T H E CYTOLOGY OF T H E TUMOR CELL I N T H E ROUS CHICKEN
SARCOMA
MICHAEL LEVINE 1
(From the Laboratory Division, Montifcore Hospital, New Yo7k C i t y )
The cytology of the Rous chicken sarcoma No. I has attracted the attention of biologists since its discovery. Considered broadly, this study has
been limited to that group of cells which responds in the animal body to invasion by foreign substances. This response to the tumor-producing substances 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 developmental 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 intravitam 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. I t 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. I t 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 O F TUMOR CELL I N ROUS CHICKEN SARCOMA
277
Rous SARCOMA
AND OTHERTRANSPLANTABLE
TUMORS
OF THE FOWL
The Rous Chicken Sarcoma N o . I (CTZ): Rous (120) in 1910 reported
the discovery of a transplantable avian tumor which he described as a spindlecell 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 dissemination 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. I t 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 diversities, the tumors grade into one another, and, since mitosis occurs most frequently 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 structure. 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 differentiated 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 processes 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 polymorphonuclear 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 concluded 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, l l l ) , 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 hemoglobin 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 T U M O R . C E L L IN ROUS C H I C K E N 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 conditions 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 transplantation 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 laboratories, 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 undifferentiated 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 myxomatous 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 becomes 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 metastatic 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 material 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 macrophages, with inclusion of cellular particles are similar to those of the parenchyma, 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 macrophages are noted. Except for their smaller size, the resemblance of these cells
to tumor cells is close. Capillaries with swollen walls penetrate the aggregation 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. I t is these free cells that are responsible
CYTOLOGY O F TUMOR CELL I N 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 ( 1 3 3 ) 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 recognized and removed at intervals for microscopic examination. I n 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 potentialities 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 between 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 TUMOR
IN EMBRYOS
OF FOWL
AND
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 extraembryonic 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 difOF
282
MICHAEL LEVINE
ferent from the cellular reactions induced by injury and by injections of infusorial earth, for in the new tissues stellate mesodermal cells and spindleshaped 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 tumors 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 ( 1 16) 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, however, 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 propagation 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
GRAFTOR FILTERED
AGENT
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 BrachettoBrian (74) believe that both the cells and the active agent in the cells are
responsible for this effect, while Ricardi ( 1 17) 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 fibroblasts 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 assumed 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 corroboration. A typical sarcoma of the fowl was described, the chief histologic constituent 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 treatment 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 ” filters, and these be regarded as responsible for tumor production. Borrel (10)
studied the growth of the Rous sarcoma in vitro and observed that the malignant 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 considered contaminants, which do not appear to interfere with the growth and
development of the tumor. Glover and his associates (47) apparently isolated 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 lacking 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 7 5 " C. for thirty minutes
to an hour. I t 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 subjected 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 preparations, 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.
THEBLOODAND 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 elements. By the use of these dyes on tissues studied in vitro and in vivo Goldmann, Tschaschin, Kiyono, and others have cleared up some of the difficulties
in identifying these elements, but even these methods, which involve considerable technical and biological difficulties, have not brought about a generally accepted view as to the origin of these cells. Cappell ( 1 2 ) 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 Mjassojedoff (90). These studies show ameboid cells of various kinds with transitional 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 applied to them such names as clasmatocytes, adventitious cells and, later, resting wandering cells (Maximow, SO). Maximow described these cells as part
of an irregularly arranged common connective tissue which is to be found everywhere 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 colloidal 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 reticuloendothelial metabolic apparatus intimately connected with the metabolism of
cholesterol. These and other studies gave rise to the theory of “ reticuloendothelium,” 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 depending 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 elements some of which are capable of taking up vital dyes, such as neutral red.
The “ active wandering cells ” embrace three cell types distinguished principally 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 kidneyshaped nuclei and ameboid protoplasm which takes up vital dyes. The smaller
wandering cells, Maximow believes, are identical with the non-granular leukocytes of the blood.
The inactive, fixed group of cells, or the resting wandering cells of Maximow, 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 processes 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 intermediate forms Maximow finds difficult to interpret. He suggests that they
may be the expression of a dual transformation of an ameboid cell into a resting 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 development of histiocytes from mesenchymal cells (81).
( 2 ) Monocytes: The monocyte, or non-granular leukocyte, or large mononuclear leukocyte, is bound up with the problem of the histiocytes. These
cells in the mammal are comparatively large, with abundant cytoplasm; occasionally 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. I t 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 lymphocyte. 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, 8 5 ) divides 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 I N ROUS C H I C K E N 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 inflammatory process or in vitro, according to Maximow, is definite. The isolated 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 workers that in inflammation not only polyblasts, but also lymphocytes and plasma
cells, may originate from the proliferations of perivascular histiocytes. Ferrata’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, differentiated and incapable of transformation, Maximow (84) holds that they
may give rise to polyblasts during an inflammatory process. Through a process 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 reacting cells of the histiocytic type and, finally, into fibroblasts with loss of phagocytic properties. A large number of the lymphocytes of the blood of the chick
grown in vitro turn into thrombocytes. Maximow (82) believes that mesenchymal 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 ( 2 5 ) 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 fibroblasts in tissue cultures of lymph of the thoracic duct of the rabbit. The
lymphocyte becomes transformed into a polyblast, which in turn is transformed 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 subcutaneous 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. Recovery 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 methods, 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, centrosphere, 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 ( 3 6 ) , 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 indefinite, 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 clasmatocytes, 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 digestion. 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, constitute races which he believes are physiologically different, though morphologically 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 derived 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 represent the usual sequences in cellular development. Transformation of fibroblasts 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 represent in vitro extreme functional states of the same cell, He (107, 108) concludes 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 fibroblasts derived from bone and concluded that these fibroblasts possess no distinguishing morphological features. Their transformation into bone was not evident under usual cultural methods but when the growth-stimulating substances
were reduced, transformations occurred. The conclusion is drawn that proliferation inhibits the normal function of the cells.
Horning ( 5 1) studied osteoblasts and muscle fibroblasts, and concluded
that cells are endowed with an inherent intrinsic mechanism and that cell behavior 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 dependent 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 believes 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 originate 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 several thousand. The locations of isolated cells were marked so that they could
be kept under continuous observation. Under certain, though indefinite, conditions 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 fibroblasts. Moen was unable to determine whether the macrophages and fibroblasts 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 injected desiccates, while the former are found distributed through the tumor
tissue. The origin of tumor giant cells is obscure. They are generally regarded 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 confused 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 i n Literature
Spleen
Lymph Node
Bone Marrow
Lambert, 1912
I Lewis and Web- Foot, 1912
Erdmann, 1917
' ster, 1921
Lambert and
Maxirnow, 1922, Grossman, 1923
Hanes, 1913
1924, 1925
Weil, 1913
Sniyth, 1916
Barta, 1925
Chlopin and
Chlopin, 1925
Erdma nn, Eisner ,
and Laser, 1925
Timofejewskij and
Benewolenska ya,
1925
Blood and
Buffy Coat
Fowl Tuniors
i n vitro and
i n vivo
Awororow and
Timofejewskij,
1914
M. K. Lewis, 1925
'rimofejewskij and
Renewolenskaya,
1925
Lewis and Lewis,
1925, 1926
Kous, 1911
Iioskin, 1926
Murray and Begg,
1930
Zweibaum, 1933
Llombart, 1935
Pires Soares, 1937
Levine, 1939
-_
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 twoday 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 believes 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 endothelium of the vessels. They ingested lamp black, while the nearby lymphocytes 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 endothelial cells together with these giant cells form a dense wall about the injected
agar, surrounded by a capsule of young connective tissue,
29 1
CYTOLOGY O F TUMOR CELL I N ROUS CHICKEN SARCOMA
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, represent fusions of large mononuclear wandering cells of endothelial or pulp cell
origin. The accumulation of granules with an affinity for neutral red dye becomes 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 correspond 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 cultures, which seem to separate themselves sharply from the tumor cells because
of their resemblance to the Langhans type of giant cell.
TISSUECULTURESTUDIESOF
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 proliferated 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 communications, Carrel (13, 14) attempted to show that the macrophage of the
chicken blood is sensitive to the tumor-producing agent or virus of Rous sarcoma, 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 macrophages, 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 expression 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 fibroblasts 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 movements 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 processes 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 indistinguishable. I t 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 (fibroblasts). In a study of the Rous chicken tumor grown in vitro (30) he observed that the cells which grow out of the explant are extremely polymorphous, 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 cultures. Stained, these cells show a distinct nucleus with one large nucleolus
and two accompanying smaller ones. Spindle-shaped cells also appear resembling 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 separate 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 reports, does not occur. I n a study of the mouse carcinoma, on the other hand,
Fischer and Parker ( 3 3 ) 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 ( 3 1 ) found that the
principal cells in the explant are the ameboid cells and fibroblast-like connective-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 ( 3 2 ) pointed out what they believe to be another physiological difference between normal and malignant cells grown in vitro. I n 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 ( 6 1 ) 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 separate 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 conditions. 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 fortyeight hours, appears to reach full development in seventy-two to ninety-six
hours, and continues to grow for several days longer. The cells are spindleshaped 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 cultivated 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 concomitant 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 ( 7 2 ) , 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 malignant 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 appearance of cells with chromosomal aberrations. In this observation he is in accord with a number of workers who have studied one or another of these
tumors independently. That malignancy is due to these chromosomaI abnormalities is at present beyond the sphere of experimental verification.
Kimura (54, 5 5 ) studied 142 generations of the Fujinami myxosarcoma
of the fowl in tissue culture. He found that only in the first and second transplant generations of the myxomatous tissue or after the addition of heart tissue
to his cultures, did round cells make their appearance. These cells degenerated in the second generation transplants and in subsequent generations
spindle cells entirely dominated the cultures. These spindle cells were morphologically like fibroblasts, differing from the latter only in their limited
growth in vitro. Inoculation of fowls with tissue cultures of various transplant generations showed, according to Kimura, that the spindle cells are the
malignant ones. When round cells are present, as in the first transplant generation 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, twentyeight, 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 I N 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 ( S S ) , 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 macrophages 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 ( 7 7 ) 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 malignant 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. Ludford 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 reproductions of these cells are suggestive of those forms that have been more
commonly designated as the intermediate or transitional forms in the transformation 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 experimentally 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 surmount 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 tumorproducing agent would be neutralized. After a given time these cells were injected 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 hematologists, tissue culturists, and those who have studied these tumors in v i m ,
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, irregular 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. I t is quite conceivable 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 reported by various workers, from in vitro and in vivo material, suggests strongly
that Ludford’s uniform results were due to the maintenance of constant cultural 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 monocytes 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, finegranular 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 cytoplasm 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 I N 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 appearance, 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 transformed 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 myoproteins.
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 wandering 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 transformation 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-
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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. I n 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-indophenol, and phenol indophenol. It was observed that the concentration necessary 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 substances. 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 containing 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 macrophages showed variation in types of pseudopodia; typical forms with one
pseudopodium were more common than the bipseudopodial type. The fibroblasts 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 O F TUMOR CELL IN ROUS C H I C K E N 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 perivascular tissue, he believes, plays an important part in the spread of the disease 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 monocytes 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. I t accumulates by the fourth day in typical
rosettes in the round cells. I n 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 impaired. 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. T o determine why the previously 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 epidermis. 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 fibroblasts 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 induced 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, subcutaneous tissue, and skin. Haddow states that at the end of the second day
evidence of tumor ‘inception was present, while specimens taken in the succeeding 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 vacuolization. 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 ( 3 7 ) 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 histiocytes 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 M H , ; rapidly growing sarcoma MH, !; slowly growing fibrosarcoma MH,,.
CYTOLOGY OF TUMOR CELL I N 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 (1 1 2 ) 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 proliferating nuclei in invaded muscle are a response to injury and not an evidence
of conversion into tumor. Foulds ( 3 9 ) 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 endothelioma MH,, except that they show no disposition to line spaces. He believes (40) that a series of tumors may be arranged in which there is an increasing 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 ( 7 5 ) 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 morphological 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 desiccates 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-L6pezcontends that the microglia and their derivatives and the ameboid elements of the Rous tumor are identical. The microscopic 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 intensively, 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 ( 7 9 ~ )study of Rous sarcoma indicates the course of development 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 believes, appears when the large, round, undifferentiated cells are transformed
into fibroblasts.
( To be continued ,)