T H E TRANSMISSION OF LEUKEMIA OF M I C E W I T H
A SINGLE CELL
WITH THE ASSTSTAKCE OF
CHARLES BREEDIS
(From the Drpnrtm~rzts of Yntholngy, Public Nralth aizd Prrventivr Mediciizr, Corizrll
University Mrdiral Cnllrgr, N r w Y o r k , N . Y . )
Nunlerous experiments have been performed to determine the number of
cells necessary for the transmission of malignant tumors of mice. These
studies, reviewed by de Gaetani and Blothner ( I ) , indicate that more than a
hundred thousand cells are necessary to transmit mouse carcinoma and mouse
sarcoma. These experiments, however, have not been made with inbred animals, and it is probable that under favorable conditions a I I I U C ~ smaller number of neoplastic cells would transmit the disease. Richter and Mcl>owell ( 2 )
observed that several thousand cells were required to transmit leukemia among
their inbred mice. We have found that under favorable conditions both
lymphoid and myeloid leukemia can be transmitted with an estimated number of from 1 to 100 cells (3, 4, 5 ) . I n our studies a dense cell suspension
was prepared and counted in the blood counting chamber, dilutions were made
for the injection of small numbers of cells, and the actual number of cells injected was estimated.
I t seemed desirable to obtain more accurate information concerning the
relation of the number of cells introduced to the duration of the disease and
the character of the lesions produced. This was accomplished with the aid
of the micromanipulator and is the subject of this communication. Furthermore, the micromanipulator was used to secure for inoculation material that
had been examined under the microscope in micro-droplets and found to contain no cells.
The micromanipulator and the technic of operation have been described (6, 7 ) .
Leukemic tumors produced by subcutaneous inoculation were removed
under aseptic conditions, cut up in "I'yrode solution, and filtered through
cotton to remove the larger particles. The cell suspension was kept in a test
tube immersed in iced water throughout the procedure. Microscopic droplets
containing none, a single, or a few cells, were sucked u p in a micro-pipette and
injected subcutaneously or intraperitoneally.
In the earlier experiments the cells were suspended in Tyrode solution and
direct injections were made with the micro-pipette. In later experiments
Tyrode solution containing 10 per cent mouse serum was used; the cells were
deposited on depression slides containing 0.2 C.C.Tyrode-serum and injected
1 This study has been supported by grants from the Lady T a t a Memorial Trust and the International Cancer Research Foundation, and by a Fund for the Study of Leukemia.
276
with the aid of a syringe and needle. Successful injections with single cells
were made only with the latter method. T h e mice used were young inbred
relatives of the mouse from which the transmissible strain took its origin.
The inoculations, unless otherwise stated, were intravenous, and the strain
of leukemia used was that known as S2 ( 5 ) . This atypical form of leukemia
has been employed in most of these studies because previous experiments (5)
have shown that a few cells are capable of transmitting it, and the question of
cell-free transmission of leukemia of this strain has not been definitely estab.
T a s r . ~I : Experiments I and 2
---
-
-
1
1 1
Number
of mice
injrctrtl
Material injected
Ccll-frce
From 14 t o 100 cells
Large nunibcrs ol cells injected with
syringe anrl needle
/
Experiment 1
Number of
success~u
injections
Exprinicnt 2
Number
of n i c e
injected
Number of
successfu~
injections
9
3
3
TABLE
I I : Experiment 3
Material injected
Number
of mice
injected
Number of
successful
injections
Length
of life
in days *
Known numbers of cells injectcd with the aitl of
micro-pipette
Cell-free material
102-1 17 cells per mouse
Estiniatetl numbers of cell, injrctccl with 5)ringe
and needle
1000 cells per mouse
10 cells per mouw
0.1 cells per niousc
* The length of life after inoculation refers t o that of successfully injected III~CC. The figure
in parentheses gives the average. One mouse injected with cell-free material and killed 95 days
after inoculation had leukemic infiltration of the spleen with cells different from those of Strain S2.
lished. I n addition, a strain of lymphoid leukemia (Akf5) associated with
conspicuous enlargement of lymph nodes and spleen and moderate increase of
large lymphocytes in the blood has been studied.
Experiments 1 and 2: In the first two experiments the cells suspended in
Tyrode solution were drawn into the micro-pipette and injected directly into
the peritoneal cavity of mice. These experiments were unsuccessful, probably because the cells were either injured by the medium or were too fragile
to withstand the micromanipulation. The results of inoculations were as
shown in Table I.
278
JACOB
PURTH A N D MORTON C. KAHN
Experiment3: The failure recorded in the first two experiments induced us
to add 10 per cent serum to the Tyrode solution. T h e dilutions were made
with the red cell counting pipette and the mice were injected with syringe and
needle. I n addition to injection with known numbers of cells, material found
to be cell-free under the microscope was sucked up into the micro-pipette, deposited on depression slides and injected intravenously with the aid of a syringe
and needle. Each mouse received approximately twenty cell-free droplets.
The results of these inoculations were as shown in Table 11.
Experiment 3 has shown that mice injected with from 102 to 117 cells
died with leukemia from thirteen to nineteen days after inoculation, those receiving an estimated number of 10 cells died after sixteen days. Material
containing an estimated number of 0.1 cells or inaterial which after examination under the nlicroscope was found to be cell-free failed to produce the
disease.
Experiments 4-7: After these preliminary tests, attempts were made to
produce leukemia with a few cells with the improved technic and the micromanipulator as in Experiment 3 . The results are set forth in Table 111.
TABLEI 11: Experiments 4-7
I
1
I
Number
of mice
injected
Number of
successful
injections
Length
of life
in clays
EXI'ERIMENT
4
Cell-free material
Single cell per mouse
3 cells per mouse
10 cells per niou\c
53-70 cells per moust.
EXPERIMENT
5
Single cell per mouse
Large number of cells (approsi~nately150,000)
E X P E R I ~ I B6N T
Single cell pcr mouse
Subcutaneous inoculation with tunlor particles
EXPERIMENT
7
Single crll per mouse
Subcutaneous inoculation with turnor particles
These experiments indicate that leukemia can be produced with regularity
by the introduction of from 53 to 107 cells and inoculations are occasionally
successful with single cells.
Injections with Single Cells of Strain Akf 5: Inoculations with single cells
of lymphomatosis of Strain Akf 5 were made in two experiments with the aid
of the micro-pipette. T h e results are shown in Table IV.
These experiments indicate that lymphocytosis of Strain Akf 5 can occasionally be transmitted by the introduction of a single cell. The small number of successful injections with single cells made it desirable to determine if
inoculations with estimated small numbers of cells of Strain Akf 5 made with
syringe and needle are likewise more successful than inoculations with the aid
of the micro-pipette.
T.\BLEIV: Experiments with Lymphomatosis, Strain Afk 5
Number
of mice
injected
-
EXI'ERI;\II..NT
1
Single cell injections
Largr numbrr of cells (approsin1;ltely 200,000)
Number ol
succrssful
injections
1 '4
Lcngth
of l ~ f e
in days
p
pp
p
P
-
-
14-16 ( , 5 )
EXPER.IIIENT
2
Single cell injections
Subcutaneous injection of spleen particles
The cell suspension containing approximately 10,000 cells per c.mn1. was
prepared as with Strain S2, counted, and diluted with Tyrode-serum. T h e
estimated number of cells were injected intravenously in volume of 0.1 C.C.
T A I ~ LVE
--
-
Number of cells
I
--
. -
Number of
successful
injections
Number
of mice
injrctecl
Length
of life
in days
These experinlents indicate that the introduction of an estimated number
of from 1 to 10 cells produce leukemia fatal in from twenty-four to twentynine days, but inoculations fail in the absence of malignant lymphocytes.
Sz~mmaryof the Experinzents with Mice
The results obtained with lymphomatosis Akf 5 and the atypical form of
leukemia S2 are similar, and the experiments made with these two strains of
leukemia are summarized in Table VI.
TABLE
VI : Results of Experiments with Mice, Summarized
-.
-
-.
Strain
-
I
Strain
Atf 5
i
-
Total
Number of experiments
Number of mice injected with single cells
Nunlber of mice that developed leukemia
The percentage of successful inoculations with single cells is very low,
only 5 of the 97 mice injected having developed leukemia. I t is not certain,
2 80
JACOB
FURTH A N D MORTON C. ICAHN
however, that every mouse actually received a single cell, for this might have
remained adherent to the glassware. Moreover, the possibility of injury to
the leukemic cells during the procedure described is great, and injured cells do
not produce the disease. I t is likewise possible that single cells perish in the
new host unless they meet conditions favorable for their multiplication. The
difficulty of transmitting by subcutaneous injections certain strains of leukemia
which are readily transmissible by intravenous injections and the success of
inoculations of an estimated small nunlber of cells made with the aid of a
syringe and needle suggest the assumption that leukemic cells are either injured or lost during the process of micromanipulation.
Leukemia in the five mice after injection with single cells introduced by
means of the micromanipulator was the result of inoculations and not spontaneous disease, for the following reasons. T h e type of disease conformed
with that of the strain whose cells were introduced. The disease developed
after the expected incubation period. Only one instance of leukemia occurred
in the 52 mice that received cell-free material, and the predominant type of
cell in this case was different from that introduced.
Inoculation with Cells Purposely Injured
Numerous experiments that have been described (2-5) indicate that live
cells are necessary for the transn~issionof leukemia of mice. Nevertheless,
this view is not generally held and the possibility of transmitting leukemia by
cell-free agents has been suggested recently by several investigators ( 8 ) .
The cells were isolated in micro-droplets in the usual manner. A new
micro-pipette was then prepared, but the fine tip was fused into a ball by
gentle application to the flame of the micro-burner. The cells were impinged
against the under side of the cover slip and rubbed about with this ball-tipped
micro-pipette until crushed.
Seven mice were injected with from 19 to 3 5 crushed cells each and all remained healthy, while all of 6 mice that received a similar number of uncrushed cells died with leukemia after from fifteen to eighteen days.
TABLE
VI I : Results of Experiments with Chickens
Nunrber
of
rxpcrinlcnt
Strain usctl
N u m b r r of
chickerls
injcctpcl
Sarcoma virus No. 11
Sarcoma virus No. 15
Leukosis virus No. 1
Sarcoma-leukosis virus No. 13
EXPERIMENTS
WITH CI-IICKENS
Failure of cell-free transmission of mouse leukemia with the technic described suggested control experiments showing that with a known tumorproducing virus the disease is transmitted under similar conditions. Such ex-
periments made with chicken sarcoma and leukosis met with little success, as
shown in the summary of results in Table VII.
The strains of chicken sarcoma and leukosis used in these studies have
been described ( 9 ) . Experimental conditions for the transmission of chicken
sarcoma and leukosis are less favorable than those of mouse sarcoma and
leukosis, especially when the micro-pipette is employed. Inbred animals are
not available for inoculation and the dose necessary for the transmission of
these diseases is large. Although exact data on the number of neoplastic cells
or virus particles necessary to transmit chicken leukosis or sarcoma are not
available, it seems to us that the required dose is large. I t is significant that,
in spite of these adverse conditions, leukemia was produced in 1 of 9, and
sarcoma in 2 of 3 1 chickens injected.
DISCUSSION
In the experiments described a small number of mice inoculated with single
leukemic cells developed leukemia. These mice died within fifteen to fifty
days. This finding indicates that if leukemia results from the malignant
transformation of a single normal leukocyte the mice will die within that
period. Mice of our stock died with spontaneous leukemia at the age of
seven to fifteen months; we may suppose that in these animals the malignant
transformation of normal cells has occurred within approximately seven weeks
before death.
When mice received a dose larger than approximately 20 cells, all of them
died of leukemia; but inoculations with smaller numbers of cells were successful only in a small percentage of inoculated animals. This result may be explained by supposing that many of the cells used for inoculation were not
viable, and that many cells were lost or injured during the process of manipulation. In the experience of one of us (M. C. K.)microinanipulation does not
injure bacteria, histiocytes, and polynuclear leukocytes, but leukemic cells appear to be more fragile than normal leukocytes. I t may be supposed, furthermore, that not all cells are capable of reproduction and that cells which do not
reach an organ favorable for their growth perish. The latter assumption is
supported by the finding that with several strains of leukemia subcutaneous
inoculations with large numbers of cells fail to produce the disease, while intravenous inoculations made with a smaller number of cells are successful.
Attempts to transmit mouse leukemia with cell-free material and with
injured cells have already been reported in previous communications, but the
absence of intact cells in the inoculunl had thus far not been ascertained by
direct method. In the experiments described in the present communication,
the cell-free material to be injected was examined microscopically and the
cells were crushed while watched under the microscope. Failure to transmit
the disease in these experiments supports the assumption that transmission of
leukemia of mice is due to the implantation of living leukemic cells into
susceptible hosts.
Single cell inoculations were made with the aid of the micromanipulator
into 97 mice, of which 5 developed leukemia. All of 52 mice inoculated under
282
JACOB PURTH AND MORTON C. ICAHN
similar conditions with cell-free material remained healthy. Crushed leukemic cells introduced into 7 mice produced no ill effects, while all of 6 mice
receiving a similar number (approximately 20-25) of uncrushed cells died
with leukemia. The virus of chicken leukosis or sarcoma introduced into 40
chickens with the aid of the micro-pipette produced the disease in 3 birds.
Leukemia may be explained by assuming that at least a single cell undergoes malignant transformation in an adult individual. In mice developing
leukemia a t the age of seven to fifteen months, this transformation may take
place within four to seven weeks before the death of the animal. A single
malignant white blood cell is capable of producing the systemic diseaseleukemia-which has hitherto been regarded by many workers as having a
multicentric origin. 'Transmission of mouse leukemia is due to the implantation of living malignant leukocytes.
1. DE G A ~ T A NG.
I , I;., AND BLOTIINER,E . : Ztsch. f . Krebsforsch. 44: 108, 1936.
E. C.: J. Exper. Med. 57: 1, 1933.
2. RICHTER.M. N., A N D MCDOWELL,
3. FURTH,J., SEIBOLD,
H. R., A N D RATHBONE,
R. R.: Am J. Cancer 19: 579, 1933.
1.FURTH,J.: J. Exper. Med. 61: 423, 1935, and unpublished data.
W. A,. A N D FURTH,J . : Am. J. Cancer 30: 75, 1937.
5. BARNES,
6. KAHN,M. C.: Am. Rev. Tuberc. 20: 150, 1929.
7. KAHN,M. C., A N D S C H W A R Z K ~H.:
P F , J. Bact. 25: 157, 1033.
8. RASK-NIELSEN,
H. C., A N D R . : Act3 pntl~.microbial., Scand. 13: 243, 1936, and references
cited.
9. FURTH,J . : J. Exper. Med. 63: 127, 1936 (viruses I1 and 1 5 ) ; J. Exper. Med. 53: 243,
1931 (virus 1).
STUBBS,E. L., A N D FURTH,J. : J. Exper. Med. 61 : 53, 1935 (virus 13).