THE CYTOLOGY OF THE TUMOR CELL IN THE ROUS CHICKEN SARCOMA

MICHAEL LEVINE 1 (From the Laboratory Division, Montifcore Hospital, New Yo7k City)

The cytology of the Rous chicken sarcoma No. I has attracted the at- tention of biologists since its discovery. Considered broadly, this study has been limited to that group of cells which responds in the animal body to in- vasion by foreign substances. This response to the tumor-producing sub- stances from the Rous tumor, modified by the normal developmental tendencies of the cells, results in a neoplasm which in its well developed stages consists generally of cells of spindle or stellate shape, of a fibroblast-like nature, though round cells of the monocyte type are also present. The origin of these cells has been studied in vivo only after the tumor anlage has been well established. The work is laborious and trying, for all fowls do not respond uniformly to the tumor agent. Sequence in develop- mental stages is difficult to obtain since the tumor-producing substances vary in activity and the fowl may show a reaction akin to resistance. The use of intra- vitam dyes as an aid in identifying certain cell types adds another difficulty, for these stains, while serviceable, may produce toxic or even lethal effects. Added to these difficulties are those more fundamental barriers to a proper interpretation of histologic preparations. At present the hematologists are divided as to the ontology of the various groups of cells that make up the reaction tissue induced by an invading foreign substance. There are at least three different well established views regarding the origin of these cellular elements. Most of our knowledge pertaining to the cytology of the Rous tumor is based on material observed in tissue cultures. It appears, however, from a study of the voluminous literature, much of which is conflicting, that the cells in vitro are no longer parts of a tumor tissue, but merely subsisting isolated elements. The interactions of these cells as they exist in the body have to a large measure been destroyed. It seemed obvious, therefore, that only by standard cytological methods applied to a systematic study of tissue in vivo could a possible solution of the complex problem be attained. The following is a report of a study of the Rous chicken sarcoma from a period shortly after the tumor-producing material is introduced into the body of the fowl to the time when the bird succumbs. The various problems involved in the Rous tumor have been reviewed separately in a number of papers. Foulds’ (40) critical review deals with many of the important phases of this study. Here it will be necessary to refer only to those papers which have a direct hearing on the cytological origin of this neoplasm and the behavior of the cells involved. ’ Aided by grants from the Chemical Foundation and the International Cancer Research Foundation. 276 CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 277

Rous SARCOMAAND OTHERTRANSPLANTABLE TUMORS OF THE FOWL The Rous Chicken Sarcoma No. I (CTZ): Rous (120) in 1910 reported the discovery of a transplantable avian tumor which he described as a spindle- cell sarcoma capable of growth only in a relatively small percentage of young animals and in a stock of barred Plymouth Rock birds intimately related to that in which the tumor had originally occurred. The tumor remained true to type and by frequent transfers to other animals within the varietal stock its malignancy was increased and its invasive and metastatic powers were augmented (121). Within three days after a graft had been implanted, it was found to be vascularized and had spread into the tissue of the host. Metastases to the lungs, liver, kidney, and heart are recorded as of frequent occurrence. ROUS,in his early paper, described changes which occur in the first few days after inoculation. The lymphocytes rapidly accumulate about the nearby blood vessels and around the graft, until, after a period of a week, the latter is entirely enclosed by mononuclears, plasma cells, and fibroblasts, producing the appearance of a lymph node. In the following year, Rous (122, 123) reported the transmissibility of this tumor by cell-free extracts of the neoplasm. The tumors produced by the cell-free filtrates differ from those produced by the introduction of inocula into fowl only in that the former are slower to develop. Histologically, the growth consists of spindle cells arranged in bundles, with a slight vascularizing framework. Small giant cells were observed, especially in the degenerating portions of the tumor. Amitotic and mitotic divisions of the spindle cells are recorded as of frequent occurrence. Metastasis occurs by the dissemina- tion of tumor cells through the blood or, occasionally, through the lymph channels. ‘Rous found it difficult to obtain early stages in the development of the tumor resulting from the injection of the cell-free filtrate. That glycerinization or desiccation and powdering of the tumor tissue still leaves it an active tumor-producing substance when introduced into a susceptible fowl was demonstrated by Murphy and recorded by ROUS,but their efforts to establish the nature of the agent (23, 127) have up to the present been unsuccessful. The filtrate retains its activity for relatively short periods, while the powdered material kept in sealed capsules may remain potent indefinitely. Yet they hold (128) to the opinion that the tumor graft is responsible for the new tumor in the susceptible host; for histologic studies, they contend, show the origin of the new growth to take place in the graft. In a comparative study of the behavior of the tumor inocula and the filtrable agent of this fowl tumor made by ROUS,Murphy, and Tytler (131, 132) it was shown that the latter produces a neoplastic change in the tissue only after a long time, as compared with the proliferation of the implanted neoplastic tissue. The agent seems to depend upon a special set of conditions in order that it may induce a malignant change. It is further contended (132) that the filtrable agent requires some cell-proliferating or cell-deranging mechanism, such as is induced by injury or by the introduction of a foreign substance. Continued histologic studies brought to light variations in the type of cell found in the tumor. Rous and Murphy (129) observed tumor giant cells 278 MICHAEL LEVINE as well as spherical cells which they considered imperfectly differentiated stages of the spindle cell. They expressed the belief that, despite any diver- sities, the tumors grade into one another, and, since mitosis occurs most fre- quently in the spindle cells, they consider the neoplasm essentially a spindle-cell sarcoma. Other forms of chicken tumors transmissible by a cell-free filtrate as well as by the tumor graft have been described from the same laboratory. Rous and Lange (124) described a chicken sarcoma fissured by blood sinuses. The principal cell type, a spindle-shaped structure with a short longitudinal axis, has a large vesiculate nucleus surrounded by a sparse cytoplasmic struc- ture. This tumor grows better in an alien host (125) than in the variety of fowl from which it was originally obtained. The filtrable agent does not survive in dried or glycerinated tissue; its activity is not constant, and it produces tumors sporadically several months after injection. This tumor, designated as CTXVIII, is thus more sharply differentiated from the original Rous chicken sarcoma (CTI) by its cultural behavior than by its histologic structure, though this difference might be minimized had CTXVIII been transplanted and studied as frequently and as intensively as CTI. Tytler (138) described a chicken tumor (CTVII) which is sharply dif- ferentiated on histologic grounds from all other known tumors of the fowl. This is a slow-growing osteochondrosarcoma, transmissible by a cell-f ree filtrate. Osteoblasts appear to play no part in the development of this tumor. Spindle cells which occur in globular form with few or no cytoplasmic proc- esses are the principal constituents. All these cells have a similar nuclear and cytoplasmic structure, resembling the Wanderzellen myeloblasts of Maximow. Mononuclear cells filled with eosinophile granules and the ordinary poly- morphonuclear eosinophile leukocytes occur. Tytler suggests the possibility of the origin of the myelocyte type of cell from a basophile cell which, in turn, seems to be related to the globular connective-tissue cell. Rous and Murphy (130) surveyed the field of the fowl tumors and con- cluded that, while CTI, CTXVIII, and CTVII are distinct entities and are produced by distinctly different agents, they possess many characteristics in common. Claude and Murphy (23) have summarized the extensive studies on the transmissible tumors of the fowl, and to their review the reader is referred for the nature of the agent in these tumors. Pentimalli (109, 110, lll), in a series of papers based on experimental and spectrographic observation, states that the hemoglobin in the fowl bearing the Rous tumor adsorbs the active tumor-producing agent, and that this phenomenon is favored by a low temperatue to the point that even the red corpuscles of the rabbit succeed in fixing the agent. Embryonic tissue is also capable of this adsorption. Pentimalli contends no molecular change in the hemoglobin is involved, but a physical-chemical liaison; for when the hemo- globin is analyzed spectrographically the normal absorption bands are unchanged. The Fujinami and Inamoto Tumor: In the same year that Rous reported his discovery of the spindle-cell fowl sarcoma, Fujinami and Inamoto (42) reported a myxosarcoma of the fowl, which has since been widely studied. This tumor bears a close resemblance to the Rous sarcoma. Microscopically, CYTOLOGY OF TUMOR.CELL IN ROUS CHICKEN SARCOMA 2 79 it consists of large spindle cells of the fibroblast type, with admixtures of small round cells. It is readily transpIantable by grafts, desiccated tumor, and Berkefeld filtrates of the tumor tissue. Tissues free from metastases and organs of tumor-bearing birds were found to be capable of producing the tumors when implanted in a healthy bird (46). Infusorial earth, Lycopodium spores, and finely powdered charcoal, when introduced into the muscles of fowl bearing this tumor also produced tumors biologically and morphologically identical with the original neoplasm. Fujinami and Suzue believe, therefore, that the etiologic agent is widely diffused throughout the body and that con- ditions suitable for tumor production exist in the region of inflammatory granulation processes. Charcoal fragments imbedded in a fowl bearing the tumor for four or five days are said by Fujinami (41) to be capable of inducing the tumor when transferred to the muscle of a healthy bird, and Fujinami and Sonoda (43, 44, 45) showed that charcoal introduced into a tumor-bearing bird absorbed this sarcoma agent even if it were wrapped in absorbent cotton and sealed in the cavity of a cocoon. Transplants of this tumor to alien species, such as the duck, have succeeded for 76 tumor generations. Teutschlander (137) added another fowl tumor to the list of chicken neoplasms. This tumor, arising spontaneously and capable of transplanta- tion by graft, by tumor powder, and by cell-free filtrates, he describes as a myxomatous angiomatous mixed-cell sarcoma. Giant cells which make their appearance in this tumor are not as common as small lymphocyte-like cells. Between these two cellular extremes many transitional forms are found. Teutschlander observed that the growth of graft transplants is similar to that of the Rous tumor and of typical mammalian tumor transplants; that is, the peripheral portion of the inoculum grows while the central portion becomes necrotic. Rous and Murphy and their associates, as well as workers in other labora- tories, have attempted to establish the neoplastic nature of these avian tumors, while others have contended that, since the growths are transmissible by desiccates and cell-free filtrates, they are more representative of granulomas. Teutschlander describes a zone of small round cells in the peripheral layer of his tumor, with nuclei possessing dense chromatic material. These he believes to be of the lymphocytic cell type and to represent the elementary undifferen- tiated tumor elements. The mature blastoma presents cells with abundant cytoplasm; yet in a series of cellular elements of the tumor, in which the lymphocyte and the tumor cell represent the two extremes, many imperfect or transitional cells are recognized. The invasion of the muscle fibers by this tumor is similar to that observed in the Rous and Fujinami tumors. Llambias and Brachetto-Brian (74) made an intensive study of another fowl tumor which occurs in the connective tissue and muscle of the fowl. This tumor invades and destroys tissue and is associated with hemorrhage of varying degree. It metastasizes frequently to the parenchymatous organs. In microscopic preparations spindle and round cells seem to be the most abundant cellular elements, with the former predominating. The nucleus in this cell is described as monstrously large, with one or a number of nucleoli, and surrounded by an abundance of cytoplasm. Llambias and Brachetto- 2 80 MICHAEL LEVINE

Brian believe that in its structural character and its reproduction their tumor is identical with the sarcomas of other animals, especially with the myxom- atous types. In studying the fate of the implants of this tissue in a series of birds, they removed the inocula at daily intervals up to the first week and then after the lapse of a week. The inoculated tumor fragment always be- comes surrounded by a plasmatic covering and in some respects it takes on the aspect of a granulation tissue. The covering soon disappears, however, for it is invaded by the neoplastic elements. There is also an invasion of the normal muscle tissue. The Fowl Endothelioma: Murray and Begg (98) have described still another type of fowl tumor which appears to be a distinct pathological entity. In contradistinction to the Rous and Fujinami tumors, the Murray tumor, referred to frequently as MH,, is derived from the endothelial lining of the blood vessels. It originated in the abdominal cavity of a chicken and meta- static nodules were found simultaneously in the liver and kidney. Histologic examination of the tumor reveals a variety of cell types represented by clusters of round cells and spindle cells, together with areas of polymorphic and giant cells. Walls of giant cells form about the necrotic tissue, similar to those found in the Rous tumor produced by injections of powder. The tendency to giant cell formation is very strong, and even in the absence of necrotic mate- rial enlarged binucleate cells are common. The nuclei of the giant cells are similar to those of the other parenchyma cells. The nuclei of the round, polymorphic, and spindle-shaped cells are eccentric, large, and spherical, with little chromatin and slightly enlarged nucleoli. The cytoplasm is alike in all types, and is characterized by delicate vacuolization. Cells, probably macro- phages, with inclusion of cellular particles are similar to those of the paren- chyma, and for that reason their histogenesis is difficult of interpretation. Murray and Begg studied the histogenesis of the tumor by inoculation with powdered tumor and cell-free filtrates, though variations in infectivity of both kinds of inocula, coupled with varying susceptibilities of the inoculated fowls, beset the task with difficulties. The first stages in the development of the endothelioma, after injection of a cell-free filtrate of the tumor, are manifested by the accumulation of lymphocytes about the capillaries, six days later. These cells enlarge, and at nine days aggregations of macro- phages are noted. Except for their smaller size, the resemblance of these cells to tumor cells is close. Capillaries with swollen walls penetrate the aggrega- tion of macrophages, and at thirteen days the endothelial cells of the capillary system present the appearance of tumor parenchyma cells. Earlier stages were studied after the injection of powdered tumor tissue. Polymorphonuclear and mononuclear leukocytes infiltrate the mass of powder in twenty-four hours, following which little or no change occurs for three or four days. Up to seven days there is no invasion of the inoculated area by angioblasts; after that the capillaries proliferate and newly formed vessels result. It is only during the second week after inoculation of the powder that the cells forming the capillaries present the nuclear characters of the neoplasm. The nuclei are large and vesiculate, with hypertrophied nucleoli. Free cells about the capillaries are produced by the endothelial cells, and multiply to form loose cell masses. It is these free cells that are responsible CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 281

for the growth of the tumor, Later the capillaries in the tumor area revert to a normal thin-walled character. Grafted tumors grow much more rapidly than those produced by cell-free material, for at the end of a week the new tumor is established and beginning to invade the surrounding tissue. Murray and Begg suggest that the macrophages become ameboid and throw off parts of their cytoplasm; that this process tends to increase the tumor agent and so influences the newly formed capillary endothelium. ROUSSY,Oberling, and GuCrin (133) studied the Murray and Begg tumor and corroborate the observations of the latter workers on the development of this neoplasm. They observed that slowly growing transplants invariably produced large numbers of metastases, especially in the ovaries. The results of their study of the histogenesis of this tumor are not conclusive. They soaked pieces of pith from elder stems in the tumor filtrate and inserted these into the pectoral muscle of the chicken. These fragments were easily recog- nized and removed at intervals for microscopic examination. In these prep- ;irations Roussy and his associates substantiated the findings of Murray and Begg, but since cells of the same type appeared in their control inoculation, the authors were unable to indicate the specific type of cell to which the origin of the neoplasm was attributable. They believe that in all probability very young mesenchymal cells, endowed with considerable evolutionary poten- tialities and capable of giving rise to monocytic, lymphocytic, myeloblastic, fibroblastic, and angioblastic elements, are the mother cells from which the tumor tissue develops. Such a young cell, they believe, is the hemohistioblast of Ferrata, and they suggest the name hemohistioblastoma for the tumor. Roussy and his co-workers point out the gross histologic resemblance be- tween Murray and Begg’s tumor and the sarcoma of Rous. They stress the polymorphic nature of the tumor, its macrophage and giant cell types, and its frequent regression. The general appearance is more that of a granulation tissue than of a sarcoma. Since structurally this endothelioma has all the cellular characteristics of the control reaction tissues, it would seem possible for the cytologist to find some substantial differences between the normal and the tumor cells : so far, only variable, ephemeral characteristics, not generally accepted, have been suggested. GuCrin and Bonciu (48) did not study the histogenesis of the endothelioma of the fowl first described by Murray and Begg, but observed the presence of mononuclear cells which they contend were derived from macrophages, and which they believe to be the tumor cells.

Rous TUMORIN EMBRYOSOF FOWLAND OF ALIEN SPECIES Murphy and Rous (96, 97, 126) jointly and independently studied the behavior of the fowl sarcoma implanted in the membranes of developing embryos of chicks, ducks, and pigeons. They found that in the membranes of the chick the behavior of the inoculum depends upon the blood supply. In the more vascularized membranes, the implanted tissue is supplied with blood vessels in four days, and at the end of a ten-day period develops into a tumor of appreciable size. When inoculations are made into the extra- embryonic cavity and yolk sac, many scattered growths appear on the surface of the allantoic membrane, the amnion, and yolk sac. The tumors do not differ to any extent from those of the adult fowl and are not markedly dif- 282 MICHAEL LEVINE ferent from the cellular reactions induced by injury and by injections of in- fusorial earth, for in the new tissues stellate mesodermal cells and spindle- shaped cells are present in addition to the polymorphonuclear leukocytes and mononuclear cells. The tumor tissue in these embryos, however, is composed of loose columns of cells of a homogeneous structure, The sarcomatous cells are distinguished from the normal connective-tissue cells by their greater size and more deeply stained cytoplasm, The nucleus is characterized by a large vesiculate structure with delicate chromatin network and large nucleolus. Murphy and Rous state that implantations of this tumor in embryos are successful only when the mesodermal layer is exposed to the implant. The filtrate or desiccate of the Rous tumor is equally effective in producing tu- mors in the chick embryo. The tumor is not transplantable to adult pigeons or ducks, but in the embryos of these birds almost half of the number of tests made gave positive results. Tumors from embryos, when transplanted to the young of the species, do not grow. Murphy (96) later showed that the chick embryo can be used as host for transplants of rat, mouse, and human tissue. Various tissues of the chicken could likewise be grown for a period of a week on this medium. In the early study of the propagation of the Rous tumor it was shown that the tumor at first grew only in blood-related animals. As the number of implantations increased, the transplanted tumor survived in the same pure stock, and then in related breeds. Purdy (116) investigated the propagation of grafts of the Rous sarcoma in ducklings. In the same year, Des Ligneris (24) succeeded in transplanting the tumor to the turkey and guinea-hen, while Andrewes (1) showed that it would grow in the pheasant. 'In no case, how- ever, was the tumor transplantable for more than two tumor generations. Purdy, by injecting minced Rous chicken tumor into day-old ducklings of the Khaki Campbell variety, produced tumors in this alien species. The growth is very marked in twenty-four hours and continues for six days. If the duckling survives the two days following this period, the tumor begins to regress and has disappeared completely at the end of the second week. Purdy produced five tumor generations in ducklings, and contends that the propa- gation could be continued indefinitely. Filtrates of this duckling-grown Rous sarcoma are effective in producing tumors only in the fowl and not in other ducklings. Purdy (115) earlier studied the behavior of the Fujinami myxosarcoma in the duckling and observed that the age of the host and the dose of injected tumor were significant factors in the production of the tumor in this bird. He contends that a large dose of tumor mince is effective not because of any greater growth energy conferred upon the cells of the resulting tumor, but because the tumor starting at a number of foci attains a greater size before regressive changes have time to appear. These multiple tumors prove fatal before changes occur that cause regression, while the effects of a single tumor are insufficient to cause the death of the host. Fujinami and Suzue (46) found the Fujinami myxosarcoma to be transplantable in the quail, duck, and pigeon, but not in the guinea-pig or rabbit. The histology of the tumor in the alien species is identical with that of the original fowl tumor. They were able to transplant this sarcoma in ducks for sixteen tumor generations. CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 283

THETUMOR GRAFT OR FILTEREDAGENT The transplantability of the tumor both by tissue grafts and by Berkefeld filtrates of tumor extracts raises the question as to the r6le played by the two sources in tumor production. Rous and Murphy (121, 126) believe that the peripheral cells of the tumor graft proliferate and ultimately give rise to a tumor, which destroys the host in three weeks. Llambias and Brachetto- Brian (74) believe that both the cells and the active agent in the cells are responsible for this effect, while Ricardi (117) concluded that the cells do not share the responsibility for the new growth. More recently Irwin, Gairns, and Banting (52) set up a series of carefully devised experiments to test this point. Since the proliferating fibroblasts of the host so closely resemble the tumor cells, an attempt was made to isolate the graft without interfering with the fluid exchange between the host and grafted tissue. Grafts enclosed in sacs of fresh peritoneum were studied daily up to and including the 96th hour. In these studies there was no evidence of survival of the transplanted cells. These were destroyed by the leukocytes of the host, while the fibro- blasts adjacent to the graft became malignant, When silver-impregnated peritoneum of the hen was used as sac material for the tumor grafts, the results were the same. On the basis of these experiments, it must be as- sumed that powdered desiccate of the tumor tissue encased in a membrane would produce tumors in the host. This has not been attempted. The results of ROUS’studies on the chicken tumor found wide corrobora- tion. A typical sarcoma of the fowl was described, the chief histologic con- stituent of which is a spindle-shaped cell with morphological variants. The tumor metastasizes principally to the lung, liver, kidney, and heart, and may be transmitted to other fowls. Other tumors of the fowl closely related to this sarcoma are transmissible by grafts, by Berkefeld filtrates of the fresh tumor tissue, and by tumor desiccates. These concepts aroused considerable opposition, for no mammalian tumor is known to be capable of propagation by means of filtrates, desiccates, or glycerinated tissue. Nakahara (100)’ subjected the Rous chicken tumor to drying or treat- ment with glycerine, and claimed that these processes failed to destroy all the tumor cells. He believed (99, 101, 102, 103) that filtrates free from cells are incapable of producing the fowl tumor, for his preparations revealed minute ameboid cells that readily passed through the Berkefeld “ V ” and ‘‘ N ” fil- ters, and these be regarded as responsible for tumor production. Borrel (10) studied the growth of the Rous sarcoma in vitro and observed that the malig- nant cells are hypertrophied to excess, containing an enormous mitochondria1 net. Strange corpuscles, which he believed to be parasites, were observed in these cultures. In an earlier paper Borrel (9) had laid emphasis on the appearance of an intergranular substance arranged in a characteristic delicate network formation. This he believed to be the product of specific living elements which induce the hypertrophy of the macrophages and fibroblasts. Many workers have observed intercellular bodies but these have been con- sidered contaminants, which do not appear to interfere with the growth and development of the tumor. Glover and his associates (47) apparently iso- lated bacteria of a polymorphic nature from the Rous tumor, but they are not to be considered the etiologic agents. 284 MICHAEL LEVINE

Nakahara and Nakajima (104) returned to this problem later and showed that small quantities of the chicken tumor filtrate drawn into capillaries could be examined directly. for non-filtrable cells. 'These filtrates were found lack- ing in cellular elements, but when injected in the suitable fowls they produced tumors. Sumikoshi ( 136) believes that powdered tumor material contains living cells, and can withstand temperatures up to 75" C. for thirty minutes to an hour. It has been shown by Rous and Murphy that the viability of the tumor material is destroyed at 55" C., although Levine and Baumann (65) produced growths of the fowl with powdered tumor dried and heated in vucuo at 60" for half an hour. Growth failed when this material was sub- jected to higher temperatures for the same length of time. Formol, which is prevalently used as a routine fixing agent, differentiates the nucleus from the cytoplasm but does not give sufficiently critical results for determination of the viability of the cell. Stained smears of powdered tumor tissue show many intact cells that stain like the formol-fixed prepara- tions, but these are not viable when cultured on favorable media. Isibasi and Sinohara (53) reported that no living cells could be found in dried and powdered tumor tissue of the fowl. They found that this powder, kept in the cold, without air and light, for 1000 days, remained potent as a tumor producing-material in the fowl.

THEBLOOD AND LOOSECONNECTIVE-TISSUE ELEMENTS At present no theory of the origin of the blood and connective-tissue elements has been universally accepted. A number of views, supported by experimental data and extensive observations, have been advanced as to the origin and developmental tendencies of the various elements in these tissues. Since the cellular components of the Rous tumor consist principally of some of the elements of common connective tissue, a brief review of the recent literature should be of interest. Maximow, whose contributions are numerous, has adequately reviewed (84, 85) most of the work in this field, and his studies, as well as his reviews, have been drawn upon. One factor which has contributed to the confusion that prevents a clearer understanding of the histogenesis of these cellular elements has been the nomenclature. Clarification of this point can hardly be accomplished here, but an effort has been made, where possible, to use a uniform terminology so that the difficulties arising from this source may be avoided. For the last score or more years, intravitam and supravital dyes have been used in the study of the histogenesis of the blood and connective-tissue ele- ments. By the use of these dyes on tissues studied in vitro and in vivo Gold- mann, Tschaschin, Kiyono, and others have cleared up some of the difficulties in identifying these elements, but even these methods, which involve consider- able technical and biological difficulties, have not brought about a generally ac- cepted view as to the origin of these cells. Cappell (12) has given a clear and comprehensive review of the dyes and methods used in the intravitam and supravital staining processes. Observations on the development of the blood and loose connective-tissue cells have been made chiefly on mammalian organisms. That analogous cell CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 285 types prevail in the fowl is shown by Kiyono and Nakanoin (57) and Mjas- sojedoff (90). These studies show ameboid cells of various kinds with tran- sitional stages. ( 1) Ndrmal Macrophages and Fibroblasts: Macrophages were described by Metschnikoff as phagocytic cells of the connective tissue, their principal r61e being that of a defense mechanism, especially in inflammatory processes. These cellular structures were observed and studied by others, who have ap- plied to them such names as clasmatocytes, adventitious cells and, later, rest- ing wandering cells (Maximow, SO). Maximow described these cells as part of an irregularly arranged common connective tissue which is to be found every- where in the mammalian body. He believed them to be easily identifiable through the agency of intravitam stains, such as isamin blue or trypan blue, for they segregate colloidal dye and are capable of transforming themselves into a variety of cell forms, such as ameboid cells, giant cells, and epithelioid cells. Their phagocytic properties are distinct from their ability to store col- loidal substances. From 1909 to 1914, according to Maximow (84), the conception of the macrophage was extended by the use of vital staining methods. It was shown that not only do the resting wandering cells take up the dye, but that intensive accumulations of dye occur in certain cells in the spleen, liver, lymph nodes, and bone marrow. In the latter part of this period, it was further shown that the macrophages, or the resting wandering cells of the loose connective tissue, are a part of a cell system distributed in various organs which function in the general metabolism of the body. Kiyono (56) applied the name “ histiocytes ” to the cells capable of segregating vital stains. Landau and McNee (62) called attention to the presence of a reticulo- endothelial metabolic apparatus intimately connected with the metabolism of cholesterol. These and other studies gave rise to the theory of “ reticulo- endothelium,” or the system of “ histiocytes.” Connective-tissue cells which take vital stains are considered part of the histiocyte system. Maximow points out that the histiocytes show structural variation depend- ing upon their location, while their microscopic appearance is dependent upon their physiological activities. Accordingly, two forms of cells occur in the loose connective tissue of the mammal, which he calls “ the resting wandering cells ” and “ the active wandering cells.” Both groups contain cellular ele- ments some of which are capable of taking up vital dyes, such as neutral red. The “ active wandering cells ” embrace three cell types distinguished prin- cipally by size, although other pertinent cytological differences are present. The smallest cells resemble the lymphocytes of the blood. These do not take up the vital stain. Larger cells have the morphological characteristics of monocytes and show the typical neutral red rosette after treatment with the dye. The largest cells in the series are identical with the free ’histiocytes or with the polyblasts of inflammation. They have eccentric, oval or kidney- shaped nuclei and ameboid protoplasm which takes up vital dyes. The smaller wandering cells, Maximow believes, are identical with the non-granular leuko- cytes of the blood. The inactive, fixed group of cells, or the resting wandering cells of Maxi- mow, belong to the histiocytes and are known, as mentioned above, under vari- 286 MICHAEL LEVINE ous names, the most common of which is ‘‘ macrophage.” These are of many shapes and vary from rounded bodies to elongated forms with branched proc- esses forming spindle-shaped structures. The nucleus also is varied in shape; it may be round, oval, or kidney-shaped. The cytoplasm takes up isamin blue and trypan blue as well as particulate bodies such as India ink and carmine. The other elements which play an important r81e in the loose connective tissue are the fibroblasts, sometimes referred to as fibrocytes or desmocytes. These cells are of many forms and are involved in the formation of fibers. They are long, flat elements, taking the shape of slender spindles when seen in profile. They do not take up dyes except in the presence of inflammation or when cultivated in vitro. The fibroblast nucleus is large, oval in shape, with fine granular chromatin material, and one or two well differentiated nucleoli. Bloom (8) states that fibroblasts are not always structurally different from mesenchymal cells. They form connective-tissue fibers which, however, are not characteristic of these cells alone, for reticular cells, smooth muscle cells, perivascular cells, osteoblasts, and chondroblasts may give rise to fibers both in vivo and in vitro. Transitional forms between the various cell types in the loose connective tissue are generally accepted. The significance of these in- termediate forms Maximow finds difficult to interpret. He suggests that they may be the expression of a dual transformation of an ameboid cell into a rest- ing wandering macrophage cell, and then into a fibroblast, or that they may represent interrupted development or a transient functional condition. The presence of transitional forms between fibroblasts and resting wandering macrophage cells in the normal connective tissue further suggests the develop- ment of histiocytes from mesenchymal cells (81). (2) Monocytes: The monocyte, or non-granular leukocyte, or large mono- nuclear leukocyte, is bound up with the problem of the histiocytes. These cells in the mammal are comparatively large, with abundant cytoplasm; oc- casionally they show a granular consistency and an archoplasmic body near the indented side of the kidney-shaped nucleus. There is no accepted view as to their origin. It is believed by some hematologists that no sharp distinction exists between the monocytes and the lymphocytes. Others believe that the monocytes arise from myeloid tissue of the bone marrow. Still others hold that they take their origin from the endothelium. Aschoff and Kiyono (2) and Kiyono (56) regard them as free histiocytes. Bloom (8) points out that the monocytes of fish, amphibian, reptile, chick, and mammalian blood undergo marked changes, developing into macrophages, epithelioid cells, giant cells, and fibroblast-like cells. The transformations of the leukocytes, he believes, are as variable as the technic used in culturing them. Maximow (83, 84) believes that the monocyte originates from the lympho- cyte. The relationship between the histiocytes and the other elements of the loose connective tissue and the blood is manifested under abnormal conditions, such as inflammation, and in tissues grown in vitro. Maximow (84, 85) di- vides the elements in these tissues into three groups : fibroblasts, histiocytes, and hemocytes. The fibroblasts are differentiated elements and are incapable of morphological transformations ; they elaborate the intercellular substances, or, as endothelium, form the lining of blood vessels. The histiocytes are involved CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 287 in the general defense mechanism of the body. The hemocytes or blood cells, which circulate in the blood and lymph, consist of the hemocytoblasts or stem cells, and give rise to such differentiated elements as granulocytes, monocytes, erythrocytes, and megakaryocytes. The behavior of the histiocytes in an in- flammatory process or in vitro, according to Maximow, is definite. The iso- lated cells mobilize as free cells, and thus become a part of the mononuclear exudate cells or polyblasts of inflamed tissue, or a part of the polyblasts or macrophages which are found in cultures of connective tissue of any origin. These cells retain their power to store vital dyes. The term “ polyblast ” is Maximow’s and describes a type of cell capable of undergoing varied transformations. The fibroblasts and the endothelium, as pointed out above, represent highly differentiated elements incapable of transformation into other cell types. The fixed histiocytes are readily shown to be capable of transformation into ameboid polyblasts or macrophages in inflammation and in tissue cultures. Polyblast cell types, such as epithelioid cells, foreign-body giant cells, pus phagocytes, and free blood macrophages, can be traced back to histiocytes. The fixed histiocytic polyblasts can be transformed into typical fibroblasts. It has been contended by earlier work- ers that in inflammation not only polyblasts, but also lymphocytes and plasma cells, may originate from the proliferations of perivascular histiocytes. Fer- rata’s hemohistioblasts have been considered the first primitive blood cells of the embryo, and it is believed they are capable of giving rise to many types of blood cells. Maximow holds that these elements are ordinary storing and phagocytizing, clasmatocyte-like histiocytes. (3) Cell Transformations: The small lymphocytes of the hemocyte group are variously regarded. While some authors believe they are fixed cells, dif- ferentiated and incapable of transformation, Maximow (84) holds that they may give rise to polyblasts during an inflammatory process. Through a proc- ess of growth, they attain the size and shape of polyblasts and store dyes in a vitally stained animal. These cells are capable of developing into ameboid re- acting cells of the histiocytic type and, finally, into fibroblasts with loss of phag- ocytic properties. A large number of the lymphocytes of the blood of the chick grown in vitro turn into thrombocytes. Maximow (82) believes that mes- enchymal cells give rise to the hemocytoblast, or lymphocyte, and the histiocyte, both capable of forming active polyblasts in tissue culture or inflammation. This cell is now capable of forming a histiocyte, which may be transformed into a fibroblast. Downey (25) considers the lymphocyte a polyvalent cell with capacity to differentiate in various directions, depending largely on external conditions. Bloom (7) traced the transformation of lymphocytes into fibro- blasts in tissue cultures of lymph of the thoracic duct of the rabbit. The lymphocyte becomes transformed into a polyblast, which in turn is trans- formed into a fibroblast. The transformation of fibroblasts into macrophages has been championed by a number of workers. W. and M. von Mollendorff (93, 94) based their evidence on extensive studies of connective-tissue preparations of the mouse and rabbit stimulated by light. M. von Mollendorff (92) studied subcutane- ous connective tissue of the mature rabbit and showed that by application of heated needles to the culture, the fibroblasts could be changed into rounded 288 MICHAEL LEVINE cells producing cells of the lymphocytic or histiocytic type. Rounded cells when gently warmed were induced to spread out and give rise to cell types which resembled fibroblasts. The addition of 1 per cent solution of trypan blue to pure cultures of fibroblasts induces the retraction of the pseudopodial structures, and the cells take on the characteristic form of histiocytes. Re- covery is observed when the cells are returned to a dye-free medium. Evans and Scott (27) believe that the fibroblast is capable of phagocytizing dyes. Sabin, Doan, and Cunningham ( 134), by employing supravital staining meth- ods, recognized a fundamental morphological characteristic of the monocyte and the clasmatocyte. Neutral red is segregated in the monocytic cell in a distinct pattern, as a rosette of small bodies that surround the vacuoles, centro- sphere, or phagocytized cells. The clasmatocytes show no such orientation of neutral red bodies. Other differences of histological significance are pointed out by these investigators. Forkner (36),who studied the lymph node of the rabbit for the purpose of determining the origin of the monocyte, adds a new term to the cluttered terminology--“ premonocyte,” signifying a stage intermediate between the monoblast and the monocyte. The monoblast, the existence of which is indefi- nite, is characterized, according to Forkner, by its failure to segregate neutral red. He believes the free macrophage takes its origin from the common endothelial cell which gives rise to the monoblast, premonocyte, and monocyte, or more directly from the macrophages lining sinuses, reticulo-endothelial cells. Lewis (7 1) holds that mononuclears or monocytes, macrophages or clasmato- cytes, and epithelioid or endothelioid cells are merely different manifestations of the same cell type. The markedly different characters which they present are dependent upon extrinsic factors, such as ingested material and its diges- tion. The difficulty of establishing a relationship between these cells, which show size and structural differences, is due to their migratory character, their tissue-forming habit, and their phagocytic powers. Parker (105, 106), in in vitro studies, pointed out the existence of various races of fibroblasts in the same organism. These cells, when isolated, consti- tute races which he believes are physiologically different, though morphologi- cally indistinguishable. Each cell race, according to its origin, constitutes a specific cell type characterized by its nutritional properties. From a study of chick embryos thirteen to fifteen days old, he concludes that fibroblasts de- rived from the heart muscle, skeletal muscle, perichondrium of cartilage, and periosteum of bone, represent different cell strains. Variations in the media induce abnormal changes in these cells so that the forms produced do not repre- sent the usual sequences in cellular development. Transformation of fibro- blasts into monocytes may occur as a result of a low nitrogenous content of the media. Parker believes that the form which the cell takes is an expression of its physiological state at a given moment; the fibroblast and the macrophage rep- resent in vitro extreme functional states of the same cell, He (107, 108) con- cludes that the physiological properties of fibroblasts depend solely upon their origin, No two races of fibroblasts react identically to the same environment, even though removed from the same part of two individuals of the same age and species. CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 289

Fischer and Parker (34) studied, in tissue cultures, the behavior of fibro- blasts derived from bone and concluded that these fibroblasts possess no distin- guishing morphological features. Their transformation into bone was not evi- dent under usual cultural methods but when the growth-stimulating substances were reduced, transformations occurred. The conclusion is drawn that pro- liferation inhibits the normal function of the cells. Horning (51) studied osteoblasts and muscle fibroblasts, and concluded that cells are endowed with an inherent intrinsic mechanism and that cell be- havior in vitro is not determined solely by the environmental conditions of the medium. His observations of the disintegration processes in cells suggest a teleological interpretation without demonstrable proof, His conclusions are in accord with those of Parker. He notes, also, that the rate of cytolysis is de- pendent upon the inherent growth energy of a given strain. Santesson (135) cultivated, in vitro, 77 spontaneous tumors of mice, of which 28 were adenomas, 31 adenocarcinomas, and 18 carcinomas. He be- lieves that cells of the ameboid type, resembling the monocyte, are the first to migrate from the tumor fragment in culture; these are followed by fibroblasts. The ameboid cells degenerate after three or four days, while the fibroblasts cease to migrate and then disintegrate. Fibroblasts and macrophages origi- nate from blood cells and the stroma present in the tumor. The adenomas in culture form an even single layer of cells without cell irregularities or mitoses. The carcinomas grow irregularly with a tendency to form double layers. Cell irregularities and mitoses are common. Moen (91) studied the origin of fibroblasts from isolated mononuclear cells of the guinea-pig. Using Carrel’s microflask technic, he found the number of mononuclear cells which appear in the cultures to vary from a hundred to sev- eral thousand. The locations of isolated cells were marked so that they could be kept under continuous observation. Under certain, though indefinite, con- ditions these cells were observed to divide mitotically and to form colonies of morphologically typical fibroblasts. The greater number of these specially selected cells, however, were seen to form macrophages. Many of these macrophages died, while others slowly developed into spindle or stellate forms. These cells showed oval or rounded nuclei with several nucleoli; they rarely divided, but frequently degenerated; they did not migrate but changed their form. Moen noted transitional stages between the typical macrophages and fibroblasts. Colonies of fibroblasts originating from mononuclear cells were easily transplanted and maintained the morphological characteristics of fibro- blasts. Moen was unable to determine whether the macrophages and fibro- blasts originated from two different cell types, such as the monocyte and the lymphocyte, or whether they represented merely different developmental stages of a common type. (4) Giant Cells: Hyperchromatic cells, which most likely represent tumor giant cells in division stages, have interested the oncologists because of their possible diagnostic value. In the Rous sarcoma, tumor giant cells as well as foreign-body giant cells are present. The latter occur constantly about the in- jected desiccates, while the former are found distributed through the tumor tissue. The origin of tumor giant cells is obscure. They are generally re- garded as hypertrophied cells in which nuclear division occurs without cell 290 MICHAEL LEVINE

division. The relation of the foreign-body giant cell to the tumor giant cell is also not clear. These two types of cell, Levine (64) believes, have been con- fused by many workers as identical structures. They differ, however, in origin, size, number of their nuclei, chromatin content, and nuclear behavior, The foreign-body giant cell is not known in division stages. It is frequently produced in tissue cultures in the presence of a foreign body, the cover glass often serving this purpose. Tissues in which foreign-body giant cells have been observed are listed in the accompanying table. The factors which induce their formation are not known, but the weight of evidence seems to indicate that they are produced by fusion of cells of the monocytic group.

TABLEI: Occurrence of Foreign-body Giant Cells as Recorded in Literature

Blood and Fowl Tuniors Lymph Node Bone Marrow Spleen Buffy Coat in vitro and in vivo - -_ Lambert, 1912 I Lewis and Web- Foot, 1912 Awororow and Kous, 1911 Lambert and ' ster, 1921 Erdmann, 1917 Timofejewskij, Iioskin, 1926 Hanes, 1913 Maxirnow, 1922, Grossman, 1923 1914 Murray and Begg, Weil, 1913 1924, 1925 M. K. Lewis, 1925 1930 Sniyth, 1916 Barta, 1925 'rimofejewskij and Zweibaum, 1933 Chlopin and Renewolenskaya, Llombart, 1935 Chlopin, 1925 1925 Pires Soares, 1937 Erdma nn, Eisner , Lewis and Lewis, Levine, 1939 and Laser, 1925 1925, 1926 Timofejewskij and Benewolenska ya, 1925 I

Forbes (35) made an intensive study of the development of giant cells in vivo in the rabbit. Following subcutaneous injections of agar, examinations were made beginning at the 18th hour, daily for the first ten days and at two- day intervals up to the 22d day. The oldest inoculation was of 44 days' standing. Forbes observed the development of young blood vessels about the injected mass at the end of the first day, and these continued to develop until the fifth day. Mononuclear cells appeared close to the agar, and could be seen migrating through the blood-vessel walls. The endothelial cells in the tissue spaces about the agar underwent rapid proliferation. The source of these rapidly multiplying cells has not been determined, although Forbes be- lieves that they may arise from endothelial cells already present in the tissue spaces or from cells migrating through the vessel walls. That the mononuclear cells about the agar are endothelial is supported by the changes occurring in their nuclei and cytoplasm, clearly paralleling the reaction in the fixed endo- thelium of the vessels. They ingested lamp black, while the nearby lympho- cytes took up none. The endothelial cells lined the surface of the agar and within four days had become so compactly placed that they coalesced to form giant cells. Eight to ten nuclei are sometimes seen in a single section through one of these cells, but no nuclear divisions have been observed. Single endo- thelial cells together with these giant cells form a dense wall about the injected agar, surrounded by a capsule of young connective tissue, CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 29 1

Lambert (58, 59) showed that the introduction of Lycopoda’um spores into cultures of chick embryo spleen brought forth wandering cells, which gathered about the spores and formed foreign-body giant cells. He suggests that these giant cells, when found in spleen cultures in the zone of wandering cells, repre- sent fusions of large mononuclear wandering cells of endothelial or pulp cell origin. The accumulation of granules with an affinity for neutral red dye be- comes marked with age. The addition of spores to cultures of chick embryo heart, where the outgrowth consists of diffuse connective tissue, does not lead to the formation of foreign-body giant cells. Lewis and Webster (73) described, in cultures of human lymph node tissue grown in auto or homo plasma, the appearance of small wandering cells, large wandering cells, giant cells, spindle and triangular cells, and occasionally large lymphocytes, polymorphonuclear leukocytes, and eosinophils. These authors state that giant cells and large wandering cells are identical in structure and general appearance. They differ, however, in size. The large wandering cells are the smaller of the two and contain one or two nuclei. These cells corre- spond to epithelioid or endothelioid cells. The giant cells arise by fusion of large mononuclear cells or by amitotic division without cytokinesis. Lewis (69) does not seem to distinguish the tumor giant cells from the foreign-body giant cells. His discussion includes only such giant cells as arise in tissue cul- tures, which seem to separate themselves sharply from the tumor cells because of their resemblance to the Langhans type of giant cell.

TISSUECULTURE STUDIES OF THE Rous CHICKENSARCOMA . Carrel and Ebeling (19) studied pure cultures of mononuclear leukocytes isolated from the blood of adult chickens. They found that these cells pro- liferated at a slower.rate than fibroblasts and, unlike the latter, showed no tendency to form a tissue. Under certain conditions these mononuclear cells changed in form so as to assume the appearance of fibroblasts. In later com- munications, Carrel (13, 14) attempted to show that the macrophage of the chicken blood is sensitive to the tumor-producing agent or virus of Rous sar- coma, and is transformed by it into the malignant cell. Thus the macrophage becomes the propagating cell of the malignant tumor, while the fibroblasts do not differ from the normal cell of the same type and generdly remain incapable of producing the disyase. Pure cultures of these ameboid cells, or macro- phages, when injected into suitable chickens produce sarcoma. Carrel (16, 17) studied in vitro tissue from spontaneous chicken sarcomas, a mouse sarcoma, some tar sarcomas, and teratomas produced by arsenic and other chemicals. Pure cultures of macrophages were obtained, and these, or the fluid in which they were growing, produced sarcomas when inoculated into susceptible fowls. Carrel believes that the malignancy of the cell is an ex- pression of a disturbed metabolism. The agent of the disease propagates itself once it is produced by such chemicals as tar, arsenic, and certain substances produced by bacteria, helminths, x-rays, and other agents. Carrel (15) also made a comparative study of normal and transformed malignant macrophages in vitro. The normal macrophages (under which he includes mononuclear leukocytes, endothelial cells, and monocytes) are large, 292 MICHAEL LEVINE

active, ameboid cells with clear cytoplasm, which emigrate and reproduce in the coagulum. These cells are non-tumor producing. Their transformation into fibroblasts occurs under certain conditions which are associated with the appearance of necrotic cells. Carrel (18) concludes that the Rous sarcoma is a disease of the macrophages, though under certain conditions the fibro- blasts may also become infected. He is unable, however, to define or point out the essential characteristics of a diseased macrophage. Morphologically it is no different from a normal macrophage; yet its lifetime is shorter. Carrel and Ebeling (20) studied the conditions under which monocytes are transformed into fibroblasts, by the tissue culture method. In 1922 they (19) had observed these changes, but were unable to reproduce the conditions that favored them. At that time they described the cytoplasmic processes in these transformed cells as long and pointed, and showing none of the move- ments which characterize the pseudopodia of monocytes. The nuclei were said to be oval and to contain one or two nucleoli, and staining with neutral red showed the typical granules of the normal fibroblast. In their later study (20) these investigators brought about this transformation by treating a culture of monocytes with Rous sarcoma extract. Fibroblasts thus obtained are stellate in form with one or two nucleoli in a large oval nucleus, about which are distributed small granules of neutral red. The cytoplasmic proc- esses are fixed and resemble those of normal fibroblasts. Carrel and Ebeling point out that the transformation of monocytes into fibroblasts takes place through an enormous increase in the size of the cells, whereby giant monocytes are formed, resembling the tissue macrophages, from which they are indis- tinguishable. It is of interest to note that in tissue cultures of Jensen rat sarcoma and mouse sarcoma No. 10, these workers (21, 22) recognize the malignant cell as a fibroblast, which they state is coarser and more refringent than the normal cell, though possessing the cytological characteristics of the latter without any structural abnormality. They stress the view that these may be healthy cells, for no detectable morphological characteristic can be considered specific for malignancy. Fischer (28, 29) observed that fragments of inert muscle tissue added to the culture media in which ameboid cells (monocytes) are growing, readily induce the transformation of these cells into connective-tissue cells (fibro- blasts). In a study of the Rous chicken tumor grown in vitro (30) he ob- served that the cells which grow out of the explant are extremely polymor- phous, the smallest being the size of bacteria.. He recognized various cells which he classified as of medium and of small size which, in life, showed marked activity but no distinguishing cytological traits. Large ameboid cells of slow movement, spherical in shape, with highly granular cytoplasm and nuclei rarely visible during life, constituted another group of cells in these cul- tures. Stained, these cells show a distinct nucleus with one large nucleolus and two accompanying smaller ones. Spindle-shaped cells also appear re- sembling large fibroblasts. Intermediate cells, having cytoplasmic projections suggesting transition forms between mononuclear lymphocytes and fibroblasts, are also observed. The nucleoli show an increase in number. Fischer stresses the activity of these cells as though this characteristic were sufficient to sepa- rate the malignant from the normal cells. " Amalgamation " with ultimate CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 293 separation into the original units is another trait of malignant fibroblasts which was not observed among the normal cells. Mitotic cell division, Fischer re- ports, does not occur. In a study of the mouse carcinoma, on the other hand, Fischer and Parker (33) found that the percentage of mitosis in the malignant tissue exceeded that in the normal tissue. In a later study of the Rous tumor, Fischer and Laser (31) found that the principal cells in the explant are the ameboid cells and fibroblast-like connec- tive-tissue cells. The ameboid cells are synonymous with Carrel’s monocytes; they resemble the monocytes of the blood stream and the macrophages of the spleen. Carrel’s findings are corroborated by Fischer and Laser, who observed that these cells are bound up with the malignant elements; as long as they remained in the cultures, tumors could be produced with them. The ameboid cells of the sarcoma give rise to pure cultures of fibroblasts. Both types of cells are phagocytic, though the normal fibroblast does not possess that property. While these cells are morphologically identical, physiologically they differ in that the normal fibroblasts will not store dyes. Fischer and Laser (32) pointed out what they believe to be another physiological dif- ference between normal and malignant cells grown in vitro. In a study of a variety of induced tumors cultured over a period of months, they found that malignant tissues proliferate much more slowly than normal cells. In pure cultures of fibroblasts grown in vitro Ephrussi and Hugues (26) claim to have observed a transformation into macrophages in the form of free ameboid cells with characteristic undulating membranes. These changes are similar to and corroborative of those first described by Fischer and others, and appear to be conditioned by changes in the concentration of embryonic extract in the medium used. Rounded macrophages appear in the presence of a high concentration of the extract, while forms with large, undulating, pseudopodial structures (clasmatocytes) appear in weak concentrations. Lambert and Hanes (61) observed in cultures of rat and mouse sarcomata two types of cells, which they described as spindle cells and macrophages. They believe that these are morphological variations and not generically sep- arate types. Both sarcoma and carcinoma cells cultivated in vitro showed active phagocytosis. Lambert (60) compared the behavior of sarcomatous tissue of the mouse and rat with that of normal cells under tissue culture con- ditions. He noted that the growth of the malignant cells is more rapid and that within twenty-four hours the cultures have reached maximum activity and ceased to grow. The normal tissue does not begin to grow before forty- eight hours, appears to reach full development in seventy-two to ninety-six hours, and continues to grow for several days longer. The cells are spindle- shaped and frequently show active division stages. Transplants from normal cells are more successful than from the malignant ones. Tumor cells are less hardy than normal cells and require a larger food supply. Lewis’ (70) study of sarcoma cells in tissue cultures presents a rational interpretation of the significance of the various types of cellular structure found in these neoplasms. Two types of cells migrate from the explant culti- vated in vitro, namely, mononuclear cells and spindle cells. The former, Lewis states, are like normal cells in behavior and structure, while the spindle cells differ from normal cells because of their large granular nuclei, which are 294 MICHAEL LEVINE embedded in a dense cytoplasmic structure bearing inclusions of small neutral red granules, Lewis suggests the possibility that these spindle cells are the tumor cells and represent permanently modified cells. He regards them as somatic mutations since they maintain their kind by division. He is not in accord with Carrel, who believes the macrophage to be the malignant element. The proof upon which Carrel bases his contention, Lewis holds, is inconclusive since the transfer of the macrophages to a susceptible host is not accomplished without the simultaneous transfer of the tumor agent in the medium in which the cells are growing, Lewis further suggests that the macrophages are con- comitant cells associated with the injured tissue, and present a phenomenon not unlike that which occurs in trauma. The proof that the spindle cells are the active tumor-producing cells has not been generally accepted. Lewis (72), in his comparative studies of mouse sarcoma cells and normal cells grown in vitro, maintains that the malignant fibroblast is similar to the norma1 one, In a general summary of the cytological structure of the malig- nant cell, he pictures it as one with larger nuclei, heavier nuclear membranes, and larger and more irregularly shaped nucleoli than are found in the normal cell. Phagocytosis is more common among malignant fibroblasts than among normal ones. Lewis believes that malignancy is due to cytoplasmic changes, a disease of the cytoplasm. It may be noted that there is little consistency in the observations reported by the various investigators as to the cytoplasmic and nuclear structures of the Rous tumor cells. There is, on the other hand, marked agreement as to the similarity between the normal and the so-called malignant cell, though the latter is generally characterized as of larger size than a normal cell of the same type. Levine (63, 64), however, in a cytological study of a series of mammalian and avian tumors grown in vivo, pointed out the constant appear- ance of cells with chromosomal aberrations. In this observation he is in ac- cord with a number of workers who have studied one or another of these tumors independently. That malignancy is due to these chromosomaI ab- normalities is at present beyond the sphere of experimental verification. Kimura (54, 55) studied 142 generations of the Fujinami myxosarcoma of the fowl in tissue culture. He found that only in the first and second trans- plant generations of the myxomatous tissue or after the addition of heart tissue to his cultures, did round cells make their appearance. These cells degen- erated in the second generation transplants and in subsequent generations spindle cells entirely dominated the cultures. These spindle cells were mor- phologically like fibroblasts, differing from the latter only in their limited growth in vitro. Inoculation of fowls with tissue cultures of various trans- plant generations showed, according to Kimura, that the spindle cells are the malignant ones. When round cells are present, as in the first transplant gen- eration after the addition of heart tissue, tumors fail to appear or the tumor formed is a slowly growing one, causing death in fifty-five days. Kimura cites only two examples illustrating this important phase of his experiment. The second generation transplants, where round cells are still to be expected, produced malignant tumors causing death in twenty-five, twenty-six, twenty- eight, thirty-three, and thirty-seven days, while the third generation, where no round cells are present, produced tumors causing death in twenty-nine, thirty- CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 295 two, or eighty days. With each of these generations there was one case in which a fatal tumor did not develop. Inoculation with fourth generation tissue ‘cultures caused death in twenty-nine days, and with fifth generation cultures, in forty-two days. Kimura (SS), nevertheless, contends that the cultures consisting almost entirely of cells of the fibroblast type produce tumors from which the hosts die and that there is no relationship between the malignancy of the tissue cultures and the round cells. The experiments cited are unfortunately too few to be conclusive, especially with the first generation transpIants where round cells represent the more abundant cell type. Round cells seem to be associated with slowly growing tumors; striking support of these observations is presented below. Burrows ( 11) studied the Rous sarcoma in tissue culture and found macro- phages and fibroblast-like cells. He was able to render the macrophages malignant by the addition of the tumor filtrate, but not the fibroblasts, thus confirming the observations of Carrel. Bisceglie (5, 6) studied in vitro cultures of spleens of seventeen- and eighteen-day old chicks. He believes that the transformation of the normal cell into a malignant one is attributable to a change in the mechanism of metabolism, due to a substance which he calls ‘‘ blastine.” He holds that the neoplasm is not caused by a specific agent but is the result of a metabolic process which arises out of changes in cells or tissues which may be produced by a variety of different agents. Ludford (77) applied intravitam stains to the study of the Rous tumor grown in tissue culture. Chick fibroblasts in seven-day-old cultures to which trypan blue has been added on the second day are filled with colored droplets. Rous tumor cells grown in vitro show the migration of small irregular shaped cells which stain intensely twenty-four hours after the addition of trypan blue to the culture. In older cultures these cells become hypertrophied and multinucleate, probably as a result of fusion. Ludford believes them to be macrophages and contends that they are not restricted to the tumor. Carrel has regarded them as malignant cells. Ludford believes they are not the ma- lignant cells but considers them comparable to the polyblasts and macrophages of Maximow in mammalian tumors. These cells infiltrate the tumor tissue while it is growing in vivo and emigrate from the explant grown in vitro. Lud- ford is of the belief that certain cells characterized principally by their large nucleoli and by their failure to store vital dyes are the real malignant cells. These cells do not emigrate from the explant and are best shown in teased and sectioned material. Their resemblance to mammalian tumor cells, however, is Ludford’s chief criterion for designating them the tumor cells. His re- productions of these cells are suggestive of those forms that have been more commonly designated as the intermediate or transitional forms in the trans- formation of the macrophages into fibroblasts. Ludford is convinced that cancer cells grown in vittro do not segregate trypan blue in the same way as do their non-malignant prototypes. Pursuing the tissue culture technic as a means of determining experi- mentally the malignant cells in chicken sarcomata, Ludford (78) studied the effects of a Berkefeld filtrate of Fujinami and Rous tumors on cultures of the buffy coat of the blood of the fowl, and on cultures of fibroblasts from explants 296 MICHAEL LEVINE of pectoral and heart muscles of young chicks, The buffy coat cultures and the supernatant fluids of the tumor extracts are incapable of producing tumors when injected into young chicks. Tumors, however, were produced’ when cultures of fibroblasts were injected into similar young birds but in only one case when the supernatant fluid was injected. Ludford attempted to sur- mount the criticism (70) that the free active agent in these cultures is the source of tumor-producing substances rather than the cells. He added to cultures, in which the tumor filtrates had an opportunity to bring about changes in normal cells, tested immune sera so that effects of the free tumor- producing agent would be neutralized. After a given time these cells were in- jected into chicks. The fibroblast cultures alone yielded tumors. Ludford’s studies have led him to the conclusion that transformed fibroblasts are the malignant cells of the Fujinami and the Rous tumors, for these cells only are “ infected ” by the tumor agent. Contrary to the observations of the hema- tologists, tissue culturists, and those who have studied these tumors in vim, Ludford contends that there is no evidence of any transformation of the mononuclear cell types into cells resembling fibroblasts. Of his normal buffy coat cultures and those treated with filtrates of the tumors, less than one per cent showed fibroblasts, and these, he believes, were accidently introduced. The cultures, however, showed, after a growth of fourteen days in vitro, ir- regular mononuclear cells, giant cells, and epithelioid cells. The absence of fibroblasts seems to indicate, as Ludford suggests but is not prone to accept, that the monocytes in his cultures were not in the phase at which they are most susceptible to the influences that induce transformations. It is quite con- ceivable that fibroblasts may be introduced into cultures of buffy coat of blood, but in view of the variation in cell forms observed in tissue cultures, these changes may be attributed to culture conditions. The fact that forms intermediate between the monocyte and fibroblast cell types have been re- ported by various workers, from in vitro and in vivo material, suggests strongly that Ludford’s uniform results were due to the maintenance of constant cul- tural conditions which failed to favor transformations. Zweibaum (141) studied the cells of Rous sarcoma in vitro, comparing them with normal cells of the spleen and subcutaneous tissue, and the mono- cytes of the blood of an adult fowl. He is in accord with the earlier workers on the nature of the cells in the Rous tumor. The macrophages he arranged in three groups, small, middle-sized, and large. The tumor macrophages differ from the normal cell. They show two types of nuclear structure: in one the chromatin is granular, the distribution is uniform, and the staining capacity feeble; in the other the chromatin granules are voluminous, few in number, and the cell stains heavily. The normal macrophage has a uniform, fine- granular structure with one or two nucleoli and stains with acid dyes. The number of chromosomes in the sarcomatous macrophages, Zweibaum believes is larger than in normal cells. Further distinctions exist between the cyto- plasm of the normal and tumor macrophages. The structure of the fusiform cells of the sarcoma is analogous with the normal fibroblast. Zweibaum agrees with Carrel that the macrophage is the malignant cell, basing his contention on careful cytological studies. Malignancy at present seems to be a function which has no definite morphological expression in the cell. The variation CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 297

described by Zweibaum and others has not been definitely associated with malignancy. Pires Soares’ ( 114) studies on fowl leukocytes in vitro differed from those of other investigators in that only homologous plasma was used without the addition of extracts and without transplanting or subculturing the growth. In the first twenty-four hours his cultures showed the migration of small leukocytes and lymphocytes; the latter were identified by their characteristic movements. Only after forty-eight hours do monocytes make their appear- ance, and these are soon transformed into cells of the macrophage type, which are recognized by ingested dCbris. Giant cells of the Langhans type also appear. By the seventh or eighth day the greater part of the cells have trans- formed themselves into epithelioid cells. Both the giant cells and epithelioid cells are formed by fusion of the macrophages, the epithelioid cell contains an oval nucleus, eccentrically placed with an archoplasmic body close to it. A vacuolar system is often recognized, The Langhans’ giant cells have usually two or three nuclei, while others have as many as twelve to fifteen, arranged about the periphery of a rosette of vacuoles. Pires Soares is in accord with Fischer’s conception of the transformation of monocytes into fibroblasts, and believes the macrophage to be an intermediate stage in the absence of myo- proteins. In vitro studies of cell transformation in pathological tissue from human beings are of great interest. Awrorow and Timofejewskij (3) studied the blood elements in a case of myelogenous leukemia. They observed that the red blood cells degenerate and are phagocytosed by the macrophages. The poIymorphonuclear leukocytes show no progressive changes; they break up and are ingested by the macrophages. The myelocytes are longer lived; they wander like leukocytes and divide mitotically, but ultimately disintegrate, become pyknotic, and are taken up by the macrophages. The changes in the myeloblasts are varied. Changes in form and size occur in three ways. Thus the myeloblast may undergo hypertrophy, transform itself into a wander- ing cell of irregular shape, become phagocytic in nature, and resemble the polyblasts of Maximow; or it may become a spindle-shaped cell with an elongated nucleus and a reticular or honeycomb cytoplasmic structure; or, in the third place, it may become hypertrophied and capable of further trans- formation into a giant cell, macrophage, or wandering cell with cytoplasmic projections. The giant cells are formed in the presence of foreign bodies. Some of the wandering cells belong to the group of fibroblasts; others to the clasmatocytes and to the fixed polyblasts. Rinehart (118) studied a case of monocytic leukemia in man by the silver impregnation method, giving special attention to the nucleus of the monocyte. The stem cell of the monocyte he believes to be Ferrata’s hemohistioblast, an undifferentiated mesenchymal cell. His observations in the case recorded showed hemohistioblasts with intermediate forms and mature monocytes. Elqect of Dyes, Metals, and Other Chemicals: Lewis and Andervont (67), in an attempt to follow the r61e of the leukocytes in the production of the Rous tumor, treated these cells with carmine and injected them into susceptible fowls. The leukocytes failed to produce the tumor due, according to these investigators, to the adsorption of the active agents of the tumor by the car- 298 MICHAEL LEVINE mine granules. Later M. R. Lewis (66) studied the effect of 36 different dyes on the Rous tumor agent in 100 chickens. Eighteen of the dyes tested inactivated the tumor-producing power of the filtrate, showing a greater effect on the agent than carmine. In 5 of these the effect was shown to be due to a change in the pH of the agent rather than to the specific composition of the dye. Toluidin blue was found to have a very marked inactivating effect on the agent. In another study of this type, Lewis and Lewis (68) tested 80 different dyes and obtained inactivation of the tumor agent by relatively small concentrations of toluidin blue, phenol-3-indopheno1, 4’-bromo phenol-3-indo- phenol, and phenol indophenol. It was observed that the concentration neces- sary to inactivate the tumor in vitro was not administrable in vivo. MCnCgaux, Odiette, and Moyse (88) studied the effects of various metals on fibroblasts in cultures and arranged the metals tested in three groups: (1) very toxic, as copper, iron, and magnesium; (2) moderately toxic, as zinc, silver, tantalus, tin, nickel, and tungsten; (3) non-toxic, as gold, aluminum, and lead. They did not study the mechanism of the toxicity of these sub- stances. Vogelaar and Erlichman (140) were able to keep human fibroblasts alive for three months in a synthetic medium containing copper chloride (CuC1, . 2Hs0), Human thyroid tissue grew actively in a medium containing 0.0075 mg. copper per C.C. for a period of two months, but a medium con- taining 0.0112 copper per C.C. proved to be decidedly toxic. Baker (4) showed that liver ash, glutathione, and hemoglobin stimulate the growth of fibroblasts in vitro. Casein digest, glycocoll, and nucleic acid provide a medium in which fibroblasts of the rat sarcoma proliferate for a considerable time as rapidly as in embryonic juice. Ludford (76) studied the effects of vital new red and trypan blue on transplantable mouse tumors and concluded that the effect of these dyes is to lower resistance in the cells which segregate them. This indicates a possible danger of overidosage in the treatment of cancer with colloids and semicolloids, namely a breaking down of the mechanisms which may be offering resistance to the malignant growth. Kiyono (56) believes the sarcoma cells of the chicken store granules of carmine which remain unchanged after many cell generations.

Rous CHICKENSARCOMA IN VIVO Studies on the development of Rous chicken sarcoma in vivo are relatively few. Roskin ( 119) directed his efforts toward a cytological interpretation of the phenomena observed in carefully fixed and stained sections of the tumor. He found two basic cell types, round cells or macrophages and elongated cells or fibroblasts; tumor giant cells were also present in abundance. The macro- phages showed variation in types of pseudopodia; typical forms with one pseudopodium were more common than the bipseudopodial type. The fibro- blasts are described as elongated cells, and forms intermediate between these and the macrophages were observed. Roskin believes the transformation may be in either direction. The commonly accepted direction is from macrophage to fibroblast. McGowan (86), recognizing the difficulties in interpreting the histogenesis of the Rous tumor, outlined a scheme of the origin of blood cells which he CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 299 assumes will throw light on this problem. Using Aschoff’s and Maximow’s terminology and classification, modified by his. own observations, he states that the mesenchymal cell gives rise to the hemohistioblast. From this type of cell the monoblastic plasma cells originate, and these in turn give rise successively to monoblasts and monocytes, which include the histiocytes, macrophages, free histiocyte wandering cells, and finally the fibroblasts. McGowan holds that the Rous tumor is essentially a local manifestation of a disease of the reticulo-endothelial system involving the monocyte. The peri- vascular tissue, he believes, plays an important part in the spread of the dis- ease since this tissue represents a store of reticulo-endothelial elements. The effects of intravitam stains on the Rous tumor cells led McGowan to conclude that free histiocytes of the reticulo-endothelial system are the affected elements, and their differentiation into fibroblasts determines their malignancy. McJunkin (87) recognizes a similarity between the cellular elements in the chicken sarcoma and in the granuloma of Hodgkin’s disease. The mono- cytes of the blood are identical, he claims, with the chief reacting cells in Hodgkin’s granuloma. Similarly both the round cells and the spindle cells of the chicken sarcoma resemble the chicken monocyte. McJunkin found that the round cells and spindle cells alike are capable of phagocytosing carbon particles. Though, on the whole, fewer of the spindle cells ingested the carbon, many of them were laden with it. Neutral red was also introduced into tissue cultures of the tumor. It accumulates by the fourth day in typical rosettes in the round cells. In older cultures the spindle cells likewise show accumulation of the dye. With the aid of aniline blue, the spindle cells were shown to produce collagen, and fibroglia fibrils were demonstrable coursing along their surfaces; no fibroglia fibrils were seen in the round ameboid cells. The phagocytic activity of the spindle cell leaves McJunkin in doubt as to its qualities as a fibroblast. Mottram (95) investigated the response of the Rous tumor tissue to beta radiations from radium. He found that cultures of the tumor are resistant to beta rays, while in vivo the cells are destroyed. The tumor-producing power of a desiccate or of a cell-free filtrate of irradiated tumor tissue was not im- paired. Mottram further observed that a filtrate of the tumor applied to previously irradiated skin produced no reaction, while unirradiated skin in the same birds reacted with tumor formation. To determine why the previ- ously irradiated areas were not receptive to the tumor filtrate a study was made of scarified tissues. In such tissue, non-irradiated and non-treated, fibroblasts were found filling the zone of scarification, while epidermis was growing out under the crust of serum which had replaced the original epi- dermis. At the same period (four days) the histology of scarified tissues to which tumor filtrate had been applied showed loosely packed fibroblasts not unlike the normal cells, but larger and dividing abundantly. Seven days after scarification and application of filtrate, small Rous tumors were seen composed of spindle cells. Mottram believes that the tumor arises from normal fibro- blasts which lie in the dermis. Comparison of irradiated scarified areas with non-irradiated areas showed a great difference. The Rous tumor does not appear in the irradiated area because there are no healthy fibroblasts from which it can arise. 300 MICHAEL LEVINE

Haddow (49) made an attempt to solve the question of the histogenesis of the Rous tumor by the application of vital stains to fowls bearing tumors in- duced by injections of a cell-free filtrate. He believes that the Rous sarcoma is a disease of the reticulo-endothelial system, more strictly of that portion of the mesenchyme represented by the histiocytic elements of Kiyono. Haddow used the usual dilutions of trypan blue in sixteen injections at intervals of three to four days, waiting a week after the last injection before introducing the supernatant fluid of the tumor extract into 15 birds. From one of these, injected tissue was excised at the end of the 48th hour, daily fixations being made from the remaining birds. The tissue included muscle, subcu- taneous tissue, and skin. Haddow states that at the end of the second day evidence of tumor ‘inception was present, while specimens taken in the suc- ceeding days showed increasing signs of neoplastic growth, culminating in a mass of bluish white tissue at the end of seven days. In the earliest stages the intermuscular fasciae showed numerous young tumor cells undergoing rapid division. Haddow states that the free histiocyte, initially affected by the agent, shows marked morphological changes, the most obvious of which are the formation of protoplasmic processes, pseudopodia and cytoplasmic vacu- olization. These cells Haddow believes undergo division and their daughter cells, through recognizable intermediate stages, give rise to fibroblasts. There is no doubt, in his mind, that the tumor is derived from the action of the filtrate on previously normal resident cells recognizable in his studies by their content of segregated dye. He agrees with earlier workers that the monocyte is the basic cell from which the tumor arises. The resident macrophages are altered by the tumor agent and the bulk of the tumor results from the multiplication of these cells, as is indicated by the fact that the majority of the tumor cells show no visible segregation of trypan blue. Foulds (37) also used trypan blue in a study of six of the better known filtrable tumors of the fowl, which differ both in histologic structure and in rate of growth. Frozen sections and teased preparations of the tissues were obtained from birds injected with the stain. In the Rous sarcoma the great majority of the spindle cells were found to contain no detectable dye, Round cells, some of which showed segregation of the dye, were irregularly distributed throughout the tumor; the stained cells Foulds believes to be infiltrating his- tiocytes or polyblasts and not true tumor cells. Like Ludford, he believes that the tumor cells rarely segregate trypan blue and are so distinguishable from the cells of extraneous origin which infiltrate the growth. The results obtained with the Rous sarcoma were duplicated with the Fujinami tumor. The tumor cells of endothelioma MH, failed to segregate dye, but proved to be strongly phagocytic under suitable conditions. Foulds concludes that failure to segregate intravitam dyes is common to the tumors studied and is independent of the cell type, rate of growth, or degree of differentiation. Vital staining with trypan blue reveals no difference between avian and mammalian neoplasms. Striking analogies also exist between tumor dissemination in fowls and mammals (38). In a histologic study of the filtrable tumors of the fowl, Foulds (39)

f Rous sarcoma No. 1 ; Fujinami myxosarcoma; endothelioma MH,; fibrosarcoma MH,; rap- idly growing sarcoma MH, !; slowly growing fibrosarcoma MH,,. CYTOLOGY OF TUMOR CELL IN ROUS CHICKEN SARCOMA 301 refers to invasion of the muscle fibers, with survival of the sarcolemma sheath. Changes in the muscles consist of enlargement of the muscle fibers with loss of striations and multiplication of muscle nuclei. Peyron (112) believes that there is a conversion of muscle cells into tumor tissue. Millar (89) studied the regeneration tendencies in injured muscle in the rabbit and described cell and nuclear changes which Foulds believes resemble closely those occurring in muscle injured by tumor invasion. He believes, therefore, that the pro- liferating nuclei in invaded muscle are a response to injury and not an evidence of conversion into tumor. Foulds (39) regards the Rous chicken sarcoma as predominantly a spindle-cell tumor, though it may contain sections consisting almost entirely of rounded cells which are scarcely distinguishable from endo- thelioma MH,, except that they show no disposition to line spaces. He be- lieves (40) that a series of tumors may be arranged in which there is an in- creasing variability in structure due, probably, to increasing susceptibility to external conditions. Arranged in order of simplicity these are : fibrosarcoma MH,, Rous chicken sarcoma CTI, endothelioma MH,, and leukosis. Foulds believes that these all arise from a common cell type, but in the transformation into tumor tissue the normal cells are altered differently or to a different extent in each strain. Llombart (75) made an extensive study of the histopathology of the Rous tumor. His method involved formol fixation of tumors induced by grafts, staining with hematoxylin and eosin, and silver impregnation. His control fowls were inoculated with tissues of the Jensen rat sarcoma. He holds that in every new growth two types of cells make their appearance, macrophages and spindle cells. In the first developmental stages of the tumor, spindle cells are seen surrounding the implant. Macrophages are few in number and are like the reactive cells in the human sarcoma. Silver impregnation showed the absence of mitochondria in the macrophages and their presence in varying counts in the fibroblasts. Division stages are abundant in the first days of tumor growth, but later become infrequent. Intermediate stages between the two cell types are also recorded. By sacrificing birds on successive days after implantation with Rous tumor grafts, Llombart studied the gross morpho- logical changes in the surrounding areas. The changes that take place on the day following implantation are slight; only hemorrhage and leukocytic infiltration with intermuscular edema occur. On the second and third days giant cells are formed which envelop the graft. This Llombart interprets as a defense mechanism, a barrier between the healthy tissues and the transplant. He finds no difference between the macrophages of the tumor and those of inflammatory process. His sections are identical with those observed in tissue cultures. Vksquez-L6pe.z ( 139) implanted fragments or injected filtrates or desic- cates of the Rous tumor tissue into the intracerebral region of the fowl. Well defined nodules of tumor tissue developed, which invaded the brain along the perivascular Virchow-Robin spaces. .These tumors are said to arise through the concentration of compound granular corpuscles derived from the microglia cells. V&squez-L6pez contends that the microglia and their deriva- tives and the ameboid elements of the Rous tumor are identical. The micro- scopic characters of the Rous sarcoma in the brain are similar to those of 3 02 MICHAEL LEVINE intramuscular or subcutaneous growths, except for structural peculiarities when the tumor arises in the meninges. The cellular types are the same whether the transmission is made by graft or by injection of a cell-free filtrate. There are two cell types-the fibroblast-like form and the round cell with macrophagic tendencies. The histogenesis of the tumor was not studied in- tensively, but VBsquez-L6pez suggests that the reticulo-endothelial system in the nervous system is represented by the microglia and the perivascular cellular formations, and it is from this source that the tumor tissue is formed. Mauer’s (79~)study of Rous sarcoma indicates the course of develop- ment of the neoplasm as follows: Blood monocytes, endothelial and adventitial cells give rise to large proliferating monocytes; these form fibrocytes, which in turn develop into the sarcoma cells. The evidence of malignancy, Mauer be- lieves, appears when the large, round, undifferentiated cells are transformed into fibroblasts. ( To be continued ,)