Submicroscopic Cytoplasmic Particles Occasionally
in the Ehrlich Mouse Ascites Tumor*
C.
C.
SELBY,
C.
A.
E.
E.
GREY,
S.
MOORE,
LICHTENBERG,
AND
J. J.
C.
in
only
three
additional
instances
(2).
MATERIALS
AND METHODS
Mice inoculated with approximately one million
cells of a stock suspension of the Ehrlich mouse
ascites tumor develop within a week a peritoneal
ascites which contains a high percentage of tumor
cells that grow progressively to kill the animal
within 2—Sweeks. Specimens of these cells are col
lected by withdrawing the ascitic fluid from the
peritoneal cavity with a syringe. In our early
studies the cells were prepared for electron micros
copy by centrifuging
them free of ascitic fluid
and then adding a I per cent suspension of buffered
neutral osmium tetroxide (11, 14) or by adding a
saline solution to the ascitic fluid, centrifuging it,
and then resuspending in a mixture of saline and
osmic acid (2). Cells fixed in this manner did not
appear to be optimally fixed. Mitochondria
were
generally
swollen, and the nucleolar internal
structure
@
was not well preserved.
It was therefore
decided to eliminate the time delay involved in
centrifuging cells free of ascitic fluid before fixa
* This
investigation
was
supported
by
an
institutional
re
per cent solution
for publication
appeared
of osmium
to be to add a 2
tetroxide
buffered
at
pH 7.3 directly to the ascitic fluid immediately
after it was removed from the mouse. Equal vol
umes of fixative and fluid have been employed,
although a greater volume of fixative is desirable
when an ascitic fluid containing a particularly
high concentration
of cells (i.e., over 10 per cent)
is encountered.
Cells
fixed
in this
way obey
the
criteria of optimum preservation that are general
ly accepted today (11), as demonstrated
by the
accompanying illustrations.
The cells are fixed for from 80 to 45 minutes,
after which interval the fixative is removed by
centrifugation
and the cells resuspended in water.
They are washed with three changes of water
within 1 hour and then resuspended in 70 per cent
alcohol. In some cases specimens were stored in
this condition, while in others (2), they were
placed and stored in 5 per cent neutral formalin
before washing, although no particular merit has
been noted for this procedure. Following this they
are dehydrated in 95 per cent alcohol followed by
two changes of 100 per cent alcohol, and then em
bedded in appropriate
combinations
of n-butyl
and methyl methacrylate
according to the usual
procedures.1 Optimum penetration of fixative and
embedding is achieved in these preparations, since
all solutions are in direct contact with each cell.
Thin (0.I-@) sections were originally cut in the
modified Spencer microtome (7) or in a newer ver
sion of this microtome (6). A mechanically advanc
ing microtome of novel design has recently been
built in this Institute
(16),2 facilitating
routine
sectioning of serial sections at any thickness be
tween the limits of 0.02—I @i.Employing
this
microtome, sections of the order of 300 A (Fig. 7)
and 1 (see Figs. 1—4)in thickness have been cut
1A 8:1 ratio of butyl to methyl methacrylate was used
originally with catalyst which had been stored for a long time.
Currently a 2:1 ratio is employed with 1 per cent fresh cat
alyst (plasticizer extracted).
search grant by the American Cancer Society and by a grant
from the Lila Babbitt Hyde Foundation.
Received
Sloan-Kettering
tion. The best practice
Therefore,
little progress has been made in understanding
their function or determining their identity. De
spite our consequent lack of knowledge concerning
the biological significance of these particulates,
it
has been considered advisable to report upon their
interesting
morphology
for the information
of
other investigators
who might observe a similar
phenomenon.
FRIEND,
BIESELE
(Divisions of Experimental Pathology and Experimental Chemotherapy,
InstiMe for Cancer Research, New York, N.Y.)
Certain constant-diameter
particles were noted
in an unusually regular array in the cytoplasm of
one of the first specimens of the Ehrlich mouse
ascites tumor to be examined electron-microscopi
cally (15). Since this time, many different speci
mens of the tumor have been examined in this
laboratory, but these particles have been observed
Found
June 10, 1954.
‘C.
R. Stryker,
unpublisheddata.
790
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SELBY et al.—Partwles
@
from the same face of the tissue block, thus per
mitting parallel light and electron microscope
studies.
For phase microscopy, the 1-@ sections are
picked up on glass slides, and the methacrylate is
removed by immersion in xylene for about 10
minutes. The sections are transferred to absolute
alcohol, mounted in Diaphane, and covered with
No. 1 coverslips. For ordinary light microscopy it
was found to be useful to dehydrate the sections
in dioxane after removal of the plastic, and stain
for 10 minutes in Löffler's alkaline methylene
blue. The blue stain is picked up by all osmiophilic
areas, making them clearly visible.
The thin (0.03—0.1) jz sections for electron
microscopy are picked up on film-covered grids in
the usual way and examined, without removal of
the plastic, in an RCA EMIl 2-B electron micro
scope equipped with an intermediate lens and 25
objective aperture.
RESULTS
Cytoplasmic
masses of particles such as al
ready described (15) have been observed in only
four out of 70 specimens of the ascites tumor ex
amined. Specimens collected in two animal labora
tories over a period of 2@years, and at stages of
growth
of from 5 to 31 days following
the original
inoculum, are included in this group. Two of the
specimens containing
these masses came from
mice which had been in the same cage for 10 days,
with seven other mice whose ascites did not show
similar masses. One of the other specimens posi
tive for these particles came from a mouse which
had been inoculated,
together with five other
mice, with Bunyamwera
virus 28 hours previ
ously. The ascitic fluid of the other five inoculated
mice and of five control mice was removed at
@,4, 22, 46, and 52 hours
following
the virus
inocu
lum. None of these fluids, nor that of the control
mouse sacrificed at 28 hours, was positive for cells
containing these characteristic
cytoplasmic par
ticles. A repeat experiment
with Bunyamwera
virus did not disclose these particles in any of the
infected groups.
The two types of specimens encountered may
be compared in Figures 1 and 2. These are light
micrographs of methylene blue-stained sections of
the same specimens which were fixed and em
bedded for electron microscopy. In Figure 1 large
cytoplasmic masses of high density are indicated
in about 20 per cent of the cells. These are not evi
dent in Figure 2. Examining the unstained sec
tions under oil immersion with the phase micro
scope, one may clearly distinguish these masses
from fat droplets or other normal cytoplasmic
in Ehrlich
791
AsdI,e8 Tumor
components.
Phase micrographs
demonstrating
this are shown in Figures 8 and 4. Normal cyto
plasmic and nuclear components appear to retain
their normal relationships in the presence of these
densities, although many sections are encountered
in which the latter fill nearly the entire cytoplas
mic volume. By examining thick (I j@)sections
of all the four specimens in which such masses did
appear, it was found that they occurred in about
20 per cent of the cell sections. Only sections of
cells sufficiently large to contain a nucleus were
included in this count. It was found that such
scanning of thick sections under the phase micro
scope
was a rapid
and
accurate
method
for de
termining the presence or absence of these particles
in a given specimen and saved a considerable
amount of electron microscope operation.
An electron micrograph of a cell such as that
illustrated in Figures 3 and 4 is shown in Figure 5.
Here the cytoplasmic mass is seen to consist of
constant-diameter
spheres in a predominantly
hexagonal array. Such particles have never been
observed within the nucleus, are usually separated
by an appreciable space from the nuclear mem
brane, and may be found at any position in the
rest
of the
cytoplasm.
The morphology of the particles constituting
these arrays has appeared the same in resting, mi
totic, or degenerated cells. They are illustrated in
cells at metaphase in Figures 6 and 7. Identical
masses have also been seen free in the ascitic fluid
without any attachment
to cells (Fig. 9). They
have been seen within leukocytes, in which case
the presence of other foreign matter within the
cells suggested that the particles had been phago
cytized. The absence of a membrane surrounding
the particulate masses is illustrated in Figures 7,
8, and 9.
The particles are apparently
spherical, since
only circular profiles are observed, and generally
occur in small adjacent groups in hexagonal close
packing. In particularly
thin sections, the indi
vidual particles display a dense core and less
dense shell. Such a section is illustrated in Fig
ure 8. Here the section is thinner than the out
side diameter of the particles, so that some par
ticles are cut through their centers, and some cut
only at their periphery. The distance between the
centers of adjacent particles is 580 ±40 A, while
there is a dense core within each one. In thicker
sections only the dense core and some of the less
dense shell surrounding it are resolved so that the
actual diameters of the particles appear to be
about 400 A, as previously stated (2, 15).
In Figure 7 certain straight tubules with diame
ters of the order of 500-600 A can be distinguished
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Cancer Re8earch
792
running
through
the particulate
arrays.
These
are
believed to be longitudinal
sections of tubules,
since many oval or circular profiles may be seen
with the same diameter whose transparent interi
ors distinguish
them from the denser spheres.
These images, therefore, appear to be cross-sec
tions, and the double lines longitudinal sections of
tubules. Their presence may be related to the
particles or to normal cell components occurring
at the same site. Similar tubular profiles, with
dense particles of macromolecular dimension close
ly associated to their outer wall, appear in all
ascites tumor cells. These correspond
exactly
to the endoplasmic reticulum (12) but are much
more sparsely distributed
in these cells than in
those of the liver. They are always characterized,
in ascites cells, by the macromolecular
particles
on their outer wall, and generally occur singly
without any association to other tubules. Similar
images, without attached
macromolecular
par
tides, are commonly found in the centrosphere of
these cells. In this case they generally occur in
parallel groups of two or more tubules and are
associated with other granules and vesicles of the
centrosphere.
Such structures have been recog
nized in the centrosphere
of many cells,' but an
adequate description of them has not yet been
published.
It should be mentioned here that experiments
have been conducted upon Ehrlich mouse ascites
tumor cells infected with influenza, Newcastle
disease, and Russian encephalitis viruses, in addi
tion to those already mentioned with Bunyamwera
virus, and that the cytoplasmic particles did not
appear as characteristic
of these infections.
DISCUSSION
Cytoplasmic masses of submicroscopic particles
with a characteristic
morphology have thus been
observed in certain specimens of a mouse tumor
under rare, and so far unreproducible,
conditions.
Their identity is unknown, since no similar cellu
lar component or contaminant has been described
previously.
These particles must have been present in all,
or nearly all, the cells of the four specimens where
they were observed to account for their high inci
dence (20—30per cent) in the 1-j@sections of these
specimens. Since they occurred in approximately
the same incidence per cell and in the same large
masses in these specimens, it is extremely unlikely
(although not impossible) that they were present
but in much smaller quantity in the 66 specimens
where they were not detected.
‘G.
E. Palade, personalcommunication.
The morphology of these particles may be con
sidered to be “virus-like―
for several reasons:
a) Many small animal viruses are constant
diameter spheres in purified
dition (9).
or intracellular
con
b) Some viruses have been identified intracellu
larly in close-packed array (3, 5).
c) Some larger viruses (5, 10) have been demon
strated to consist of a dense core and less dense
shell.
If these particles do represent a virus, it may
be (a) a virus carried by the host mouse or (b) a
virus carried by the Ehrlich ascites tumor. There
is little or no information
concerning the first
alternative,
since no electron microscope studies
of mouse viruses in their host cell have yet been
reported. Consideration
of their identity as any
particular virus would, therefore, be purely specu
lative. It is unfortunate
that no other tissues of
the mice harboring the ascites positive for these
particles has been preserved, so that we have no
information concerning the presence or absence
of particles in other tissues. Studies of this tumor
in mice experimentally
infected with various
known
mouse viruses
are under
way. If their corre
lation with a mouse infection is considered, it must
be remembered
that in two cases the particles
were encountered in only one or two mice out of
several in the same cage and that in one case they
appeared in addition to a Bunyamwera infection.
Thus far, the particles have been established as not
due to the experimental viruses (Russian encepha
litis, Newcastle disease, Bunyamwera and influen
za viruses)
commonly
employed
in one
of the
laboratories from which the specimens were ob
tamed.
Concerning the second possibility, experiments
have been under way for some time4 to investigate
the possibility that there may be a filtrable agent
intimately involved in the growth of the Ehrlich
ascites tumor. This possibility is suggested from
the facts that the tumor originated as a mammary
carcinoma (8) and that the particles are so similar
to those already described in cultured mouse
mammary tumor cells (13). They are also similar
to those observed in another tumor of viral etiol
ogy, the Rous chicken sarcoma (4). In all three
tumor types, the particles are cytoplasmic
and
consist of a dense core and less dense shell whose
outer dimensions are in the same size range (50—
60 m@sin the Ehrlich ascites tumor; 130 m@ in the
mouse mammary tumor [13]; 60-80 mp in the
Rous sarcoma [4]). However, morphological simi
larities need have no special significance, since
@C.Friend and C. C. Selby, unpublished data.
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SELBY
et al.—Partwles in Ehrlich
many viruses with radically different activities
share common morphologies.
There are two aspects of the particles in the
Ehrlich tumor cells which make it difficult to con
sider them as etiologic agents : (a) their high mci
dence in some specimens and complete absence in
others, all of which contain cells in every stage of
development;
(b) their presence in comparable
number and morphology in cells varying in condi
tion from mitotic to degenerating.
It is not yet
known
whether
the particles
observed
in the mam
mary carcinoma and Rous sarcoma follow a similar
pattern of incidence, since the only observations
reported have been on cultures of these tumors.
Electron microscope studies of tissue cultures are
limited to the well spread edges of cells at the
periphery of the cultures, so that it is seldom pos
sible to estimate the condition (resting, mitotic,
or degenerating) of the cell or to estimate the per
centage of cells in each culture which contain
particles. Some authors (1) consider that their
failure to repeat the observation of particles in the
Rous sarcoma indicates an extremely low incidence
of cells containing visible particles in a given cell
population.
They argue that this is consistent
with the virus titers obtained from the tumor cells.
However, in the case of the Ehrlich ascites tumor,
the complete absence of particles in most speci
mens and nearly 100 per cent incidence in others
cannot be explained on the basis that only one out
of so many cells would contain virus in visible
form. If they are to be considered as related to the
tumor rather than to anintercurrent
mouse infec
tion, some other explanation must be found for
their irregular appearance.
Otherwise it seems
more obvious to attribute them to an irregularly
appearing mouse infection.
It must be mentioned, however, that, until di
rect evidence is found, it need not be assumed that
these particles represent virus of any sort. Their
morphology is admittedly
suggestive, but by no
means conclusive, evidence for their identity as a
virus.
Their organization
in hexagonal close-packing
need not be considered as evidence of crystallinity,
since it is the form that any number of constant
diameter spheres would take when compressed by
drying
or cellular
forces.
Although
the possibility
that these spheres represent some abnormal and
possibly crystalline form of normal cell compo
nents is extremely unlikely, it cannot be ignored.
However, their presence in mitotic cells argues
against their being a degeneration product, while
their absence in cells prepared at the same time,
in the same manner, and with the same solutions
as those in which they were present, argues against
793
Ascites Tumor
their originating from the action of abnormal solu
tions or treatment upon normal cell components.
The final possibility that must be mentioned and
should be investigated is that these masses are an
intracellular form of contaminating
fungus.
Although it is evident that our knowledge of
normal and tumor cell structure under unusual
conditions and of its relation to micro-organisms
and fungi is still fragmentary
enough that these
particles should not be discounted as a nonviral
component, their identity as a virus is admittedly
the most plausible hypothesis at the present time.
Until a fresh tissue specimen known to contain
these interesting particles is at hand, little more
can be investigated
concerning their incidence,
presence in other mouse tissues, or chemistry. It is
hoped that by bringing these observations to the
attention of other investigators more progress can
be made on the next occasion that they are ob
served.
SUMMARY
1. Striking cytoplasmic
masses of submicro
scopic spheres have been observed to be present in
nearly all cells of four and completely absent in 66
of the Ehrlich mouse ascites tumor specimens
which have been extensively studied.
2. The
individual
particles
consist
of a dense
osmiophilic core surrounded by a shell with an
outer diameter of 580 ±40 A.
3. The particles are exclusively cytoplasmic and
aggregate in groups often in hexagonal close
packing without an enclosing membrane.
4. The particles occur in identical morphology
in resting, mitotic, or degenerating cells and may
occur extracellularly in the ascitic fluid.
5. Smooth-walled tubules with diameters simi
lar to those of the particles are often associated
with them and may be a normal cell component.
6. The identity of these particles is unknown.
Various possibilities which have been considered
include altered cell component, degeneration prod
uct, tumor agent, fungus, or virus, of which the
last is thought to be the most likely.
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W., and OBERLINO,
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Cancer, 40:178—85, 1953.
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8. BUNTING,
H. Virus-likeParticles in a Human Skin Papil
loma.Proc.Soc.Exper.Biol.& Med., 84:827—32,
1953.
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of a section
of 1.@&thickness,
stained with methylene blue, of a specimen of the Ehrlich
mouse ascites tumor showing the unusual cytoplasmic deposits.
The latter are indicated by arrows where they are large
enough to be evident. They are distinguished from normal lipid
droplets by their large size and nonspherical shape. X900.
FIG. 2.—Light
micrograph
of a section
of 1-ti thickness
of a
different specimen of the Ehrlich mouse ascites tumor prepared
at the same time and in an identical manner to that of Figure 1.
No unusual cytoplasmic masses are evident. X900.
FIG. 8.—Phase
objective)
micrograph
of an unstained
(dark
section
M contrast
oil immersion
of a specimen
similar
to
that illustrated in Figure 1. Mitochondria (m) and cytoplasmic
masses (p) are indicated. A nucleolus (n) shows the nucleolem
mar coiled structure common in electron micrographs
(18).
The central cell illustrates the case when the cytoplasmic
masses nearly completely encircle the nucleus without actually
touching the nudear membrane. Taken at X1,455 and enlarged
to X2,910.
FIG.
4.—Phase
micrograph
of the
same
section
as that
illustrated in Figure 1. Lipid droplets (1) and cytoplasmic
masses
ture are
stained
plasmic
(p) are indicated. Mitochondria
and nucleolar struc
not so dearly seen in this stained section as in the un
one shown in FigureS. The great density of the cyto
mass is evident. Taken at X1,455 and enlarged to
X2,91O.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1954 American Association for Cancer Research.
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FIG. 5.—Electron
micrograph
of a section
of O.O5-j.@thick
ness of an Ehrlich ascites cell from a specimen similar to that
of Figures 1, 3, and 4, to demonstrate that the large cytoplasmic
masses are composed of constant-diameter
spheres. An area
where they are in particularly regular array is outlined. Mito
chondria (rn) and lipid droplets (1) are indicated. X15,000.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1954 American Association for Cancer Research.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1954 American Association for Cancer Research.
FIG. 6.—Electron micrograph of a thin section of a cell in
metaphase, showing sections of chromosomes (c) in the upper
half and of arrays of the particles in the lower half of this view.
The particles are best recognized when they are in hexagonal
array (p). A lipid droplet (1) is indicated. X16,600.
FIG. 7.—A higher
magnification
view
of the particles
in a
cell at metaphase.
A section of a doublet chromosome
is
shown on the right (c) with typical hexagonally-packed
groups
of particles on the left (p). X30,000.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1954 American Association for Cancer Research.
4
S..
I
I
6
p
S
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1954 American Association for Cancer Research.
FIG. 8.—Electron micrograph of a section of O.O2—O.O3-@
thickness of the same specimen illustrated in Figures 1 and 3.
The portion included in this highly magnified view is of a cyto
pIaSnIiC
mass.
The
internal
structure
of the constituent
par
ticles is evident in this section, thinner than the outside
diameter of the particles. The variation froln a rigidly hexago
nal arrangement
is evident from the failure of all particles to
lie in exactly straight lines, although it can be shown that
some of the areas are thin sections of hexagonal arrays. Pro
files of tubules in longitudinal
(1) and transverse section (1)
are indicated. Taken at X8,600 and magnified photographi
cally
to X60,000.
FIG. 9.—Electron
the particles.
particularly
micrograph
of an extracellular
Their external
shell and internal
well seen in the outlined
portion.
Photographs
printed
group of
dense core are
X36,000.
by Mr. Peter Menegas.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1954 American Association for Cancer Research.
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Submicroscopic Cytoplasmic Particles Occasionally Found in
the Ehrlich Mouse Ascites Tumor
C. C. Selby, C. E. Grey, S. Lichtenberg, et al.
Cancer Res 1954;14:790-794.
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