SPINDLE-CELLED TUMOURS AND LEUCAEMIA I N MICE AFTER
INJECTION WITH A WATER SOLUBLE COMPOUND O F
1:2 :5 :6-DIHENZANTHRACENE '
H.4KOLD RUKIIOWS
AND
J . W. COOK
( F r o m The Rctrarch In\titzrt~, The Caizc cr Hospital ( F r e e ) , Londoiz)
Since the observation was first made (Rurrows, Hieger and Kennaway)
that spindle-celled tumours having the characters of sarcomata could be induced by the subcutaneous injection of 1:2 :5 :6-dibenzanthracene into rats
and mice, several other compounds prepared in this Institute have been found
to have similar properties. I n all the experiments with these compounds
three features have been constant: (1) the compounds used have been insoluble in water and have been administered either in solution in lard or olive oil,
or as colloidal suspensions in water (Boyland) ; ( 2 ) the tumours have consisted mainly or entirely of spindle cells; (3) the primary tumours have
appeared only in the immediate neighbourhood of the site of injection.
Sodium 1 :2:s: 6-dibenzanthraccne-9: 10-rndo-nB-succinatc
I t seemed of interest to examine the behaviour of a water-soluble carcinogenic compound, which might be expected to give rise more readily to
tumours at some site other than that of the injection. T o this end attempts
have been made to prepare various water-soluble derivatives of 1 :2 :5 :6dibenzanthracene. The present communication describes some preliminary
observations concerning the Effects on mice of one such compound, namely,
sodium 1 :2 :5 :6-dibenzanthracene-9 : lO-endo-c&succinate (Cook).
An aqueous solution (0.3 per cent) of this compound was prepared and
it was observed that on standing the solution deposited a small amount of a
fine crystalline precipitate, possibly an acid salt formed by slight dissociation.
The suspension containing this precipitate was used for the following experiments, male stock mice of no special strain being employed.
EXPERIMENT
1 (Commenced June 8, 1931; last mouse killed March 10, 1 9 3 2 ) : After
the preparation had been shaken to distribute the precipitate, 0.2 C.C.was injected subcutaneously in the right groin and flank of each of 20 male mice, twice a week. These injec1
Submitted for publication May 28, 193.5.
267
20s
HAROLD BURROWS A N D J . W . COOK
tions c,iuserl some local inflammation and scarring; otherwise they did not appear to have
,my immediate ill effect on the health of the animals. Of the 20 mice (Table I), 4 eventu,illy had ascites, on which account they were killed; 3 of them were found t o have spindlecelled tuinours of the diaphragm, and one of the mice had lcucaeinia but no tumor. ?'he
IN IS^ -mortem records are
RS
follows :
Fro. 1. LIVEROF
"
MAI.EIC
" M o r i s ~16
IJ:irjie iiumbcrs of luucocytes occupy the hepatic sinusoids aiid form masscs surrountlinx thc
portxl vussc~ls. X 150.
" .I.1dcic " M o m c 76: Killed on the 3 181h day.
Sanguineous ascites is present ant1
thc liver i s enhrged and pale, with rounding of the lobules. The spleen is greatly enlarged,
;inti there is a general enl'irgeinent of the lymph nodes. Microscopic sections show the liver
hinusoids ( rowded with whitts corpuxlths, I ; q e collections of which are massed around the
por1,iI vesscls (Fig. 1 1. 'The spleen is crowtlctl with similar cells which at one point appear
to hcive infiltr'ited through the capsule Great numbers of leucocytes are present in lhc
I)lootl vessels of the organs examined microscopically.
SPINDLE-CELLED TUMOURS AND LEUCAEMIA I N MICE
269
“ Muleic ” Mouse 6 : Killed on the 381st day.
There is sanguineous ascites, and a pale
mass of growth involves the whole of the diaphragm and the ensiform process; other smaller
nodules are present in the peritoneum of the right hind quadrant of the abdomen. The
liver is enlarged. Microscopical examination shows that the main tumor is composed of
spindle-cells which are invading the muscle of the diaphragm (Fig. 2 ) .
The liver i s
“ lMnleic” Mouse 9 : Killed on the 423rd day on account of ascites.
enlarged, with rounding of the lobules. A tumour of the diaphragm is present (Fig. 3 ) ,
and numerous smaller nodules of growth are scattered over the parietal and visceral surfaces
of the peritoneum. Microscopical sections show the tumours to consist of polymorphic cells
varying much in size; invasion of the diaphragm has occurred.
“ Mnleic ” Mouse 20: Killed on the 423rd day.
Chylous ascites is present. The lohes
of the liver are rounded. A spindle-celled tumour involves the diaphragm and nodules of
growth are scattered over the peritoneum. The diaphragm has been completely traversed
by the tuniour. which extends into the pleural cavity (Fig. 4 ) . A sepirate widely spread
mass is invading the walls of the stomach and intestine (Fig. 5).
EXPERIMEKT
2 (Commenced Oct. 13, 1931; last mouse died Nov. 28, 1932 ) : In this
experiment, before the injections were commenced small pieces of sterile sponge were introduced aseptically into the subcutaneous tissue of the right flank in each mouse. The purpose of this was to ohserve whether, by setting up a chronic inflammation. a local fixation
of the compound injected might occur. I t was thought possible that a water-soluble carcinogenic substance might cause tumours at the site of injection in these circumstances,
whereas in the absence of local fixation it might induce tumours in distant regions of the
body. This experiment was begun before any tumours had appeared in the mice of the
first experiment. Ten mice prepared in this way were given injections twice a week into
or around the fragments of sponge, the dosage used being the same as in Experiment 1.
The fragments of sponge ulcerated out in most cases after some weeks or months; in one
instance tht. sl)onge W:LS retained for niore than a year. T w o of the mice subjected to this
treatment eventually showed tumours at the site of injections. One o l these mice was
killed on the 380th day of the experiment. The tumour consisted mainly of spindle cells,
and was regarded as a granuloma. No significant lesions were seen elsewhere in the body.
270
SPINDLE-CELLED TUMOURS AND LEUCAEMIA I N MICE
271
Sponge Mouse 9 : Killed on the 397th day. There was a tumour at the site of the injection, consisting mainly of spheroidal or oval cells with hyperchromatic nuclei, invadin:
the subjacent muscles (Fig. 6 ) . On opening the body it was noticed that the blood was
pale and showed little tendency to clot. The liver, spleen, and lymph nodes were enlarged.
Microscopical sections show the liver sinusoitls to be crowded with round cells (Fig. 7). The
cells composing the t umour resemble those occupying the hepatic sinusoids and composing
the greater part of the enlarged spleen and lymph nodes.
FIG.6 .
"
SPOSCE
" MOLSE0 : KOUNU-CEI.I.ED
T I J M O ~TW.\DINC
JK
X 500
MUS~IX
AT SITE OF INJECTIOS.
EXPERIMENT
3 (Comniencetl Oct. 4. 1032; last mouse killed Sept. 29, 1933): Each of
twenty male mice was given 0.5 C.C.of the preparation intraperitoneally twice a week. This
procedure was stopped a t the eighth week because the injection caused the formation of
adhesions in the peritoneum with partial obliteration of the pxitoneal cavity. Fifteen injections were given in all. Among these mice two tumours eventually a p p m w l .
Spindle" Mal" M o u w 1 6 : Killed on the 163rd day on account of chylous ascites.
celled tumours were growing from and invading both halves of the diaphragm (Figs. 8 and
9 ) , arid small plaques of similar cells were scattered about the peritoneum. Under the
microscope the liver appears almost normal except for an occasional small collection oi
lymphocyte-like cells adjacent to the lobular branches of the portal vessels.
" Ma1 " Mouse 19: Died on the 273rd day of the experiment with congestion of the
lungs and sanguineous pleural effusion. The liver was not enlarged. hut was adherent to
the stomach and diaphragm. Attached to and invading the stomach is a large tumour consisting mainly of spindle cells of various sizes, together with some giant cells and some
smaller cells of indefinite shape (Fig. 1 0 ) . The liver seems normal exLept for a iew small
periportal collections o i lymphocytes.
EXPEHIMEUT
4 (Commenced May 1 2 , 1 9 3 3 ) . Ten mice were given biweekly subcutaneous injections of 0.5 C.C.of the preparation used in the preceding experiments. into the
right groin and flank.
" Blood coiwt " Mouse 10: On the 133rd day a tumor 2 cm. in lenglh was present a t
the site of injection in one of these mice, which was killed. There was no ascites and the
liver appeared normal. The spleen was turgid, firm, and paler than normal. Apart from
272
1IARC)LII BURROWS A N D J. W. COOK
Sit(, of
injectirrn
SlIII(.utanl.oIIs
right groin
illlll fl'lllk
... ~~.
Sul~cn~'lllel~ns
~
sanL!llinl~-
riglit groin
atid flank
011sascites
Subcutaneous (a) Diapliragm
(6) I'eritoniwin
right groin
and flank
Ascitcs
Subciitiineou.; ( a ) Diaphragm
(6) Stomach
right groin
anrl Aank
(0 I'eritoneum
Cliylous
ascites
Groin
~Ub~Ll~llll'lJllS
right groin
ilnd flank
.
Spleen :lIIlI
lytnpll
nodes
enlnrgerl
cII,~lous
6 . "hlul"
Il1ol1sc'
-
1Snl;rrzrd
sinrisoids
crowded
witli white
cells
nscites
16
Stomncll
No ascites
I<igllt groin
Tllums
and flank
[his no signs of leucaemia were observed. The lymph nodes were not enlarged. Microscopic
sections show the tumour to consist of cells of various shapes and sizes with spindle-cells
predomin,iting (Fig. 11 I Portions of this tumour were grafted into 20 normal stock mice,
with h I ' takes." and subsequent graftings have carried on the tumour, which is now in the
48th generation. (Mny 1035). The tumours produced by these grafts are spindle-celled
(Fig 12 ). In mice bearing grafts of the fourth and subsequent generations, Dr. Parsons
h;~sfound that most of those in which the grafted tumours have attained a large size show
the liresence of a leucaemic condition with great enlargement of the spleen. Her observ,i~ i o n son this have already been published.
-
--
~
_
T.eucocytes, per cent
lied cell
('0u I1 t
(tiiillions)
-
8.26
8.5
10.3
11.0
6.6
8.9
9.2
8.2
8,125
18,218
7,900
.30,81)0
2 1,500
19,500
2 1,900
1 7,000
25.1
12.6
10.9
6.3
14.3
5.7
I 1.5
30.3
60.4
78.4
84.1
87.3
81.9
87.5
85.3
66.1
12.2
8.0
3.1
2.7
2 .o
2.3
I .3
3.3
0.5
1.0
1.0
1.8
1.8
1.4
1.9
0.2
1.8
0.0
0.9
1.9
0.0
3.1
0.0
0.1
SPINDLE-CELLED TUMOURS AND LEUCAEMIA I N MICE
273
I3lootl counts were niade hy 1)r. Parsons :it intcrwls throughout the experimental treatment of this mouse, and she has kindly supplied the figures given in Table 11.
The eight blood counts recorded in this tahle show that the leucocytes rose to 18,000
by the 19th day, then fell and rose agnin, to 30,800, by the 47th day. Blood counts carried
out by Dr. Parsons upon all 10 mice of this series showed a diphasic change of this type,
which is therefore due in a11 probability to the injections. Between the 83rd day and the
131st day, when the blood was examined for the last time, " blood count mouse 10 " showed
a fall in total leucocytes from 21,000 to 17,000, a fall in lymphocytes from 18,600 to 11,300,
and a rise in polymorphs from 2500 to 5100. This type of change (fall in lymphocytes
and rise in polymorphs) is that seen in the form of leucaemia found later on (in the 4th
grafted generation) to accompany the transplantable sarcoma derived from this particular
FK,.7
"
SPONGE
" M o r s ~
9 , SLCTION
OF LIVER
Sinusoid\ are crowtled with leucocytes :tiid liver cells are undergoing destruction.
X 120.
mouse. The blood of the mouse in question showed no abnormality when first examined,
upon the fifth day of the experiment, and hence the association of this tumour with leucaemia
did not arise from the production of the primary tumour in a mouse already bearing a
distinct leucaemia. However, the possibility cannot now be excluded that the leucaemia
might have been introduced with the mice used for grafting in the early generations. Hence
the question whether 1 , 2 , 5 :6-dihenzanthracene in any form can produce leucaemia de novo,
;IS well as sarcoma. must await the results of further experiments.
" Blood c n i m t " Mozise 2 : This mouse was found to be sick on the 164th day and was
killed. A large pale growth was found occupying the upper part of the thoracic cavity, and
thcre was a general enlargement of the lymph nodes. The thoracic tumour consisted of
cells resembling lymphocytes which were invading the heart muscle (Fig. 13), trachea, and
other neighhouring structurcs.
T h e general results of these experiments are shown in Table I.
No controls were used with Experiments 1 and 2. As controls to Experiment 3, 20 mice were given intraperitoneal injections of 0.5 C.C.normal saline
solution. The first doses were given on Oct. 4, 1032, and no further injections were given after the eighth week, when the treatment with the dibenz-
2 74
HAROLD BURROWS AND J. W. COOK
anthracene compound was stopped. No tumours or ascites occurred in these
mice, the last six of which were killed on Oct. 13, 1933, all being found to be
free from tumours, ascites or other macroscopic lesion,
It was conceivable that in the body the compound used for these experiments might undergo fission into 1 :2 :5 :6-dibenzanthracene and sodium maleate. Therefore, as an additional control, 10 mice were given biweekly
intraperitoneal injections of 0.5 C.C. of a 0.033 per cent aqueous solution of
275
SPINDLE-CELLED TUMOURS AND LEUCAEMIA I N MICE
sodium maleate, this concentration corresponding with that of the dibenzanthracene compound. The first injection was made on Dec. 9, 1932. These
mice remained healthy and none of them suffered from tumours or any lesions
suggesting leucaemia. The last two survivors were killed on April 24, 1934,
at which time they were still free from any gross signs of disease. A summary
of the results in the treated and control animals is presented in Table 111.
-.- ..__..-
-
-
.-
-- -
Sodium 1 : 2 :5 :6-tlibenzanthra
cene-9 l O - ~ n d o - 0 1 ~
succinate
Sodium Chloride
Sodium hlaleate
--
~
~
60
20
8
3 (associated with
tumours in two
cases)
-
10
L)ISCUSSION
Two points of interest arise out of the foregoing experiments: ( a ) the
development of tuiiiours following injection of a water soluble compound, and
(0) the appearance of leucaemia and lymphosarcoma. Hitherto it appears
that, with one exception, all the pure chemical substances with which malignant tumours have been induced experimentally in animals have been insoluble
in water. The exception is the observation recorded by Browning and his
collaborators of sarcoma formation in a mouse following the subcutaneous
276
HAROLD BURROWS AND J. W. COOK
injection of 2 (p-amino-styryl)6 (p-acetylamino benzoy1amino)quinoline methoacetate dissolved in water.
Regarding the leucaemia that appears to have been present in some of
these mice, it is unfortunate that more exact details of the blood changes are
not available. The experiments, however, were of a preliminary and exploratory nature, and in such work, especially when many different substances
are being tested for carcinogenic properties, it is not always possible to provide beforehand the means for observing precisely every possible development
that may arise. An association between tumours and leucaemia has been
recorded by several observers (Oberling and Gukrin, Furth, and others),
and it might perhaps be suggested that the experiments now under discussion
were performed on mice of a leucaemic stock. Against this are the following
facts: ( 1) In all the four sets of experiments tuinour formation was seen,
and leucaeniia or lyniphosarcoma occurred in three of them; ( 2 ) in none of
the controls did these abnormalities ensue; ( 3 ) in mice obtained from the
same source, and used for other experimental work, no such heavy incidence
of spontaneous leucaeniia has been observed.
Though it might be unjustifiable at the present stage to assert that the
chemical compound injected was the cause of both the local tumours and the
leucaemia, yet it seems possible that this was the case.
At the outset of the experiment it had been thought possible that tumours
might appear in places distant from the site of injection. However, the
tumours which arose elsewhere than at the site of injection-with the exception of the lymphosarcoma in ‘‘ blood count ” mouse 2-had their origin in
FIG. 12. SECTION
OF SmwI~E-CErxDTUMO~JR
OF 4 1 s ~GENERATIOX
OF GRAFTSDERIVE[)
FROM
“ BLOODC O L ~ T
MOCJSE
”
10. X 150
FIG. 13.
‘I
BLOODCOUNT” MOUSE2. ROUND-CELLED
MEDIASTINAL
TUMOUR
INVADING
THE
HEARTMUSCLE. X 120
277
278
HAROLD BURROWS AND J. W. COOK
the peritoneal cavity and principally affected the diaphragm, growing from its
abdominal surface. The probable explanation seems to be that, in giving
what were intended to be subcutaneous injections, the needle sometimes
passed into the peritoneal cavity. The abdominal wall of the mouse is thin,
and such a mistake can be made easily enough, especially when, as in the
present instance, the earlier injections cause scarring in the subcutaneous
tissues. In some previous experiments (Burrows) in which intraperitoneal
injections of 1:2 :5 :6-dibenzanthracene dissolved in emulsified olive oil were
used, the diaphragm was found to be a common site for the resultant tumours.
SUMMARY
The repeated injection of a water soluble compound, namely sodium
1 :2 :5 :6-dibenzanthracene-9 :10-cndo-up-succinate, into mice was followed
sometimes by the growth of spindle-celled or polymorphic-celled tuniours
having malignant characters, sometimes by leucaemia, and sometimes by both
these conditions in the same individual. In one instance a condition of
lymphosarcoinatosis occurred.
REFERENCES
E.: Colloidal solutions of 1 :2 .5:6-dibenzanthracene, Lancet 2 : 1108, 1932.
BOYLAND,
BROWNING,
C. H., COHEN,J. B., COOPER,K. E., ELLINGWORTII,
S., A N D GULBRANSEN,
R.:
The antiseptic and trypanocidal action of some benzoylamino quinoline anil and styryl
compounds, Proc. Roy. SOC.,Ser. €3, 113:300, 1933.
BURROWS,
H.: hlesoblastic tumours following intraperitoneal injections of 1 :2 : 5.6-dibcnzanthracene in a fatty medium, Proc. Roy. SOC.,Ser. B, 111: 238, 1932.
BURROWS,
H., HIEGER,I., A N D KENNAWAY,
E. L.: The experimental production of tumours
of connective tissue, Am. J. Cancer 16: 57, 1932.
COOK,J. W. : Polycyclic aromatic hydrocarbons. Part I11 : The chemistry of 1 :2 : 5 :6-dibenzanthracene, J. Chem. SOC.1931, p. 3277.
FUKTII, J. : Lymphomatosis. myelomatosis, and endothelioma of chickens caused by a filtrable agent, J. Exper. Med. 58: 253, 1933; 59: 501, 1934.
OHERLING,
C., A N D G U ~ R I NM.
, : Lksions tumorales en rapport avec la leuckmie transmissible des poules, Bull. Assoc. franc. p. 1’Ctude du cancer 2 2 : 180, 1933.
I’AHSONS, L. I).: Leukaemia coincident with and transmissible by a spindle-celled sarcoma
in the mouse, J. Path. & Bact. 40: 45, 1935.