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 48°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.— 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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1958 American Association for Cancer Research. 1168 Cancer Research Vol. 18, November, 1958 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 mß (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 ß about 12 imj thick enclosing a clear central zone in length and 0.6 /¿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.—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.—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 SUGIHARA—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.