Electron Microscopy of Tumor Cells and Tumor Viruses
A Review
W. BERNHARD
(Institut de Recherchessur le Cancer, Vittejuif, Seine, France)
I.
H.
III.
IV.
V.
CONTENTS
Electron microscopy of normal cells
The ultrastructure of cancer cells
1. The resting nucleus
2. Mitosis
3. The cytoplasm
a) Mitochondria
6) The study of basophilia
c) The Golgi apparatus
d) The centriole
e) Various other cytoplasmic structures
/) The ground substance
g) The cell membrane
The study of virus tumors and tumor viruses
1. The Shope fibroma virus
2. The Shope papilloma virus
3. The Rous sarcoma and its presumed
agent
4. Virus-like particles in other avian tumors
5. The myeloblastosis and erythroblastosis
virus
Virus-like particles in normal chicken
tissues
The mammary tumors of mice and the
problem of agent identification
8. Virus-like particles in other neoplastic
tissues of mice
9. The Lücketumor virus
10. Human tumors of known viral etiology
11. Suspect inclusions in human neoplasms
of unknown etiology
Concluding remarks
References
FOREWORD
The successful use of the thin sectioning method
and high resolution electron microscopy in general
cytology and virology developed in the last 5 years
has impressed even those who, fearing artefacts
everywhere, attributed the most limited credit to
these new methods of morphological investigation.
Received for publication February 11, 1958.
A considerable number of cancer institutes have
their laboratories of electron microscopy as units
already fully integrated with the other depart
ments for team work on a common research plan.
The question may arise, however, whether such
laboratories should be enlarged, in view of ex
pected further developments in ultrastructure re
search and their possible contribution to the can
cer problem. On the other hand, investigators
working on a variety of problems, for whose solu
tion they might need the help of an electron
microscopist, are interested to know the possibili
ties and limitations of ultrastructure research in
cancer. This review on the recent achievements in
this field is designed to contribute to their orienta
tion. However, the author is aware of the fact that
such an attempt is likely to be rapidly out of date
and incomplete; numerous questions posed in this
article may soon find an answer in new discoveries
resulting from the many studies which are now
under way.
The application of electron microscopy to the
solution of specific cancer problems (cancer cells
and cancer viruses) was not possible until a better
knowledge of the ultrastructure of cells and viruses
in general had been obtained. Although very far
from being complete, this knowledge is now broad
enough to allow comparative studies of normal
and abnormal structures. Therefore, a summary
of the contributions of electron microscopy to
normal cytology is presented in the first part of
this review. In a second part, the general aspect
of cancer cells as observed with the electron micro
scope is described, and the variations in size, num
ber, or ultrastructure of their cell organdÃ-es are
discussed. In a third part, the results of recent
cellular studies of virus tumors are reported, con
cerning specific lesions due to the presence of
virus particles or their precursors. Finally, an at
tempt is made to outline future research. The
place of the electron microscope, not only in basic
research but also in pathology and as a possible
aid to pathological diagnosis, will be evaluated.
491
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492
Cancer Research
I. ELECTRON MICROSCOPY OF
NORMAL CELLS
Recent valuable summaries on this general topic
have been given by Porter (122), Howatson and
Ham (85, 86), and Oberling (105). Also, the Ar
den House Conference of Tissue Fine Structure
in 1956, edited by Porter (123), and the Stock
holm Conference of Electron Microscopy in 1957,
edited by Sjöstrand and Rhodin (141), should be
consulted. An abundant bibliography can be
found in these papers. A stranger to this field may
rapidly be lost facing an extraordinary diversity
of images representing nuclear and cytoplasmic
structures of many varieties of cells. As far as
the cytoplasm is concerned, the electron micro
scope has demonstrated a considerable structural
complexity where the light microscope a short time
ago revealed nothing because of its low resolution.
The most important discoveries made with the
electron microscope concern, in fact, the cyto
plasm and not the nucleus.
The investigation of nuclear structures has
thus far been rather disappointing. Chromosomes
of interphase nuclei in somatic mammalian cells
are revealed in ultra-thin sections as granular,
structureless masses; even during mitosis they
show far more instructive details when studied in
the light microscope. One should not forget that
the present technic of ultra-thin sectioning allows
only what is practically two-dimensional cytology.
Chromosomes are located in numerous geometrical
planes, and furthermore their sub-units are prob
ably twisted to various degrees in their axis, from
the molecular range up to ranges within the
resolution of the light microscope; consequently,
their image, as seen in sections of only 200-500 A
thickness, can scarcely be expected to reveal a
regular structural pattern (127). The "genes" still
remain to be located and morphologically an
alyzed with the electron microscope. Recent
progress was made in the study of nuclei from
protozoa and invertebrates (102, 130) in which
highly organized structures could be described. Al
most simultaneously with the light microscope
(54), the electron microscope showed the nucleolus
to have largely a filamentous structure (24, 29).
These filaments form a loose network, their ultrastructure very much resembling that of chromo
somes. This organized part of the nucleolus is
often embedded in a more diffuse granular matrix,
perhaps corresponding to what Caspersson called
the "nucleolus-associated chromatin" (32). The
nuclear membrane was revealed to have two
layers, the outer being often in close connection
with the cytoplasmic membrane system. Between
these two sheets, numerous porelike formations
Vol. 18, June, 1958
were found (2, 5, 64, 149), which are believed to
play an important role in the nuclear-cytoplasmic
exchange.
As mentioned, the attention of the electron
microscopist has been drawn to cytoplasmic organelles rather than to nuclear structures. Among
these, the mitochondria have been the subject
of many studies. They have a well defined submicroscopic architecture: the mitochondrial body
is surrounded by a double membrane parti
tioned by "cristae" (112, 140), thus representing a
considerable surface of membranes. With minor
variations, the same structural organization was
found in mitochondria of the whole animal king
dom and even from plants. Therefore, it has be
come very easy to identify immediately on electron
micrographs, without staining procedure, not only
normal mitochondria but also their normal or
pathological derivatives.
The ergastoplasm, called by the Rockefeller
school "endoplasmic reticulum" (114, 115, 120),
was the subject of a rediscovery (23, 43). Known
since the end of the last century, it was thought to
be found mainly in oocytes or glandular cells.
The electron microscope has shown its ubiquity
in almost all cell types, although its quantity
varied considerably. Its ultrastructure was studied
in detail (114, 115). Aside from the membrane
system, a hitherto unknown granular constituent
was described (113) and its relationship to cyto
plasmic basophilia and RNA metabolism demon
strated. For bibliography, see Haguenau (72).
Another important contribution to the knowl
edge of the ultrastructure of cells was the descrip
tion of the Golgi complex (39-41). After 50 years
of controversy about its existence or nonexistence,
its structural organization was defined and its
presence demonstrated without stains in most
animal cells.
The centriole (3, 81, 142, 146) is identifiable as
a short cylinder, the "coat" of which is formed
by approximately nine fine tubules, single or
multiple, resembling closely the basal corpuscles
and the appearance in cross-section of cilia from
mammalian cells or ciliates (57, 132). Spindle
fibers of the achromatic figure were shown to have
a tubular appearance (81, 121). Completely un
known cytoplasmic components were found re
cently in invertebrates and batrachian oocytes,
and in mammalian sperms as well; they were
called "annulate lamellae" (145). Their signifi
cance is still a riddle.
This very brief summary cannot mention the
large number of other ultrastructural elements
recently described and peculiar to different tissues
such as nerve cells, muscles, blood, glands, kidney,
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BERNHARD—Electron Microscopy of Tumor Cells and Viruses
gonads, and so on. It can be considered fortunate
that at the very moment when fractional ultracentrifugation technics are widely used to explore
the biochemistry of the cell, electron microscopy
is ready to give an accurate description of cellular
organdÃ-es whose existence was already known,
but which were not well defined because of the lack
of resolution of the light microscope. Thus, the
biochemist can verify the "purity" of his nuclear,
mitochondrial, and microsomal fractions and subfractions by means of ultra-thin sectioning of his
centrifugation pellets.
II. THE ULTRASTRUCTURE OF
CANCER CELLS
The first temptation met by the electron microscopist working on the cancer problem was, of
course, to demonstrate a structure specific for
the malignant process, therefore allowing an easy
distinction between normal and cancer cells.
In the light of the work which has been done, re
vealing the extraordinary complexity of proto
plasmic structures, this optimism already appears
to be somewhat naïve.However, if on the one
hand the light microscopist, after one century of
intense research, has failed to find a characteristic
morphological feature common for all cancer cells,
on the other hand biological experiments have un
doubtedly proved, by the transmission of cancer
through one cell (63), that the mechanism of
malignancy is located at the cellular level. This
seemed to justify the belief that a high-resolution
microscope might rapidly reveal specific lesions.
Ten years of research, initiated by the very
stimulating paper of Claude, Porter, and Pickels
(34), have brought much disillusionment. Cowdry's statement (38) that cancer cells do not have
any morphological features visible in the light
microscope which can be exclusively demonstrated
in tumors remains true for the electron micros
copist. He faces considerable difficulties, since,
in addition to the great variety of cancer tissues
and cells as shown by the classical methods, he
deals with the varieties of their submicroscopic
architecture.
Nevertheless, it has been possible to recognize
certain tendencies which characterize cancer cells
in general. The most important phenomenon is
dedifferentiation or anaplasia, well known to the
pathologist at the histological and cytological
level; the electron microscopist encounters this
loss of structural organization throughout the
submicroscopic range (15, 106). Anaplasia can be
exaggerated, ending with the complete loss of cell
organdÃ-es.In this case, the cytoplasm is essential
493
ly made up of the ground substance alone. Ana
plasia may also be accompanied by dysplasia,
but one can agree on the whole with Dalton
and Felix's statement (42) that the differences be
tween the ultrastructure of normal and malignant
cells are essentially of a quantitative and not a
qualitative nature.
The more mature the tumor examined, the
more complex is the ultrastructure of its cells, a
fact which could be expected. In those tumor cells
that still function (e.g., mammary tumors secret
ing milk [22], plasmocytomas elaborating pro
teins [30], myxosarcomas synthesizing a myxoid
substance [89]), the products of this activity may
be detected in their cytoplasm together with the
hypertrophy of the cellular organdies involved in
the process. However, no explanation can be given
for cases in which entirely dedifferentiated
cells show hypertrophied Golgi apparatus (e.g.,
some mammary tumors of mice [21]) or when
metastasizing tumor cells are still able to form a
ciliated border with a complex ultrastructure (92).
A description of the different cell organdies as
they appear in various types of cancers illustrates
in detail the polymorphism of their submicroscopic
structures.
1. THE RESTINGNUCLEUS
For the same reasons which apply in the study
of the normal nucleus, mentioned above, the
exploration of the nucleus of cancer cells with the
electron microscope is disappointing and has
added almost nothing to the wide knowledge of
the irregularities of its volume, form, and chromatin structures, accumulated by many careful
light microscopical observations since Virchow.
The abnormalities of shape are perhaps still more
striking on electron micrographs, since the thin
sections show all the deep invaginations of the cell
membrane in the nuclear body, a phenomenon
which is of course not specific for cancer, but where
it is found very frequently (Fig. 1). These ir
regular infoldings of the nuclear surface can
form small canals which are seen to be connected
with the nucleoli (4,131,152). They are, therefore,
likely to play a role in the transport of nucleolar
material into the cytoplasm. The bigger the
nucleus, the more the nuclear membrane is folded,
an observation which suggests an important in
crease of the nuclear surface.
The fine structure of the ckromatin in these
nuclei cannot be distinguished from the chromatin
threads and clumps of normal cells. It has granu
lar or filamentous components of about 80-150 A
in diameter. Surrounded by clumps of chromatin,
pathological, strongly PAS-positive inclusions of
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Cancer Research
unknown origin have been described in a chicken
sarcoma (61). These zones appear in the electron
microscope as formed by coarse, closely packed
filaments or tubules.
The ultrastructure
of the nucleoli does not seem
to be visibly altered in cancer cells (16). They are,
as is well known, mostly hypertrophie, but the ap
pearance of both their filamentous component and
the diffuse matrix is unchanged. In certain types
of cancer cells, the filamentous network is pre
dominant or even exclusively present, e.g., in the
liver tumor of rats (16), in the Yoshida sarcoma
(152), and in the Shope fibroma (18); in others,
the matrix is more important and may completely
mask the presence of the "nucleolonema"
(16,
151). These differences are linked to special types
of cancers and refer perhaps to a particular
metabolism of these cells; nothing is known of the
cause of this behavior.
The nuclear membrane of cancer cells does not
differ from that of normal cells (74). Its two sheets
are equally present in all tumors, and its thickness
is unchanged. The pore-like formations are also
present, and their size and number per surface
unit do not seem to be altered. However, as the
nuclei of cancer cells have a more lobulated sur
face, these pores are more likely to be cut in
tangential
sections, and therefore they may be
more easily visible there than in most normal cells.
2. MITOSIS
The electron microscope has not yet been able
to demonstrate any ultrastructural
differences be
tween the chromosomes of normal and malignant
cells. As has been pointed out above, the investiga
tion of such possible differences is not facilitated
by the present technic of thin sectioning or of
staining and fixation. Ultrastructure
research has
added nothing to the many well known earlier
observations on chromosomal abnormalities with
regard to number, shape, position during mitosis,
and physicochemical properties.
3. THE CYTOPLASM
a) Mitochondria.—Since these organdÃ-es are the
site of oxidative cellular respiration, they are also
of special interest in view of a frequently discussed
cancer theory (148). Their study with the elec
tron microscope was of particular
importance,
since the accurate observation
of their varia
tions in cancer cells was beyond the limita
tions of the light microscopical technics. The first
question concerned their quantitative
differences,
frequently
discussed
in the earlier literature.
Electron microscopic examinations of many sam
ples of identical or different cancers prove that the
Vol. 18, June,
1958
number of mitochondria not only varies from one
type to another, but also from one specimen to
another of the same type, and even from one
cancer cell to another in the same tissue. Each
electron microscopist may find examples of tumor
cells which have an increased number of mito
chondria (4, 89) (Fig. 3), others which seem to be
normal in number, and others which have fewer
(26, 84, 103) (Fig. 4). But the general impression
is that cancers have fewer mitochondria than normal
cells. This is only a general rule, and it is easy to
find exceptions to it.
The shape of mitochondria in tumor cells does
not seem to be of more importance than in normal
cells. All transitions
between
elongated
and
spherical bodies are found, and their shape prob
ably depends far more on the metabolism of the
cell than on the origin of the tumor.
The size of mitochondria is equally variable and
may reach extremes in tumor cells, from giant
mitochondria
of 4 n in diameter, found in an
Ehrlich ascites tumor (152), to very small granules
of apparently
mitochondrial
nature measuring
less than 200 HIMin diameter (121, 152). All inter
mediate sizes are found. Many tumors have mito
chondria of normal size, but the malignant ceil ap
pears to have a tendency toward small-sized mito
chondria.
The density and inner structure of the chondrioma are more variable in tumor cells than in
most normal cells. As mitochondria
in tumor cells
often appear swollen, the density decreases rapid
ly and the characteristic
double membranes
of
their outer walls and cristae may disappear com
pletely. The swelling of mitochondria,
preceded
by ultrastructural
defects, is very frequently en
countered (26, 129, 131, 134, 151) (Fig. 4). In ap
pearance, it cannot be distinguished
from the
nonspecific tumefaction
of mitochondria
as seen
in various injuries (cloudy swelling [65]). It would
be of great interest to know whether injured and
swollen mitochondria
in cancer cells are linked
with the malignant process and represent a pri
mary lesion, or if these defects are only the result
of the rapid aging and degeneration of these cells,
or of poor nutrition and respiration, very common
in necrobiotic areas of tumor tissues. This prob
lem is not easy to solve; but it would seem at the
present moment that, whereas mitochondrial
de
fects in cancer cells may often be attributed
to
secondary
causes accompanying
the intensive
growth of tumors, the lesions of these cytoplasmic
organdÃ-es may also be more directly linked with
malignancy. The author has observed many tumor
cells which were well preserved in all respects and
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BERNHARD—Electron
Microscopy of Tumor Cells and Viruses
probably very actively growing when being fixed;
the only visible lesion was the swelling of some of
their mitochondria.
An apparently opposite process is also observed
in many tumor cells : tumor mitochondria are not
swollen, but, on the contrary,
may be much
denser and smaller than normal. The smaller they
are, the denser their body. Their "crests" may
thus become scarcely visible or even be absent.
Among these small and dense mitochondria
one
finds all intermediate
aspects between typical
mitochondria
and dense, spherical formations
which have been called "microbodies"—very dense
particles without any visible inner structure and
limitated by a single membrane. They have been
considered as precursors of mitochondria (65,128).
These microbodies can be found in many normal
and pathologically altered cells, and they can also
be reproduced experimentally
(126). Their very
frequent presence in cancer cells may perhaps be
interpreted
as an expression of an intense mitochondrial regeneration, a process stimulated by the
gradual disappearance
of the functionally active
chondrioma.
This is a hypothesis which still re
mains to be corroborated.
However, some facts
reported in the earlier literature of electron micros
copy can be interpreted
in favor of such an ex
planation. Porter's "growth granules" (124, 133)
and Oberling's "ultrachondrioma"
(107, 108), de
scribed mainly in cancer cells but also in rapidly
growing embryonic tissues and in cells of inflam
matory processes, were detected in the cytoplasm
of spread cells before the era of thin-sectioning.
The close relationship between these formations
and mitochondria
was admitted (80, 120). Since
these particles have not been sectioned, their inner
structure is not known, but it seems likely that
"growth granules," "ultrachondrioma,"
and "mi
crobodies" are similar or identical and represent
stages in mitochondrial
regeneration.
The storage of various products in mitochondria
of normal cells was often observed (65, 97, 126).
Occasionally, tumor mitochondria
also store vari
ous substances. This was observed in hepatomas
induced by dimethylaminoazobenzene
in rats and
also in primary human liver cancers (unpublished
observation).
The nature of the dense granular
masses filling the enlarged mitochondrial
body in
these cases has not been determined. On the other
hand, ferritin-hemosiderin
granules can be found
within microbodies in pathological
paraerythroblasts of fowl erythroblastosis
(14). Even viruses
may be observed within some mitochondria
of
this disease (13). Whether the agent as such has
penetrated
incidentally
or whether it has been
495
formed in a matrix, filling previously the mito
chondrial body, is still unknown.
Summarizing all the observations reported on
tumor mitochondria,
one is struck by the extraor
dinary variations in their numbej-, size, form, and
density, and by the frequent lesions they present.
These facts should be taken into account by all
biochemists using ultracentrifugation
methods for
isolation of the mitochondrial
fraction. They will
probably encounter many more difficulties in ob
taining purified material than for normal mito
chondria, since tumor mitochondria not only vary
more in size and density, but also are likely to be
more fragile than normal. Therefore, a critical
morphological
control of such pellets is highly
desirable.
6) The study of basophilia.—Since basophilia is
one of the characteristics
of the cytoplasm
of
cancer cells, it was particularly
important
to
examine the ultrastructural
substrate of this stain
ing property. However, extensive research on the
ergastoplasm
of a large number of normal cells
had to be done first in order to classify the striking
functional variations of these structures and to get
an idea of what can be considered normal or ab
normal.
The observations with the electron microscope
prove again the very great variability of these
structures in cancer cells. All transitions between a
densely packed lamellar ergastoplasm, or "nebenkern" formations encountered in benign (76) or in
malignant
(53, 58, 109) tumors, and cells of ex
tremely anaplastic tumors with complete absence
of such membranes or granules may be encountered
(Figs. 5-7). One may also find many cancer cells
which do not show any quantitative
difference
when compared with homologous normal cells. All
the structural details seem to be identical; but,
in general, malignant cell-s have a tendency to lose
their organized lamellar ergastoplasm, and baso
philia is more often linked to RNA granules only,
which are, as in normal embryonic cells, diffusely
scattered in the cytoplasm or grouped in small
clusters (84, 129) (Fig. 7). No lamellar support of
these granules is then visible. The term "unorgan
ized ergastoplasm" could be proposed to designate
this apparently
more primitive form of cytoplasmic basophilia (85). Cancer cells in the cyto
plasm of which no basophilic structures can be
detected, may correspond to the extreme form of
the B-cells described by Caspersson and Santesson
(33).
The ergastoplasmic
membrane system is fre
quently altered in cancer cells. The elongated
tubules or lamellae can be disrupted to form small
or large spherical vesicles, similar to those pro-
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496
Cancer Research
duced experimentally
(150) or simply by poor
fixation. The ergastoplasmic sacs can also be swol
len and contain a homogeneous, apparently struc
tureless substance of medium density. Such pic
tures have been described in cells from various
tumors (27, 121, 131), but since they can also be
found in cells participating
in inflammatory proc
esses (118), they do not seem to merit particular
attention.
As a conclusion to all these observations,
one
may stress the quantitative
variability of basophilic structure in the cytoplasm of cancer cells
and the increased basophilia, which parallels an
increase of diffusely dispersed RNA granules
rather than an extended lamellar system; the
latter, on the contrary, tends to disappear. Conse
quently, the biochemist will realize that "microsomal fractions"
from cancer cells can be mor
phologically different from normal and be subject
to greater variations in their chemical composi
tion.
c) The Golgi apparatus.—Only 3 years ago,
Cowdry (38) wrote that "the knowledge of the
Golgi apparatus in normal
nant cells, is disappointing."
cells, let alone malig
The situation, as we
have noted, has changed. However, if the Golgi
complex in normal cells is now morphologically
well defined and can also be demonstrated easily in
most cancer cells, no constant ultrastructural
dif
ference between normal and tumor cells has been
described (42,73,85). The Golgi zone may be either
hypertrophied
or reduced in size, but the archi
tecture remains essentially the same: bundles of
smooth double membranes,
microvesicles
(gran
ules), and vacuoles. The vacuolar component is, as
a rule, less developed, and the microvesicles often
predominate
in tumor cells, thus resembling
more the Golgi zone of embryonic cells (42, 85)
(Fig. 2). Its development in a given tumor is more
or less constant. As was well known, certain benign
tumors, induced by continuous hormonal stimula
tion, may show an extreme hypertrophy
of the
Golgi apparatus, easily demonstrable in ultra-thin
sections with the electron microscope (76). Ma
lignant mammary tumors of mice may sometimes
also have a considerably enlarged Golgi apparatus
(21) but, curiously enough, this is not true for the
strain T422 of the mammary adenocarcinoma
of
the rat (131). In a hepatoma of the rat (85), in
the virus-induced
erythroblastic
leukemia
of
fowls (14), and also in some human leukemias (25),
the pathological cells are also characterized by an
abnormally developed centrosphere. In Rous sar
coma cells, this organelle is not much developed
(27, 53), and it is even less visible in ascites cells
of the Ehrlich and Yoshida tumors (152). In an
Vol. 18, June, 1958
anaplastic uterine carcinoma of the rat and in the
Brown-Pearce carcinoma of the rabbit, the Golgi
zone was found to be constantly hypotrophic (per
sonal observation). It can be concluded that neither
the hypertrophy nor the hypotrophy of this organelle
is characteristic for all neoplasms. The case of each
tumor has to be considered separately, and various
aspects may be observed.
An unusual observation has been made on the
Golgi apparatus in mammary tumors from strains
of mice considered to be infected with Bittner
milk factor.
Cytoplasmic,
virus-like
particles
visible in the electron microscope are often found
to be in close connection with the Golgi structures
(21). The inclusions which are formed later on
occupy the cell area ordinarily filled with Golgi
material (see Part III).
d) The centriole.—Thanks to the above-men
tioned hypertrophy
of the Golgi area in certain
tumors, it is particularly easy to demonstrate this
tiny organelle in electron micrographs from such
material
(Fig. 2). Closely associated
with the
Golgi zone during the interphase, its size does not
seem to change with the hyper- or hypotrophy of
this zone. Its ultrastructure
in cancer cells has
not been distinguishable
from the centrioles of
normal cells (81). Nothing is yet known about the
ultrastructure
of "bird's eye" inclusions or "Plimmer bodies," the existence of which was related
to an abnormal growth or multiplication
of the
centriole (see Cowdry [38]).
The position of the centriole, together with the
Golgi zone, determines
the polarity of a cell.
Cancer cells, as is well known, may gradually lose
this polarity on their way to complete anaplasia.
This phenomenon, easily visible under the light
microscope, can be demonstrated
probably with
greater accuracy on low-power electron micro
graphs of certain mammary tumors of mice (per
sonal observation).
e) Various other cytoplaxmic structures.—As al
ready mentioned above, a hitherto unknown cytoplasmic formation has been found in certain nor
mal cells: the "annulate
lamellae"
(145), also
called "membranae
fenestratae"
(131). These
structures have also been seen very frequently in
certain tumor cells, e.g., in a mammary tumor of
the rat (131), in Ehrlich and Yoshida ascites tu
mor cells (152), and in chicken sarcomas.1 They
probably
also exist in other types of cancer.
Their presence may be the expression of a cer
tain degree of dedifferentiation,
or they may have
some relation to rapid cell growth. New facts have
to be reported before any correct interpretation
of their significance in tumor cells can be given.
1M. Friedlaender, personal communication, 1958.
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BERNHARD—Electron
Microscopy of Tumor Cells and Viruses
Tumor cells are still capable of elaborating cilia
with the same ultrastructure as that found in nor
mal organisms. Well known in some benign tu
mors, this is probably an exceptional observation
in malignant cells; nevertheless, it was noted in
the hormone-induced kidney tumor of the hamster
whose cells, even after having metastasized, still
form basal corpuscles of a fragmentary ciliated
border (92).
Cytoplasmic inclusions of various different types
are often visible on electron micrographs. Thanks
to the high resolving power of the electron micro
scope, their nature is more easily defined than
with the light microscope, where many inclusions
of general origin have been interpreted in the past
as "specific" and linked to the presence of para
sites of all kinds (for literature, see Ewing [55],
Cowdry [38], Masson [94], and Hamperl [78]).
As in normal cells, these inclusions are some
times due to the persistence of secretory functions
in cancer cells of glandular origin. Other inclusions
are purely degenerative (fat, complex myelin
figures also being shown for other, nonmalignant
cells [119, 143]). Among this group may be men
tioned a special type of ring or tubule-like struc
ture, appearing in delimited cytoplasmic areas of
a fowl sarcoma.1
Cytoplasmic inclusions may also consist of
debris of phagocytosed bodies (erythrocytes [4,
152]), observed in different tumors. Finally, cyto
plasmic inclusions of viral origin are frequent in
some virus tumors. They are described in the next
section.
/) The ground substance.—No special feature is
visible in the hyaloplasm of osmic acid-fixed
cancer cells. It looks homogeneous, dense, with
areas in which a texture of very thin fibrils
may sometimes be discovered. Similar fibrils of
30-80 A in diameter are also ranged parallel to
one another and, united in bundles, form limited
regions in human leukemic cells (28), in avian
sarcoma cells (129), and in the Yoshida ascites
tumor cells (152). These characteristics may repre
sent a degenerative process of the ground sub
stance; however, no explanation for their exact
significance can be given.
g) The cell membrane.—Its thickness, measur
ing approximately 70-100 A, and its appearance do
not differ from normal cell membrane. Like homol
ogous normal cells, the free surface of cancer cells
(mainly of epithelial, but also of mesenchymal,
origin) may form cytoplasm protrusions, "microvilli." These formations can either be shorter and
less numerous or, on the contrary, much longer
than normal (131). Similar digitations also exist
between some cells of various malignant epithelio-
497
mas where they form a closely interdigitated con
tact line between one another. It will be interesting
to learn whether these protrusions regress in more
dedifferentiated tumors and whether the strangely
reduced coherence of cancer tissues (35) may be
explained by their gradual disappearance.
The desmosomal connections found between
normal epithelial cells are also observed in epithe
lial tumors, either well developed or partially dis
rupted as shown in a human cervix carcinoma
(147).
III. THE STUDY OF VIRUS TUMORS
AND TUMOR VIRUSES
The absence of specific lesions in the abovedescribed cancer cells is, of course, disappointing.
Tumors of known viral origin, however, offer the
possibility of detecting the causative agent. From
the very beginning of these investigations, Claude,
Porter, and Pickels" work on the Rous sarcoma
(34) opened the way for all future studies in this
field. Since then, virus tumors appeared to repre
sent ideal objects for the electron microscopist.
Besides their being interesting from the point of
view of general and tumor virology, they offer to
the morphologist possibilities of research some
what comparable to the radioactive tracer technic
in biochemistry. The entry of such particles into
the cells can be followed, their place and method
of multiplication studied, and their relationship to
cell organdÃ-esdetected. Therefore, a better knowl
edge of the mode of action of the most rapidly ef
fective carcinogens might have a more general sig
nificance in carcinogenesis. The identification of
viruses in cancer cells has become possible, how
ever, only since technical procedures were fur
ther improved—in particular, since thin-section
ing of typical and well known classical viruses
has given a first solid basis for their comparison
(C. Morgan et al. [98-101], Melnick et al. [67, 96];
for further literature, see R. Williams [153]).
Among the virus-like particles found in virus
tumor cells, few have been identified as specific
tumor agents; others were only supposed to be
related to tumor growth, but no experimental
proof could then be given. Finally, an increasing
number of other particles were claimed to be
present in various cancers which are not known to
be of viral origin. These particles more or less re
semble viruses, but they could not be proved to be
infectious agents and to have anything to do with
the disease itself. Therefore, one has to be extreme
ly careful about the interpretation of such findings
and, consequently, about the use of terms. The
morphologist's task is to describe what he sees
and to be as critical as possible in the choice of
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498
Cancer Research
Vol. 18, June, 1958
micrographs for illustrating his articles to avoid
controversies about terminology and meaning of
suspected particles. The rather harmless word
"virus-like" particles is now often used in the
literature and means that, at the present stage of
our knowledge, the investigator has not been able
to take a clear-cut position.
which of the different elements represents the
active virus carrying the infection from one cell
to another.
Interesting attempts to time the evolution of
the agent within the cytoplasm have recently
been made with tissue cultures (37, 59). In normal
fibroblasta from rabbit testis infected with the
Shope virus, the first lesions appear 4 hours after
1. THE SHOPEFIBROMAVIRUS
infection as a small area of diffuse, strongly
Benign in adult rabbits, malignant in newborn Feulgen-positive viroplasm which undergoes fur
or cortisone-treated animals (50, 79), these tu
ther growth and partial changes in staining af
mors are relatively easy to study with the electron finity. Feulgen-positive zones are gradually trans
microscope.
formed into pyronine- positi ve areas. The diffuse
Important structural modifications of the cyto
viroplasm is probably the morphological expres
plasm, owing to the presence of the large fibroma sion of a soluble antigen which can be recovered
virus, have been found in all the tumors (18, from the supernatant (37).
91). Many cells show various kinds of lesions,
The first virus corpuscles with a single mem
which can be classified roughly as three distinct brane appear between the 8th and the 10th hour
types of patterns, corresponding to subsequent
after infection. After 24 hours, hundreds of such
phases of the development of the virus: (a) In the bodies have been formed. Later, double-membrane
paranuclear cytoplasm of a variable number of "doughnuts" appear, and, after several days of
tumor cells, homogeneous zones containing a fine culture, crystal-like structures may be found.
granular mass appear. Such regions ("viroplasm")
Finally, other peculiar bodies may appear in old,
are more or less sharply defined and correspond
infected cells, of mitochondrial size, but with an
to the Feulgen-positive inclusion bodies visible interior formed by densely packed concentric
in similar areas in the light microscope (36) membranes giving an onion-like appearance.
(Fig. 8). They often contain crystal-like ele
No differences have been observed between the
ments composed of elongated, parallel lamellae viruses of benign fibroma and malignant sarcoma
about 70 A thick, (o) Within the viroplasm cells. Furthermore, micrographs of the Shope
spherical corpuscles appear, about 220 mju in fibroma virus are very similar, if not identical, to
diameter, bounded by a single membrane and those of the pox group previously described (67,
containing the same granular material also visible 99). This proves that the fibroma virus belongs to
around them (Fig. 9). Some of the corpuscles may the same family, as has been suggested before
remain empty, others become denser and form a (60). The striking resemblance of cytoplasmic
core. These virus bodies seem to develop progres
lesions, as found in cells infected with the vaccinia,
sively. At the beginning, segments of spheres ap
ectromelia and molluscum contagiosum virus (49,
pear in contact with condensed clumps of the 68, 99), to those described in fibromas of the rabbit
granular mass, outlining the future elementary
shows the limitations of present ultrastructure re
body. They grow to include a separate portion of search. Although the appearance of these intrathe viroplasm. Empty bodies are supposed to be cellular viral inclusions does not, so far, appear to
abortive forms of the virus whose membranes be essentially different from those in tumor cells,
were closed before the granular mass could be in the biological behavior of the various agents from
cluded. In more advanced stages, the viroplasm the pox group is completely dissimilar.
disappears almost completely, and what appears
then to be the "inclusion body" is entirely com
2. THE SHOPEPAPILLOMAVIRUS
No study on thin-sectioned papilloma tissue has
posed of hundreds of elementary bodies, each en
closed by a single membrane, (c) In a still further yet been published, but electron microscopic in
advanced stage, the membranes of the virus vestigations on this subject are under way in
particles become double.
various laboratories, and results are eagerly
All these aspects may be found separately, in awaited. The causative agent has already been
shown in shadow-cast preparations from highly
different cells, or coexisting within the same cyto
plasm. Thus, the electron microscope has demon
purified ultracentrifuged tumor extracts (87, 138,
strated that the inclusion bodies formed by this 139). The average size of the spherical particles
virus and visible in the light microscope are not was found to be about 44 imi in diameter, but
uniform, but many have a variable ultramorpholnothing could then be said about their inner struc
ogy. It is difficult, at the present time, to say ture and their relationship to the tumor cells.
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BERNHARD—Electron Microscopy of Tumor Cells and Viruses
3. THE Rons SARCOMA
ANDITS PRESUMEDAGENT
Claude, Porter, and Pickels' paper (34) on the
ultra-structure of cultured Rous sarcoma cells was
not verified immediately. The results were con
firmed only in 1953 (20), and quantitative work
combined with a bio-assay suggested the identity
of the virus-like particles present in the cells with
the causative tumor agent (51). Thin sections of
this tumor revealed the presence of particles with
a well defined inner structure, found both intraand extracellularly (66). This study was then con
firmed on a large scale (27). Among 75 tumors,
about two-thirds were found to reveal the same
particles, of 70-80 rn.fi in diameter, characterized
by the presence of a central core of 30-40 m/x, sur
rounded by two concentric membranes (110) (Fig.
14). A virus structure differing from this has also
been proposed but is believed to be based on obser
vation of altered particles (52).
These elements seem to be formed within the
cytoplasm, either in vacuoles of unknown origin
or in particular areas characterized by a diffuse
granular material, representing perhaps a "viroplasm." However, these lesions were found rarely
(27), and further data will have to be assembled
before the life cycle of this virus is understood.
Identical, virus-like corpuscles have also been
shown in thin sections of centrifugation pellets
from very active tumor extracts (27). More recent
ly, a micrograph of a much further purified
preparation was published (77); there exists a
striking relationship between the number of
virus particles present in sectioned pellets and
the virulence of the same material in bio-assays
(75). Thus, the identification of the bodies visible
in the electron microscope with the Rous virus
seems to have been established.
499
guished from the viruses associated with the Rous
sarcoma (Fig. 15). Their occurrence in the tumor is
the same, and their presence either in cytoplasmic
vacuoles or on the cell membrane is as character
istic as for the Rous tumor. No cytoplasmic
lesions comparable to the presumed "viroplasm"
of the latter sarcoma have been seen.
Finally, virus-like particles which also are in
all respects similar to these corpuscles have been
found in the spleen and bone marrow of chickens
presenting the typical symptoms of neurolymphomatosis (personal observation) and probably
also of visceral lymphomatosis, though the ultrastructure of the presumed virus particles is not
clearly visible on the published pictures (46, 47).
In a previous paper (137), particles similar to those
found in chicken leukemia were already shown on
shadow-cast preparations from plasma of fowls
with lymphomatosis.
5. THE MYELOBLASTOSIS
AND
ERYTHROBLASTOSIS
VIRUS
The purification of the myelo- and erythroblastosis viruses from the plasma of leukemic
chickens by Beard et al. (IO, 136) was an impor
tant achievement in tumor virus research. The
centrifuged pellets obtained from this material
were prepared by Sharp's method, and electron
micrographs revealed that these virus preparations
were approaching a monodisperse phase of
spherical particles of about 120 m/x in diameter.
It was furthermore shown that a correlation exists
between the quantity of particles and infectivity.
The authors concluded that these particles could
be identified as the agent of that disease. This
method, necessitating the drying of the viruses on
a formvar membrane and shadow-casting, reveals
only opaque bodies without inner structure. When
4. VIRUS-LIKEPARTICLESIN OTHER
thin sections of such pellets were made, it was easy
AVIANTUMORS
to show that all these particles measuring about
Similar corpuscles have also been found in thin 80 mju in diameter were morphologically indis
sections of tumor cells from the Murray-Begg en- tinguishable from those present in the Rous sar
dothelioma (129). The particles were present in coma and other fowl tumors (19) (Fig. 18). The
all these tumors, but they were not very numerous difference in size of the agents is certainly due to
differences in the technic used in the previous
and were found to be outside the cells only, adher
ing to the cell membrane. Their diameter meas- studies. It, therefore, becomes evident that all
sured 110 m^, on the average, so that the virus chicken tumor viruses belong to the same family
corpuscles seemed to be bigger than in the Rous when classified by morphological criteria. The
sarcoma. Whether, however, this difference in size plasma from animals with myeloblastosis as well as
erythroblastosis contains the same bodies, but,
is constant or due to differences in magnification
calibrations of the electron microscope has yet to whereas the pellets from myeloblastosis may con
be elucidated. The inner structure of these agents, sist almost exclusively of virus particles, the
preparations from erythroblastosis are more often
at any rate, is absolutely identical.
Similar bodies have been observed in the Fuji- contaminated with disintegrated cell structures,
and viruses are less numerous. The identification
nami myxosarcoma (93). They measure approxi
of the erythroblastosis virus in the tissues is thus
mately 80 m¿Ãin diameter and cannot be distin
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500
Cancer Research
facilitated. The same particles found in the plasma
were previously shown in spleen and bone marrow
cells of leukemic chicks (13). It is an interesting
fact that the agent is very rarely seen in the ab
normal erythroblasts of the peripheral blood but
mainly in the undifferentiated reticular cells,
either in vacuoles within the cytoplasm or in the
intercellular spaces (Figs. 16, 17). It may also be
present in the interior of some mitochondria.
6. VIRUS-LIKEPARTICLESIN NORMAL
CHICKENTISSUES
The problem of the specificity of the abovedescribed virus-like bodies, present in neoplastia
tissues, becomes more embarrassing if one con
siders the fact that similar particles have also been
found in normal controls. They appear generally
in very low numbers, but they have probably
been seen in considerable quantities in three out
of 150 different cultures of normal chick embryo
tissue (68). An occasional contaminant may have
produced these findings, and it was also sug
gested that the lymphomatosis virus was present
in these cells. Later on, such particles have been
seen repeatedly in thin sections of normal tissue
(129), and, recently, a systematic study by Bene
detti of the spleen and bone marrow from numer
ous normal chickens and chick embryos (11) has
proved that the same virus-like particles may in
deed be present in a low percentage (10 per cent)
of the examined specimens (Fig. 19). They have
even been found in the embryos, which proves
that such agents can be transmitted by the "nor
mal" egg (12). It appears that in certain races the
corpuscles occur more frequently than in others,
where they are either very rare or absent. The sig
nificance of these findings is not yet clear, but it is
assumed that the viruses are present in an inactive
form, either as a nonspecific saprophytic agent
showing the same ultrastructure as the oncogenic
viruses or as a latent form of the lymphomatosis
virus. It is well known that this disease is extreme
ly widespread in chicks, without necessarily giv
ing clinical symptoms. Its transmission through
the eggs has been proved biologically (31). One
can conclude that in neoplastic and normal tissues
of chicken, virus-like particles of similar morpho
logical appearance have been shown with the
electron microscope. The fact that, on the one
hand, millions of identical bodies are present in the
purified and highly active plasma of leukemic
chickens (19) and that, on the other hand, a good
correlation exists between their number in tumor
extracts of the Rous sarcoma and the infectivity
tested in bio-assays (75, 77) proves that they are
capable of playing an important role in carcino-
Vol. 18, June, 1958
genesis. For the electron microscopist they all
have the same morphological feature, and he can
not determine whether or not they are active or
whether they cause this or another disease. He
therefore has to be constantly guided by bio
logical tests.
7. THE MAMMARY
TUMORSOF MICE ANDTHE
PROBLEMOF AGENTIDENTIFICATION
Porter and Thompson's paper (125), dealing
with the presence of virus-like particles in mam
mary tumor cells cultivated in vitro, inaugurated
a long series of studies by various authors which
have not yet permitted the morphological identi
fication of Bittner's milk factor, but which have
brought to light numerous new facts. When the
electron microscopic observations of shadow-cast
preparations from ultracentrifuged milk failed to
elicit useful information about the specific agent
(70, 116), a first attempt was made to identify it
in thin sections (88). Further interesting observa
tions followed (7, 44), and, with rapidly improv
ing technic, more details became available (6, 8,
9, 17, 22, 45, 48, 144). It was established that
in all tumors from mice of strains with high tumor
incidence, two different kinds of virus-like bodies
are present: those that are predominantly intracytoplasmic, measuring about 65-70 m/z in
diameter (Fig. 11); and others, predominantly
extracellular, measuring 105 m/i approximately
(Figs. 12, 13). Both types may, however, be found
within or outside the cell. The intracellular form
(here called type A, to simplify the terminology) is
characterized by a concentric double membrane;
the extracellular corpuscles (called type B) have
an eccentric "nucleoid." Both types are easily dis
tinguishable from casein bodies of the normal milk
(22). Their number is extremely variable, as it
can form opaque inclusion bodies, visible in the
light microscope (72), or be present sporadically,
visible after days of search only in a few cells of
the tumor. Type A is believed to be transformed
into type B according to a well defined mechanism
(6, 22) (Chart 1). The occasional appearance of
particles of type B within cytoplasmic vacuoles
suggests a transformation from type A without
the use of a microvilli system.
Since these particles have been constantly found
in various laboratories of three continents and in
all strains of mice known to carry the milk factor,
and since no other virus-like structure has hitherto
been revealed in the same material, it seems logical
to admit that they are related to the Bittner virus.
However, no comparative biological study based
on the titration of extracts from tumors with a
variable amount of viruses has yet been carried
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BERNHARD—Electron Microscopy oj Tumor Cells and Viruses
501
out. On the other hand, it is known that artificially
induced mammary tumors of mice, believed to be
free of the milk factor, also contain such particles
(9, 22, 45). Though a quantitative difference seems
to exist (22), physiological factors influencing the
secretion of such particles with the milk may play
a role and could explain the divergences between
morphological and biological observations. A fur
ther discussion of this problem is found elsewhere
(21). The virus-like bodies revealed in the mam
mary cancers are, in a latent phase, perhaps, just
as ubiquitous in mice as the viral bodies in
chickens. Further observations on various neo-
diameter (58 mji), aggregated in exact hexagonal
array, were occasionally found in the cytoplasm of
these cells (in four of 68 tumors only). They
consist of a central dense core of about 35 rn.fi in
diameter, which is surrounded by an outer mem
brane. Tubular structures may also be visible be
side these crystal-like inclusions, which seem to be
linked with the evolution of the virus. A compa
rable observation was reported later (152), made in
only one out of 32 Ehrlich tumors examined. The
particle size and fine structure were apparently
identical, but no geometric arrangement of the
bodies and no tubular elements were found. Their
CHART1.—Presumed mechanism of transformation of intracellular A particles to extracellular B particles in mam-
mary tumors of mice. Passage through a cell membrane
with microvilli; dense casein particles on the right.
plasms of mice should rapidly contribute
pate our ignorance
in this field.
occasional presence in this tumor leads to the con
clusion that the particles represent a nonspecific
viral contaminant and have nothing to do with the
etiology of this cancer.
Another form of particles, similar to the A-type
of the mammary tumors, was also found in the
Ehrlich ascites cells. First described as probably
linked with the infection of Anopheles A virus
(62), they were later also encountered in control
cells (61). These bodies are not arranged in clusters
but appear more or less scattered, close to the
Golgi zone; they are found within the "endoplasmic reticulum." The particles measure about
60 m/i in diameter and are bound by a double
membrane. No differentiated core can be distin
guished.
These observations have been confirmed on a
to dissi
A particular observation reported since 1955
(17) and recently confirmed on a wider scale (21)
has already been mentioned above. The A par
ticles have a close topographical relationship with
the Golgi zone. Inclusion bodies appear in close
contact with it and may finally occupy the whole
area at the expense of the Golgi material. Instead
of milk, the cell then would secrete the patho
logical bodies (Chart 2).
8. VIRUS-LIKEPARTICLESIN OTHER
NEOPLASTICTISSUESOF MICE
An interesting finding in Ehrlich ascites tumor
cells is reported by Selby et al. (134,135). Clusters
of spherical, virus-like particles of fairly constant
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502
Cancer Research
large scale and on various strains of Ehrlich
ascites cells (1); identical bodies have also been
found in the cytoplasm of the melanoma S91 (42),
in Friend's dedifferentiated acute leukemia, trans
missible by filtrate (82), and in cells from a
granulocytic leukemia of mice, transplantable in
an ascitic form (46). Cells of another experi
mentally produced myeloblastic leukemia (SOF16)
also contain particles of 60-70 m/¿in diameter,
which can probably be classified in this group, but
whose structural pattern has still to be shown in
detail (83).
Vol. 18, June, 1958
plasm, or in the intercellular spaces. These bodies
seem to evolve according to a complex and yet
poorly understood mechanism. The complete
particles, which may be considered as "mature,"
are spherical, of uniform size (90-100 m/u), and
surrounded by a thick capsule which includes an
eccentrically placed dense core 35-40 m/< in
diameter. They are mainly present in the cyto
plasm but may also be seen in the nucleus and in
the extracellular spaces. Besides these elements,
others considered as incomplete forms are found in
the karyoplasm. They are hollow spheres sur-
CHABT2.—Golgizones of two mammary tumor cells, in which A particles (thick membranes) are found to be closely asso
ciated with Golgi vesicles.
Particles supposed to be viruses are found in
electron micrographs of cervical lymph nodes and
thymus glands of AKR and G58 mice, with
spontaneous lymphoid leukemia, and also in the
Gross C3H strain with induced lymphoid leu
kemia (46). The size of such bodies is said to vary
from 90 to 180 m^. They show a dense core; their
morphological classification is not yet possible. A
shadow-cast preparation from a filtered extract of
this leukemia showing "doughnuts" of 30-60 mjt
in diameter suggests the existence of a concentrical
double membrane in the suspect particles (70).
9. THE LÃœCKE
TUMORVIRUS
A very careful study has been carried out on the
renal adenocarcinoma of the Leopard frog (56).
A third of all the tumors examined contained
virus-like bodies, either in the nucleus, the cyto-
rounded by a thin membrane which seem to
arise in a finely granular matrix. Some of these
particles also contain a dense inner body. Such
lesions probably correspond to the nuclear inclu
sion bodies seen in the light microscope. Further
more, bundles of thick and very osmiophilic fila
ments, as well as vacuolar inclusions, have been
encountered in the cytoplasm. Their significance is
as yet unknown (Chart 8).
Although the author's interpretation is very
cautious and although no bio-assay study has
been carried out, it seems highly probable that
the particles visible in the electron microscope
represent, indeed, the causative agent of this
cancer. The fact that they were not seen in twothirds of the cases examined is, of course, no argu
ment against this assumption.
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BERNHARD—Electron Microscopy of Tumor Cells and Viruses
10. HUMANTUMORSOF
VIRALETIOLOGY
This chapter concerns only small and benign
tumors: moUuscum contagiosum, laryngeal papüloma common warts, and venereal warts (condylomas). The latter have not yet been examined
with the electron microscope, and no recent study
on the virus of the common warts has been pub
lished. A previous work, however, based on ultracentrifugation and shadow-casting technic (96),
has led to the isolation of the verrucae virus. In the
centrifuged sediment of the ground cells, pseudocrystalline clusters have been seen, formed by
NUCLEAR
503
vaccinia (99) and fibroma virus (18) is striking.
An observation of virus-like inclusions in the cyto
plasm of two cases of the human laryngeal papilloma (95) has still to be confirmed on a larger scale
and with more structural details before the iden
tification of the virus will be possible.
11. SUSPECTINCLUSIONSIN HUMANNEO
PLASMSOF UNKNOWNETIOLOGY
Up to now it has not been possible to demon
strate the presence of virus particles in human
malignant tumors; thus, the main postulate of the
virus theory of cancer (104, 111) has not yet been
CYTOPLASMIC
H
EXTRACELLULAR
CHART3.—Diagram showing nuclear, cytoplasmic, and extracellular virus-like particles and unknown struetures'associated
with theJLuckeJtumor cells (courtesy of Dr. Fawcett).
many'spherical particles, 50-59 m/* in diameter;
when scattered as single bodies, they have a diam
eter of 68 m/i. Since these preparations have not
been sectioned, no information is yet available on
the inner structure of the viruses and on their
behavior within the intact cells.
MoUuscum contagiosum lesions have also been
studied both by ultracentrifugation and thin sec
tioning (67, 96). When the virus is isolated, it
appears as brick-shaped and, after treatment with
pepsin, shows a central "nucleoid" like other
viruses of the pox group (117). In thin section, the
cytoplasmic inclusions show many elementary
bodies identifiable with the virus itself (220 m/i in
diameter). New investigations have shown further
details of the evolution and ultrastructure of this
agent (49) (Fig. 10). Its resemblance to the
confirmed. Three recent papers deal with this sub
ject, but none proves more than the existence of
structures in tumor cells which might be related to
viruses. In benign and malignant rectal polyps,
Feulgen-positive cytoplasmic inclusions are de
scribed which do not seem to be nuclear frag
ments (90). The electron microscope shows well
defined areas in the cytoplasm of a structure dif
ferent from the karyoplasm. These inclusions
might be viroplasm, but no virus-like particles
have been detected within them. On the other
hand, a very malignant human myxosarcoma con
tained hitherto unknown cytoplasmic inclusions of
a mixed fibrillar and granular ultrastructure (89).
Their histochemical nature could not be clearly
defined, but it was concluded that their presence
must be related to the abundant production of
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504
Cancer Research
mucopolysaccharides
in this tumor. Finally,
particles of about 100 m/x in diameter have been
found in lymph nodes in a case of acute lymphatic
leukemia (46). Since the ultrastructure of these
bodies is not yet well defined, it is not clear
whether they are related to particles such as seen
in tumors of a known viral origin.
IV. CONCLUDING REMARKS
If one considers electron microscopy as the
principal tool of a science which should bridge the
gap between morphology and biochemistry, it is
evident that there remains much work for all
those already involved or only beginning in this
field. In particular, cancer research needs ultrastructure research not only to acquire a much
better knowledge of the architecture of the normal
cell, but also to build up a pathological anatomy
of tumors and tumor cells at the submicroscopic
level. Recent years have already brought to light
a considerable number of new facts, but we may
be sure that this is only the beginning of a further
important development in morphology. If cells
from various dedifferentiated cancers have a
rather uniform appearance on electron micro
13
15
Vol. 18, June, 1958
graphs and thus are hardly distinguishable from
one another, it is also a striking feature of many
tumor cells that they present a great variety of
ultrastructural patterns. Whether the electron
microscope will be used in the near future for the
diagnosis of neoplastic tissues is rather doubtful.
However, its use may already be very interesting
in special cases of tumor diagnosis when the light
microscope cannot clearly separate cancers of
similar appearance but of varied origin. Thus, a
systematic morphological description of all experi
mental and human tumors, regardless of their
etiology, is very desirable. Nothing is yet known
about the submicroscopic alterations in precancerous lesions. Here again, the possible clinical ap
plication of the electron microscope depends en
tirely on systematic studies of this problem which
have to be carried out first. The bridge will be
crossed when we arrive.
The biochemist, when analyzing centrifuged
fractions from cancer cells, should notice that the
variations in the composition of cancer cells are
considerably greater than in their homologous
normal cells. It appears most important to analyze
accurately the ultrastructure of a given tumor be-
19
O 2
CHABT4.—Diagram representing the main types of viruses
or virus-like particles hitherto shown in tumor cells. 1-5,
pox group: Shope fibroma and Molluscum contagiosum virus;
1-2, early stages of their development; 6-12, particles found
in mouse tumors; 6, in Ehrlich ascites cells (Selby, Wessel);
7-10, A—particles in mammary tumors; 10, found in Ehrlich
ascites and in leukemia cells as well; 11-12, B—particles in
mammary tumors; 13-18, particles shown by Fawcett in
the frog kidney tumor; 19, chicken "virus," as shown in
all chicken sarcomas, leukemias, lymphomatosis, and also
in some normal tissues.
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BERNHARD—Electron
Microscopy of Tumor Cells and Viruses
fore using it for chemical studies. The cell fraction
should be concurrently submitted to morpho
logical checking with the electron microscope, if
precise and long-lasting dosages are intended to be
made on homogeneous pure pellets.
The search for viruses in tumor cells has already
led to very encouraging results as far as tumors of
known viral etiology are concerned (Chart 4).
Complex virus structures and unexpected morpho
logical relations existing with normal cell organelles have been discovered; but the life cycle
of such particles has been only partially revealed,
and many questions are still open.
In the present situation, electron microscopists
may feel like Ulysses and his companions between
Scylla and Charybdis. On the one hand, some are
tempted to find viruses everywhere in tumors and
to claim their discovery before the ultrastructure
of such bodies is shown to resemble that of known
viruses. They should, furthermore, remember the
number of bacteria isolated from cancers in the
past and believed to be directly related to the
disease. Other investigators, on the contrary, do
not pay any attention to the presence of virus
particles in tumors. They label them, perhaps too
quickly, as secondary factors of only little interest.
Virus tumors offer a unique possibility for ultrastructural research and give an interesting basis
for future studies on human cancers. But, what
ever the working hypothesis of the cancer sci
entist may be, if he wants to carry out investiga
tions in which cell structures are involved, he will
sooner or later have to deal with electron micros
copy.
ACKNOWLEDGMENTS
The author is very much indebted to Dr. Marlene Friedlaender and Dr. Robert Dourmashkin for their help in the
correction of the English text.
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FIG. 1.—Portion of a nucleus in a Yoshida sarcoma cell.
The arrows (—>)indicate cross-sections of deep invaginations
of the nuclear membrane (MN), as they are frequently en
countered in cancer cells. Magnification X 26,000.
FIG. 2.—Hypertrophie microvesicular type of a Golgi zone
as seen in a mammary tumor of the mouse; c, centriole.
123.
Magnification X40.000.
FIG. 3.—Increased number of dense mitochondria in a
human myxosarcoma. Magnification X26.000.
FIG. 4.—Swollenmitochondria in a Ehrlich ascites tumor
cell. N, nucleus. Magnification X 26,000.
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.Jpj
f-.f¿ w¿ 'V
L--¿*
*
•
*j i
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Examples of cytoplasmic deditferentiation in cancer cells.
FIG. 5.—Portion of the cytoplasm of a Rous sarcoma
cell. Lamellar or canalicular ergastoplasm well developed.
Mag. X5a,000.
FIG. 6.—Cellof a Brown-Pearce carcinoma. Ergastoplasm
less developed, visible as numerous small vesicles. Mag.
X ¿6,000.
FIG. 7.—Ehrlich ascites tumor l'eli. Cytoplasmic basophilia
almost exclusively represented through RNA granules diffusely
scattered in the ground substance. N, Nucleus. Mag.
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FIGS.8, 9.—Shopefibroma virus of the rabbit.
FIG. 8.—Inclusion body in the cytoplasm characterized
by a dense diffuse mass (viroplasm). Early stage of develop
ment. Mag. X50,(M)0.
FIG. 9.—Appearance of the virus bodies within the viro
plasm. Mag. X 40,000.
FIG. 10.—Molluscum contagiosum virus from the human
skin. Two elementary bodies, the one with a "nucleoid"
as shown with high magnification (X 155,000). Micrograph
from Dr. Dourmaskin.
FIGS. 11-13.—Spontaneous mammary tumor of the mouse.
FIG. 11.—Group of intracellular virus-like "A"-particles
as found in many cancer cells. Mag. X 80,000.
FIG. 12.—Extracellular "B"-particles of the same tumors.
Mag. X 42,000.
FIG. 13.—High magnification (X 150,000) of same extra
cellular particles, showing their inner structure.
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FIGS. 14-19.—Virus-like particles associated with nrnlignant or normal chicken tissues.
FIG. 14.—Particles shown in the Rous sarcoma. Map.
X 58,000.
FIG. 15.—Highmagnification (X 160,000), showing typical
ultrastructure of similar particles found in the Fujinaini
sarcoma.
FIG. 16.—Particles found in crytlirohlastosis. Extracellular
position. Mag. X34.000. (Micrograph from Dr. Benedetti).
FIG. 17.—Krythroblastosis. Some particles in a cytoplasmic
inclusion. Mag. X85,000.
FIG. 18.—Purifiedcentrifugaron pellet of the myeloblastosis virus (specimen from Dr. Beard). Morphologically identi
cal particles in the highly infective plasma. Mag. X58,000.
FIG. 19.—Afew similar particles found in a normal chick
einliryo (micrograph from Dr. Benedetti). A",Nucleus; m, mi
tochondrion. Mag. X50,000.
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O.Ã-S'LL-
15
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BERNHARD—Electron
Microscopy of Tumor Cells and Viruses
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Electron Microscopy of Tumor Cells and Tumor Viruses: A
Review
W. Bernhard
Cancer Res 1958;18:491-509.
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