Cytoplasmic inclusions in cells of lymphosarcoma 6C3 HED

Experimental

280

CYTOPLASMIC

INCLUSIONS

LYMPHOSARCOMA II.

ELECTRON

RIICROSCOPIC

Cell Reseurch 26, 280- 289 (19G2)

IN CELLS

OF

6C3 HED’ OBSERVATIONS

L. H. SOBIN Department

of Pathology,

The New York Hospital-Cornell New York, N.Y., U.S.A.

Me&al

Center,

Received June 28, 1961

inclusions containing tieoxyribose nucleic acici and a basic in the cytoplasm of neoplastic cells of lymphosarcoma BC3HED, these inclusions as have been recently reported [ 101. This paper describes they appear with the electron microscope. Because these hodies were more numerous in areas of degeneration and necrosis, mice \vere treated with guinea pig serum or arsenic-azo-protein, materials previously founti to bring about necrosis of ll\mphosarcoma KYHEI) cells in uirro [‘i, HI. BASOPIIILIC

protein,

MATERIALS

AND

METHODS

Sterile suspensions of lymphosarcoma 6C3HED cells [6] in Ringer’s solution were implanted subcutaneously into each groin of adult ZBC mice (500,000 cells per site). Between six and twelve days following implantation, some of the tumor-bearing mice were given I ml intraperitoneal doses of either arsenic-azo-bovine albumin (containing 1 mg As per ml) for 1, 2 or 3 consecutive days or guinea pig serum for 1 or 2 consecutive days. Both agents cause complete regression of lymphosarcoma 6C3HED, as shown by Kidd [7, 81. Seven to 21 days following implantation, the tumors, which were then 1 to 2 cm in diameter, were excised using ether anesthesia. Portions of the tumors were fixed for 1 hr in buffered 1 per cent osmium tetroxide, pH 7.4 [9] containing 3 per cent sucrose 141, dehydrated in graded alcohols and embedded in a 9 : 1 mixture of n-butyl and methyl methacrylate monomers containing 0.07 per cent uranyl nitrate and 2 per cent catalyst (Luperco CDB). Polymerization occurred in 24 hr at 47°C. Thin (50 to 100 mp) and thick (2 to 4 p) sections were cut with glass knives on a Servall, Porter-Blum microtome. The thin sections, supported on Formvar or carbon-coated copper mesh grids, were examined with the electron microscope either unstained or employing 7.5 per cent uranyl acetate, 10 per cent phosphotungstic acid or saturated lead acetate [l&14]. Thick sections were examined 1 Supported in part by Institutional Research Grant No. IN-73 from the American Society and by Kesearch Grant No. CY-2573 from the National Cancer Institute. Experimental

Cell Research 26

Cancer

Cytoplasmic inclusions in tumor cells 1”

281

Fig. I.-Lymphoma cell with intracytoplasmic nucleoprotein inclusion indenting the nucleus. Cellular debris (arrows). From tumor of mouse treated with arsenic-azo-protein. Hematoxylin and eosin. y 4000. Fig. 2.-Lymphoma cell containing a whole degenerated tumor cell. The pyknotic nucleus (,V) of the phagocytosetl cell is partially retracted from its membrane (lines). Mitochondria (arrows) are still recognizable in the cytoplasm of thr included cell. Nucleus of phagorytic cell (5’). Extracellular debris (II). This and all subsequent figures are electron micrographs from mice treated with arsenic-azo-protein. j 13,000.

with a phase microscope. In addition, tissues fixed in buffered 10 per cent formalin, pH 7, were similarly embedded in methacrylate; thick and thin sections were examined in the light and electron microscopes. An RCA-EMU 3b electron microscope was used with operating voltages of 50 and 100 k\‘.

L. H. Sobin OBSERVATIONS

A single type of cytoplasmic inclusion was described in previous light microscopic studies of lymphosarcoma (iC3HED cells: this vvas a deeply basophilic mass which stained positively for deoxyribose nucleic acid and basic protein; these inclusions were present in up to 20 per cent of tumor cells in areas of necrosis [IO] (Fig. 1). Electron microscopy, however, reveals three relatively distinct groups of structures not ordinarily found in the cytoplasm of the tumor cells. They are: whole cell inclusions, large dense inclusions and small membranous inclusions. Whole cell inclusions.-These are whole cells vvith completely or partially intact cell membranes, pyknotic nuclei and varying numbers of mitochondria (Fig. 2). The inclusions are similar to degenerating tumor cells and are the largest of the three types, but are the least frequently found (present in less than one per cent of cells). Large dense inclrrsions-These are large, round or ovoid, dense objects occupying up to one-third the diameter of the cell (Figs. 3 and 4). They most closely resemble the inclusions seen in routine histologic sections. The size, shape and great electron opacity of these bodies is similar to that of pyknotic nuclei. Formalin-fixed, alcohol-dehydrated, methacrylate-embedded tissues, despite their poorly preserved fine structure, exhibit round cytoplasmic in elusions of similar electron density. Therefore, it is unlikely that the electron density is primarily due to high lipid content. The internal structure of these inclusions consists of finely and coarsely granular material. However, around the periphery and penetrating into the bodies are multiple membranes (Fig. 5). Generally, the number of membranes is inversely proportional to the overall electron density and size of the inclusion, i.e. the most dense inclusions have fewer membranes (Fig. C;), whereas the less dense bodies exhibit considerable internal membranous structure (Fig. i). Smnll membranous inclusions.-These inclusion bodies arc larger than the mitochondria and consist of fine membranes arranged as vv-horls and lamellae, often with some racuolar pattern or containing electron dense material Fig. S.-Tumor cell in an area of necrosis containing a large dense inclusion (11). A mcnrbrane partially surrounds the inclusion. A smaller dense mass (arrow) may have been extracted from a defective portion of the larger mass (line). The host ccl1 shows signs of degeneration: fragmented cell membrane and edematous cytoplasm. Debris (D). Nucleus (S). x 10,000. Fig. 4.-Lymphoma cell in mitosis from a degenerating area in the tumor. A large dense inclusion (I?) lies in the cytoplasm with the mitochondria. Chromosomes (C) are at the equator of the cell. x 13.000. Experimental

Cell Resenrch 26

283

Cytoplasmic inclusions in tumor cells

Experimenfnl

Cr

arch 26

L. H. Sobin (Figs. 8 and 9). They are the smallest of the three types and are about as numerous as the large dense inclusions. In cells containing any one of the three types of inclusions, the entlogenous structures, e.g. nuclei, mitochondria, etc., may either he normal or exhibit signs of degeneration (IFig. 3). These organelles show no consistent association \vith the inclusions except that the larger ones appear to displace mitochondria and indent the nuclei. In cells undergoing mitosis, inclusions remain in the periphery of the cells and do not appear to disrupt the inner nucleoplasm (Fig. 4). As noted in previous studies [lO], cptoplasmic inclusions are present, in lesser numbers, in areas not associated with obvious necrosis. Howcvcr, inclusions in such areas do not differ from those present in regions of tiegeneration (Fig. 10). Rlacrophages are very sparsely distributed \vithin the tumor; they contain large amounts of lipid material as \\-ell as nuclear debris (Fig. 11). 13ecause of their numerical rarity and their abundant and heterogeneous contents, it has not heen possible to compare the disposition of ingested cellular material in macrophages to that of the lymphoma cells. DISCUSSION

From the morphologic data presented, it is possible to postulate that the three types of inclusions represent three stages of intracellular digestion of phagocytosed material. There can he little doubt that the whole cell inclusions are, in fact, phagoqtosed tumor cells. The large dense inclusions are, most likely, the remnants of pyknotic nuclei. This assumption is primarily sup ported by the structural resemhlanre to the pyltnotic nuclei of the whole cell inclusions. In addition, up to t\\-enty per cent of the tumor cells in foci of necrosis contained Yeulgen-positive inclusions [lo!. However, less than one per cent of the morphologically similar inclusions observed with the electron microscope were whole cell inclusions (the remainder being large dense inclusions). Therefore, it appears that the large dense inclusions rcpresent Feulgen-positive structures. The relation between the large dense inclusions and the small membranous inclusions is less easily established. It is supported by the apparent increase

Fig. S.-Dense inclusion in the cytoplasm penetrate the mass. x 73,000. Fig. B.-Dense inclusion phoma cell. x 73,000. Experimental

Cell Research

encapsulated

26

of a lymphoma

by multilayered

cell. Double membranes

membranes

surround

in the cytoplasm

and

of a lyn-

Cyfoplasmic inclusions in tumor cells

285

zh 26

of a large dense inclusion in the cytoplasm of a lymphoma cell. There is extc Fig. 7.-Part about the periphery and extending into the mass. Mitochondrion mer nbrane formation Nut :leus (IV). x 73,000. of a lymphoma cell containing a small membranous inclusion (B) about 1 ,u in Fig. S.-Part and whorls of membranes. Mitochondria (arrows). Su cleus leng [th with elaborate laminations (N). x 34,000. Experimenfal

Cell Research 26

yig. 9.-Small inclusion with laminated membranes in the cytoplasm of a lymphoma cell. Del me morphous material (arrow) is in the central portion of the body. Nucleus (N). Cell membrs me 07). x 34.000. ‘ig. lO.-Lymphoma cell with a cytoplasmic inclusion (arrow) surrounded by a membral ncound vacuolar space. From a region that shows no signs of cellular degeneration. Portions of ther lymphoma cells are seen. x 11,000. Eqxrimental

Cell Kesearch 26

L. H. Sobin

I:ig. Il.-Macrophage in tlcgenerating portion of tumor. The cytoplasm contains numerous inclusions. Homogeneous structures (arrows) appear to contain lipid. Portions of surrounding tumor cells are seen. x 8000.

in membranes that accompanies the decrease in electron density and size of the larger bodies and by the presence of electron dense material within some of the smaller membranous inclusions. It is reasonable to s~~pposc that the membranes that surround and penetrate the inclusions are in some way related to a digestive mechanism, the small membranous inclusion being essentially an end product. The morphology of these membranous structures and the concept of digestion of nuclear or cellular material raises the question of the relation between the membranous inclusions and lvsosomes [S], phagosomes [ 111, etc. This problem must remain speculative until information regarding the enzymatic properties of the membranous bodies is available. If the assumption is correct that these three inclusions represent stages of the same process, some idea may be obtained about the length of time Experimental

Cell Hesearch

26

Cytoplusmic inclusions in tumor cells

289

The comparative rarity of the whole cell involved in cellular digestion. inclusions probably indicates rapid decomposition of the cytoplasmic structures either prior to ingestion or shortly thereafter. The greater frequency of the other bodies suggests a slower process of digestion of nucleoprotein. The presence of inclusions in cells undergoing mitosis indicates that intracellular digestion can extend beyond one generation. If the inclusions were prerenting the completion of mitosis, a larger number of dividing cells would contain these bodies. This finding is in agreement with reports of cell division folloving phagocytosis [a]. These cells possess an extraordinary degree of phagocytic ability despite the fact that they are both lymphoid and neoplastic-the types of cells least often, if eycr, associated with phagocytosis [l, 5, 12, 131.

SUMMARY

Basophilic nucleoprotein-containing inclusions in the cytoplasm of neoplastic cells of lymphosarcoma AC3 HEI) are examined in the electron microscope. Three relatively distinct, but probably interrelated, cytoplasmic bodies arc described: whole cells with pyknotic nuclei; large electron dense masses encapsulated and penetrated 1,~ membranes; and small structures with abundant membranes and vacuoles sometimes containing electron dense material. It is postulated that these groups represent various stages of intracellular digestion following phagocytosis of tumor cells. The author gratefully

acknowledges

the technical

assistance of Miss Lucille

Nelson.

REFERENCES 1. BARSES, LT. A. and FURTH, .J., Am. J. Cancer 30, 75 (1937). I<. G., SIMBONIS, S., HILL, R. B. and \VING, D. IT., Kuture 183, 4i6 (19.59). 2. Bmsm, 3. DR DUVE. C., ~ESSMIAN, B. C., GI.INETTO, R.. \\'TTIA~~x, R. and !WPELM.ASS, F., J~iochem. J. 60. 604 (1955). J., j. Bio@gs. Biochem. Cytoi. 3, 827 (1957). 4. CAULFIELD; 5. Cowonu, E. V., Cancer Cells, pp. 48, 61-62. W. B. Saunders, Co., Philadelphia, 1955. 6. GARDNER, 11'. LT., DOCGIIERTY, F. F. and \VILLIAYS, IV. L., Cnncer Kesenrch 4, 73 (1944). 5. KIOO, J. G., J. Exptl. Merl. 98, 565 (1953). H. -ibid. 108, 665 (1958). 9. PALADE. G. I:., J. Ezptl. Med. 95, 2X5 (1952). 10. SOBIN, I,. H., Ezptl. Cell I
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