneal Cavity. The factor is produced
in a challenged but not in an unchallenged immune lympho
cyte culture, as is already mentioned. As shown in other
experiments, SL2 cells are rejected from the peritoneal cavity
about I 0 days after immunization (6); therefore, the arming
factor can be expected in the peritoneal cavity of immunized
mice at the time of collection of the peritoneal cells.
Five ml of medium were injected into the peritoneal cavity
of immune C57BL mice and the cavity was syringed out after
gentle massaging of the abdomen. The fluid withdrawn from
the cavity was spun down, and the supernatant was designated
as “1
00% peritoneal exudate.― This 100% peritoneal exudate
and serial dilutions were added to monolayers of normal
peritoneal macrophages for 24 hr. The monolayers were
washed and challenged with 2 X 10@ SL2 cells in 3 ml of
growth
medium.
The
factor
was present
in the peritoneal
exudate and the dilution end point was 1/25 (Table 7).
The Number of Lymphoma Cells Coated by the Factor. To
investigate the number of lymphoma cells that can be coated,
ios , 106 i0@, and 108 SL2 cells were suspended in 1 ml of
the undiluted arming factor containing supernatant at 4°for 4
hr. These SL2 cells were washed 3 times and added to normal
macrophage monolayers at a concentration of 2 X 10@ SL2
cells in 3 ml of growth medium. Coating of the cells is
incomplete or borderline at concentrations of iO' and 108
SL2 cells/ml, respectively (Table 8).
Following the coating of lymphoma cells with the factor
containing medium , the supernatant of this medium was tested
for its macrophage-arming capacity. Table 9 shows that only
undiluted supernatant from medium in which S X l0@ SL2
cells/ml were coated retained a part of the growth-inhibiting
capacity.
1974
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3091
E. Pels and W. Den Otter
Table S
The factor coats macrophages as well as lymphoma cells
inhibition
at 24 hr
lymphomaGrowth
Macrophage culturesChallenge
(%)NormalSL20NormalSL2
exposed to the
factor
for
15mm
lhr
4hr32±Sa
53±4Normal
40±8
for15mmSL218±3lhrSL240±84hrSL257±3Normal
exposed to the factor
the04hrfor4hrNomacrophagesSL20No
exposed to the factor forSL2
macrophagesSL2
exposed
to
exposed
to the
factor for 4 hr0
a Mean
± S.E.
Table 6
Reversible loss ofgrowth inhibition of coated @nacrophagesby trypsina
treatment
inhibition
at 24 hr
(%)Normal Macrophage
culturesGrowth
58 ±
15 ±8
Normal armed with the factorb
Normal armed with the factor and
trypsinized
Normal armed macrophages, trypsinized
and rearmed'@ with the factor0
@
a
1%
trypsin
in
Fischer's
medium
59 ±2
for
1 hr.
b Exposed to the factor for 24 hr.
C Mean
±S.E.
d Incubated with the factor for 1 hr.
DISCUSSION
SMAF is producible in the growth medium when immune
peritoneal lymphocytes are incubated with the specific target
cells, inasmuch as normal macrophages are armed by this
supernatant
and
this
reaction
is
specific.
The
factor
is
cytophiic in that it binds not only to the specific target cells
but also to peritoneal macrophages, and the activity of armed
macrophages is terminated by trypsin treatment. The factor
coats target and lymphoma cells within 4 hr and disappears
from the supernatant by the end of 4 hr incubation with iO@
lymphoma cells/ml at 4°.These findings show that SMAF is
easily absorbed from the medium. Armed macrophages did not
show cytotoxicity when SMAF-coated lymphoma cells were
added to them.
This absence of cytotoxicity can be explained by the lack of
available receptor sites for SMAF on the armed macrophages
since these sites were already occupied. Added complement is
3092
not required for cytotoxicity.
This still leaves open the
possibility that macrophages or other cells may make
complement components that play a role in killing. As SMAF
is present in the peritoneal cavity of immunized mice, one
might speculate that one of the functions of immune
peritoneal lymphocytes is the production of SMAF. This
factor behaves like a cytophilic antibody, coating normal
peritoneal macrophages and rendering them specifically cy
totoxic. This is of interest, since the immune macrophage
membrane also possesses a cytophilic factor that recognizes
the specific antigen (6). The difference between immune
macrophages
and armed macrophages in this allogenic
C57BL/SL2 system is that immune macrophages cause lysis of
target cells within 9 hr [cytolytic macrophages (6)] , while
macrophages armed by SMAF only inhibit growth of the
target cells (cytostatic macrophages).
This difference between immune and armed macrophages
might be characteristic for the allogeneic system , as Evans and
Alexander (8) were unable to distinguish in their syngeneic
system between the cytotoxic action of macrophages (a)
derived from the peritoneal cavity of immunized mice and (b)
rendered cytotoxic by a factor released into the medium by
mixed cultures of immune spleen cells and antigenic cells. One
could attribute the specific cytotoxic action of immune and of
armed macrophages to coating with the same specific
cytophilic factor, but there is no direct evidence to support
this hypothesis. The study of the transformation of an armed
macrophage into an immune macrophage will be possible in an
allogeneic system since both macrophages can be distinguished
in such a system. Repeated exposure of armed macrophages
and sensitized lymphocytes to the specific target cell might
cause this transformation. Whether SMAF from challenged
immune peritoneal lymphocytes is the same as the SMAF's
released from immune spleen lymphoid cells (8, 9) remains to
be determined.
The assumption that a product of challenged sensitized
lymphoid cells can stimulate macrophages to transform into
CANCER
RESEARCH
VOL. 34
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A Cytophilic Factor from Immune Peritoneal Lymphocytes
150—.
Ui
0
I-
Chart 1. In the peritonealcavity of immunizedmice ..@
@
100j
the capacity of lymphocytes to produce SMAF disappears earlier than the immunity of macrophages meas
@
used as percentage
@
@
were measured in vitro. •,percentage of growth inhibition of immune macrophages; o, percentage ot
of growth
inhibition
(GI); both
growth inhibition of normal macrophages; ., recipro
cal of titer of supernatants from challenged immune
lymphocyte cultures; 0, reciprocal of titer of superna
tantsfromchallenge
normallymphocyte
cultures.
)0
DAYS AFTER
Table 9
The disappearance of the factor during coating of the lymphoma cells
Table 7
The factor in the peritoneal cavity
A. SL2 cells/ml
inhibition
at 24 hr
Macrophage cultures exposed―
to serial dilutions of the 100%
(%)1 exudatebGrowth
(undiluted exudate)68
3―1/552±91/2529±
7―10°
±
(no exudate)0
5x io@1
hr
mice.
The
cell-free
fluid
at 24 hr (%)
after challenge of
from
the cavity
is called
1/25
0
1/1250
7 ±
1/5
0
1/25
1/125
i
0
2±2
40±5
20±5
1/5
1/25
at 37°.
b Five ml medium were injected into the peritoneal cavity of
immune
to supernatant from
A in dilution ofGrowth
±
41
24
inhibition
Macrophage exposed
supernatant―
during B1061/5
incubation@'B.
121/12512±51/62510
a For
IMMUNIZATION
“100%
exudate.―
0
1/125
2±2
Normal control18±8
0
c Mean ±S.E.
The number ofSL2
Table 8
cells per ml that can be coated with the factor
a Supernatant
from
2 X
106 immune
peritoneal
lymphocytes
(CS7BL) lymphocytes immunized against DBA/2 lymphoma cells)
challenged with 2 x 10' SL2 cells for 24 hr.
b At 4°for 4 hr.
Macrophage cultures
challenged .vith
2 x 10°ofSL2
(%)Coated
cells/mi
supernatant
during
inhibition
at 24 hr
incubation―Growth
±Uncoated
SL210820
SL21080Coated
3UncoatedSL210@33
SL210@0Coated
±
SL2l0@59
3UncoatedSL21060Coated
±
SL2l0@56
7Uncoated
SL210@0
±
a At
4° for
b Mean
4 hr.
± S.E.
NOVEMBER
the highly activated cell found in infected animals was first
raised by Mackaness (16). Asherson and Zembala (3)
concluded from their experiments that peritoneal lymphocytes
interacting with antigen convey a factor to macrophages that
enables macrophages to transfer dermal delayed reactions in a
mouse. Lohmann-Mattheset a!. (14) suggestedthat macro
phages are sensitized by an antibody-like factor of lymphocyte
origin, which strongly adheres to their surface and enables
them to exert specific cytotoxicity following addition of the
antigen. Many other substances are released into the culture
medium from challenged sensitized lymphocytes that cause
inhibition of migration (2, 5), activation as measured by
increased spreading ( 19), blastogenesis of nonsensitized
1974
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3093
E. Pels and W. Den Otter
lymphocytes (17), and the aggregation of macrophages (15).
The relationship of SMAF to these substances is unknown.
The migration inhibitory factor is reported to be a
nonimmunoglobulin (2). On the other hand, macrophages bear
a receptor for IgG that attaches through its Fc portion, leaving
active sites free to interact with antigen (4, 10). Antibodies
cytophilic for macrophages have been identified as 7S
72 -globulins
capable
of
binding
complement
and
lysing
cells.
However, our results seem to exclude the possibility SMAF is a
complement-dependent
cytophilic antibody causing lysis of
the cells. Evans et a!. (9) described SMAF from T-cells
consisting of 2 major components, the one being> 300,000
daltons, the other 50 to 60,000 daltons. These findings suggest
that SMAF might consist of 2 components,
an im
munoglobulin and a component that is too small to be an
intact immunoglobulin.
Transplantation,
14: 220—226, 1972.
7. Evans, R., and Alexander, P. Cooperation
of Immune Lymphoid
Cells with Macrophages
in Tumour
Immunity.
Nature,
228:
620—622, 1970.
8. Evans, R., and Alexander, P. Rendering Macrophages Specifically
Cytotoxic
by a Factor from Immune Lymphoid Cells.
Transplantation,
12: 227 —
229, 1971.
9. Evans, R., Grant, C. K., Cox, H., Steele, K., and Alexander, P.
Thymus-derived Lymphocytes
Produce an Immunologically
Specific Macrophage-arming Factor. J. Exptl. Med., 136:
1318—1322,
1972.
10. Gelfand, E., Abramson, N., Merler, E., and Rosen, F. The
Monocyte Receptor: Inhibition of Receptivity by Low Molecular
Weight Peptides of the Fe Fragment. Clin. Res., 19: 44 1, 1971.
11. Granger, G. A., and Weiser, R. S. Homograft Target Cells: Specific
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97—99,1966.
ACKNOWLEDGMENTS
We thank
support
Professor
13. Hoy, W. E@,and Nelson, D. S. Studies on Cytophilic Antibodies v.
G. J . V. Swaen and Professor G. Bras for
and advise and Dr. R. Evans and Professor
P. Alexander
for
their stimulating discussions.
Alloantibodies
Cytophilic
for Mouse
Macrophages.
Australian
J.
Exptl. Biol. Med. Sci., 47: 525—539, 1969.
14. Lohmann-Matthes, M. L., Schipper, H., and Fischer, H. Macro
phage-mediated
Cytotoxicity
against
Allogeneic
Target
Cells in
Vitro. European J. Immunol., 2: 45—49,1972.
15. Lolekha, S., Dray, S., and Gotsoff, S. P. MacrophageAggregation
invitro:
aCorrelate
ofDelayed
Hypersensitivity.
J.Immunol.,
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CANCER RESEARCH VOL. 34
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The Role of a Cytophilic Factor from Challenged Immune
Peritoneal Lymphocytes in Specific Macrophage Cytotoxicity
Elisabeth Pels and Willem Den Otter
Cancer Res 1974;34:3089-3094.
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