Spontaneous Rat Malignant Tumors of Fibrohistiocytic Origin: An Ultrastructural Study

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1 Vet. Pathol. 19: (1982) Spontaneous Rat Malignant Tumors of Fibrohistiocytic Origin: An Ultrastructural Study P. GREAVES, J.-M. MARTIN, and M.-T. MASSON Centre de Recherche, Laboratoires Pfizer, Amboise, France Abstract. The ultrastructural features of a spontaneous malignant fibrous histiocytoma and three malignant histiocytomas in Sprague-Dawley rats are described. The ultrastructural features of the malignant histiocytomas (or histiocytic sarcomas) support origin from cells of the monocyte series. The histogenesis of more fibrous tumors (fibrous histiocytomas) remains uncertain. The malignant fibrous histiocytoma has been recognized increasingly in man since its early description nearly 20 years ago [ 141. Although malignant histiocytomas and malignant giant cell tumors of soft parts are considered as separate entities [12, 131, there is considerable histological, tissue culture, and ultrastructural evidence that all these tumors possess a common histogenesis [5, 151. Although the precise nature of their histogenesis still remains in doubt, these tumors do contain both fibroblasts and histiocytes in various proportions, accounting partially for a wide spectrum of different histological appearances [24]. Despite a variety of appearances, the various subgroups have been associated with a characteristic, yet unpredictable, biological behavior in man [ 191. Although soft-tissue sarcomas in rodents have been induced by a variety of agents and studied for many years [3], it only has been recognized recently that some of these induced tumors resemble human fibrous histiocytomas or malignant histiocytomas [4, 10, 11, 181. They also have been described recently occurring spontaneously in aging Osborne-Mendel and Sprague-Dawley rats [6, 201. A clinicopathological study of spontaneous rat soft tissue tumors revealed the usefulness of the fibrohistiocytic concept in the experimental situation [7]. It was confirmed that a few aged rats spontaneously develop fibrohistiocytic neoplasms and that these tumors possess a broad spectrum of histological appearances, ranging from being composed principally of spindle cells arranged in a characteristic storiform or cartwheel pattern (fibrous histiocytoma) to tumors of principally histiocytic cell-type (malignant histiocytoma or histiocytic sarcoma). Furthermore, it was shown that their biological behavior is related to their histological appearances and this behavior appears similar to that of corresponding fibrohistiocytic neoplasms in man. 491

2 498 Greaves, Martin, and Masson In the present study, the electron microscopic appearances of four of these rat neoplasms were evaluated to characterize the component cells and help elucidate their histogenesis. Materials and Methods The four tumors in this study were obtained from four Sprague-Dawley-derived (Cr1:COBS- CD(SD)BR) rats (Charles River France, 59, rue de la Paix, St Aubin-Lks-Elbeuf) that were used in carcinogenicity studies in Amboise. One female, rat 1, was from a group treated with a low oral dose of a noncarcinogenic compound. Two males, rats 2 and 3, and one female, rat 4, were rats from untreated groups. Housing conditions, diet, autopsy, and histological methods were as described previously [7]. For electron microscopy, tumor fragments measuring approximately 1 cu mm were prefixed in phosphate-buffered, 4% gluteraldehyde for two hours at 4 C and washed in phosphatebuffered, 2% osmium tetroxide for two hours at 4"C, and after further washing in phosphate buffer, the tissues were dehydrated in increasing concentrations of ethanol and finally embedded in epoxy resin. Sections were cut ultrathin and stained with uranyl acetate and lead citrate. The tissues were screened with the ZEISS E 109 electron microscope (Carl Zeiss, Oberkochen, West Germany) and photographed using 35-mm black and white pan film. Results One female, rat 1, was euthanatized at 13 months of age because of weight loss associated with the presence of a mass 10 cm in diameter situated in the proximal right foreleg. The other female, rat 4, also was euthanatized because of weight loss but in association with a mass 3 cm in diameter situated on the upper abdomen. Both males, rats 2 and 3, were 25 months of age at euthanasia, and both had tumors situated around the bone of the right hindleg. One tumor measured 8 cm in diameter (rat 2), and the other tumor measured 4 cm in diameter (rat 3). Each tumor was solid, white, and firm and infiltrated the surrounding soft tissues, including adjacent abdominal wall or thoracic cage. Metastatic deposits were seen at autopsy in the liver and lungs in rats 3 and 4. Light microscopy The light microscopic features of these tumors have been described previously in more detail [7]. The tumor arising in the foreleg of the female rat 1, however, appeared largely fibroblastic and was composed principally of plump or elongated spindle cells arranged in a storiform or cartwheel pattern characteristic of the fibrous histiocytoma in man (fig. 1). In addition, there were foci composed of rounded cells with indistinct eosinophilic cytoplasm and containing dark, oval, or rounded nuclei. Well-defined zones of necrosis and a scattering of inflammatory cells also were present. Mitoses were quite common in cellular areas. There was definite histological evidence of infiltration of surrounding muscle and fat but no metastases were found. The other three tumors (rats 2, 3, and 4) were predominantly histiocytic and were composed of rounded cells with irregular or rounded nuclei within abundant eosinophilic cytoplasm (fig. 2). Benign-looking multinucleated giant cells were found in

3 Malignant Rat Fibrohistiocytic Tumors 499 Fig. 1: Predominantly spindle cell tumor (rat 1) with storiform pattern characteristic of fibrous histiocytomas. HE. Bar = 50 pm. Fig. 2 Malignant histiocytoma (rat 3) composed of fairly uniform histiocyte-like cells with abundant eosinophilic cytoplasm and irregular to rounded nuclei. Benign-looking multinucleated cell also present. HE. Bar = 50 pm. Fig. 3: Spindle cells in fibrous histiocytoma (rat 1 ) with elongated indented nuclei, numerous mitochondria, some rough endoplasmic reticulum. Cells separated by pale ground substance and collagen fibers. Bar = 2 pm.

4 5 00 Greaves, Martin, and Masson all tumors (fig. 2). Focal necrosis was striking. The tumor cells infiltrated widely between tissue planes and into skeletal muscle, and formed pulmonary metastases. Rats 3 and 4 also had metastatic deposits in the liver, pancreas, peritoneum, lymph nodes, kidney, and testes. Electron microscopy The general spindle-cell nature of the first tumor (rat 1) was confirmed on electronmicroscopic examination. These cells had a few elongated, fine cytoplasmic processes, and nuclei were generally oval or spindle-shaped but had some infolding (fig. 3). Nuclear euchromatin was abundant with nucleoli sometimes prominent and marginated. The granular endoplasmic reticulum, present in various amounts, possessed cisternae somewhat dilated with amorphous, moderately electron-dense material (fig. 3). Some free-lying polyribosomes were scattered in the cytoplasm and a fairly prominent Golgi apparatus was seen usually. Variable numbers of mitochondria were present and appeared swollen in some cells. Homogeneous, moderately electron-dense membrane-bound bodies, acceptable as lysosomes, were seen in some cells as well as very occasional lipid droplets. The cells generally were well separated by abundant electron-lucent ground substance and collagen (fig. 3). Although apparently simply composed of uniform histiocytes with a scattering of multinucleated giant cells on light microscopic examination, the cells in the other three tumors (rats 2,3, and 4) had variable ultrastructural appearances. Some spindlecell foci were seen and these spindle cells resembled those in rat 1, possessing fairly abundant rough endoplasmic reticulum and smooth-cell margins (fig. 4). Many cells, however, although characteristically rounded, had extremely irregular and interdigitated cell margins separated by little or no ground substance or collagen fibers (fig. 5). Some nuclei in these cells were oval or rounded (fig. 4), but a large proportion were irregular and some had deep invaginations (fig. 6). Nucleoli often were large and marginated. Rough endoplasmic reticulum was present in many cells but appeared commonly as sparse, short profiles (fig. 6). Variable numbers of polyribosomes were seen. Mitochondria, present in variable numbers, were swollen distinctly in some cells. The Golgi apparatus was sometimes quite prominent (fig. 7). Some cells were particularly histiocytic in appearance, containing numerous dense membrane-bound lysosomal bodies (fig. 6), while others contained few cytoplasmic organelles. Occasional lipid droplets and myelin bodies were present in some cells. Some cells in rats 2 and 4 also contained fine cytoplasmic filaments, devoid of focal densities (fig. 7). No Langerhans granules were seen. The foreign body-type giant cells were observed in close proximity to both histiocyte and fibroblast-like cells. In general, these giant cells possessed both the nuclear and cytoplasmic features of the histiocyte-like cells, except for the presence of variable numbers of peripherally arranged nuclei. Central parts of the cell

5 Malignant Rat Fibrohistiocytic Tumors 50 I Fig. 4 Part of cytoplasm of spindle cell in predominantly histiocytic tumor (rat 3). Smooth cell boundaries and dilated cysternae of rough endoplasmic reticulum. Bar = I pm. Fig. 5 Low power view of malignant histiocytoma (rat 3 ). Rounded, irregular nuclei, interdigitated cell margins, little intercellular ground substance. Bar = 2 pm.

6 502 Greaves, Martin, and Masson Fig. 6 Histiocytic cell from rat 3 with highly irregular nucleus and numerous lysosome-like bodies. Bar = 1 pm. Fig. 7: Part of cell from histiocytic tumor, rat 2. Part of large nucleolus and cytoplasmic filaments. Bar = 1 pm.

7 Malignant Rat Fibrohistiocytic Tumors 503 cytoplasm contained variable numbers of unordered microfilaments as well as vacuoles, lysosome-like or phagosome-like structures. Discussion Spontaneously arising fibrous histiocytomas and malignant histiocytomas or histiocytic sarcomas only recently have been described in the rat [6,7,20]. This diagnosis can be difficult and the large number of fibrosarcomas in the literature may reflect the common habit of pathologists to classify bizarre, seemingly undifferentiated, spindle cell tumors as fibrosarcomas [22]. The ultrastructural features of spontaneous rat fibrohistiocytic tumors do not seem to have been well characterized, although some workers who have studied both fibrohistiocytic tumors in man and induced rat sarcomas at the ultrastructural level have noted their similarity [ 10, 111. Very recent work using formalin-fixed material also has shown fibrohistiocytic features at ultrastructural level in some tumors [20, 281. The spindle cells, which formed the major component of the first tumor (rat 1) and were present focally in the other two neoplasms, had smooth cell margins and contained rough endoplasmic reticulum, often dilated with dark material; these features are similar to fibroblasts. Some spindle cells contained lysosomal structures which could be regarded as evidence of histiocytic differentiation. There was no evidence of myofibroblast or endothelial cell differentiation in the spindle cells, although some authors report the presence of myofibroblasts [ 1,8,22] and endothelial cells [2] in fibrohistiocytic tumors in man. Thus, in general, the tumor in rat 1 was composed principally of spindle cells and morphologically resembled the dermatofibrosarcoma protuberans in man [ 151. It is important to note that this tumor in man, composed mainly of spindle cells arranged in a storiform manner, has been regarded as a tumor of fibrohistiocytic origin for many years, mainly on the basis of tissue culture studies [ 151. Although this concept has been questioned [8], realization of the similarity of these tumors in both rat and man may help in their understanding. The tumors in rats 2, 3, and 4 were focally fibroblastic in appearance. Some cells in these two tumors were particularly histiocytic or macrophage-like in character and this finding supports the histiocytic nature of this neoplasm. Many more cells in these tumors were relatively nonspecific in appearance, but by virtue of their round to irregular indented nuclei, abundant cytoplasm containing short profiles of rough endoplasmic reticulum, lysosomes, and ruffled cell borders, they closely resemble cells described in fibrohistiocytic tumors in man [5,22]. Another author [8] also noted abundant cytoplasmic filaments without focal densities in the histiocytic cells of a malignant fibrous histiocytoma in man, similar to those seen in our rats 2 and 4. The giant cells in rat 4, although variable in appearance, did possess fine structural features in common with murine inflammatory multinucleated giant cells, which are thought to be derived from fusion of circulatory monocytes [ 171. The large number of filaments observed in some multinucleated cells may be a phenomenon related to

8 5 04 Greaves, Martin, and Masson aging [21]. Thus, the presence of these cells also provides further evidence for the histiocytic nature of these neoplasms. In general, these histiocytic tumors do appear to form a distinctive group in the rat, such that some authors who have recently studied a large number of these neoplasms have designated them separately as histiocytic sarcomas [20]. However, these rat histiocytic tumors (rats 2, 3, and 4) not only bear a remarkable biological and light-microscopic resemblance to the malignant histiocytoma in man [ 161, but based on the few existing reports [9], also seem similar at the ultrastructural level. The fact that foreign-body type or osteoclast-like cells also are seen in some rat tumors also may explain their close ultrastructural resemblance to the malignant giant cell tumor of soft parts in man which is thought to be a special variant of malignant fibrous histiocytoma. Others [ 11 showed that most cells in these giant cell tumors in man also were either undifferentiated mesenchymal cells or mononuclear histiocytic cells and additionally, they found spindle cells and macrophage-like cells in small numbers. In conclusion, our findings confirm both the similarity of these rat tumors to the group of fibrohistiocytic and histiocytic tumors in man and this fact may be important in their understanding. The electron microscopic features of the malignant histiocytoma (histiocytic sarcoma) do suggest origin from cells of the monocyte-histiocyte series. The ultrastructural features of the more fibrous tumors (fibrous histiocytoma) are less specific and thus their histogenesis remains to be more clearly defined both in man and in rats. Acknowledgements The authors are grateful to Dr. A. M. Monro, Director of the Research Centre, and Dr. J. M. Faccini, for their encouragement in this study, and to Mrs. D. Cubilie, for assistance in preparation of the manuscript. References 1 ALGUACIL-GARCIA, A,; UNNI, K.K.; GOELLER, J.R.: Malignant giant cell tumour of soft parts. Cancer , CARSTENS, P.B.H.; SCHRODT, G.R.: Ultrastructure of a sclerosing hemangioma. Am J Pathol , CARTER, R. L.: Tumours of soft tissues. In: Pathology of Tumours in Laboratory Animals, vol. 1, Tumours of the Rat, ed. Turusov, V. S., Part 1, p International Agency for Research on Cancer, Lyon, DAMJANOV, I.; SUNDERMAN, F.W. JR.; MITCHELL, J.M.; ALLPASS, P.R.: Induction of testicular sarcomas in Fischer rats by intratesticular injection of nickel subsulfide. Cancer Res , Fu, Y.S.; GABBIANI, G.; KAYE, G.I.; LATTES, R.: Malignant soft tissue tumors of probable histiocytic origin (malignant fibrous histiocytomas): General considerations and electron microscopic and tissue culture studies. Cancer , GOODMAN, D.G.; WARD, J.M.; SQUIRE, R.A.; PAXTON, M.B.; REICHARDT, W.D.; CHU, K.C.; LINHART, M.S.: Neoplastic and non-neoplastic lesions in aging Osborne-Mendel rats. Toxicol Appl PharmacolS , 1980

9 Malignant Rat Fibrohistiocytic Tumors GREAVES, P.; FACCINI, J.M.: Fibrous histiocytic neoplasms spontaneously arising in rats. Br J Cancer 43: , HARRIS, M.: The ultrastructure of benign and malignant fibrous histiocytomas. Histopathology , JOHNSON, W. W.; COBURN, T.P.; PRATT, C.B.; SMITH, J.W.; MAHESH KUMAR, A.P.; DAHLIN, D.C.: Ultrastructure of malignant histiocytoma arising in the acromion. Hum Pathol , KATENKAMP, D.; STILLER, D.: Cellular composition of the so-called dermatofibroma (histiocytoma cutis). Virchows Arch [Pathol Anat] , 1975 I I KATENKAMP, D.; STILLER, D.: Structural patterns and histological behavior of experimental sarcomas. 11. Ultrastructural cytology. Exp Path 11: , KAUFMAN, S.L.; STOUT, A.P.: Histiocytic tumors (fibrous xanthoma and histiocytoma) in children. Cancer , MACKENZIE, D.H.: Miscellaneous soft tissue sarcomas. Recent Advances in Pathology , O BRIAN, J.E.; STOUT, A.P.: Malignant fibrous xanthomas. Cancer 17: , OZZELLO, L.; HAMELS, J.: The histiocytic nature of dermato-fibrosarcoma protuberans. Tissue culture and electron microscopic study. Am J Clin Pathol , OZZELLO, L.; STOUT, A.P.; MURRAY, M.R.: Cultural characteristics of malignant histiocytomas and fibrous xanthomas. Cancer , 1963 I7 PAPADIMITRIOU, J.M.; ARCHER, M.: The morphology of murine foreign body multinucleate giant cells. U Ultrastruct Res , PRADHAN, S.N.; CHUNG, E.B.; GHOSH, B.; PAUL, B.D.; KAPADIA, G.J.: Potential carcinogens. 1. Carcinogenicity of some plant extracts and their tannin-containing fractions in rats. JNCI 52: , SOULE, E.H.; ENRIQUEZ, P.: Atypical fibrous histiocytoma, malignant fibrous histiocytoma, malignant histiocytoma and epithelioid sarcoma. A comparative study of 65 tumors. Cancer , SQUIRE, R.A.; BRINKHOUS, K.M.; PEIPER, S.C.; FIRMINGER, H.I.; MA, R.B.; STRAND- BERG, J.D.: Histiocytic sarcoma with a granuloma-like component occurring in a large colony of Sprague-Dawley rats. Am J Pathol , SUTTON, J.S.; WEISS, L.: Transformation of monocytes in tissue culture into macrophages, epithelioid cells and multinucleate giant cells. J Cell Biol , TAXY, J.B.; BATTIFORA, H.: Malignant fibrous histiocytoma, an electron microscopic study. Cancer 40: , WARD, J.M.; KULWICH, B.A.; REZNIK, G.; BERMAN, J.J.: Malignant fibrous histiocytoma. An unusual neoplasm of soft tissue origin in the rat that is different from the human counterpart. Arch Pathol Lab Med , 198 I 24 WEISS, S.W.; ENZINGER, F.M.: Malignant fibrous histiocytoma. An analysis of 200 cases. Cancer 41: , 1978 Request reprints from Dr. P. Greaves, Centre de Recherche, Laboratoires Pfizer, B.P. 109, Amboise Cedex (France).