Mesotheliomas of Peritoneum, Epicardium, and Pericardium Induced by Strain MC29 Avian Leukosis Virus1

Home , Mesentery, Mesothelium, Peritoneum

[CANCER RESEARCH 30, 1287 1308, May 1970] Mesotheliomas of Peritoneum, Epicardium, and Pericardium Induced by Strain MC29 Avian Leukosis Virus1

J. F. Chabot, Dorothy Beard, A. J. Langlois, and J. W. Beard

Department of Surgery, Duke University Medical Center, Durham, North Carolina 27706

SUMMARY myeloid hematopoietic tissue neoplasms distributed in bone, liver, and other sites, primary renal tumors occur Injection of strain MC29 avian leukosis virus into the in high incidence, erythroblastosis is occasionally seen, peritoneal, pericardia!, and air sac cavities of the chicken and, in small proportion, growths arise from hepatic pa- resulted in high incidence of tumors of the mesothelium renchymal cells (9). A singular influence of the MC29 of the respective structures. As determined by light and strain is the infection and rapid morphological alteration of CEC" in tissue culture not observed with other leuko electron microscopy, the growths arose as papillomas or expanding tumors by alteration of the squamous mesothe sis agents (4, 8, 16). Recent studies initiated with the in lium to spheroidal or cuboidal cells characterized by u/7ro-altered CEC revealed still another unusual host re rounded nuclei with clear nucleoplasm and very large sponse to this leukosis strain. Cells inoculated into the nucleoli and cytoplasm deeply stained with hematoxylin. chicken by different routes gave rise to tumors simulating With continued rapid growth, the nuclear features les transplants at various sites in the peritoneum. However, sened somewhat, cytoplasmic staining diminished greatly, introduction in the same way of cell-free fluid from CEC and the cell limits became indistinct. A second stage of cultures altered by MC29 virus resulted in the appear metaplasia was marked by frequent alteration of the ance of growths at similar sites and, also in the pericar epithelioid cells to cartilage which increased both by con dium and epicardium. Further investigation showed that tinued alteration of peripheral mesothelioma cells and certain of the tumors were the result of neoplastic re proliferation of chondrocytes. The tumors, sometimes sponse of peritoneal and pericardial mesothelium to in isolated but usually coalesced in masses, were poorly en fection with the MC29 virus. Studies have been made of capsulated, contained little fibrous stroma, and showed the occurrence, pathomorphology, and ultrastructure of no necrosis. They readily invaded contiguous visceral tis some of these tumors, and the results are described in sues but did not metastasize to distant sites. Numerous this report. For comparison there are considered, also, 2 tumors in subcutaneous tissue and myelocytic1 growths virus particles were observed in the tumors together with occasional budding of the particles from the cell mem in visceral organs. Growths induced by response to prep branes. The mesotheliomas were an addition to the al arations of morphologically altered cells will be consid ready broad spectrum of responses to strain MC29 virus ered in another report. consisting of myelocytic growths, high incidence of renal tumors, primary tumors of the liver parenchyma, and singular aspects of morphological alteration of chick em MATERIALS AND METHODS bryo cells in vitro. These virus-induced tumors in the chicken closely resembled mesotheliomas of unknown Virus. Isolation of strain MC29 leukosis agent and etiology in man and bovines in both morphology and be chicken response to virus obtained from blood plasma havior. from birds diseased with the strain were described (12, 18). The virus for the present studies was in 3 pools from CEC cultures infected with strain MC29 of previous pas INTRODUCTION sage in vitro. Passage was in primary cultures (14) of embryos from eggs of RIF-free White Leghorn hens4 Strain MC29 avian leukosis virus infection in the (22). Fluids in contact with cells of altered morphology chicken results in the induction, principally, of myelocy- for 4.5 to 6 hr in cultures 6 to 15 days following exposure toma and myelocytomatosis (12, 18). In addition to these "The abbreviations used are: CEC, chick embryo cells: RIF, re 1This work was aided by USPHS Grant C-4572, by the Annie Ma sistance-inducing factor; WW, subcutaneous wing web tissue; i.e.. intra- bel Sherris Memorial Grant for Cancer Research from the American cardiac: dpi, days postinoculation. Cancer Society, Inc., by National Defense and Education Act Title IV ' The term myelocyte is used in descriptions of strain MC29 virus Fellowship 67-08530.0, by IN-611 American Cancer Society International disease to designate nonmyeloblast myeloid elements at any level of Research Grant, and by the Dorothy Beard Research Fund. The paper differentiationâ€”with or without granules (15, 18)â€”lessthan that of the is part of the studies submitted by J. F. C. in partial fulfillment of the mature granulocytes. requirements for the degree of Doctor of Philosophy in Zoology in the 4The chick embryos were from White Leghorn eggs provided by Graduate School of Arts and Sciences of Duke University. Dr. Roy Luginbuhl, University of Connecticut, through the Research Received September 12, 1969; accepted November 19, 1969. Resource Program of the National Cancer Institute Bethesda, Md.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1970 American Association for Cancer Research. Chabot, Beard, Langlois, and Beard to virus (14) were harvested and passed through 0.45-^ nitely related to dose as measured by numbers of virus Millipore filters. Virus particle content and infectivity of particles. Introduction of virus into the heart through the the fluids were measured by electron microscopy (24) chest wall gave neoplasms of the epicardium or pericar and focus assay (13), respectively. dium or both in 4 of 19 birds. Injections into the wing Chickens and Examinations. White Leghorn chicks of webs of 19 chicks resulted in l (Y148) apparently virus- line 15 (28) were inoculated 1 day after hatching with induced subcutaneous growth. Another tumor in subcu virus amounts and by WW, i.e., or i.p. routes as indicated taneous tissue (Y623) arose in the abdominal wall near in Table 1. Blood smears made routinely as in earlier the site of i.p. inoculation. Generalized response of the studies (5) were stained with May-Grunwald-Giemsa. myeloid hematopoietic cells consisted principally of At the onset of illness or after appropriate periods of diffuse infiltration of the liver by nongranulated myelo- study, birds were killed by bleeding from the heart. Tis cytesâ€”myelocytomatosisâ€”with few circumscribed cell sues for light microscopy were taken in Zenker-formol aggregatesâ€”myelocytomas; few of the cells contained solution, and the sections were stained with hematoxylin eosinophilic granules. Except in birds receiving inocu and eosin. Some sections were stained with Alcian blue lations i.e. (into the circulating blood), myeloid cell re and by Masson's technique. Cells from peritoneal fluid sponse was relatively small. Line 15 chickens were less were stained with May-Grunwald-Giemsa or hematoxy- responsive to strain MC29 in this respect than the Shaver lin-eosin. birds previously used (18). Moreover, as with BAI strain Specimens for electron microscopy were fixed for 2 to A (myeloblastosis) virus (6), the i.p. inoculation route was 4 hr in 5% glutaraldehyde (Fisher G-151, Fisher Scien much less effective in the induction of generalized mye tific Co., Silver Spring, Md.) buffered at pH 7.2 with loid response than the i.v. or i.e. routes. sodium cacodylate (20, 23); transferred to 0.1 M sodium Peritoneal Tumors. Birds with tumors in the peritoneal cacodylate buffer, pH 7.4, containing 0.3 M sucrose (23); cavity exhibited general signs of lassitude, droopiness, and held for 2 to 5 days at 4Â°.The tissues were postfixed disinterest, and ruffled feathers. The skin and mucous for 1 hr with 1% osmic acid buffered at pH 7.2 with Vero membranes were pale, and the blood smears of many of nal (19), embedded in Maraglas (7), sectioned with glass the birds showed large mononuclear primitive cells (15, knives on a Porter-Blum ultramicrotome, stained with 18) of the myeloid series. Peritoneal fluid in volumes of uranyl acetate (29) and lead citrate (21), and examined about 8 to 80 ml was found in a large proportion of the with a Siemens Elmiskop I at 80 kV equipped with a birds, and some animals showed pronounced abdominal double condenser and a 50-/Â¿objectiveaperture. distension. The murky, greenish fluid contained large numbers of mesothelial cells and other mononuclear ele ments. RESULTS Figs. 1 to 4 illustrate the gross appearance of minute beginning MC29 virus-induced growths (Fig. 1) and that Table 1 summarizes 3 experiments yielding the results of large (Fig. 2) and smaller (Figs. 3 and 4) developing described here (Bird Y879, Figs. 18 to 21, from a differ neoplasms. An impressive feature of the process was the ent study was given an injection i.p. of 8.5 X 10' virus high growth response of the mesothelial cells in the loose particles). Of 148 birds receiving inoculations into the folds of mesentery. This was indicated by the numerous peritoneal cavity in the 3 studies, 52 had tumors of de tiny individual growths (Fig. 1) and emphasized by the monstrable mesothelial cell origin. Incidence was defi multitude of nodules coalescing into the extensive nodu-

Table I Chicken response to tissue culture-grown MC29 virus inoculated into the wing web. into the peritoneal cavity (i.p.), and into the heart (i.e.) Cor. no., number of chicks surviving early nonspecific deaths; MYE. myeloid growth: PCS, pericardial or epicardial neoplasms; PTS, tumors of peritoneal mesothelium: ERY, erythroblastosis; SUBC, tumor in subcutaneous tissue; and ASC, ascites.

Exper-Virus . . . r particles no.19191716181619172223MYE15563141530PCS4000000000PTS1012105121714ERY1000000000SUBC0100000010ASC221141021710 iment v6(X (ml)0.10.050.50.50.50.50.50.50.250.5Routei.e.WWi.p.i.p.i.p.i.p.i.p.i.p.i.p.i.p.ChicksinoculatedY100-I19Y139-157Y158-176Y347-364Y365-383Y384-401Y402-421Y422-440Y602-624Y625-648(20)(19)(19)(18)(19)(18)(20)(19)(22)(23)Cor )961 10

2513127971

115234.60.90.18985

280560Volume

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1970 American Association for Cancer Research. Mesotheliomas and Strain MC29 Avian Leukosis Virus lar masses of neoplastia tissue in Figs. 2 to 4. Tumors ap tributes but which retained the prominent features (Figs. peared anteriorly in the loose mesentery in the region of 12 and 22) of spheroidal shape and large nuclei and nu the triangular dorsal ligament attachment of the gizzard. cleoli. The cytoplasm was not notably deeply stained, and Throughout essentially the whole length of the gut, the cell boundaries were indistinct, especially in the com growths were widely distributed in the mesenteric layers. pact growths. Tumor architecture varied relatively little, Tumors of like appearance occurred also in the mesothe- showing a loose arrangement of cells in some growths lial cells of the abdominal air sacs where virus was inocu (Fig. 12) and a very compact arrangement in others (Fig. lated i.p. or in the axillary air sacs following virus injec 22). Small numbers of other cells were spindle shaped tion (intended i.e.) into the chest. Very few and only with elongated nuclei which were of a structure resem small growths arose in the mesothelium covering the wall bling fibroblasts and greatly different from the predomi of the intestine; none was seen in the free surfaces of the nant mesothelial tumor cells as well demonstrated in liver, spleen, or kidney or in the parietal peritoneum. In electron micrographs (see Figs. 42 and 43). The origin of contrast, the delicate pancreas lying in relatively loose these stremai cells as seen in Fig. 11, for example, was mesothelium-covered areolar tissue was largely covered not clear, but the framework probably represented non- over by or incorporated in the tumor masses. The numer neoplastic spindle cells of the original mesenteric areolar ous growths did extend readily onto the intestine and tissue stimulated to proliferation rather than the result other viscera, however, when they originated at or close of further change in the morphology and function of the to the junction of the mesothelium covering the organs mesothelial tumor cells. However, appreciable numbers with that of the mesentery. As illustrated below (see of cells of these characteristicsâ€”but not necessarily the Figs. 27 and 28), such tumors invaded and infiltrated the same typeâ€”appeared to participate in the formation of contiguous organ tissues. The singular distribution of re cartilage (see Fig. 13 and the electron micrographs of sponding cells is strikingly emphasized by the lack of tu Figs. 42 and 43). mor nodules in the mesothelium along the vascular loops A second metaplastic process marked by alteration of of the mesentery (Fig. 1). the epithelioid mesothelioma cells to cartilage was of Mesothelium of both parietal and visceral peritoneum very frequent occurrence, not only in different growths consists of simple squamous cell monolayers which are but in multiple foci of the same growth (Fig. 13). Carti closely applied to the viscera and abdominal wall and lage formation began apparently by morphological and form both sides of the thin mesentery sheets and air sac functional alteration of typical mesothelioma cells and walls. Response of the peritoneal mesothelium to MC29 progressed both by continued change of contiguous tu virus infection was marked (Figs. 6 and 8 to 11; see Fig. mor cells at the periphery of the tissue and by multiplica 7 for comparison with epicardial response) by widespread tion of cells embedded in the matrix. As noted before, metaplasia marked by rounding of the cells on both sides extensive growths in some birds, as exemplified particu of the sheets (Fig. 10). Although morphological changes larly by Y160 (Figs. 8 and 9), consisted largely of carti seemingly occurred in a large proportion of the cells (Fig. lage. Transitional alteration to cartilage in these growths 6), growth resulting in tumor formation appeared limited seemed to proceed almost directly from altered mesothe to relatively widely separated foci (Figs. 1, 6, 8, and 11). lial cells with but fleeting intervention of neoplastic ele In many instances, rapid focal cell multiplication exerted ments of the predominant type such as those of Figs. 12 pressure laterally, causing outfolding of the tissue to form and 22. Fig. 9 illustrates a cartilage growth with only a pendunculated, papillomatous, frequently intricately few of the usual tumor cells. branched cauliflower or wart-like growths (Figs. 6, 8, 10, The cartilage was of somewhat aberrant form, different and 11). In the initial stages, the tumors in the large pro from the normal structure and from that occurring in portion of birds consisted principally of rounded pear- other avian virus tumors (10, 11). Cell arrangement was shaped or cuboid mesothelial cells with delicate spindle irregular without lacunae or distinct cell pairing such as cell supporting tissue (Fig. 11). Growths in some hosts, that evident in the BAI strain A virus-induced nephro- however, consisted almost wholly of cartilage (Figs. 3, 8, blastoma (10). With Alcian blue, intercellular material and 9). Such neoplasms were, in fact, cartilage papillo showed varying intensities of stain (Fig. 14), most pro nnas. nounced at the cell surface and light in areas of widely Mesothelial cells newly altered morphologically, as il spaced cells. With few exceptions, the blue-staining ma lustrated in Fig. 11, exhibited characteristic features re terial, cartilage, was sharply separated from the other lated to deeply blue-stained cytoplasm, relatively large tumor structure. Frequently, however, deeply blue- spheroidal nuclei with clear nucleoplasm, and large nu stained strands formed small or extensive branched net cleoli, all of which resulted in a "bird's eye" appearance works between the ordinary tumor cells in the absence of of the elements. A few growths, particularly in the free recognizable cartilage. Enlargement of the cartilage nod folds of mesentery and thin flaps of omentum, main ules was attended by nutritional difficulties and damage tained the papillomatous or cauliflower structure, but to the cells away from the periphery. There was never most of the neoplasms enlarged throughout to form in any evidence either of calcification or osteoid formation. dividual or, for the most part, firm confluent nodular Amounts of connective tissue varied relatively little in masses. With such growth, cell morphology changed pro the various tumors. Encapsulation was scant, and most of gressively to that of elements of somewhat different at the growths consisted of tumor cells extending to the

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1970 American Association for Cancer Research. Chabot, Beard, Langlois, and Beard periphery (Figs. 6 and 33). The outer layer of other tu sion was usually effected by encroachment on a broad mors, particularly of those in the epicardium, was squa- tumor front (Fig. 26), but highly irregular and anaplastic mous epithelium (Figs. 7 and 31). Many of the compact growth was also evident (Fig. 27). Frequent infiltration growths, like that of Fig. 22, contained no intercellular of the intestinal wall resulted in overgrowth and destruc material stained by Masson's technique, but some of the tion of muscle with extension through the muscularis mu same growths showed branched fibers of unidentified cosa into the lamina propria (Fig. 28). Fig. 29 illustrates material stained with Alcain blue (17). Variations ranged typical invasion of the ovary. from these thin strands to distinct but narrow intercon Epicardial and Pericardia! Tumors. Like the perito necting bands (Fig. 15) stained by Masson's technique. neum, the epicardial and pericardia! mesothelium is a There were no predominantly fibrous growths as such. monolayer of simple squamous epithelium. Pathomor- Most of the tumors of loose structure (Fig. 12) did not phological response of the epicardial cells to infection show staining of the intercellular material with either with strain MC29 was similar to (Figs. 5, 7, and 30 to 34) Alcian blue or the Masson stains. Necrosis was not ob but yet different in some respects from that of the peri served in any of the tumors. toneum. The gross appearance of the epicardial growths Pseudoglandular formations were encountered in only (Fig. 5) was characterized by sheets or masses of con a few instances. The survey picture of Fig. 18 reveals fluent cauliflower-like papillomatous protrusions, as in numerous tubules formed of cells of the morphology the auricular region (upper arrow), or somewhat individ demonstrated at higher magnification in Fig. 19. The ual tumors of like appearance in the epicardium of the tubule-like arrangements consisted of epithelial cells ly ventricles (lower arrow). Similar papillary growths oc ing in a loose "stroma" of elements indistinguishable curred in the pericardium. The pronounced response of from those of the typical mesothelioma growth. Fibrous the epicardium closely adherent to cardiac muscle was in connective tissue was not discernible. In another section marked contrast to the comparatively few and minor tu of the same growth (Fig. 20), many "tubules" were cut mors in the peritoneum covering the walls of the intes both lengthwise and crosswise. This aspect of the tumor tine and other organs and the lack of growths originating has the appearance of an unusual papillomatous struc in the parietal peritoneal layer. ture. Fig. 21 illustrates another type of pseudotubular Papillomatous growth of the epicardium (Fig. 7) con structure consisting of epithelioid cells bounded in bun sisted in numerous pendunculated and filiform processes, dles or strands by well-defined stroma and traversed by some consisting mostly of epithelial cells (Fig. 31) and thin delicate capillaries. others with a preponderance of connective tissue. In Fig. With few exceptions, the mesotheliomas were of char 7 it is evident that the epicardial cells were less respon acteristic histological morphology and easily distinguish sive to the virus, since most of them that were not a part able from other MC29-induced growths. Occasionally, of the growths retained the squamous morphology. Cells however, the mesothelioma cells exhibited features sur which did respond were of low cuboidal shape (Fig. 30) prisingly similar to analogous aspects of myelocytic with deeply stained cytoplasm. The spheroidal nuclei ex growths. Fig. 22 illustrates the morphology characteristic hibited clear nucleoplasm and prominent nucleoli which, of a mesothelioma and Figs. 23 and 24 are sections of tu nevertheless, were distinctly smaller than those in cells of mors in the same bird invading the liver and spleen, re the peritoneal tumors. The tumors were often pearly spectively. The 3 growths show both similarities and white and hard and contained nodules of cartilage (Fig. differences related particularly to prominence of the nu 31). The growths invaded the cardiac muscle and formed cleoli and relatively large nuclei with clear nucleoplasm. extensions deep in the heart wall (Fig. 32). The cross- Certain attributes of these tumors are likewise similar to section histology of a pericardia! papillary process (Fig. the cells of the minute myelocytoma in the liver seen in 33) resembled that of analogous growths from the peri Fig. 25. Further comparisons are made to the mylocytes toneum (Fig. 11). of myelocytomatosis massively invading the liver as in Subcutaneous Growth. A tumor (Fig. 34) in the sub Fig. 36 and permeating the hepatic sinuses as illustrated cutaneous tissue at the site of virus inoculation occurred in Fig. 37. The tumors of Figs. 23 and 24 were of uncer in the wing web in only 1 bird. Masses of individual cells tain origin, but both arose at the respective surfaces of permeating the muscle and interstitial tissues and consti the spleen and liver. The neoplasm of Fig. 23 bore a tuting the associated solid growth were morphologically striking resemblance to the mesothelioma of Fig. 21; the indistinguishable from neoplasms like that of Fig. 22. cells of the growth in the spleen (Fig. 24) exhibited a The invading cells at the edge of the main growth were syncytial appearance singular with respect to the mor of indistinct outlines but contained large spheroidal nu phology of the other tumors exemplified. clei and large nucleoli. The origin of the tumor could not Attributes of tumor malignancy were expressed by be determined. Although the cells were like those of the rapid growth and by invasion and infiltration of contig peritoneal growths, they likewise exhibited some charac uous organ tissues but not by distant mÃ©tastases.The teristics of myelocytoma. It does not seem too remote to pancreas was regularly involved. Small circumscribed regard the tumor as a derivative of vascular endothelium growths often appeared at the surface of the organ, but in the inoculated 1-day-old chick. in many regions the tumor cells penetrated and de Another growth in the subcutaneous tissue was in a stroyed the pancreatic structure (Figs. 26 and 27). Inva bird inoculated into the peritoneal cavity and appeared

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1970 American Association for Cancer Research. Mesotheiiomas and Strain MC29 Avian Leukosis Virus in the abdominal wall near the inferior aspects of the rib nuclei were characteristics shared by various cell typesâ€” cage. This growth (Fig. 35) consisted of large spheroidal chick embryo fibroblasts (8) and myelocytes (15)â€”in re mononuclear cells. These elements enmeshed among sponse to the MC29 agent. Except for size, the structure bands of connective tissue showed some resemblance to of the nucleoli was not unusual (Figs. 38 and 39). The myelocytes seen in other locations (Figs. 25, 36, and 37). organdÃes exhibited the characteristic components of Figs. 36 and 37 illustrate some characteristics of the dense ribonucleoprotein granules and the fibrillar net cells of myelocytomatosis. In Fig. 36 the cells are invad work. There were included, also, masses of amorphous ing the liver and replacing the parenchyma. Such relatively electron-lucent material which closely resem growths not infrequently show large blood-filled sinuses. bled the substance of the nearby nucleoplasm. The nu The myelocytes in Fig. 37 are crowding the liver sinuses clei were approximately spheroidal with somewhat irreg with attendant damage to and replacement of liver pa ular outline. Few cells contained more than traces of renchyma. In these growths as in the myelocytoma of condensed chromatin; rather, the material was distrib Fig. 25, the myelocytes (15) show the same large sphe uted largely in the diffuse state with much of it as fibrils roidal nuclei with clear nucleoplasm and prominant nu of the dimensions of deoxyribonucleoprotein strands as cleoli as the cells of the mesothelioma. seen in altered chick embryo cells (8). In the cytoplasm, Fibrosarcomas comprise no part of the strain MC29 on- ribosomes and polysomes were numerous, and, in most cogenic spectrum. In neither the present studies nor in cells, the rough endoplasmic reticulum was rather sparse. others including many hundreds of birds inoculated with A notable feature was the formation of fibers of the di MC29 leukosis virus has there been a single tumor mensions of collagen at the peripheral membranes of the ascribable to primary growth of fibroblasts. This is the cells of morphology typical of the mesothelioma. more remarkable in view of the profound influence of A characteristic of the mesothelioma cells of both peri the strain in the morphological alteration of chick em toneal (Fig. 40) and epicardial (Fig. 41) origin was the bryo cells (8, 16). formation of hyaline substance and collagen fibers in Peritoneal Cells. Fluid associated with the peritoneal varied amounts. In some tissue (Fig. 41) there was little growths was slightly viscid and of a greenish tint. Fluids or no intercellular hyaline material, and collagen, like from different birds contained variable numbers of cells wise, varied greatly (Figs. 40 to 43). In some sections which occasionally were sufficiently numerous to form a (Fig. 40) there was much lightly stained intercellular ma definite buffy coat on centrifugation. Most of the cells in terial with little collagen. Sections of other peritoneal smears prepared from the buffy coat and stained with (Figs. 42 and 43), as well as of epicardial, growths con May-Griinwald-Giemsa were large, squamous, mesothe- tained large amounts of collagen. In addition, cells typi lial elements of normal appearance (Fig. 16) with thin cal of both peritoneal and epicardial mesotheliomas widespread pink-stained cytoplasm containing small vac (Figs. 40 and 41) showed more or less widely distended uoles and indistinct peripheral cell membranes. The cisternae containing material like that in the intercellular ovoid nuclei were distinct, pink stained, and of uniformly spaces. granular appearance. Cells of other morphology occur Electron micrographs emphasized, in part, differences ring in smaller numbers and staining densely blue with between the cells of epicardial growths (Fig. 41) and May-Griinwald-Giemsa were of spheroidal shape with those of peritoneal tumors (Fig. 40) suggested in the his- well-defined limiting membranes and cytoplasm. The tological examinations. In the former, the nucleoli were cytoplasm containing numerous small vacuoles stained somewhat smaller and more compact than those in the deeply also with hematoxylin-eosin (Fig. 17), in contrast latter. There was a distinctly greater proportion of con to the faint coloring of the mesothelial cells in the same densed chromatin distributed in small masses throughout smears. The nuclei were relatively large, and nucleoli the nucleoplasm (Fig. 41), and in smaller quantities at were not notable. The identity of the cells was obscure. the nuclear membrane. Other aspects of morphological There was no correlation between cells in the peritoneal differences from the peritoneal growths were the appar fluid and those in the circulating blood. Although it ently smaller proportions of cytoplasm and the possibly seemed likely that the cells were altered mesothelium, smaller concentration of cytoplasmic ribosomes. the morphology of the cells could not be unequivocally Collagen fibers clearly arose from the mesothelial tu identified with that of the tumor elements viewed in sec mor cells (Figs. 38 and 43). At least one other type of cell tions. occurred, also, in association with the larger collagen Ultrastructure. A notable feature of mesothelial re deposits. Cells of this sort (Figs. 42 and 43) were spindle- sponse to MC29 virus was a relative uniformity in the shaped and presumably represented the fibroblasts de sequential alterations in morphology from the simple rived either from the supporting stroma or conceivably squamous cell type to the rounded epitheloid forms and by metaplastic processes from mesothelioma cells. While the further metaplastic change to cartilage-producing some of the heaviest deposits of collagen were associated cells. Fig. 38 illustrates some of the finer aspects of the with the spindle cells, it was evident that the mesothelial tumor cell morphology already indicated by the histo- tumor cells likewise actively elaborated fibers (Figs. 38 pathological findings. In the growths occurring most com and 43). The light micrographs (Figs. 9 and 13) gave evi monly, such as those of Figs. 12 and 22, the large nucleoli dence that the intercellular hyaline material was elabo and the paucity of condensed chromatin in the spheroidal rated principally by altered or metaplastic mesothelium.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1970 American Association for Cancer Research. Chabot, Beard, Langlois, and Beard and Fig. 40 illustrates cisternae of such cells greatly dis susceptibility of the epicardium and pericardium as com tended with material like that outside the cell. The spin pared with the peritoneum. dle-shaped cells (Figs. 42 and 43) likewise showed dis In contrast, much evidence of specificity of virus in tended cisternae. fluence on the characteristics of cell alteration or meta plasia is evident from consideration of certain MC29 ef fects. Infection of chick embryo cells results in rapid DISCUSSION morphological alteration (4, 8, 16) of a type not apparent with other leukosis viruses. Specificity of MC29 virus Mesothelial cell tumors induced by strain MC29 virus action on myeloid hematopoietic tissue both in tissue cul represent the addition of a distinctly different neoplastic ture and the host is well demonstrated (15) by the differ process to the increasingly broad recognized spectrum ence between the morphology and growth potentials of (2) of oncogenic responses to the avian leukosis viruses. myeloblasts resulting from BAI strain A virus infection Unusual aspects of strain MC29 oncogenic activity have and the analogous attributes of the myelocytes respond already been evident by differences from other strains ing to MC29. In both cases the cells initially affected by not only in the spectrum of tissues affected but in the the respective viruses were the same. Another example characteristics of cell response (4, 8, 9, 13, 15, 16, 18). In is the marked difference between the complex BAI this respect, responses to this agent are of special signi strain A virus-induced nephroblastoma (10, 11) consisting ficance in distinguishing elements of influence specific to of both differentiated and primitive mesenchymoma, the virus from manifestations of common intrinsic cell well-developed fibrosarcoma, cartilage, osteoid forma potential for alterations of morphology and function. tion, and keratinization in comparison with the simple, As other tissues affected by leukosis agentsâ€”except relatively well-differentiated glomerular and tubular re hepatic parenchyma (9), which is derived from endo- nal carcinoma associated with MC29 infection (18). The dermâ€”mesothelial cells are of mesodermal origin. In tissues affected by both viruses are the nephrogenic cell contrast to the relatively primitive hematopoietic cells rests in the kidney after hatching of the host. Particular affected by all leukosis strains, the mesothelium is a characteristics of several types of cellsâ€”chick embryo "mature" well-differentiated tissue. Virus infection re fibroblasts (8) and myeloid (16) and mesothelial cellsâ€” sults in initial alteration of squamous to, likewise, well- infected with MC29 virus are the much enlarged nucleo- differentiated cuboidal, low columnar, or spheroidal epi lus, the occurrence of chromatin in the diffuse state, and thelial cells constituting the tumors. Progressive transi the high ribosome content of the cytoplasm. Influence of tion of some growths to cartilage results from a second the cell per se, however, is still manifest by variation in distinct process of neoplasia progressing from the initial these attributes as between the epicardial and peritoneal differentiated metaplastic progenitor to the terminal tumors. stage of differentiation to cartilage. Derivation of chon- Another significant feature of the virus-induced meso- drocytesâ€”i.e., cells producing fibers and intercellular theliomas is their remarkable similarity to tumors occur hyaline materialâ€”from neoplastic mesothelium was eas ring in mammals, particularly in man. These neoplasms ily traced by the electron micrographie evidence of for (1, 25-27) of unknown etiology characteristically exhibit mations of these components by typical tumor cells. multiple foci of growth both in the peritoneum and the There was some indication that associated nonneoplastic pleura in association with accumulation of fluid. Growths spindle cells also contributed to the fibers and, perhaps, as described in man and bovine (1) may consist of papil- hyaline material. In principle, the alterations implied lomatous primary epithelial formations or of principally systematic directional change representing the resultant fibrous processes with small amounts of epithelium. A of the individual vectors of virus effects, cell genetic po possible explanation of the multiple origin of the mam tential for morphological and functional expression, and malian growths is spread by tumor cell implantation, but the influence of tissue microenvironment on both. consideration of a possible virus etiology of the conditions Without an established basis for comparison, it is dif should not be neglected. Epithelial cell morphology in the ficult to distinguish between specific virus effects and mammalian tumors resembles closely that of the chicken manifestation of cell potential in the alterations to virus- neoplasms. Nuclei and nucleoli are described as large, induced neoplasms. Some aspects of cell influence on at but neither is as prominent as the corresponding organ- tributes of metaplasia, nevertheless, are readily apparent. elles in the chicken tumors, and much of the chromatin Most prominent are selective susceptibilities of various in the mammalian growths is of the condensed type. cells or tissues to infection with different leukosis agents. Notable differences are the apparently slow growth, the Especially impressive in the present studies are the var frequent excess of fibrous tissue, and the absence of car iations in the susceptibility of mesothelial cells from the tilage in the growths in man. Spread by invasion of con- high response of those of the mesentery to the absence tigous tissue but not by distant metastasis occurs alike in of growths arising in the mesothelial integument of the both avian and mammalian conditions. viscera such as the liver, kidney, and spleen. Even in the In the study of pleural and peritoneal mesotheliomas mesentery where mesothelium was almost uniformly in man, the tissue of origin of the growths was not ini affected, growth occurred only in widely separated foci. tially clear, but diagnosis was aided by tissue culture (27). A further aspect of cell potential was the distinctly lower Origin of the neoplasms in the chicken was obvious, since

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the numerous tumors at different stages of development (Wilms' Tumor): Ultrastructure. J. Nati. Cancer Inst.. 29: 41 105. afforded ample opportunity for tracing the growth. There 1962. was no reason to doubt the virus etiology of the condition. 11. Ishiguro. H.. Beard, D., Sommer. J. R.. Heine, U., de ThÃ©.G.. The tumors were not observed in the absence of virus and Beard. J. W. Multiplicity of cell response to the BAI Strain A (Myeloblastosis) Avian Tumor Virus. I. Nephroblastoma inoculation; incidence was related to virus dose deter (Wilms' Tumor): Gross and Microscopic Pathology. J. Nati. Can mined by virus particle count; particles of the morphol cer Inst.. 29:1 39, 1962. ogy typical of that of the RNA leukosis agents (3) were 12. Ivanov. X., Mladenov, Z., Nedyalkov. S., Todorov, T. G., and frequently seen; and virus buds occurred occasionally in Yakimov, M. Experimental Investigations into Avian Leucoses. the peripheral membrane of the characteristic tumor V. Transmission, Haematology and Morphology of Avian Myelo- cells. Opportunity for further studies is afforded by cytomatosis. Bull. Inst. Path. Comp. Anitn.. 10: 5 38, 1964. ready growth of the tumor cells in vitro. In work already 13. Langlois, A. J., and Beard, J. W. Converted-Cell Focus Formation begun, it was observed that at least 2 and probably 3 in Culture by Strain MC29 Avian Leukosis Virus. Proc. Soc. distinct types of cells emerge in culture. A prominent Exptl. Biol. Med., 126: 718 722, 1967. culture component consists of cells of epithelioid appear 14. Langlois. A. J., Bolognesi. D. P.. Fritz, R. B.. and Beard. J. W. ance. Others, however, are of the same morphology as Strain MC29 Avian Leukosis Virus Release by Chick Embryo Cells Infected with the Agent. Proc. Soc. Exptl. Biol. Med., 131: that of chick embryo cells altered in vitro by MC29 virus. 138-143, 1969. Continuation of the experiments should provide further 15. Langlois, A. J., Fritz, R. B., Heine, U.. Beard. D.. Bolognesi, D. P., information of the metaplastic potentials of the mesothe- and Beard, J. W. Response of Bone Marrow to MC29 Avian Leu lial cells under the influence of strain MC29. kosis Virus in Vitro. Cancer Res., 29: 2056-2074, 1969. 16. Langlois. A. J.. Sankaran. S.. Hsiung, P. L.. and Beard, J. W. Massive Direct Conversion of Chick Embryo Cells by Strain REFERENCES MC29 Avian Leukosis Virus. J. Virol.. /: 1082 1084. 1967. 17. Lison. L. Alcian Blue 8 G with Chlorantine Fast Red 5 B. A 1. Ackerman. L. V. Tumors of the Retroperitoneum, Mesentery, Technic for Selective Staining of Mucopolysaccharides. Stain and Peritoneum. In: H. I. Firminger (ed.). Atlas of Tumor Pa Technol.,29:13l-138, 1954. thology, pp. 97 136. Washington, D. C.: Armed Forces Institute of 18. Mladenov, Z., Heine, U.. Beard. D.. and Beard. J. W. Strain Pathology, 1954. MC29 Avian Leukosis Virus. Myelocytoma, Endothelioma, and 2. Beard. J. W. Introduction to Avian Leukemia. In: M. A. Rich Renal Growths: Pathomorphological and Ultrastructural Aspects. (ed.). Experimental Leukemia, pp. 205 232. New York: Apple- J. Nati. Cancer Inst.. 38:251 285, 1967. ton-Century-Crofts, 1968. 19. Palade. G. E. A Study of Fixation for Electron Microscopy. J. 3. Beard, J. W., Bonar, R. A., Heine, U., de ThÃ©,G.,and Beard, D. Exptl. Med., 95:285 298. 1952. Studies on the Biological. Biochemical, and Biophysical Proper 20. Powell. T. E., Ill, Philpott, C. W.. and Maser. M. D. On the ties of Avian Tumor Viruses. In: Viruses, Nucleic Acids, and Hydrogen Ion Concentration and Osmolality of Fixative Compo Cancer, The University of Texas-M. D. Anderson Hospital and nents. J. Cell Biol.. 23: 110A. 1964. Tumor Research Institute at Houston, pp. 344 373. Baltimore: 21. Reynolds, E. S. The Use of Lead Citrate at High pH as an Elec The Williams & WilkinsCo., 1963. tron-opaque Stain in Electron Microscopy. J. Cell Biol.. 17: 208 4. Bolognesi, D. P., Langlois, A. J.. Sverak, L., Bonar, R. A., and 212. 1963. Beard, J. W. In Vitro Chick Embryo Cell Response to Strain 22. Rubin. H. A Virus in Chick Embryos Which Induces Resistance MC29 Avian Leukosis Virus. J. Virol.. 2: 576-586, 1968. in Vitro to Infection with Rous Sarcoma Virus. Proc. Nati. Acad. 5. Eckert. E. A., Beard, D., and Beard, J. W. Dose-Response Rela Sci. U.S.,46.1105 1119. 1960. tions in Experimental Transmission of Avian Erythromyeloblastic 23. Sabatini. D. D.. Bensch, K.. and Barrnett, R. J. Cytochemistry and Leukosis. III. Titration of the Virus. J. Nati. Cancer Inst., 14: Electron Microscopy. The Preservation of Cellular Ultrastructure 1055-1066, 1954. and Enzymatic Activity by Aldehyde Fixation. J. Cell Biol.. 17: 6. Eckert, E. A., Beard, D., and Beard, J. W. Dose-Response Rela 19-58, 1963. tions in Experimental Transmission of Avian Erythromyeloblastic 24. Sharp, D. G., and Beard, J. W. Counts of Virus Particles by Sedi Leukosis. V. Influence of Host Age and Route of Virus Inocula mentation on Agar and Electron Micrography. Proc. Soc. Exptl. tion. J. Nati. Cancer Inst., /5: 1195-1207. 1955. Biol. Med.. 81: 75 79, 1952. 7. Freeman, J. A., and Spurlock. B. O. A New Epoxy Embedment 25. Stout, A. P. Solitary Fibrous Mesothelioma of the Peritoneum. for Electron Microscopy. J. Cell Biol.. 13:437-443. 1962. Cancer, 3:820-825, 1950. 8. Heine, U., Langlois. A. J.. Riman, J., and Beard, J. W. Ultra- 26. Stout. A. P. Mesotheliomas of the Pleura and Peritoneum. J. structure of Chick Embryo Cells Altered by Strain MC29 Avian Tennessee Med. Assoc.,44:409 411, 1951. Leukosis Virus. Cancer Res., 29:442 458, 1969. 27. Stout, A. P.. and Murray, M. R. Localized Pleural Mesothelioma; 9. Heine. U., Mladenov, Z., Beard. D.. and Beard, J. W. Morphology Investigation of Its Characteristics and Histogenesis by the Method of Hepatoma Induced by Strain MC29 Avian Leukosis Virus. ///. of Tissue Culture. Arch. Pathol...J4. 951 964. 1942. Program of the 24th Annual Meeting of the Electron Microscopy 28. Waters, N. F. Breeding for Resistance and Susceptibility to Avian Society of America. B-21. San Francisco. August 22 to 25, 1966. Lymphomatosis. Poultry Sci., 24:259-269, 1945. 10. Heine. U., de ThÃ©.G.. Ishiguro, H., Sommer. J. R., Beard, Dâ€ž 29. Watson. M. L. Staining of Tissue Sections for Electron Micros and Beard, J. W. Multiplicity of Cell Response to the BAI Strain copy with Heavy Metals. J. Biophys. Biochem. Cytol., 4: 475 478, A (Myeloblastosis) Avian Tumor Virus. II. Nephroblastoma 1958.

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Fig. 1. Loop of intestine (Y357, 39 dpi) illustrates numerous small, early mesotheliomas in the mesentery. The minute tumors (Figs. 6 and 11) were distributed in widely separated foci and were not evident on the surface of the intestine. Very few were in the mesentery along the vascular channels. Reference lines in Figs. 1 to 5 represent 1 cm. Fig. 2. Large and small individual growths (Y636, 46 dpi) occupy almost all of the mesentery and incorporate the pancreas (P) in the confluent tumor masses. Even with such massive growth, the surface of the intestine showed only occasional tiny tumors. Nevertheless, growths were nu merous at the junction (J) of the mesenteric with the intestinal mesothelium. Fig.3. Growths of varying sizes (Y160, 42 dpi) in the mesentery and involving the pancreas (P). Thin, loose tags of omentum (O) or mesentery were sheets of cartilage papillomas as illustrated in Figs. 8 and 9. Fig. 4. Growths similar to those of Fig. 2 (YI74, 30 dpi). Fig. 5. Tumors (Y104, 43 dpi) arising in confluent masses in the epicardium ensheathing both the auricles (upper arrow) and the ventricles (lower arrow) (see Fig. 7). Fig. 6. Low-power survey section of growths in mesentery (Y357, 34 dpi) shown in Fig. 1. A specimen of the mesentery was fixed and em bedded flat and sectioned in planes parallel to the surface. Both sides of the mesentery (A/) are covered with uniformly rounded or pear-shaped altered mesothelial cells (see Fig. 10). Numerous papillomas (PA) arise in complex structural arrangement from narrow pedicles (Z.) (see Fig. 11). X 50. Fig. 7. Sectionâ€”made in the same way as that of Fig. 6â€”ofpapillomatous processes arising from epicardium (Y330, 87 dpi) covering the auri cles as in Fig. 5. The growths arose from narrow pedicles (L). Relatively few of the mesothelial cells responded: the others remained as thin, squamous epithelium (SE). The micrograph shows the relationship of the papillomas to muscle of the auricle (AM) (see Fig. 41). X 50. Fig. 8. Papillomatous growths (Y160, 42 dpi) consisting almost wholly of cartilage (C) arising from narrow (L) and broad (LL) mesenteric pedicles (see Fig. 9). X 50. Fig. 9. Higher magnification of a part of a growth like that in Fig. 8. The tissue is predominantly cartilage (C) with few unaltered mesothelioma cells (ME). X 340. Fig. 10. Perpendicular section of mesentery (M) of Y357 shown in Figs. 1 and 6 illustrating response to MC29 virus activity of mesothelial cells altered to spheroidal or pear shapes on both sides of the membrane. There is the suggestion of a beginning papilloma (arrow), x 340. Fig. II. Papilloma arising by a narrow pedicle from the mesentery of Y357 (Figs. I and 6) illustrating the complex branching of the growth consisting of spheroidal, altered mesothelial cells with large clear nucleoplasm, and large, prominent nucleoli which have a "bird's eye" appear ance. X 340. Fig. 12. Well-developed mesothelioma (Y350. 34 dpi) arising in the peritoneal mesentery showing loose structure of characteristic cells. The cells of variable external conformation from spheroidal to elongated shapes contain nearly round nuclei with clear nucleoplasm and large nucleoli (see electron micrograph of Fig. 38). A growth with compact structure and closely packed cells is illustrated in Fig. 22. X 340. Fig. 13. Micrograph (Y363, 28 dpi) illustrates a focus (O of beginning progressive metaplasia of mesothelioma cells of typical morphology to cartilage (see Fig. 14). The cells vary from spheroidal to spindle shape, and many contain the prominant nucleolus in clear nucleoplasm. X 340. Fig. 14. Portion of tumor (Y104, 43 dpi) stained with Alcian blue showing hyaline cartilage (C) with chondrocytes (CH) in the process of dif ferentiation from unaltered typical mesothelioma cells (ME). X 340. Fig. 15. Portion of compact mesothelioma (Y379, 48 dpi) like that of Fig. 22 stained by Masson's technique illustrating the content of connec tive tissue (ST). X 340. Fig. 16. Smear from buflfy coat obtained by centrifuging peritoneal fluid from bird (Y361, 28 dpi) with mesothelioma and stained with May- Grunwald-Giemsa. Almost all of the cells are typical, unaltered squamous mesothelium. They contain fairly regular, ovoid nuclei with pink-stain ing granular chromatin. The cell limits are indefinite, and the broad, thinly spread cytoplasm contains many small vacuoles. X 725. Fig. 17. Smear prepared as that for Fig. 16 (Y363, 28 dpi) but stained with hematoxylin-eosin. Cells with distinctly stained vacuolated cyto plasm contain relatively large nuclei (N) eccentrically placed. Origin of these elements is obscure but may represent morphologically altered me senteric mesothelial cells like those of Fig. 6. Other cells only faintly stained are of a morphology resembling that of the mesothelial cells of Fig. 16. X 725. Fig. 18. Survey micrograph of mesothelioma of Bird Y879, 41 dpi (8.5 X 10' virus particles i.p.) illustrating a pseudoglandular type of growth with numerous large and small tubules and cyst-like structures. X 150. Fig. 19. Higher magnification of a portion of the same growth as that of Fig. 18. Cells forming tubules are the deeply stained, "bird's eye" type epithelium like the altered mesothelium illustrated in Fig. 11. The interstitial cells have the morphology of the commonly occurring meso thelioma (Figs. 12 and 22). Organized connective tissue stroma is not evident. X 340. Fig. 20. Tubular or papillomatous architecture of epithelium in the same growth as that of Fig. 18. X 340. Fig. 21. Another form of pseudotubular or pseudoacinar arrangement of epithelial cells in same growth as that of Fig. 18. The tubules or strands of cells are supported by a delicate spindle cell stroma. X 340. Fig. 22. Mesothelioma (Y641. 60 dpi) of typical compact tumor architecture occurring as separate tumor for comparison with Figs. 23 to 25. X 340. Fig. 23. Neoplastic cells (IT) invading liver from the surface in the same bird (Y641) yielding the growth of Fig. 22. Only vestiges of com pressed parenchyma cells (LP) remain. X 340. Fig. 24. Neoplastic cells invading spleen from the surface of the organ in the same bird (Y641) yielding the growth of Fig. 22. The cells appear in a syncytial-like arrangement with indeterminate peripheral outlines. Almost all of the splenic tissue (SP) has been replaced. X 340. Fig. 25. Minute myeloma in the liver (YIOO, 41 dpi) consisting of myelocytes without granules exhibiting morphology resembling mesotheli oma cells (Fig. 22), the cells of the invading tumors of the liver (Fig. 23) and spleen (Fig. 24), and the elements of myelocvtomatosis of the liver (Figs. 36 and 37). X 340. Fig. 26. Micrograph of pancreas (Y363, 28 dpi) illustrating tumor cell (IT) invasion with permeation of normal acinar structure (A) by neoplastic cells. X 340. Fig. 27. Disorganization of pancreatic structures (A) and invasion by highly irregularly distributed anaplastic mesothelioma cells (IT) (Y169, 30 dpi). X 340.

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Fig. 28. Invasion of intestinal wall (Y173. 30 dpi) by tumor cells (IT) penetrating the muscularis mucosa (OS) and continuing into the lamina propria. X 340. Fig. 29. Invasion of the ovary (Y165, 41 dpi) by mesothelial tumor cells (IT) and distribution of the cells about the follicles (OÃ.).X 340. Fig. 30. Micrograph of epicardium (Y330, 87 dpi) enveloping the region of the auricles illustrating mesothelial response (ES) to virus. The cells are of spheroidal or low cuboidal shape with prominent although not greatly enlarged nucleoli in spheroidal nuclei. Despite evident similari ties, the response of the epicardial cells differs from that of the mesenteric serosa in nuclear and nucleolar morphology (compare with Fig. II). The edge of tumor cell invasion of the subepicardial tissue is shown above (IT). X 340. Fig. 31. Epicardial tumor (Y104, 43 dpi) invading deeper tissues (IT) adjacent to auricular muscle (AM). Tumor consists principally of sphe roidal cells of nuclear characteristics resembling those of the cells in Fig. 30. Epicardial cells (arrow) enclosing tumor and adjacent musculature are flat and show no evidence of response to virus (see Fig. 7). The tumor cells lie separated in a smooth matrix staining lightly blue with the appear ance of cartilage (C) which was better defined in other epicardial growths. X 340. Fig. 32. Portion of tumor (IT) (Y108, 43 dpi) which had penetrated and was completely surrounded by auricular heart muscle (AM). Mor phology of the tumor resembled closely that of growths in mesentery (see Figs. 12 and 22). Invasion of the heart muscle and incorporation of mus cle cells into the growth are evident at the periphery of the tumor (see Fig. 41). X 340. Fig. 33. Papilliform growth with capsule cells bounding the growth (arrow) (Y330, 87 dpi; see Fig. 7) consisting of altered epicardial cells with features of morphology resembling those described for Fig. 30. Blood-filled channels (BO were not infrequent in mesotheliomas and myelocytic growths induced by MC29 virus (see Fig. 36). X 340. Fig. 34. Tumor in subcutaneous tissue (Y148, 32 dpi) at the site of virus injection into the wing web. Micrograph shows the edge of large growth similar to that illustrated in Fig. 12. Cells capillary (A1)with the endo- thelial cell (D) and pericyte (W). A few virus particles (K) are in intercellular spaces. X 12,300.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1970 American Association for Cancer Research. Mesotheliomas of Peritoneum, Epicardium, and Pericardium Induced by Strain MC29 Avian Leukosis Virus

J. F. Chabot, Dorothy Beard, A. J. Langlois, et al.

Cancer Res 1970;30:1287-1308.

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