Download DIBENZANTHRACENE MOUSE SARCOMAS

DIBENZANTHRACENE MOUSE SARCOMAS
HISTOLOSY
WARREN H. LEWIS
(From the Department of Embryology, Carnegie Institution of Washington, and the Johns Hopkins
University)
This paper is concerned with the histology of the first 50 neoplasms produced in this laboratory in pure strain mice by one injection of 0.8 mg. of
1 :2 :5 :6-dibenzanthracene dissolved in lard or olive oil, into the axilla from the
back. The tumors were of usable size at from 95 to 183 days after the injection. Twenty-four were in BA strain mice and 26 in C,,H strain. All gave
100 per cent growth in mice of the strain of origin and were transferred without difficulty from mouse to mouse. Some of the tumors were carried for a
few, others for many generations. A few of the tumors were temporarily lost
by death of the host but were recovered by the inoculation of colonies of cells
from roller tube cultures. Bouin, dioxan, paraffin, hematoxylin-eosin technic
was used. Sections were usually cut from both ends of the block.
The histologic sections of 28 of the primary (first-generation) tumors show
principally spindle cells and invaded host tissues, the latter in varying amount
and condition. The remaining 22 show, in addition to the above elements,
modified skeletal muscle fibers, muscle giant cells, and myoblasts. The muscle
elements, with rare exceptions, disappear in later generations. The latter consist ultimately of spindle cells somewhat similar to those of the first group.
The origin of the spindle cells and the significance 05 their histologic variations are obscure. These cells are evidently the malignant elements. Presumably many of them are permanently modified connective-tissue cells, but
some may be derived from modified skeletal muscle and myoblasts. Their
origin cannot be settled with certainty from histologic sections. The histologic
variations produced by the growth of the spindle cells may be due to slight differences in their malignant modification, to environmental factors in the host,
or to a combination of the two. Other factors such as continued modification,
elimination and selection of malignant cells may also play a part. This, again,
cannot be determined from histologic sections.
All of the tumors contained necrotic areas of various sizes and showed
various fortuitous histologic changes as a result of invasion and contact with
host tissues. The primary growths were usually more complicated in the latter
respect than the later generations, partly because of the effects of the dibenzanthracene.
Most of the 28 primary sarcomas without modified muscle have many of
their spindle cells arranged in conspicuous bands (Figs. 1 and 1 3 ) ; some have
areas without much band formation, and various gradations between the two
conditions are seen. Differences exist in the size of the cells, in the size of the
1 Aided
by a grant from the International Cancer Research Foundation.
52 1
FIG. 1 ( 2 4 5 4 ~ ) . TUMORC3, STRAIN BA, FIRSTGENERATION:LARGE SPINDLECELLS IN CONSPICUOUS BANDS; No MODIFIEDMUSCLE
IN SECTIONSOF THISOR LATERGENERATIONS
;
SIMILAR TO FIG.6. x 200
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DIBENZANTHRACENE MOUSE SARCOMAS
523
nuclei, and in the number of spindle cells with two or more nuclei. Giant
nuclei are common.
The sections of the second and later generations are similar in some cases
to those from the primary tumors; in other cases they are not (Figs. 2 and 15).
The cells and bands of cells are frequently somewhat coarser in the primary
than in the later generations, though often the photographs of primary sections and those from later tumors cannot be distinguished at a glance. There
are undoubtedly many temporary modifications of the malignant cells and
fortuitous reactions in particular hosts and special regions.
Whether or not there are permanent differences between the malignant cells
is not clear from the sections. The biological characteristics indicate, however, that the cells are not all alike. C11, for example, is similar histologically
to some of the other tumors, yet M. R. Lewis and E. G. Lichtenstein (19363)
found that various generations of this tumor could be transferred indefinitely
in three other strains of mice, though this was true of none of the other tumors
studied. They (1936 a, b ) also found that six of the tumors, after repeated
inoculations into hosts of foreign strains, became transplantable into various
strains and retained their transplantability in subsequent generations. Twelve
other tumors could not be thus changed. Each of these groups included some
tumors with modified muscle in the primary growth and some without. Only
tumors of later generations were used. No particular histologic differences
were observed between the altered tumors carried in a foreign strain and unaltered tumors carried in the strain of origin.
All transplanted dibenzanthracene tumors produce a neutrophilia of the
host. This is most pronounced with tumors C11, C16, C35, C36, and C48
(M. R. Lewis, 1937, and additional personal communication). There is no
apparent correlation between the histologic picture or the occurrence or nonoccurrence of modified muscle in the primary sections and the neutrophilia, yet
there must be differences between the cells of the tumors which produce marked
neutrophilia and those which do not. The cultural and cytological characteristics of cells from the different tumors also indicate differences which the
histology does not reveal.
The sections of 22 of the primary (first generation) tumors contain modified skeletal muscle fibers of different sizes and in different stages of dedifferentiation, muscle giant cells, myoblasts, and spindle cells. Sections of some of
the tumors show what appear to be transitions or the splitting of multinucleated
muscle fibers into myoblasts and large spindle cells. Some of the 22 tumors
would probably be diagnosed as rhabdomyosarcoma or rhabdomyoblastoma
(Figs. 3,5, 7, l o ) , and some as spindle-cell sarcoma with modified muscle fibers
and muscle giant cells (Fig. 16) since they show areas of relatively pure spindle
cells (Fig. 17).
Second generation tumors derived from 10 of the 22 primary growths contain various amounts of modified muscle and spindle cells. In five there is a
FIG. 2 (3511). TUMORC3, STRAINBA, TWENTIETHGENERATION:
DIFFERENT
FROM PRIMARY
GENERATION
(Fro. 1) ; SIMILARTO FIGS.4 AND 20. X 200
FIG. 3 ( 3 5 4 6 ~ ) . TUMORC18, STRAINBA, FIRSTGENERATION:
MUCHMODIFIED
SKELETAL
MUSCLE
(FIBERS,GIANTCELLS,MYOBLASTS)
AND SPINDLZ
CELLS. X 200
No MODIFIEDMUSCLEIN Tars
FIG. 4 (3547). TUMORCIS, STRAINBA, SECONDGENERATION:
SECTION OR IN LATERGENERATIONS;
SIMlLAR TO FIGS.2 AND 20. x 200
E‘IG. 5
TUMORC23, STRAINBA, FIRSTGENERATION:
Muca MODIFIEDMUSCLE;LONG
MODIFIED
MUSCLEFIBERS
IN SOME AREAS;SIMILAR TO FIG. 10. x 200
[Legend cotat. on mext Page]
524
(3557).
DIBENZANTHRACENE MOUSE SARCOMAS
525
considerable amount of modified muscle, often in the form of round cells with
one to several nuclei (Figs. 11 and 18) and areas of spindle cells (Fig. 12).
The other five show a few small inconspicuous muscle fibers and muscle giant
cells among the spindlecells. Five of the second generation tumors are typical
spindle-cell sarcomas without modified muscle (Figs. 4 and 6). For seven of
the tumors no sections of the second generation were made, but one or more of
the later generations of six of these consist of spindle cells without modified
muscle or muscle giant cells. The 7th tumor has in the 12th and 20th generations, the only later generations examined, numerous large giant cells of unknown source but suggestive of muscle origin.
Sections of the later generations of the tumors originally showing modified
muscle are with a few exceptions entirely without recognizable muscle elements,
consisting of spindle cells, invaded host tissues, and macrophages (Figs. 14
and 19). In sections of one or two but not all of the later generations of four
of the tumors a few small inconspicuous modified muscle fibers were observed
(Fig. 20). Giant cells also occur in some sections of later generations of 5 of
the tumors. Some of these may be of muscle origin but most of them are
probably multinucleated spindle cells. The sections of the fourth generation
of tumor C4, which showed much modified muscle in the primary and second
generation growths, contain, in addition to areas of relatively pure spindle cells
(Fig. 8), large compact areas of small round muscle cells, some of which are in
a degenerate condition (Fig. 9 ) . No later generations of this tumor are available, but the probabilities are that the muscle elements would have disappeared.
The histology of the spindle-cell areas of the second and later generations
of the tumors that lost all or most of the muscle elements is in general similar
to that of the tumors that never showed modified muscle. Compare Fig. 6
with Fig. 1, Fig. 20 with Fig. 2 , and Fig. 14 with Fig. 15.
The modified skeletal muscle fibers and muscle giant cells of the primary
tumors probably represent the reaction of muscle to injury by the dibenzanthracene and correspond to the early regeneration stages of skeletal muscle
described by Forbus and others. That the malignant spindle cells are responsible for the modified muscle seems unlikely, since many sections both of
tumors with and without modified muscle in the primary and subsequent generations show invasion, splitting and destruction of normal muscle by spindle
cells without modified muscle formation. Occasionally one part of a section
of a primary tumor shows transition of normal into modified muscle and another part the invasion, splitting apart and destruction of muscle by the malignant spindle cells. The two reactions are quite different.
The destructive process is almost always clear-cut. The invaded muscle is
split apart by the multiplication of the malignant cells between the fibers,
which usually show all stages from normal cross-striated fibers to small ones,
FIG. 6 (3558). TUMORC23, STRAINBA, SECONDGENERATION:
LARGESPINDLECELLSIN CONSPICUOUS BANDS;
A FEW VERYSMALL
MUSCLEFIBERSIN SOMI:AREAS;No MODIFIED
MUSCLEIN EIGHTHGENERATION;
SIMILARTO FIG. 1. X 200
MUCH MODIFIED
MUSCLEIN THIS
FIG. 7 (3.512~). TUMORC4, STRAINCsH, FIRSTGENERATION:
X 200
AND IN SECOND
AND FOURTH
GENERATIONS.
FIG. 8 ( 3 5 1 4 ~ ) . TUMORC4, STRAINC8H, FOURTH
GENERATION:
DENSEAREAOF ROUNDMODIFIED
X 200
MUSCLECELLS,MANYDEGENERATE.
FIG. 9 ( 3 5 1 4 ~ ) . AREAOF SMALLSPINDLECELLSFROM SAMESECTIONAS FIG. 8. X 200
FIG. 10 (3529). TUMORC10, STRAINBA, FIRSTGENE RATIO^: MUCHMOJXFIEDMUSCLE. X 200
FIG. 11 (3530). TUMORC10, STRAINBA, SECONDGENERATION:
MANY ROUNDMUSCLEGIANT
CELLB. X 200
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DIBENZANTHRACENE MOUSE SARCOMAS
527
without cross-striations, reduced to the limits of visibility. There is no
multiplication of nuclei and no giant cell formation in typical examples of the
destructive process. Occasional sections show areas in which the reaction is
less sharply defined and it is not clear whether the process is invasion and destruction accompanied by some traumatic effect which produces modified
muscle and giant cells, whether the areas in question represent an injury effect
only, or whether a combination of the two is responsible.
The modified muscle fibers present a varied histology. Cross-striations
are apparently soon lost, as they are present only in the slightly modified fibers.
The modified fibers vary in size, in length, in the number of nuclei, which are
often in compact rows, and in the degree of dedifferentiation. I n some areas
the muscle fibers are partly reduced to myoblasts, which are aggregated into
large coarse bands (Fig. 7 ) . The latter grade into some of the coarser patterns formed by spindle cells in tumors without muscle elements in the primary
generations (Fig. 1) and into coarse spindle-cell areas in other parts of the
same section. These in turn are similar to coarse spindle-cell areas of the
second and later generations of some of the primary tumors containing modified muscle (Fig. 6 ) .
The later generations of the tumors having mofled muscle in the primary
sections thus become indistinguishable from those without modified muscle.
The ultimate modified muscle elements either disappear or become similar to
the spindle cells that are presumably of connective-tissue origin.
It seems quite probable that most of the modified muscle fibers and muscle
giant cells seen in sections of the second generation are derived directly from
similar elements in the primary tumor and that they (the muscle elements)
possess in some cases limited powers of multiplication on transfer. The origin
of the few small modified muscle fibers which occasionally appear in later generations is obscure. They may be derived (1) from similar elements of the
preceding generations, ( 2 ) from myoblasts also arising from similar elements
of the preceding generations, ( 3 ) from injured muscle of the host, or (4) from
injured normal muscle of the preceding generation which was transferred with
the malignant cells. Many of the tumors are partially imbedded in muscle
and transplants taken from the periphery sometimes include normal muscle
which is injured during the excision of the material for transfer. Since none of
the muscle-free tumors contained modified muscle in their later generations
the last two sources are doubtful. Concerning an origin from myoblasts,
either normal or malignant, we have no definite evidence for or against.
The muscle changes encountered in the tumors seem to be those of dedifferentiation rather than redifferentiation. There is a tendency for such
adult cells as fibroblasts, endothelium, smooth muscle, and even skeletal muscle
to dedifferentiate a little in tissue cultures and assume a somewhat similar apFIG. 12 ( 3 5 3 0 ~ ) . AREA OF SMALLSPINDLEAND ROUNDCELLSFROM SAME SECTIONAS FIG. 11.
x 200
FIG. 13 (3500). TUMOR
C47, STRAINBA, FIRST GENERATION:
CONSPICUOUSBANDSOF SPINDLE
CELLS;No MODIFIEDMUSCLEIN THIS OR LATERGENERATIONS.X 200
GENERATION:
SMALLSPINDLEAND
FIG. 14 ( 3 5 3 2 ~ ) . TUMORC10, STRAINBA, TWENTY-FOURTH
ROUNDCELLS. X 200
FIG. 15 (3599). TUMOR
C47, STRAIN BA, TWENTY-SECOND
GENERATION:SIMILARTO FIG. 14.
x
200
$ 9
FIG. 16 (3.507). TUMORC2, STRAINBA, FIRSTGENERATION:
MODIYIEDMUSCLE;SPINDLEAND
ROUNDCELLS. X 200
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DIBENZANTHRACENE MOUSE SARCOMAS
5 29
pearance. Since the growth of malignant cells in vivo, where the host represents the culture medium, is analogous in some respects to in vitro cultures
similar changes may go on in tumors, especially the primary and early generations.
I t seems quite probable that the histology of many primary tumors which
have been classified as rhabdomyosarcoma or myoblastoma is a transitory one,
in that the muscle elements are in the process of dedifferentiation into myoblasts and spindle cells. The large percentage of such tumors produced in
mice by dibenzanthracene (Haagensen and Krehbiel) and in rats and mice by
benzpyrene and dibenzanthracene (Dunning, Curtis and Bullock), compared
with those occurring spontaneously in man, may be explained by the longer
duration of the latter, which would give more opportunity for any modified
muscle originally present to dedifferentiate into spindle cells.
The great variations in the histology of the rhabdomyosarcomas and
rhabdomyoblastomas both human and animal can likewise be attributed to
the changing character of the muscle elements.
It is quite possible that some of the primary mouse spindle-cell sarcomas
may already have passed through the transition stages before excision. There
may thus be many more spindle-cell sarcomas of muscle origin than we are at
present aware of, or the so-called muscle tumors may be merely spindle-cell
sarcomas with early regenerative stages of injured muscle which consist of
dedifferentiation changes.
SUMMARY
(1) The sections of 22 of our first 50 primary dibenzanthracene mouse
tumors were found to contain modified skeletal muscle fibers, muscle giant cells,
myoblasts, spindle cells, and invaded host tissues. Sections of the remaining
28 consisted principally of spindle cells without modified muscle. There are
similarities and differences in the primary and later generations of the latter
but the histologic sections do not enable one to determine whether this is due
to permanent differences .of the cells or to temporary differences imposed by
the host environment.
( 2 ) With rare exceptions all the modified muscle elements disappear from
the later generations, and sections of the latter are similar to those of the
spindle-cell sarcomas without modified muscle.
( 3 ) The disappearance of the modified muscle elements in later generations suggests that they either dedifferentiate into spindfe cells, which are malignant and indistinguishable from those of connective-tissue origin, or are
lost or die out.
FIG. 1 7 ( 3 5 0 8 ~ ) . SAME TUMOR
AS FIG. 16: SECTIONFROM OPPOSITCEND OF BLOCK,SHOWING
SPINDLEAND ROUNDCELLS. X 200
C2, STRAINBA, SECOND
GENERATION:
MANYSMALLROUNDMUSCLECELLS.
FIG. 18 (3509). TUMOR
x
200
+
FIG. 19 ( 3 8 3 7 ~ ) . TUMOR
C2, TENTIi GENERATION
(STRAINBA)
120 DAYSIN ROLLERTUBE
CULTURE
-k EIGHTHGENERATION
(STRAINBA) : SMALL,ROUNDAND SPINDLECELI.5
LIKETHOSE
SIIOWNIN FIG. 1 7 ; No MODIFIEDMUSCLE. X 200
FIG. 20 (3510). TUMORCZ, TENTHGENERATION
(STRAINBA)
120 DAYSIN ROLLERTUBE
CULTURE
4-EIGHTHGENERATION
(BA) : SECTION
SIMILARTO FIGS.2 AND 4 ; FEW VERY
SMALLINCONSPICUOUS
MUSCLEFIBERSIN SOMEAREAS. X 200
+
530
WARREN H. LEWIS
( 4 ) We cannot determine from the histologic sections either the origin of
the malignant spindle cells or how much or little they differ from one another.
LITERATURE
F. D.: Am. J. Cancer 28: 681-712, 1936.
DUNNING,
W. F., CURTIS,M. R., AND BULLOCK,
FORBUS,
WILEYD.: Arch. Path. 2: 318-339, 486-499, 1926.
HAAGENSEN,
C. D., A N D KREHBIEL,0. F.: Am. J. Cancer 26: 368-377, 1936.
LEWIS,M. R., A N D LICIITENSTEIN,
E. G.: Am. J. Cancer 27: 246-256, 1936 ( a ) .
LEWIS,M. R., AND LICHTENSTEIN,
E. G.: Am. J. Cancer 28: 746-751, 1936 (b).
LEWIS,M. R.: Am. J. Cancer 29: 510-516, 1937.
LEWIS,M. R.: Am. J . Cancer 30: 95-101, 1937.