A Comparative Electron Microscopic Study on Ehrlich Ascites Cancerous

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A Comparative Electron Microscopic Study on Ehrlich Ascites Tumor Cells, Yoshida Sarcoma Cells, and Human Cancerous Peritonitis Ascites Cells

G. YASUZUMIANDR. SUGIHARA

(Laboratory for Electron Microscope Research, Department of Anatomy, Nara Medical College,'Nara Pref., Japan)

It is known that the Ehrlich ascites tumor gave no growth. An operation was performed is of epithelial origin, while the Yoshida sarcoma at the hospital, revealing a tumor the size of originates from the reticuloendothelium (13). The a hen-egg in the pyloric region. The pancreas present paper deals with a comparative electron and retroperitoneal regions showed diffuse meta- microscopic study of uninfected Ehrlich mouse static inflammation which made gastrectomy im ascites tumor (EAT) cells, Yoshida sarcoma (YS) possible. cells, and human cancerous peritonitis ascites Approximately 0.5 ml. of ascites fluid was re (HCPA) cells. A morphological analysis of the moved by capillary pipette from the peritoneal EAT by electron microscopy had already revealed cavity and placed immediately in 1 per cent virus-like particles in the ground substance of osmium tetroxide buffered at pH 7.3 with veronal- the cytoplasm (5, 6, 8, 9, 12). Recent progress acetate. After fixation for 30 minutes the cells achieved via the electron microscope in the study were directly dehydrated, without being washed of the submicroscopic structure of the uninfected in distilled water, in a series of increasing concen EAT cells renewed interest in the virus-like par trations of ethyl alcohol and embedded in a mix ticles associated with the endoplasmic reticulum ture of methyl methacrylate and n-butyl methac- (1, 3, 11). Although Wessel and Bernhard (8) rylate. Polymerization was carried out at 48Â°C. have recently demonstrated a fibrous structure Sections were cut on a Shimadzu microtome with in the YS cells, they have stated that definite glass knives. They were then mounted on collodion discrimination between EAT and YS by means film-coated copper specimen grids and examined, of electron microscopy is impossible. without removal of the embedding plastic, in During the course of a series of electron micro an Akashi electron microscope model TRS-50. scope studies on the ascites cells mentioned above, carried out in this laboratory, an obvious differ RESULTS ence was observed between them: virus-like par Uninfected Ehrlich mouse ascites tumor cells.â€” ticles appeared primarily in the endoplasmic retic The cytoplasmic structure is individually variable, ulum but occasionally in nuclei of EAT cells; but it appears that the mitochondria enlarged fibrous elements, as well as spindle-like bodies to vacuolization, that the rough-surfaced endo of varying size, appeared in the ground substance plasmic reticulum (4) decreased, and that the of the cytoplasm of YS cells; division into endo- cytoplasmic dense particles characteristic to the and ectoplasm occurred in HCPA cells. EAT increase, as inoculation age proceeds. The rough-surfaced endoplasmic reticulum is MATERIALS AND METHODS not particularly well developed, but an even great The ascites fluids of 7-10 days' inoculation age er number of minute granules are evenly distrib of EAT, 4-5 days' inoculation age of YS, and of uted through the cytoplasm, giving it a finely HCPA were used in the present study. The patient stippled appearance (Figs. 1-3). Mitochondria oc was a 36-year-old housewife who had received cur in all regions of the cell but are particularly treatment following a diagnosis of cancerous peri numerous at the periphery of the cell center. tonitis at Nara Medical College Hospital. Para They tend to be larger than usual and finally centesis was performed, with removal of 5000 undergo vacuolization, forming a ring structure cc. of bloody fluid, with specific gravity of 1.010; (Figs. 1-3). The Golgi complex has the same there was a positive Rivalta reaction; culture basic structure as that of other epithelial cells, Received for publication April 24, 1958. being made up of aggregations of small vesicles 1167

and parallel arrays of smooth membranes or occasionally found within the vesicles. To date ganized into two or more groups that are found it has been possible to assemble from the electron closely attached to a lipide droplet (Fig. 1.) micrographs a sequence of formation stages which The nucleus is enveloped in the double- would possibly be a clue to the origin of these structured nuclear membrane. The nucleus is often particles. Most of them are less dense and smaller characterized by a deep indentation of its surface than those found in the EAT cell. A few circular (Fig. 4) or intranuclear canals continuous with or prolonged mitochondria with a typical internal the inner of the nuclear membranes (Fig. 2). structure are found surrounding the cell center The karyoplasm consists of a matrix of low density (Figs. 5 and 6). containing fine granules of three or more kinds A second type of abnormal structure found that differ in size and density and in their state in the cytoplasm is one or more bundles of con of aggregation (Figs. 2 and 4). spicuous fibrous elements ca. 150 A in thickness The most common of the abnormal structures (Figs. 5-7). Such elements are never seen in normal is a particle of uniform size and distinctive form albino rat ascites cells or in EAT cells. They are found in considerable numbers in the cytoplasm quite common, however, in the cytoplasm of YS and occasionally in the nucleus. The intracyto- cells. In Figure 7 the internal structure of the plasmic virus-like particles are detectable in the majority of mitochondria surrounding the fibrous lumina of the endoplasmic reticulum, being fre elements is in a state of dissolution. A further quently encountered in small or large groups (Figs. sign of pathological alterations is the appearance 1 and 3). It has been found that the particles pos of the lipide body. sess a characteristic internal structure consisting A third type of abnormal structure found in of a core of low density, 15-25 niju in diameter, the cytoplasm is short or long spindle-like bodies. surrounded by a dense shell 20-30 ra.fi thick, show Figure 8 is an electron micrograph of the short, ing an over-all diameter ranging from 55 to 75 spindle-like profile, 1.3 fi in length and 0.26 p mÃŸ (Fig. 2). It is interesting that the obliquely in width, which is composed of fine vesicular sectioned tubular endoplasmic reticulum reveals masses with moderate density, being surrounded continuity between the agranular portion of the by a few pairs of membranes in more or less membrane surrounding the particles and the ad parallel arrangement. The cross-sectioned profile jacent granular portion which can be definitely in an oval shape, the moderate-sized one being identified as rough-surfaced endoplasmic reticulum 1.0-1.6 p in diameter (Fig. 9), shows a few vesicles (Fig. 2). The virus-like particles are found in the of varying size and varying density embedded nucleus, being spheroidal in shape and 80-140 in an apparently homogeneous substance of low im* in diameter. They consist of a dense capsule opacity. The elongate body, more than 4.0 ÃŸ about 12 imj thick enclosing a clear central zone in length and 0.6 /Â¿in greatest width (Fig. 10) 40-70 imt in diameter, which is of very low density is composed of vesicles of varying size which and gives the appearance of a central cavity. contain a homogeneous mass of low density or Within this cavity is a round or oval, dense a few granules about 20 ra.fi in diameter. It is body 56 imt or less in diameter. In some characteristic that the peripheral portion of the planes of section the inner body is completely profile is surrounded by membranes in a more surrounded by a clear zone and thus appears or less parallel arrangement, usually with a some to be central, but when cut in other planes what wavy course. Mitochondria are swollen and it is clear that the inner body is eccentrically occasionally found along the body. placed and is fixed on one side to the inner aspect Human cancerous peritonitis ascites cell.â€”Small of the capsule. The intranuclear particles do not sized or vacuolized mitochondria and a few elec occur individually as they do in the cytoplasm tron-lucent vesicles of varying size are found sur but are geneally closely associated in a cluster. rounding the nucleus, which is situated in the Such aggregations of particles are situated near central part of the cell. The endoplasmic reticulum or within the vesicle appearing in the nucleus is occasionally visible in a linear array. The nu (Fig. 4). clear chromatin free of dense particles is composed Yoshida albino rat sarcoma cells.â€”The cell cen of fine granules aggregated into irregularly shaped ter consists of an aggregation of single membrane- masses of varying size and indefinite outline; these limited vesicles with varying size and varying occur in all parts of the nucleus. Nucleolonemata electron density. The cytoplasmic matrix is of are not clear in the irregularly shaped nucleolus rather low density. The rough-surfaced endo (Fig. 11). plasmic reticulum is not well developed. One or In the cell, in which the nucleus is shifted more spherical or irregularly shaped particles are toward the peripheral part of the cell and trans-

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. YASUZUMIAND SUGIHARAâ€”Electron Microsoopy of Ascites Cells 1169 formed into an irregularly shaped profile, remark Irregularly shaped bundles of coarse, dense fila able changes occur in the fine structure of the ments 25 m/i in diameter and 4 or 5 Min length cytoplasm. Numerous single, membrane-limited, have been found in the cytoplasm of the LÃ¼cke electron-lucent vesicles appear in a group in the renal adenocarcinoma cell by Fawcett (2). The cytoplasm. The size of the vesicles varies, the fibrous elements observed in the YS cell in the diameter being between 0.36 and 0.94 p. The present study and by Wessel and Bernhard (8) mitochondria are remarkably reduced in size. At are smaller in width and less osmiophilic than the same time, a great number of dense or less those in the LÃ¼ckecell. It is possible that the dense, fine vesicles appear in the cytoplasm. It fibrous elements are elements of the disintegrated is very interesting that numerous projections of mitochondria, because the mitochondria degener round, conical, or irregular shape are observed ate to a nonhomogeneous mass which seems to at the peripheral part of the cytoplasm, being be a precursor of the fibrous elements. However, surrounded by the cell membrane. That is, the the filaments of LÃ¼ckecellshave no relationship cytoplasm is divided into endo- and ectoplasm. to the mitochondria. The spindle-like body ap The former contains mitochondria and vesicles pearing in the cytoplasm of YS cells may also of varying size, while the latter contains only be related to the mitochondria, because it is sur fine vesicles (Fig. 12). rounded with a few double membranes, each of The cytoplasmic projections are gradually isolat which has a great similarity to the limiting mem ed from the cytoplasm, being predominantly round brane of the mitochondria. However, no satisfac in shape and surrounded with or without a delimit tory explanation can be offered for the significance ing surface membrane (Figs. 13 and 14). Concur of the fibrous elements and the spindle-like body. rently with these changes, the cell membrane dis Fibrous elements in YS cells have been observed appears, and the ground substance of the cytoplasm by Wessel and Bernhard (8), but the spindle-like is destroyed into loss of its homogeneous appear body has never been reported in light and electron ance, showing irregular aggregations of osmiophilic microscope observations. particles and formation of irregularly shaped gaps Four electron micrographs of HCPA cells have without a limiting membrane. The isolated cyto been presented to demonstrate remarkable changes plasmic body is composed of fine, osmiophilic of the mitochondria in their form and structure. particles homogeneously distributed or aggregated The internal structure of the mitochondria is into irregularly shaped masses of varying size less orderly than usual. They are of three or more and outline, mitochondria without cristae, and kinds that differ in size and internal structure. electron-lucent vacuoles (Figs. 13 and 14). The Some mitochondria are swollen, and their center osmiophilic particles are densely accumulated at is often free of ridges and is occupied by a matrix the nuclear membrane and in some areas in the of low density. The others decrease remarkably interior of the nucleus (Fig. 14). Occasionally in size but increase in number. Where there is the nucleolus and the nucleolus-associated body an increase in the quantity of vesicles or vacuoles, (10) are found attached to the nuclear membrane there is a diminution in the mitochondria. This (Fig. 13). fact has been interpreted as evidence of a relation DISCUSSION between the mitochondria and the accumulation of vesicles or vacuoles. A peculiar picture has The electron micrographs obtained in the pres been observed in the peripheral part of the cyto ent study provide the first visual demonstration of virus-like particles in nuclei of EAT cells, plasm; it looks like the characteristic picture of which are of a great morphological similarity megakaryocyte found in guinea pig bone marrow to the particles enveloped by the smooth-surfaced by Watanabe (7), because of division into the endoplasmic reticulum. Such particles have never endo- and ectoplasm. The present findings suggest been observed in the nucleus (1, 3, 5, 6, 8, 9, buddings-off from the cell surface. It is more 11, 12) or in the cytoplasm of YS cells (8). possible that the budding results from degenera However, the particles appearing in the cytoplasm tive changes in cell organdÃes.As far as is known, of YS cells are irregular in shape, size, and density, this is the first time a structure of exactly this and their source is less easily explained. There nature has come to attention in electron micros is no morphological indication to imply the pres copy. ence of such particles in HCPA cells. The rough- surfaced endoplasmic reticulum has been found SUMMARY in the EAT cell more abundantly than in the Ehrlich ascites tumor cells, Yoshida sarcoma YS and HCPA cells. cells, and human cancerous peritonitis ascites cells

were studied in thin sections with the electron the LÃ¼ckeRenal Adenocarcinoma. J. Biophys. & Bio chem. Cytol., 2:725-42, 1956. microscope. 3. FRIEDLAEN-DEB,M.,and MOORE, D. H. Occurrence of All the Ehrlich ascites tumor cells examined Bodies within Endoplasmic Reticulum of Ehrlich Ascites were found to contain virus-like particles of uni Tumor Cells. Proc. Soc. Exper. Biol. & Med., 92:828-31, form size and distinctive morphology in the endo- 1956. plasmic reticulum in the cytoplasm and occasion 4. PALADE,G. E. A Small Participate Component of the Cytoplasm. J. Biophys. & Biochem. Cytol., l:59-Â«8, 1955. ally in the nucleus. The particles appearing in the nucleus consist of hollow spheres (80-140 5. SEL.BY,C. C.; BIESELE,J. J.; and GREY, C. E. Electron Microscope Studies of Ascites Tumor Cells. Ann. New mpi) having a dense capsule about 12 m/* thick York Acad. Sc., 63:748-73, 1956. and a dense inner body 40-70 nut in diameter. 6. SELBY,C. C.; GREY,C. E.; LICHTENBERO,S.;FRIEND,C.; Such particles have never been observed in Yoshi- MOORE,A. E.; and BIESELE,J. J. Submicroscopie Cyto- plasmic Particles Occasionally Found in the Ehrlich da sarcoma cells and human cancerous peritonitis Mouse Ascites Tumor. Cancer Research, 14:790-94, 1954. ascites cells. 7. WATANABE,Y. An Electron Microscopic Observation of Parallel fibrous elements and occasional pecul Blood Cells. Acta Haem. Jap., 19:327-11, 1956. iar spindle-like bodies occur in the cytoplasm of 8. WESSEL,W., and BERNHARD,W. Vergleichende elektro nenmikroskopische Untersuchung von Ehrlich- und the Yoshida sarcoma cell. Their origin seems to Yoshida-Ascitestumorzellen. Ztschr. Krebsforsch., 62:140- be related to the mitochondria, although their 62, 1957. significance remains obscure. 9. YASÃœZÃœMI,G.,and HIGASHIZAWA,S.Electron Microscope Division into endo- and ectoplasm occurs in Study of Sections of Ehrlich Mouse Ascites Tumors (First Report). Gann, 47:527-28, 1956. the human cancerous peritonitis ascites cells, sug 10. YASÃœZÃœMI,G.;SAWADA,T.;SUQIHARA,R.;KIRIYAMA,M.; gesting a budding-off from the cell surface and and SUGIOKA,N. Electron Microscope Researches on the a fixation of some cells in degenerate condition. Ultrastructure of Nucleoli in Animal Tissues. Ztschr. Zell- forsch. (in press). 11. YASUZUMI,G., and SUGIHARA,R. Electron Microscope REFERENCES Study of Sections of Ehrlich Mouse Ascites Tumors. II. On 1. ADAMS,W. R., and PRINCE,A. M. An Electron Micro the Cytoplasmic Dense Particles of Ehrlich Ascites Tumor scope Study of the Morphology and Distribution of the Cells. Gann, 48:326-28, 1957. Intracytoplasmic "Virus-like" Particles of Ehrlich Ascites 12. YASUZUMI,G.;SUGIHARA,R.;KIRIYAMA,M.; and IKEDA, Tumor Cells. J. Biophys. & Biochem. Cytol., 3:161-70, T. Electron Microscope Studies of Sections of the Ehrlich 1957. Ascites Tumor. J. Nara Med. Assoc., 7:135-38, 1956. 2. FAWCETT,D.W. Electron Microscope Observations on In- 13. YOSHIDA,T. The Yoshida Sarcoma, an Ascites Tumor. tracellular Virus-like Particles Associated with the Cells of Gann, 40:1-21, 1949.

FIG. 1.â€”Ehrlich ascites tumor cell of 7 days' inoculation age. The mitochondrion (if) is provided with the parallel- arranged cristae, while the mitochondiia in the other part are almost devoid of cristae and contain a matrix of low density (MV) or an electron-lucent substance (V). The dense par ticles (P) 55-75 m/i in diameter are found in the lumina of the endoplasmic reticulum with smooth surface. The endoplasmic reticulum (E) with the rough surface can be seen scattered throughout all the area. Lipide droplets (L) in an irregular shape are found attached closely to the Golgi complex (GC). X 25,000.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. FIG. 2.â€”Ehrlich ascites tumor cell of 7 days' inoculation age, showing the intracytoplasmic virus-like particles (P) in the lumina of the smooth-surfaced endoplasmic reticulum near the nucleus (Ar). At the point marked by A, the obliquely sec tioned agranular portion of the membrane surrounding the particle shows continuity with an adjacent granular portion, which can definitely be identified as rough-surfaced endoplas- niic reticulum. The dense particles (P) frequently demonstrate a light core. Two ring structures (R) originated from the mito chondria can be seen. (' marks an intranuclear canal. X 28,000. FIG. 3.â€”Ehrlich ascites tumor cell at the same stage as Figs. 1 and 2, showing a small group of virus-like particles (P). Note the rough-surfaced endoplasmic reticulum (arrows) ap pearing in circular or elongated profiles. R represents a ring structure. Xao.OOO.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. FIG. 4.â€”Anarea of karyoplasm from an Ehrlich ascites tu mor cell of 10 days' inoculation age, showing a deep indenta tion at NM, virus-like particles (P), and a large intranuclear vesicle (V). The virus-like particles (/J) are enclosed by a dense membrane. The virus-like particle (I') can also he identified within the inclusion of the vesicle (I'). The nucleus is enveloped by the nuclear membrane (\M) with a double structure and paekeil with highly differentiated chromosomal elements. X 25,000. FIG. 5.â€”AYoshida sarcoma cell, showing a portion of the nucleus (.V) and a small region of the cytoplasm in which at least five mitochondria can be seen. The mitochondrion marked by ML is considerably swollen, but the enveloping membrane is complete. The mitochondrion marked by an ar row shows a clear, typical, internal structure, but its envelop ing membrane is scarcely visible. J/N represents a degenerating mitochondrion. Vesicles (!') of varying size and density appear in the cytoplasm; some of them contain single (ON) or a few- granules ((i} of different sizes. /â€¢'representsfibrous elements near the nucleus (.V). X3(i,HOO.

..: M ML

V V

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. FIG. 6.â€”Yoshida sarcoma cell showing a portion of the nucleus (.V) and a small region of the cytoplasm surrounded by the mitochondria (M). A great number of vesicles of differ ent sizes, being enveloped with single or double membrane (DM), appear in the cytoplasm, which contain single grannie (GS), a few smaller granules (G) or substance of low electron density. The elongated, rough-surfaced endoplasmi? reticnlum (E) can be seen at the left-hand upper corner. F represents fibrous elements. X'27,600. FIG. 7.â€”Yoshidasarcoma cell. Fibrous elements are clearly visible at the point marked by F Mitochondria marked by M represent degenerating stages /, shows a lipide droplet. X27.500.

V v GS

M N N

M M

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. FIG. 8.â€”Yoshida sarcoma cell. .SB marks a spindle-like profile in a small size. The mitochondria (J/) are transparent, being almost devoid of cristae. The rough-surfaced endoplasmic reticulum (K) can seldom lie seen. L depicts a lipide droplet. X46.000. FIG. 9.â€”Yoshida sarcoma cell showing cross-section through the spindle-like body (>SÃŸ),mitochondria (.I/), and circular (CP) or elongated profiles (KP) with low density. X46,000.

CP M

SB M

â€¢¿EP

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. FIG. 10.â€”Longitudinal section through tin- spindle-like body (.S'fi.) appearing in a Yoshida sarcoma cell. The body is composed of vesicles (I") of varying size which contain a homo geneous mass of low density ora few granules (G). The body is enveloped with a few lamellae, each of which consists of a double membrane (arrows). Mitochondria (.1/1 are swollen and occasionally found along the spindle-like body. XHO.OOM.

SB M

V G

M M V

Â»tx

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. FIG. 11.â€”HumanassÃ®tescellof cancerous peritonitis, show ing a round profile of the nucleus (.V) with an irregularly shaped nucleolus (.VO), cytoplasm with shrunken'mitochon dria (Mi, vaciiolizcd mitochondria (.1/1'), fine vesicles (I'), and endoplasmiÂ«'rcticiilinn (/â€¢,').XÂ¿0,000. FIG. li.â€”Human ascitcs cell of cancerous peritonitis, show ing an irregularly shaped nucleus (.V) and the cytoplasm packed with a great number of electron-lucent vesicles (!'"), dense mitochondria (J/), and fine vesicles (/â€¢'(')ofdifferent densities and sizes. The pel Â¡plieraiportion of the cytoplasm is provided with numerous projections (PJ) of varying size and shape. X 16,500.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. FIG. 13.â€”Human aseites cell of cancerous peritonitis. A portion of the nucleus (.V) with the nueleolua (A'O) and the nucleolus-associated botl.v (\A) is demonstrated Â¡itthe upper side of the figure. The cytoplasm is filled with electron-lucent vacuoles (V) of varying size, dense mitochondria (.I/), and fine granules (G). Cellular projections (PJ) and bodies (IB) iso lated from the cytoplasm can be seen at the periphery of the cell. X 18,500. FIG. 14.â€”Humanaseites cell of cancerous peritonitis, show ing a degenerating stage of the cell. The nucleus shows the ir regularly arranged chromosomal elements (CE) and osmiophilic particles (OP) densely accumulated at the periphery. The nuclear membrane disappears at the point marked by the arrow. The cytoplasm demonstrates irregularly shaped gaps (OP), mitochondria (if) in vesicular shape, and isolated bodies (IB). XI2,000.

CF P. N

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. A Comparative Electron Microscopic Study on Ehrlich Ascites Tumor Cells, Yoshida Sarcoma Cells, and Human Cancerous Peritonitis Ascites Cells

G. Yasuzumi and R. Sugihara

Cancer Res 1958;18:1167-1170.

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