Histogenesis and Morphology of Periosteal Sarcomas Induced by FBJ Virus in NIH Swiss Mice1

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(CANCER RESEARCH 36, 3985-3992, November 1976] Histogenesis and Morphology of Periosteal Sarcomas Induced by FBJ Virus in NIH Swiss Mice1

Jerrold M. Ward and David M. Young

Comparative Pathology Unit, Laboratory of Toxicology, Experimental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20014

SUMMARY ever, dose-related differences in latency and multicentnicity of tumor induction were noted. FBJ viruswas injected i.p. into 145 neonatal NIH Swiss [N: A detailed morphological and histogenetic study of neo NIH(s)] mice. Eighty mice developed a total of hO neo plasms induced by FBJ virus has not been reported. The plasms by 5 months of age. The mean latent period of the purpose of this paper is to characterize significant histoge tumors was 71 days (26 to 145) postinjection . The frequency netic development and natural history of FBJ virus-induced of occurrence of neoplasms at different sites was: dia neoplasms of bone and other tissues in mice. phnagm, 45%; ribs, 14%; vertebrae, 14%; femora, 9%; pelvic bones, 5%; tibiae, 4%; stemnebnae,3%; and inguinal area, 7%. The neoplasms were characterized histologically by MATERIALS AND METHODS elongated on rounded cells associated with an abundant connective tissue stroma. Occasional areas of bone forma Pregnant female NIH Swiss [N: NIH(s)] mice (Small Animal tion and apparent osteoid metaplasia were seen. Bone tu Section, Veterinary Resources Branch, NIH, Bethesda, Md.) mors appeared to arise from periosteal cells, to grow by were placed in individual polycarbonate cages with hard expansion, and to invade locally, but they failed to metas wood bedding and provided water and NIH Open Formula tasize. Neoplasms of the diaphragm originated in the cen Rat and Mouse Ration 5018 (Ralston Purina Co., St. Louis, tral aponeurosis and appeared histologically similar to bone Mo.) ad libitum . Neonatal mice, 1 to 2 days of age, received neoplasms. Histochemical studies demonstrated abundant injections i.p. (through skin in the medial aspect of the night alkaline phosphatase in tumor cells, and ultmastructural ob thigh)of 0.1 ml of a 1:20 or 1:30 0.85% NaCl dilutionof servations revealed subcellular characteristics of osteo FBJ virus pool 74-i (kindly supplied by Miriam P. Finkel and blasts and chondroblasts. Tumors were readily transplanta Christopher A. Reilly, Jr. , Argonne National Laboratory, ble and had histopathological characteristics similar to Argonne, III.). An uninjected control mouse was identified those of the primary viral-induced tumors. The results of in each litter by tail docking. this study indicate that the FBJ virus induces in NIH Swiss Mice were weaned at 4 weeks of age, separated accord mice a unique type of chondroosseous neoplasm derived ing to sex, weighed weekly, and maintained until death or from peniosteal cells which has a resemblance to human time of sacÃ±fice. Mice surviving 150 days after injection juxtacortical (parosteal) osteosarcoma. were sacrificed. A complete necropsy was performed on each mouse. Tissues were collected and fixed in 10% neu tral buffered formalin on Telly's fixative. Some tumors were INTRODUCTION placed in 90% alcohol, and bones were deminenalized in EDTA solution. Tissues were embedded in paraffin, sec Naturally occurring neoplasms of chondroosseous tis tioned at 6 IL, and stained with hematoxylin and eosin. sues have been reported in several strains of mice (5, i i , 23, Selected tissues were stained by the following methods: 24, 27). However, the etiology of these sarcomas was not Masson's tnichnome , reticulin , Giemsa, periodic acid-Schiff, known until FBJ virus was isolated from a spontaneous and Von Kossa. neoplasm in a mouse (8). The virus was found to induce Portions of several tumors were placed in cold (4Â°)for neoplastic proliferation of osteoblasts which resulted in the mol:calcium for 24 hr, quick-frozen in liquid nitrogen, sec production of osteoid and bone. CF-i , CFW, AKR, BALB/c, tioned on a cryostat, and stained for alkaline phosphatase NIH, and Swiss mice were very susceptible to the oncogenic (3). Sections of deminenalized femora (midshaft and epi effects of the virus, whereas C57BL mice were not (8, 10, physial plate), diaphragms, and tendons of 3 uninjected NIH 17-19, 22, 31). The induced neoplasms appeared to arise Swiss mice were also stained for alkaline phosphatase. from the periosteum and were compared to human paros Portions of tumors from i 1 mice were minced into small teal osteosarcomas (9, 10, 2i). Since the virus in the inocu pieces, mixed 1:1 with sterile 0.85% NaCI solution, and in lum cannot be quantitated with current methodology, mice jected through a 16-gauge needle into the s.c. tissues of received injections of dilutions of tissue suspensions. How weanling female NIH Swiss mice. Three transfer passages were made for some tumors. I Supported in part by GSA Contract 005-31212 to Baker Histology Labo ratories, Great Falls, va. For ultnastructural examination of primary and trans Received May 3, 1976; accepted August 9. 1976. planted tumors, tissues were placed in cold (4Â°)2%glutaral

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1976 American Association for Cancer Research. J. M. Ward and D. M. Young dehyde in cacodylate buffer, postfixed in 1% osmium te the proliferating cells assumed a spindle to stellate shape troxide, and embedded in epoxy resin . Sections were cut on with prominent nuclei (Fig. 4, a and b). In loosely arranged an ultramicrotome, stained with unanyl acetate and lead areas, predominant cells were more rounded and appeared citrate, and examined in an JEOL Model 100 B electron to be producing an intercellular eosinophilic matrix material microscope. Portions of a few tumors were prepared for (Figs. 4 to 6). In areas where matrix production was exuber ultrastructural localization of alkaline phosphatase (30). Ra ant, round cells became enmeshed in the homogeneous diographs of anesthetized on dead mice were prepared us material (Fig. 5). The matrix stained blue with Masson's ing a MINXnay 20 Delux S12ODcollimator unit (Picker Corp., tnichnome stain, and many reticulin fibers could be seen White Plains, N. V.). The target-to-film distance was 93 cm, with the use of a reticulin stain. Histopathological evalua and settingswere 68 KVP, 15 mawith 0.5-sec exposure. No tion of bone-associated neoplasms at later stages of devel screen X-ray film (NS2T; Eastman Kodak Co. , Rochester, opment revealed mixed components of fibrosarcomatous N. Y.)was used. and myxomatous tissue with areas of osteoid, chondroid, mineralization, and necrosis. In areas of differentiation, osteoid matrix formed micro RESULTS trabecular patterns entrapping small populations of round cells and associated capillaries. Mineral precipitated in The 1st mouse died with a large neoplasm of the night some areas of stroma without evident bone formation (Fig. femur 26 days after injection. The induction period (time to 6). Further differentiated foci revealed areas of mineralized appearance of the neoplasm) varied from 26 to 145 days osteoid lined by numerous osteoblasts and frequent osteo with a mean of 71 days. Eighty of the 145 injected mice had clasts (Fig. 4). These areas of neoplastic bone formation neoplasms by 150 days after injeciion, whereas only 25 of resembled abortive attempts to form woven (coarse fiber) the 87 mice sacrificed at 150 days had tumors. Thirty-nine % bone. Mitotic figures were observed, albeit infrequently. of the mice inoculated with the 1:30 dilution developed Islands of neoplastic bone and cartilage formation ap neoplasms, whereas 59% of those receiving the 1:20 dilu peared at random throughout the sections and were sum tion did so. Sixty-three % of the males and 48% of the rounded by a fibrosarcomatous stroma. Various stages of females developed neoplasms. Most mice lived 3 to 4 weeks differentiation were observed. Some islands of cartilage after the 1st appearance of the tumor. were undergoing transformation into bone with areas me Neoplasms appeared at several sites (Table 1). Diaphrag sembling the characteristic cellular subdivisions of endo matic tumors induced respiratory signs, whereas other tu chondral ossification. Neoplasms associated with vertebrae mors were associated with regional swellings. The femoral produced abundant matrix but, rarely, bone (Fig. 3), and tibial tumors occurred on the side of injection, and whereas 2 neoplasms of pelvic bones had abundant new vertebrae tumors were usually in the lumbar area. bone formation (Fig. 8). Radiognaphs revealed that the earliest lesions were asso Midsagittal sections of long bones containing the penios ciated with peniosteal new bone growth, especially of distal teal neoplasms contained various amounts of peniosteal femur and ribs (Fig. i). Lytic bone lesions were detectable, new bone production (Figs. 1 and 7). These areas were well and nadiodense areas of new bone production and soft organized and were considered to be induced reactive bone tissue swelling were observed associated with large tumors. formation rather than neoplastic in origin. Many areas of Neoplasms of the vertebrae were only associated with soft peniosteal new bone formation were developing adjacent to tissue swelling. the sites of neoplastic-induced periosteal elevations. In this respect, they resembled the formation of the well-described Histopathology Codman's triangle associated with human bone neoplasms. Muscle Neoplasms. Many neoplasms developed in the Bone Neoplasms. Early lesions consisted of small neo diaphragm on inguinal muscles (Table 1). Grossly, the more plastic foci adjacent to developing bones. Histologically, common diaphragm tumors originated in the central apo these foci were extracortical and contained foci of prolifer neuroses. There was no evidence that the tumor arose from ating periosteal cells (Figs. 2 and 3). In highly cellular areas, vertebrae. As it enlarged, the neoplasm became irregular and nodular. Small nodules were seen on the panietal pleura Table1 of those mice harboring large tumors. Neop!asms in NIH Swiss mice inoculated with FBJ virus Histologically, the earliest lesions resembled the neo plasms induced in bone and seemed to anise in tendons No.injected145No. of mice (Fig. 9). Primitive mesenchymal and osteoblastic cells pro withneoplasms80No. ofneoplasms110No. duced an abundant matrix with occasional foci of cartilage of:Diaphragm50Rib15Vertebrae15Femur10s.c.of neoplasms or bone (Fig. 10). Certain areas of tumors resembled fibro sarcomas. The tumors were invasive, and adhesions to liver, penitoneum, and pleura were noted. Transplantation. Ten bone and 2 diaphnagmatic neo tissue8Pelvis5Tibia4Stemebrae3 plasms were easily transplanted. They grew quickly in the 1st passage, resulting in large tumors and death by 20 to 40 days. However, after 1 passage, the tumors seemed to take less frequently and grew at a slower nate. Radiographs

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1976 American Association for Cancer Research. Sarcomas Induced by FBJ Virus revealed areas of bone formation in most tumors. On cross some foci of bone may appear as a metaplastic change from section, the bone was easily seen grossly and was usually fibmoblasts. The mineralization of the osteoid matrix is in present in 1 or 2 large zones. Histologically, all transplants complete and haphazard. Recent studies have demon were similar. They were composed of sarcomatous cells stratedbone mineralizationfactorsintransplantablemunine producing a variable stroma. Chondroosseous foci were osteosamcomas and other tumors (2, i3, i6). prominent, especially in association with necrotic areas. The ultrastructure of the neoplastic cell in these induced The amount of bone produced varied in the same tumor neoplasms and in osteosancomas in general reveal features linesand between tumor lines.Severaltumors had areas of characteristic of osteoblasts and chondroblasts (4, 20). stroma mineralization not associated with necrosis. Trans Abundant granular endoplasmic reticulum in association planted tumors invaded muscle and, occasionally, bone, with extracellular fibnillan material and collagen fibers are when adjacent to it, but did not metastasize. Special stains characteristic of neoplastic cells in FBJ virus-induced tu revealed the stroma had prominent collagen and reticulin moms and normal osteoblasts. Cell junctions or unusual fibers and contained foci of metachnomatic cartilage cells cytological changes were not seen in the peniosteal sarco and matrix. mas. The differentiation of these neoplastic cells from fibro Alkaline Phosphatase Histochemistry. The enzyme was blasts depended on the localization of alkaline phosphatase easily demonstrated in i2 induced and ii transplanted tu within the cells and the general polygonal shape of osteo mon lines. Usually, large focal areas of the tumors were blasts (21, 25). The peniosteal cells of mouse bone contain stained red. The stain was evident in granules of the sam abundant alkaline phosphatase, although their ultrastruc coma cells, and cell membrane staining was not seen. Ultra tune reveals a cell with little granular endoplasmic reticulum structural histochemistry indicated that large cytoplasmic (J. Wand, unpublished observation). The peniosteum has granules were electron dense (Fig. ii). With light micros been described to be composed of an outer fibrous layer copy, alkaline phosphatase was seen in normal cortical and an inner cellular (osteogenic) layer (i2). The neoplasms osteoblasts and peniosteal cells as intracellular medgman induced by FBJ virus appear to arise from the inner cellular ules, but only in endothelial cells of normal diaphragm, layer. and not in tendon or muscle tissues. The biological behavior of the primary and transplanted Uftrastructure. Nine primary tumors (bone and dia chondroosseous neoplasm was compatible with a low phragm) and 9 transplants from 4 tumor lines were studied. grade malignant tumor. The neoplasms grew by expansion Neoplastic cells were polygonal, embedded in the matrix, on and soft tissue invasion but never metastasized via lymphat elongated in more cellular areas (Fig. 13). In any case, the ics on blood vessels to distant sites. granular endoplasmic reticulum was very prominent, and a The origin of the tumors in diaphragm and inguinal mus few mitochondnia, lipid droplets, myelin figures, and lyso culatume is undetermined. The alkaline phosphatase activity somes were seen. Intracytoplasmic fibnils were prominent in in the tumors and histological and ultrastnuctural appear some cells (Fig. 14). The extracellulan matrix was composed ance suggested osteoblastic origin (20). However, normal of small fibnillar material and collagen fibers (Figs. i2 and diaphragm muscle or tendon did not have the enzyme ex i4). Occasionally, numerous mature C-type virus particles cept in endothelial cells. The tumor may have arisen from were observed embedded in the matrix (Fig. 12), and, less â€œmultipotentialsystemsâ€•present in these tissues (15). commonly, immature C-type particles budded from the As the only naturally occurring sarcoma virus of mice and plasma membrane of neoplastic cells. Mineralization of the the only characterized virus that induces a high incidence of matrix was not observed. neoplasms resembling osteosancomas, the FBJ virus offers a unique model for the study of these tumors (10). There are strain differences with regard to susceptibility to tumor DISCUSSION induction and perhaps to tumor histology. However, no The neoplasms induced by FBJ virus in NIH Swiss mice convincing evidence has been presented for the induction of typical cortical osteosarcomas in any strain of mouse (10, were sarcomas producing chondroosseous material and arising from peniosteal cells. Previous reports of these tu 21). Two additional munine sarcoma viruses inducing neo plasms of bone have recently been described (iO). mors have noted osteosarcomas, panosteal sarcomas, The transplantable tumors reported here and other tnans chondrosarcomas, etc. , in various strains of mice, although plantable munine osteosarcomas may be useful as a screen they did not describe in detail the histogenesis. Finkel me ing model for testing antineoplastic modalities for oncolytic ported X-ray studies of tumors in mice and observed their prominent periosteal origin (8, 9). Naturally occurring bone activity against these chondroosseous neoplasms. neoplasms have been reported in mice and appear as true osteosancomas of cortical bone (i, 5, ii, 14, 23, 24, 27), ACKNOWLEDGMENTS similar to those induced by polyoma virus (6, 28). Human We are grateful for the FBJ virus provided by Dr. Miriam Finkel of Argonne panosteal osteosarcomas appear to arise from periosteum National Laboratory, the excellent technical assistance of Keith Fauvie, and have a biological behavior different from that of cortical Major Reagan, and Snooky Taylor, and the editorial assistance of Madie neoplasms, although they may be similar histologically (7, Tyler. 26, 29). The panosteal neoplasms produced by the FBJ virus in mice have sparse areas of osteogenesis and prominent REFERENCES collagen production. They do not resemble fibnosancomas i . Albala, M. M. , and Esparza, A. R. Transplantable Osteogenic Sarcoma in and are not neoplasms with osteoid metaplasia, although Inbred AKR Mice. Cancer Res., 29: 1519-1522, 1969.

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2. Amitani, K., Ono, K., Sakomoto, Y., and Nakata, Y. Osteogenic Induction Bone and Chondroid Tissue. Acta Pathol. Japon., 25: 179-199, 1975. by Cell Free Material from Murine Osteosarcoma and Its Cultured Cell 17. KeIloff, G. J., Lane, W. T., Turner, H. C., and Huebner, R. J. In Vivo Line. Gann, 66: 327-329, 1975. Studies of the FBJ Murine Osteosarcoma Virus. Nature, 223: 1379-1380, 3. Burstone, M. S. Histochemical Comparison of Naphthol-As-Phosphates 1969. for the Demonstration of Phosphatases. J. NatI. Cancer Inst., 20: 601- 18. Levy, J. A., Hartley, J. W., Rowe, W. P., and Huebner, R. J. Studies of 613, 1958. FBJ Osteosarcoma Virus in Tissue Culture. I. Biologic Characteristics of 4. Cameron, D. A. The Fine Structure and Function of Bone Cells. In: G. H. the â€œC-TypeViruses.J. NatI. Cancer Inst., 51: 525-539, i973. Bourne (ed.), The Biochemistry and Physiology of Bone, Ed. 2, Vol. 1, 19. Levy, J. A., Hartley, J. W., Rowe, W. P., and Huebner, A. J. Studies of pp. 391-423. New York: Academic Press, Inc., 1971. FBJ Osteosarcoma Virus in Tissue Culture. II. Autoinhibition of Focus 5. Charles, A. T., and Turusov, V. S. Bone Tumors in CF-i Mice. Lab. Formation. J. NatI. Cancer Inst., 54: 615-619, 1975. Animals, 8: 137-144, 1974. 20. Paschall,H. A., and Paschall,M. M. ElectronMicroscopicObservations 6. Dawe,C.J., Law,L. W.,andDunn,T. B. Studiesof Parotid-TumorAgent of 20 Human Osteosarcomas. Clin. Orthopaed. Related Res., 111: 42-56, in Cultures of Leukemia Tissues of Mice. J. NatI. Cancer Inst., 23: 717- 1975. 797, 1959. 21. Price, C. H., Moore, M., and Jones, D. B. FBJ Virus-Induced Tumours in 7. Dwinnel, L. A., Dahlin, D. C., and Ghormley, A. K. Parosteal Juxtacorti Mice. Brit. J. Cancer, 26: 15-27, 1972. cal Osteogenic Sarcoma. J. Bone Joint Surg., 36A: 732-744, 1954. 22. Pritchard,D.J., Finkel,M. P.,and Reilly,C.A. TheEtiologyof Osteosar 8. Finkel. M. P., Biskis, B. 0.. andJinkins, P. B. Virus Inductionof Osteo coma. Clin. Orthopaed. Related Res., 111: 14-22, 1975. sarcomas in Mice. Science. 151: 698-701 , i966. 23. Pybus, F. C., and Miller, E. W. The Gross Pathology of Spontaneous 9. Finkel, M. P., Jinkins, P. B., Tolle, J., and Biskis, B. 0. Serial Radiogra Bone Tumours in Mice. Am. J. Cancer, 40: 47-53, 1940. phy of Virus-Induced Osteosarcomas in Mice. Radiology, 87: 333-339, 24. Pybus, F. C., and Miller, E. W. The Histology of SpontaneousBone 1966. Tumours in Mice. Am. J. Cancer, 40: 54-61, 1940. 10. Finkel, M. P., Reilly, C. A., Jr., and Biskis, B. 0. Viral Etiology of Bone 25. Shimokawa, K. Histological and Histochemical Findings of Transplanta Cancer. Frontiers Radiation Therap. Oncol., 10: 28-39, 1975. ble Osteosarcoma of Mice. Acta Histochem. Cytochem., 7: 112-125, 11. Franks, L. M. , Rowlatt, C. , and Chesterman, F. C. Naturally Occurring 1974. Bone Tumors in C57BL/lcrf Mice. J. NatI. Cancer Inst., 50: 431-438, 26. Spjut, H.J., Dorfman,H. D., Fechner,R.E.,andAckerman,L. V.Tumors 1973. of Bone and Cartilage, Fascicle 5. In: Atlas of Tumor Pathology. Wash 12. Ham, A. W. , and Harris, W. R. Repair and Transplantation of Bone. In: G. ington, D. C.: Armed Forces Institute of Pathology, 1971. H. Bourne (ad.), The Biochemistry and Physiology of Bone, Ed. 2, Vol.3, 27. Swarm, R. L. Transplantation of a Murine Chondrosarcoma in Mice of pp. 337-399. New York: Academic Press, Inc., 1971. Different Inbred Strains. J. NatI. Cancer Inst., 31: 953-975, 1963. 13. Heiple, K. G., Heruddon, C. H., Chase, S. W., and Wattleworth, A. Osteo 28. Vandeputte,M., Eyssen,H., Sobis, H., and De Somer, P. Induction of genic Induction by Osteosarcoma and Normal Bone in Mice. J. Bone Polyoma Tumors in Athymic Nude Mice. Intern. J. Cancer, 14: 445-450, Joint Surg., 50: 311-325, 1968. 1974. 14. Hilberg, A. W. Morphologic Variations in an Osteogenic Sarcoma of the 29. VanDerHeul,A.0., andVonRonnen,J. A.JuxtacorticalOsteosarcoma. Mouse When Transplanted to the Kidney. J. NatI. Cancer Inst., 16: 951- J. Bone Joint Surg., 49A: 415-439, 1967. 959. 1956. 30. Wetzel, B. K., Spicer, S. S., and Horn, R. G. Fine Structural Localization 15. Holtzer, H., Dientsman, S., Holtzer, S., and Biehl, J. Quantal Cell Cycles, of Acid and Alkaline Phosphatase in Cells of Rabbit Blood and Bone Normal Cell Lineages and Tumorigenesis. In: W. Nakahara, T. Ono, T. Marrow. J. Histochem. Cytochem., 15: 311-334, 1967. Sugimura, and H. Sugano (ads.), Differentiation and Control of Malig 31. Yumoto, T., Poel, W. E., Kodama, T., and Dmochowski, L. Studies on the nancy of Tumor Cells, pp. 389-398. Baltimore: University Park Press, FBJ Virus-Induced Bone Tumors in Mice. Texas Rept. Biol. Med., 28: 1974. 145-149, 1970. 16. Kawoda, K. A Transplantable Pulmonary Tumor Line in Mouse Forming

Fig. 1. Radiograph of mouse with neoplasm arising near costochondral junctions of several ribs. Note periosteal thickenings of 2 ribs (arrow), soft tissue swelling , and small areas of bone formation in neoplasm. Fig. 2. Early neoplasm arising from periosteal cells of the femur and producing abundant collagen stroma. x 90. Fig. 3. Large neoplasm attached to dorsal vertebral spine and growing in the s.c. tissue. X 11.5. Fig. 4. a , typical area of FBJ virus-induced tumor of bone. Elongated and rounded cells are embedded in a fibrous stroma. x 350. b , bone island in area of neoplasm with cells resembling esteoblasts. x 350. Fig. 5. Abundant production of osteoid-Iike matrix. Note structures resembling lacunae. x 140. Fig. 6. Mineralization of collagenous stroma. x 330. Fig. 7. Tumor mass attached to 3 ribs. Dense osteogenic area in center represents reactive periosteal proliferation of rib in association with tumor mass. x 10. Fig. 8. Radiodense mass in ischium composed of dense bone and sarcomatous tissue. x 14. Fig. 9. Tumor mass in diaphragm with necrotic center. x 16. Fig. 10. Mineralization of chondroosseous stroma in neoplasm of diaphragm. x 140. Fig. 11. Alkaline phosphatase (electron dense) is localized to large cytoplasmic granules of tumor cell resembling osteoblast. Note abundant osteoid collagen fibers in matrix. x 7000. Fig. 12. Numerous C-type viral particles found among the collagen fibers. x 25,000. Fig. 13. Typical tumor cell in neoplasms induced by FBJ virus. The polygonal cell contains abundant endoplasmic reticulum and resembles a normal osteoblast. The extracellular matrix is granular and contains collagen fibers. x 10,000. Fig. 14. Portion of neoplastic cell similar to a normal chondroblast with many intracytoplasmic fibrils and dilated granular endoplasmic reticulum. x 12,500.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1976 American Association for Cancer Research. Histogenesis and Morphology of Periosteal Sarcomas Induced by FBJ Virus in NIH Swiss Mice

Jerrold M. Ward and David M. Young

Cancer Res 1976;36:3985-3992.

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