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

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1 [CANCER RESEARCH 30, , 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 SUMMARY Injection of strain MC29 avian leukosis virus into the peritoneal, pericardia!, and air sac cavities of the chicken resulted in high incidence of tumors of the mesothelium of the respective structures. As determined by light and electron microscopy, the growths arose as papillomas or expanding tumors by alteration of the squamous mesothe lium to spheroidal or cuboidal cells characterized by rounded nuclei with clear nucleoplasm and very large nucleoli and cytoplasm deeply stained with hematoxylin. With continued rapid growth, the nuclear features les sened somewhat, cytoplasmic staining diminished greatly, and the cell limits became indistinct. A second stage of metaplasia was marked by frequent alteration of the epithelioid cells to cartilage which increased both by con tinued alteration of peripheral mesothelioma cells and proliferation of chondrocytes. The tumors, sometimes isolated but usually coalesced in masses, were poorly en capsulated, contained little fibrous stroma, and showed no necrosis. They readily invaded contiguous visceral tis sues but did not metastasize to distant sites. Numerous virus particles were observed in the tumors together with occasional budding of the particles from the cell mem branes. The mesotheliomas were an addition to the al ready broad spectrum of responses to strain MC29 virus consisting of myelocytic growths, high incidence of renal tumors, primary tumors of the liver parenchyma, and singular aspects of morphological alteration of chick em bryo cells in vitro. These virus-induced tumors in the chicken closely resembled mesotheliomas of unknown etiology in man and bovines in both morphology and be havior. INTRODUCTION Strain MC29 avian leukosis virus infection in the chicken results in the induction, principally, of myelocytoma and myelocytomatosis (12, 18). In addition to these 1This work was aided by USPHS Grant C-4572, by the Annie Ma bel Sherris Memorial Grant for Cancer Research from the American Cancer Society, Inc., by National Defense and Education Act Title IV Fellowship , by IN-611 American Cancer Society International Research Grant, and by the Dorothy Beard Research Fund. The paper is part of the studies submitted by J. F. C. in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Zoology in the Graduate School of Arts and Sciences of Duke University. Received September 12, 1969; accepted November 19, myeloid hematopoietic tissue neoplasms distributed in bone, liver, and other sites, primary renal tumors occur in high incidence, erythroblastosis is occasionally seen, and, in small proportion, growths arise from hepatic parenchymal cells (9). A singular influence of the MC29 strain is the infection and rapid morphological alteration of CEC" in tissue culture not observed with other leuko sis agents (4, 8, 16). Recent studies initiated with the in u/7ro-altered CEC revealed still another unusual host re sponse to this leukosis strain. Cells inoculated into the chicken by different routes gave rise to tumors simulating transplants at various sites in the peritoneum. However, introduction in the same way of cell-free fluid from CEC cultures altered by MC29 virus resulted in the appear ance of growths at similar sites and, also in the pericar dium and epicardium. Further investigation showed that certain of the tumors were the result of neoplastic re sponse of peritoneal and pericardial mesothelium to in fection with the MC29 virus. Studies have been made of the occurrence, pathomorphology, and ultrastructure of some of these tumors, and the results are described in this report. For comparison there are considered, also, 2 tumors in subcutaneous tissue and myelocytic1 growths in visceral organs. Growths induced by response to prep arations of morphologically altered cells will be consid ered in another report. MATERIALS AND METHODS Virus. Isolation of strain MC29 leukosis agent and chicken response to virus obtained from blood plasma 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 sage in vitro. Passage was in primary cultures (14) of embryos from eggs of RIF-free White Leghorn hens4 (22). Fluids in contact with cells of altered morphology for 4.5 to 6 hr in cultures 6 to 15 days following exposure "The abbreviations used are: CEC, chick embryo cells: RIF, re sistance-inducing factor; WW, subcutaneous wing web tissue; i.e.. intracardiac: dpi, days postinoculation. ' The term myelocyte is used in descriptions of strain MC29 virus disease to designate nonmyeloblast myeloid elements at any level of differentiationâ with or without granules (15, 18)â lessthan that of the mature granulocytes. 4The chick embryos were from White Leghorn eggs provided by Dr. Roy Luginbuhl, University of Connecticut, through the Research Resource Program of the National Cancer Institute Bethesda, Md. MAY

2 Chabot, Beard, Langlois, and Beard to virus (14) were harvested and passed through 0.45-^ Millipore filters. Virus particle content and infectivity of the fluids were measured by electron microscopy (24) and focus assay (13), respectively. Chickens and Examinations. White Leghorn chicks of line 15 (28) were inoculated 1 day after hatching with virus amounts and by WW, i.e., or i.p. routes as indicated in Table 1. Blood smears studies (5) were stained made routinely as in earlier with May-Grunwald-Giemsa. At the onset of illness or after appropriate periods of study, birds were killed by bleeding from the heart. Tis sues for light microscopy were taken in Zenker-formol solution, and the sections were stained with hematoxylin and eosin. Some sections were stained with Alcian blue and by Masson's technique. Cells from peritoneal fluid were stained with May-Grunwald-Giemsa or hematoxylin-eosin. Specimens for electron microscopy were fixed for 2 to 4 hr in 5% glutaraldehyde (Fisher G-151, Fisher Scien tific Co., Silver Spring, Md.) buffered at ph 7.2 with sodium cacodylate (20, 23); transferred to 0.1 M sodium cacodylate buffer, ph 7.4, containing 0.3 M sucrose (23); and held for 2 to 5 days at 4Â.The tissues were postfixed for 1 hr with 1% osmic acid buffered at ph 7.2 with Vero nal (19), embedded in Maraglas (7), sectioned with glass knives on a Porter-Blum ultramicrotome, stained with uranyl acetate (29) and lead citrate (21), and examined with a Siemens Elmiskop I at 80 kv equipped with a double condenser and a 50-/Â objective aperture. RESULTS Table 1 summarizes 3 experiments yielding the results described here (Bird Y879, Figs. 18 to 21, from a differ ent study was given an injection i.p. of 8.5 X 10' virus particles). Of 148 birds receiving inoculations into the peritoneal cavity in the 3 studies, 52 had tumors of de monstrable mesothelial cell origin. Incidence was defi nitely related to dose as measured by numbers of virus particles. Introduction of virus into the heart through the chest wall gave neoplasms of the epicardium or pericar dium or both in 4 of 19 birds. Injections into the wing webs of 19 chicks resulted in l (Y148) apparently virusinduced subcutaneous growth. Another tumor in subcu taneous tissue (Y623) arose in the abdominal wall near the site of i.p. inoculation. Generalized response of the myeloid hematopoietic cells consisted principally of diffuse infiltration of the liver by nongranulated myelocytesâ myelocytomatosisâ with few circumscribed cell aggregatesâ myelocytomas; few of the cells contained eosinophilic granules. Except in birds receiving inocu lations i.e. (into the circulating blood), myeloid cell re sponse was relatively small. Line 15 chickens were less responsive to strain MC29 in this respect than the Shaver birds previously used (18). Moreover, as with BAI strain A (myeloblastosis) virus (6), the i.p. inoculation route was much less effective in the induction of generalized mye loid response than the i.v. or i.e. routes. Peritoneal Tumors. Birds with tumors in the peritoneal cavity exhibited general signs of lassitude, droopiness, disinterest, and ruffled feathers. The skin and mucous membranes were pale, and the blood smears of many of the birds showed large mononuclear primitive cells (15, 18) of the myeloid series. Peritoneal fluid in volumes of about 8 to 80 ml was found in a large proportion of the birds, and some animals showed pronounced abdominal distension. The murky, greenish fluid contained large numbers of mesothelial cells and other mononuclear ele ments. Figs. 1 to 4 illustrate the gross appearance of minute beginning MC29 virus-induced growths (Fig. 1) and that of large (Fig. 2) and smaller (Figs. 3 and 4) developing neoplasms. An impressive feature of the process was the high growth response of the mesothelial cells in the loose folds of mesentery. This was indicated by the numerous tiny individual growths (Fig. 1) and emphasized by the 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.. iment v6(x ) no mye pcs pts ery10 (ml) routei.e.wwi.p.i.p.i.p.i.p.i.p.i.p.i.p.i.p.chicks inoculatedy100-i19y y y y y y y y y (20)(19)(19)(18)(19)( Volume 1288 CANCER RESEARCH VOL. 30

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

4 Chabot, Beard, Langlois, and Beard periphery (Figs. 6 and 33). The outer layer of other tu mors, particularly of those in the epicardium, was squamous epithelium (Figs. 7 and 31). Many of the compact growths, like that of Fig. 22, contained no intercellular material stained by Masson's technique, but some of the same growths showed branched fibers of unidentified material stained with Alcain blue (17). Variations ranged from these thin strands to distinct but narrow intercon necting bands (Fig. 15) stained by Masson's technique. There were no predominantly fibrous growths as such. Most of the tumors of loose structure (Fig. 12) did not show staining of the intercellular material with either Alcian blue or the Masson stains. Necrosis was not ob served in any of the tumors. Pseudoglandular formations were encountered in only a few instances. The survey picture of Fig. 18 reveals numerous tubules formed of cells of the morphology demonstrated at higher magnification in Fig. 19. The tubule-like arrangements consisted of epithelial cells ly ing in a loose "stroma" of elements indistinguishable from those of the typical mesothelioma growth. Fibrous connective tissue was not discernible. In another section of the same growth (Fig. 20), many "tubules" were cut both lengthwise and crosswise. This aspect of the tumor has the appearance of an unusual papillomatous struc ture. Fig. 21 illustrates another type of pseudotubular structure consisting of epithelioid cells bounded in bun dles or strands by well-defined stroma and traversed by thin delicate capillaries. With few exceptions, the mesotheliomas were of char acteristic histological morphology and easily distinguish able from other MC29-induced growths. Occasionally, however, the mesothelioma cells exhibited features sur prisingly similar to analogous aspects of myelocytic growths. Fig. 22 illustrates the morphology characteristic of a mesothelioma and Figs. 23 and 24 are sections of tu mors in the same bird invading the liver and spleen, re spectively. The 3 growths show both similarities and differences related particularly to prominence of the nu cleoli and relatively large nuclei with clear nucleoplasm. Certain attributes of these tumors are likewise similar to the cells of the minute myelocytoma in the liver seen in Fig. 25. Further comparisons are made to the mylocytes of myelocytomatosis massively invading the liver as in Fig. 36 and permeating the hepatic sinuses as illustrated in Fig. 37. The tumors of Figs. 23 and 24 were of uncer tain origin, but both arose at the respective surfaces of the spleen and liver. The neoplasm of Fig. 23 bore a striking resemblance to the mesothelioma of Fig. 21; the cells of the growth in the spleen (Fig. 24) exhibited a syncytial appearance singular with respect to the mor phology of the other tumors exemplified. Attributes of tumor malignancy were expressed by rapid growth and by invasion and infiltration of contig uous organ tissues but not by distant mã tastases.the pancreas was regularly involved. Small circumscribed growths often appeared at the surface of the organ, but in many regions the tumor cells penetrated and de stroyed the pancreatic structure (Figs. 26 and 27). Inva sion was usually effected by encroachment on a broad tumor front (Fig. 26), but highly irregular and anaplastic growth was also evident (Fig. 27). Frequent infiltration of the intestinal wall resulted in overgrowth and destruc tion of muscle with extension through the muscularis mu cosa into the lamina propria (Fig. 28). Fig. 29 illustrates typical invasion of the ovary. Epicardial and Pericardia! Tumors. Like the perito neum, the epicardial and pericardia! mesothelium is a monolayer of simple squamous epithelium. Pathomorphological response of the epicardial cells to infection with strain MC29 was similar to (Figs. 5, 7, and 30 to 34) but yet different in some respects from that of the peri toneum. The gross appearance of the epicardial growths (Fig. 5) was characterized by sheets or masses of con fluent cauliflower-like papillomatous protrusions, as in the auricular region (upper arrow), or somewhat individ ual tumors of like appearance in the epicardium of the ventricles (lower arrow). Similar papillary growths oc curred in the pericardium. The pronounced response of the epicardium closely adherent to cardiac muscle was in marked contrast to the comparatively few and minor tu mors in the peritoneum covering the walls of the intes tine and other organs and the lack of growths originating in the parietal peritoneal layer. Papillomatous growth of the epicardium (Fig. 7) con sisted in numerous pendunculated and filiform processes, some consisting mostly of epithelial cells (Fig. 31) and others with a preponderance of connective tissue. In Fig. 7 it is evident that the epicardial cells were less respon sive to the virus, since most of them that were not a part of the growths retained the squamous morphology. Cells which did respond were of low cuboidal shape (Fig. 30) with deeply stained cytoplasm. The spheroidal nuclei ex hibited clear nucleoplasm and prominent nucleoli which, nevertheless, were distinctly smaller than those in cells of the peritoneal tumors. The tumors were often pearly white and hard and contained nodules of cartilage (Fig. 31). The growths invaded the cardiac muscle and formed extensions deep in the heart wall (Fig. 32). The crosssection histology of a pericardia! papillary process (Fig. 33) resembled that of analogous growths from the peri toneum (Fig. 11). Subcutaneous Growth. A tumor (Fig. 34) in the sub cutaneous tissue at the site of virus inoculation occurred in the wing web in only 1 bird. Masses of individual cells permeating the muscle and interstitial tissues and consti tuting the associated solid growth were morphologically indistinguishable from neoplasms like that of Fig. 22. The invading cells at the edge of the main growth were of indistinct outlines but contained large spheroidal nu clei and large nucleoli. The origin of the tumor could not be determined. Although the cells were like those of the peritoneal growths, they likewise exhibited some charac teristics of myelocytoma. It does not seem too remote to regard the tumor as a derivative of vascular endothelium in the inoculated 1-day-old chick. Another growth in the subcutaneous tissue was in a bird inoculated into the peritoneal cavity and appeared 1290 CANCER RESEARCH VOL. 30

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

6 Chabot, Beard, Langlois, and Beard and Fig. 40 illustrates cisternae of such cells greatly dis tended with material like that outside the cell. The spin dle-shaped cells (Figs. 42 and 43) likewise showed dis tended cisternae. DISCUSSION Mesothelial cell tumors induced by strain MC29 virus represent the addition of a distinctly different neoplastic process to the increasingly broad recognized spectrum (2) of oncogenic responses to the avian leukosis viruses. Unusual aspects of strain MC29 oncogenic activity have already been evident by differences from other strains not only in the spectrum of tissues affected but in the characteristics of cell response (4, 8, 9, 13, 15, 16, 18). In this respect, responses to this agent are of special signi ficance in distinguishing elements of influence specific to the virus from manifestations of common intrinsic cell potential for alterations of morphology and function. As other tissues affected by leukosis agentsâ except hepatic parenchyma (9), which is derived from endodermâ mesothelial cells are of mesodermal origin. In contrast to the relatively primitive hematopoietic cells affected by all leukosis strains, the mesothelium is a "mature" well-differentiated tissue. Virus infection re sults in initial alteration of squamous to, likewise, welldifferentiated cuboidal, low columnar, or spheroidal epi thelial cells constituting the tumors. Progressive transi tion of some growths to cartilage results from a second distinct process of neoplasia progressing from the initial differentiated metaplastic progenitor to the terminal stage of differentiation to cartilage. Derivation of chondrocytesâ i.e., cells producing fibers and intercellular hyaline materialâ from neoplastic mesothelium was eas ily traced by the electron micrographie evidence of for mations of these components by typical tumor cells. There was some indication that associated nonneoplastic spindle cells also contributed to the fibers and, perhaps, hyaline material. In principle, the alterations implied systematic directional change representing the resultant of the individual vectors of virus effects, cell genetic po tential for morphological and functional expression, and the influence of tissue microenvironment on both. Without an established basis for comparison, it is dif ficult to distinguish between specific virus effects and manifestation of cell potential in the alterations to virusinduced neoplasms. Some aspects of cell influence on at tributes of metaplasia, nevertheless, are readily apparent. Most prominent are selective susceptibilities of various cells or tissues to infection with different leukosis agents. Especially impressive in the present studies are the var iations in the susceptibility of mesothelial cells from the high response of those of the mesentery to the absence of growths arising in the mesothelial integument of the viscera such as the liver, kidney, and spleen. Even in the mesentery where mesothelium was almost uniformly affected, growth occurred only in widely separated foci. A further aspect of cell potential was the distinctly lower susceptibility of the epicardium and pericardium as com pared with the peritoneum. In contrast, much evidence of specificity of virus in 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 morphological alteration (4, 8, 16) of a type not apparent with other leukosis viruses. Specificity of MC29 virus action on myeloid hematopoietic tissue both in tissue cul ture and the host is well demonstrated (15) by the differ ence between the morphology and growth potentials of myeloblasts resulting from BAI strain A virus infection and the analogous attributes of the myelocytes respond ing to MC29. In both cases the cells initially affected by the respective viruses were the same. Another example is the marked difference between the complex BAI strain A virus-induced nephroblastoma (10, 11) consisting of both differentiated and primitive mesenchymoma, well-developed fibrosarcoma, cartilage, osteoid forma tion, and keratinization in comparison with the simple, relatively well-differentiated glomerular and tubular re nal carcinoma associated with MC29 infection (18). The tissues affected by both viruses are the nephrogenic cell rests in the kidney after hatching of the host. Particular characteristics of several types of cellsâ chick embryo fibroblasts (8) and myeloid (16) and mesothelial cellsâ infected with MC29 virus are the much enlarged nucleolus, the occurrence of chromatin in the diffuse state, and the high ribosome content of the cytoplasm. Influence of the cell per se, however, is still manifest by variation in these attributes as between the epicardial and peritoneal tumors. Another significant feature of the virus-induced mesotheliomas is their remarkable similarity to tumors occur ring in mammals, particularly in man. These neoplasms (1, 25-27) of unknown etiology characteristically exhibit multiple foci of growth both in the peritoneum and the pleura in association with accumulation of fluid. Growths as described in man and bovine (1) may consist of papillomatous primary epithelial formations or of principally fibrous processes with small amounts of epithelium. A possible explanation of the multiple origin of the mam malian growths is spread by tumor cell implantation, but consideration of a possible virus etiology of the conditions should not be neglected. Epithelial cell morphology in the mammalian tumors resembles closely that of the chicken neoplasms. Nuclei and nucleoli are described as large, but neither is as prominent as the corresponding organelles in the chicken tumors, and much of the chromatin in the mammalian growths is of the condensed type. Notable differences are the apparently slow growth, the frequent excess of fibrous tissue, and the absence of car tilage in the growths in man. Spread by invasion of contigous tissue but not by distant metastasis occurs alike in both avian and mammalian conditions. In the study of pleural and peritoneal mesotheliomas in man, the tissue of origin of the growths was not ini tially clear, but diagnosis was aided by tissue culture (27). Origin of the neoplasms in the chicken was obvious, since 1292 CANCER RESEARCH VOL. 30

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