FROM T H E DEPARTMENT OF FORENSIC MEDICINE (HEAD: PROF. UNTO UOTILA, M.D.) A N D THE DEPARTMENT OF PATHOLOGY,

SECTION I1 (HEAD: PROF. HARALD TEIR, M.D.) , UNIVERSITY OF HELSINKI

OBSERVATIONS ON THE SERIAL TRANSPLANTATION AND GROWTH CHARACTERISTICS OF THE ITR ASCITES

TUMOR OF THE RAT'

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KALEVI PY~RALA Received 3O.vii.55

In recent years, ascites tumors have received a good deal of atten- tion in experimental cancer research because free tumor cells mul- tiplying in the peritoneal cavity provide excellent opportunities of cyto- logical and 'biochemical study. A considerable number of potential ascites tumors which grow in experimental animals are known today. For studies dealing with ascites tumors references can be made to G. Klein (1) & E . Klein (2 ) .

The best known among the ascites tumors of the rat is the Yoshida sarcoma (3) . Other such tumors are MTK sarcomas I and I1 (4), the Takeda sarcoma ( 5 ) , sarcomas developed from malignant rat hepa- tomas (6, 7, 8) , and the leukemic lymphosarcoma of the rat reported by Nakamiira ( 9 ) .

The ITR tumor is a malignant neoplasm of the retroperitoneal cavity of the rat discovered during a series of experiments in which an attempt was made to induce skin tumors in rats by applying methyl- cholanthrene into the skin and by simultaneous injection of rat skin suspension into the peritoneal cavity (10) . Ascitic fluid containing tumor cells was found in connection with the neoplasm. The tumor was of inesenchymal origin, arising either in the mesothelium or in the reticular cells of the peritoneal cavity. The ITB tumor proved to be transplantable both subcutaneously and intraperitoneally by using minced ascites tumor suspended in saline and by ascitic fluid alone (11). In the majority of animals intraperitoneal transplantation by the former method produced-apart from solid neoplastic growth-ascitic fluid, which, however, contained only a small number of tumor cells in proportion to other ascites cells.

1 Aided by a grant from the Damon Runyon Memorial Fund (DRG 291), New York, and the Sigrid Jus6lius Foundation, Helsinki.

1 ACTA PATH. XXXVIII, 1 1

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The object of the experiment now reported was to develop the ITB tumor into a true ascites tumor by continuous ascites transplantation and to study its characteristics of growth, particularly alterations in the composition of the peritoneal fluid and the occurrence of solid tumor infiltration during the development of the ascites tumor.

M A T E R I A L A N D M E T H O D S

The animals were rats of the Wistar strain. The strain used, however, was not pure inbred. The weights of the animals ranged from 80 to 200 g. Both sexes were represented, but the majority were females. Standard diet was used.

Ascitic fluid smears were immediately fixed and stained by Papanicolaou’s method (12) . The differential count of the ascites cells was made from these pre- parations by counting 500 cells.

The total number of ascites cells per millimetre was counted by the Riirker- Tiirck hemocytometer. The ascitic fluid was diluted to 1:lOO by a solution contain- ing 0.05 g of methyl violet in 100 ml of 0.5 per cent acetic acid, The staining of the nuclei and the disintegration of the erythrocytes faciliatcd the count, particularly in hemorrhagic ascitic fluid. The results were in agreement with samples diluted with saline. When it was found desirable to estimate also the number of of necrotic cells, the count was made according to the method of Goldie & Felix (13) . From ascitic fluid diluted by saline to 1: 100 a sample was taken into a hemocytometer treated by a 0.02 per cent alcoholic solution of neutral red. The count was made five minutes later. The cells with even slightly stained nuclei were considered as heing necrotic. The necrotic cell count was expressed as per cent. The total number of tumor cells per millilitre was counted from the total number of cells and from the percentage of tumor cells computed from the smear preparations.

The histological samples taken from the tumor tissue of the peritoneal cavity and from surrounding organs were fixed with 10 per cent formalin, embedded in paraffin, sectioned at 5 P , and stained with hemalaun-eosin.

Details of the procedure will also be mentioned in the experimental part of the present account.

E X P E R I M E N T A L

Differentiation of ITB Tumor Cel ls f r o m other Ce l l s o f the Peritoneal Fluid. Cells occurring in the peritoneal fluid under physiological conditions were ob-

tained by aspiration through a capillary glass pipette from 14 rats. The classifica- tion of the cells was carried out according to the principles recommended by the Vincent Memorial Laboratory S ta f f for the classification of cells in peritoneal fluid (12) . The relative proportions of the different cell types ranged within the follow- ing limits:-mesothelial cells 5.4-49.2 per cent (with a mean of 18.9 per cent), histio- cytes 8.0-27.8 per cent (20.3 per cent), polymorphonuclears 0.6-36.1 per cent (10.0 per cent), lymphocytes 20.6-73.2 per cent (49.2 per cent), and mast cells 0.3-4.0 per cent (1.6 per cent). Fig. 5 shows mesothelial cells and lymphocytes in normal peritoneal fluid. Nearly all the polymorphonuclears were eosinophil, as indicated by May-Griinwald-Giemsa stains, Yoshirla ( 3 ) , whose school follows different prin- ciples of classification, finds that the normal cell composition of the peritoneal fluid of the rat is as follows: monocytes 64-70 per cent, lymphocytes 1 6 1 7 per cent, eosinophil granulocytes 13 per cent, and mast cells 3-5 per cent.

The total number of cells in one millilitre of normal peritoneal fluid was deter- mined in 20 rats. It was found to range between 27.9 and 111.2 X 106 cells per millilitre, with an average of 70.8 x 106 cells.

In addition to uormal peritoneal fluid, cells occurring in connection with sterile peritonitis were also studied. The peritonitjs was induced according to G. Klein (14) by injecting intraperitoneally 2 ml of Omnadin “Hoechst” into 7 rats and “Kiesel- guhr” (finely ground diatomaceous earth) mixed with saline intraperitoneally into another 7 rats, On examining samples obtained by puncture from the peritoneal fluid both substances were found to have produced initially a violent polymorpho-

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nuclear response; when it was suhsiding, proliferation of mesothelial cells and histiocytes set in. Omnadin produced in the first place an increase in the numher of mesothelial cells, while Kieselguhr injection was followed hy the presence of numerous active histiocytes full of phagocytic crystals.

Mitoses were rare in the cells of normal peritoneal fluid and of sterile peritonitis. In bbth cases the frequency of mitosis was less than 0.5 per cent.

Careful niorphological study of cells in normal peritoneal fluid and in con- nection with sterile peritonitis provided a hasis for the differentiation of ITH tumor cells from those occurring in normal exudate.

ITB tumor cells are clearly different from the other cells of the ascitic fluid (Figs. 5-8). They vary in size, being usually bigger than normal cells except mast cells. The nucleus is big in comparison with the size of the cell, frequently kidney-shaped and located eccentrically. The chromatin is unevenly distributed and stains intensely. The nu- cleoli, one or more than one in number, are relatively large and ir- regular in shape. The cytoplasm is strongly basophilic. These features distinguish the ITB tumor cells froin normal exudate cells, particularly from mesothelial cells, to which they hear the closest resemblance. The difference is great enough to exclude any uncertainty. ITB cells with two and more nuclei are quite frequently seen, as are normal and abnormal mitoses. Cells undergoing mitosis were regarded as tumor cells in the present work because mitosis in other cells is rare in coni- p r i s o n with its occurrence in tumor cells.

Development of ITB Ascites Tumor and its Serial Transplantation. When the ascites transplantation of the I T H tumor was hcgun, the original

tumor had passed through 11 transfer generations achieved hy intraperitoneal in- jection of a mixture of ascitic fluid and minced tumor tissue diluted hy saline (11). 'fo the rat with which the transplantation was begun the tumor had heen trans- ferred 31 days previously. The rat was killed hy decapitation, the ahdominal skin was removed and the ascitic fluid collected through a small opening made in the wall of the peritoneal cavity. Of the fluid thus collected, 12 per cent of the cells of which were tumor cells, 2 ml were injected intraperitoneally into 5 rats of the first transplantation generation, altogether 4.4 X 100 tumor cells to each. The rats were sacrificed after 21 days. Three individuals werc found to have no tumor a t all, and one had a couple o f solid tumor nodes in the upper part of the peritoneal cavity a t the point of mescnteric attachment. Onc of the animals, however, was found to havc 20 ml of slightly bloody ascitic fluid and abundant solid tumor growth in the omentum and mesentery. Of the cells of the ascitic fluid, 16.5 per cent were tumor cells. Four millilitrcs of this fluid, altogether 11.2 X 106; tumor cells, were trans- ferred into 5 rats of the second generation, which were killed after 12 days. Al l individuals were found to have 10-35 ml of ascitic fluid and solid tumor growth. The relative numhcr of tumor cells ranged from 10.4 to 26.0 per cent. From one of the rats 4 ml of ascitic fluid was transferred to 6 rats of the third generation, alto- gether 18.8 x lo(; tumor cells. Twelve days later the tumor was again transferred to rats of a new gcneration.

While transferring tumor cells in ascitic fluid at intervals of 12 days their relative number was found to increase gradually (Fig. 1 ) . In the seventh generation the relative number of tumor cells was 56.6- 60.4 per cent..From this generation onwards the tumor cells constituted the majority of the ascitic cells. As the proportion of tumor cells in the ascitic fluid increased, the transplanted number of these cells also

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I a4X ' . . . . . . ' . ' . . . . ' . . ' ' ' G"f#t;?Zl 2 3 4 5 6 7 8 9 I0 If 22 f3 I4 15 16 I7 I8 19 i o ' r c r u 11. t i ? tab ( 0 4 236 JV JIZ 4 1 1 bzo &Q? 616 mr A? 1076 918 411 @A 971 *6 1272

Fig. i. Increase in the relative number of tumor cells in ascitic fluid during thc first transplantation. The transplantations were carried out between the second and twentieth generations at intervals of 12 days. The dots stand for values obtained

with different rats. The inoculated number of tumor cells is given at each generation.

,bWwa,J

increased (Fig. 1). I t was this that was obviously responsible for the acceleration in neoplastic growth noted between the tenth and twentieth generations, on account of which the transplantation interval was re- duced to 6-8 days after the twentieth generation and the amount of ascitic fluid injected reduced to 2 ml, the number of tumor cells trans- ferred ranging from 50 to 100 X 166.

In the thirty-fourth generation lower tumor cell counts than usual were un- expectedly encountered, and the ascitic fluid contained abundance of polymorpho- nuclears. The relative number of tumor cells remained low as a rule even in the following generations, although values exceeding 50 per cent were occasionally re- gistered. A large proportion of the tumor cells of the ascitic fluid were degenerative, and in the solid tumor infiltration of the peritoneal cavity more hemorrhages and necroses were seen than usual. The ascitic fluid contained numerous fibrinous masses. Bacterial culture was carried out from the forty-ninth generation; Salmo- nella t y p h i murium grew from all samples. The rats of the 50th generation received streptomycin and penicillin for 7 days (0.01 g of dihydrostreptomycin and 10,000 units of procaine-G-penicillin daily) ; according to sensitivity tests this combina- tion could be expected to be most efficient against the bacterium in question. The samples obtained from the fifty-second generation were sterile again, and the relative number of tumor cells exceeded 60 per cent. In the sixty-fifth generation signs suggestive of infection were again detected, and in most ascitie fluid samples the number of inflammatory cells exceeded that of tumor cells. Bacterial culture yielded Salmonella enteritidis. The rats of the sixty-seventh generation received streptomycin and penicillin for 7 days ; infection subsided, and the ascites tumor developed in the usual way, with tumor cells prevailing among the cells of the fluid. I t was decided to alter the transplantation technique because collection of ascitic fluid by the method used made the samples liable to bacterial infection. From now on the samples were taken by aspiration through a thick needle, and more attention was paid to sterility during transplantation. Bacterial cultures were made from each of the following generations down to the seventy-fifth, after which bacteriological examinations were made occasionally. The tumor remained sterile after the sixty-seventh generation; during the infections, its nature remained com- pletely unchanged.

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36 1 2 3 HOURS DAYS

Fig. 2. Alterations in the cell composition of the peritoneal fluid during the development of the ITB ascites tumor (60 X 106 tumor cells inoculated). The curves are mean values for 16 rats. The relative number of tumor cells in different individuals are

marked with dots. Death is indicated by a cross a t the day when it occurred.

By the time of the writing of this report the ITB ascites tumor has been transplanted through 100 generations. A total of 547 rats has been used for the process. Estimating from the total number of the genera- tions the proportion of successful transplantations has been 82 per cent. The tumor grew as well in male as in female rats. With the exception of a few transient alterations in the cell composition of the ascitic fluid caused by bacterial infections, no changes has been noted in the growth properties of the ITB ascites tumor during the transplan- tation process, nor in the morphology of the tumor cells nor in the histological features of the solid tumor infiltration of the peritoneal cavity.

Alterations in the Cell Composition of Peritoneal Fluid during the Development o f ITB Ascites Tumor.

In the thirty-first generation, 60 X 100 tumor cells were inoculated intraperitone- ally into 20 female rats (age 3 months, weight 140-172 g). From the peritoneal cavity of each of these rats samples were aspirated through capillary glass pipettes 3 and 6 hours after the inoculation and after that daily down to the death of the rat. The differential cell count was made from smear preparations of these samples. The total number of tumor cells per millilitre and the percentile proportion of the necrotic cells were also counted. The frequency of mitosis was determined by counting 1,000 tumor cells from each smear preparation. Sixteen rats showed in- creasing ascites formation from the fourth day onwards. The first ra t died on the thirteenth day. By the nineteenth day 16 rats had died of typical ascites tumor. No tumor developed in the remaining four individuals. Their peritoneal cavities were entirely normal when opened 30 days after the inoculation.

Fig. 2 illustrates the alterations in the cell composition of the peri- toneal fluid in those individuals in which a neoplasm developed. The

F i g . 3. Alterations in the tumor cell concentration of the peritoneal fluid during the development of the ITB ascites tumor (60 X 100 tumor cells inoculated). The curve illustrates the mean of 16 rats. The dots stand for values obtained with different

individuals.

tumor cell inoculation was followed by an increase in the number of polymorphonuclears, which was most intense in samples taken 6 hours later (Fig. 6 ) . As the polymorphonuclears began to decrease, a relative increase in the mononuclear inflammatory cells set in; this was most intense on the second day (Fig. 7 ) . The relative number of the tumor cells, which had decreased on the first two days, began to increase rapidly on the second day, to reach its maximum on the sixth day after inoculation (Fig. 8) , when the ascitic fluid gave an almost pure tumor cell culture. After this the relative number of the tumor cells fell gradually and the proportion of the mononuclear inflammatory cells increased. The relative number of tumor cells varied considerably from rat to rat, particularly during the final phase of neoplastic develop- ment, when many of them were karyorrhectic. Some giant cells were also seen, with a large number of micronuclei. The number of poly- morphonuclears did not rise noticeably even a t the terminal phase of tuinor development. The mast cells, a few of which occurred down to the third day disappeared entirely from the peritoneal fluid when the neoplastic growth became more intense. After the seventh day the ascitic fluid was often somewhat hemorrhagic.

The number of necrotic cells was less than one per cent down to the seventh day, after which it increased and averaged 5 per cent after the ninth day. The frequency of mitosis varied between one and three per cent and reached its maximum on the second and third days after inoculation.

Fig. 3 shows the concentration of the tumor cells in the peritoneal fluid during the development of the neoplasm. The concentration fell

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Fig. 4. Alterations in the cell composition of the peritorleal fluid in those rats which did not develop a tumor (60 X 106 tumor cells inoculated). The curves are mean values for 4 rats. The relative numbers of tumor cells in the different individuals have

been marked with dots.

until the second day, after which it rapidly increased and reached the maximum on the sixth day of the inoculation. As the ascitic fluid increased, the concentration gradually decreased. The values varied a great deal in different individuals.

Fig. 4 illustrates the alterations of cell composition in the 4 rats which developed no tumor a t all. These individuals also showed an initial polymorphonuclear response followed by a n increase in the numher of mononuclear inflammatory cells. The number of tumor cells decreased gradually, and they completely disappeared by the sixth day. On the seventh day the cell composition of the peritoneal fluid was normal again.

Tumor Infiltration of Peritoneal Cavity During the DerJelopment of ITB Ascites Tumor.

A dose of 60 x 106 tumor cells were inoculated intraperitoneally into 36 female rats (age 2 months, weight 100-130 g). The animals were sacrificed in groups of 5 individuals 2, 3, 4, 5 , 6 and 8 days after inoculation. Two of the remaining rats died of ascites tumor on the twelfth day of the inoculation. On the same day the four surviving were also killed. The cell composition of the peritoneal fluid was studied in the same way as in the experimental series reported in the preceding chapter, with similar results for the various separate phases of neoplastic development. Histological samples were taken from different parts of the parietal peritoneum, diaphragm, omentum, mesentery, pancreas, liver, spleen, kidneys, and lungs.

Microscopic tumor cell infiltration was visible in the omentum as early as 2-3 days after inoculation (Fig. 9 ) . The neoplastic omental growth progressed rapidly, and on the sixth day the whole omentum was infiltrated hy tumor tissue. From the sixth day onwards neoplastic growth was also seen in the mesenterium and pancreas and in the peri- toneal folds of the pelvis. On the eighth day necrotic areas and hemor- rhages were found here and there in the omental tumor tissue. On the

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Figs. 5-20. Fig. 5.-Mesothelial cells and lymphocytes in normal peritoneal fluid of the rat.

X 800. Fig. 6.-ITB tumor cells in peritoneal fluid 6 hours after inoculation. Several

polymorphonuclears. X 800. Fig. 'I.-Two ITB tumor cells among histiocytes on the second day after inoculation.

X 800. Fig. 8.-ITB tumor cells in ascitic fluid on the sixth day after inoculation.

Fig. 9.--Incipient tumor cell infiltration in the omentum on the third day after inoculation. X 60.

One of the tumor cells undergoes mitosis. X 800.

Fig. 10.-Solid ITB tumor tissue. X 800.

twelfth day, when the volume of ascitic fluid amounted to 30-40 ml, some tumor cell infiltration was seen in the parietal peritoneum, the muscles of the wall of the abdominal cavity, and the diaphragm. A

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number of rats developed a small subcutaneous tumor mass a t the point of inoculation. No neoplastic growth was visible in the liver and spleen, although the hilar regions of both organs showed plenty of neoplastic tissue during the terminal phase of tumor development, when the renal capsule was also infiltrated by tumor cells, though no neoplastic growth was noted in the kidney tissue itself.

The histological features of solid ITB tumor tissue were similar to those of original ITB tumors ( l o ) , with the closest resemblance to a highly anaplastic reticular cell sarcoma (Fig. 10) .

Lung metastases occurred in none of the rats. A search for tumor cells in the peripheral blood of 7 rats on 8 days

and of 3 rats on 12 days after inoculation yielded a negative result.

D I S C U S S I O N

G . Klein ( 1 4 , 15) & E . Klein ( a ) , who have studied the development of solid transplantable mouse tumors into ascites tumors in intraperi- toneal transplantation, found that in some cases the tumor grew as an ascites tumor even in the first generation (immediate conversion). Some of the tumors assumed the ascitic form only gradually, after several transplantations (gradual conversion). The majority of the tumors studied by these two authorities did not, however, develop into ascites tumors a t all. They drew the conclusion that immediate con- version into the ascitic form takes place only if the cells nioculated into the peritoneal cavity include plenty of cells viable in peritoneal fluid. A gradual transformation into the ascitic form, on the other hand, is the result of a selective overgrowth of a small number of cells which differ from ordinary tumor cells in that they are more capable of multiplying in peritoneal fluid. When the number of these cells exceeds a certain critical limit during the process of transplantation, the tumor begins to grow in the ascitic form. Sat0 & A r u j i ( 6 ) and Tanaka & Kano ( 7 ) succeeded in transforming a malignant ra t hepa- toma into an ascites tumor after a few generations by transplantation of minced hepatomatous tissue. When isolated cell islets found in the peritoneal cavity during the terminal phase of the hepatoma were im- planted, an ascites hepatoma developed in the first generation ( 6 ) . Isaka (8) succeeded in transforming an OAT hepatoma into an ascites tumor in the first generation by intraperitoneal injection of minced hepatomatous tissue.

The Yoshida sarcoma (31, MTK sarcomas I and I1 (4), and the Takeda sarcoma (5) are primary ascites tumors, i.e., tumors originat- ing i n the peritoneal cavity, in connection of which ascitic fluid con- taining abundance of tumor cells was encountered.

The ITB tumor resembles these sarcomas in that it was originally discovered in the peritoneal cavity and that ascites containing tumor cells was found in addition to solid tumor tissue (10) . The number of

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tumor cells in the ascitic fluid was not, however, studied in detail. The tumor was first transplanted intraperitoneally hy a mixture of ascitic fluid and minced tumor tissue (11 ) . In the A strain, a t the eleventh generation of which the development of the ITB ascites tumor hegan, the transfer had heen carried out a t intervals of 21-44 days because the tumor developed rather slowly. The neoplasm grew in 66 per cent of the animals, and 41 per cent of these developed only solid tumors without formation of ascites. In the eleventh generation the numher of tumor cells in the ascitic fluid was found to he 13 per cent of all cells. This is to say that the ITB tumor when transplanted in the ordinary way did not grow as an ascites tumor proper but as a tumor of the peritoneal cavity in connection with which ascites formation was present. The ascitic fluid contained a numher of active tumor cells, but only in a low proportion. During ascites transplanta- tion the number of tumor cells in the ascitic fluid rose gradually until, from the seventh generation onwards, the neoplasm grew as a true ascites tumor. G. Klein ( 1 ) has shown that the inoculated number of tumor cells must be above a certain critical level to enable the tumor to grow in the ascitic form. When smaller amounts of tumor cells are inoculated, mainly solid tumors or ascitic fluid containing mainly in- flammatory cells are produced. As the relative number of tumor cells gradually increased during the transplantation process, the growth of the neoplasm in the ascitic form hecame possible. I t is also possible that selective overgrowth of cells capable of multiplying in peritoneal fluid took place during ascites transplantation much in the same way as found by G . Klein (14, 15) and E . Klein ( 2 ) in developing ascites tumors in the mouse. As an ascites tumor the ITB tumor was found to grow in a larger number of rats than when the earlier method of transplantation was used, to judge from the fact that the proportion of successful cases among ascites transplantations was 82 per cent.

The fact that the tumor did not grow in all individuals was pro- bably due to the heterozygotic character of the rat strain used. The same phenomenon has heen seen in transplanting other tumors into heterozygotic rats. In the case of the Yoshida sarcoma the rate of successful transplantation was 91 per cent (16 ) , of MTK sarcoma I 85.5 per cent, and of MTK sarcoma I1 88 per cent ( 1 7 ) Safo (181, who developed inbred rat strains from non-susceptible individuals, has shown that the decisive factor i n non-susceptibility is the hereditary constitution of the animal.

During infection by Salmonella tgphi murium and SaZmonella enfe- rifidis the proportion of tumor cells to inflammatory cells in the ascitic fluid was rather low, and the tumor cells showed abundance of degenerative changes, These alterations in the cell composition of the ascites fluid may be merely ascribable to the fact that the inflam- matory cell response caused by the infection camouflaged the increase of the tumor cells. On the other hand the plentiful occurrence of

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hemorrhages and necroses in the solid tumor infiltration of the peri- toneal cavity is possibly to be associated with the many reports on the necrosis and hemorrhage producing effect of bacterial toxins on neo- plastic tissue (e .g . , 19, 20, 21 ) . The best known among these toxins is so-called Shear’s polysaccharide (21 ) , isolated from a Serratia mar- cescens culture. A toxin which affects neoplastic tissue has also been isolated from S. lyphi murium and S . enteritidis ( 2 3 ) . The effect of bacterial toxins has been found to be based on the vascular lcsions caused by them, to which tumor tissue is more liahle than normal one (24, 25 ) . Some authorities claim that bacterial toxins also have a direct effect on tumor cells (26, 27 ) . G . Klein ( l ) , however, did not find that Shear’s polysaccharide had any effect on S 37 sarcoina cells in ascitic fluid, although the preparation produced extensive necroses in solid tumors. In this connection there is reason to mention the change towards greater malignancy noted in the Takeda sarcoma following infection by Salmonella enteritidis ( 2 8 ) . Nothing of this kind was ob- served in the case of the ITB tumor.

The development of cell composition in peritoneal fluid in connec- tion with the Yoshida sarcoma ( 3 ) , Ehrlich ascites tumor (14), and sarcoma S 37 (13) differs from that noted in connection with the ITB ascites tumor in that the tumor cells begin to develop earlier, after the first 24 hours while the period of latency after inoculation of ITR tumor cells is longer even when using large doses. The tumor cells begin to develop noticeably only after 2 days. When the maximum has been reached on the sixth day, the relative number of tumor cells in the ascitic fluid hegins to decrease gradually, while that of necrotic cells increases. Regressive changes of varying degrees have been re- ported also at the terminal phase of other ascites tumors. The multi- plication of cells of the Ehrlich ascites tumor is slowed down and that of MCIM ascites tumor cells is stopped and necrotic cells increase in number (29 ) . During the terminal phase of the mouse ascites lym- phoma the concentration of tumor cells in ascitic fluid falls and pyk- notic cells increase ( 3 0 ) . Similar changes are found also when sar- coma S 37 grows in the ascitic form (13 ) . The ITB ascites tumor hegins to show a regressive tendency at a relatively early stage in comparison with other ascites tumors. The occurrence of polymorphonuclears, mesothelial cells, and histiocytes a t the different stages of the develop- ment of the ITB ascites tumor is, on the whole, in agreement with changes noted in other ascites tumors. The mast cells, a few of which were seen on the first three days after inoculation, disappeared when the number of tumor cells began to show a marked increase, in agree- ment with earlier reports (3 , 13, 1 4 ) .

As the ITB asrites tumor developed, incipient tumor cell infiltration of the omentum is noticeahle even as early as 2-3 days after the ino- culation. The heginning of solid tumor growth and its extent varies in different tumors. In the case of Ebrlich ascites tumor infiltration of

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tissues by tumor cells is seen 4-5 days after the inoculation (14, 29) , while solid tumor growth remains scanty for the whole period of neo- plastic development. Cells of the mouse ascites lymphoma grow in the tissues as early as 1-2 dayes after inoculation (30) . Solid tumor growth is seen in the case of the Yoshida sarcoma (16) ; during the terminal phase it is very abundant, as in the case of the ITB ascites tumor. Distant metastases are not usually encountered in the Yoshida sarcoma either, which Yoshida ascribes to the rapid fatal issue of the disease. Like the ITB tumor, the Yoshida sarcoma has a histological resem- blance to the reticular cell sarcoma ( 1 6 ) .

S U M M A R Y

When transplanting the ITB tumor by means of ascitic fluid, in which the number of tumor cells was small at first in comparison with that of inflammatory cells, the number of the tumor cells contained in the ascitic fluid rose gradually during the process of transplantation until, from the seventh generation onwards, the neoplasm began to grow in the way typical of ascites tumors.

The developed ITB ascites tumor has now been transferred through 100 generations. The rate of successful transplantation has been 82 per cent. The properties of growth, the morphology of the tumor cells in the ascitic fluid, and the histological features of the solid tumor infil- tration have remained unchanged during the process of transplanta- tion.

Inoculation of tumor cells produced an initial polymorphonuclear response in the peritoneal cavity followed by proliferation of inono- nuclear inflammatory cells. The tumor cells began to increase greatly in number after the second day, until on the sixth the ascitic fluid was an almost pure culture of tumor cells. Regressive changes were seen in the tumor cell proliferation soon after this, the relative number of the tumor cells showing a remarkable fall during the terminal phase of tumor development.

In connection with the ITB ascites tumor there is solid tumor growth in the peritoneal cavity which is very abundant during the terminal phase. Infiltration of the omentum by tumor cells begins 2-3 days after inoculation.

R E F E R E N C E S

1. Klein, G.: The Production of Ascites Tumors in Mice and their Use in Studies on Somc Biological and Chemical Characteristics of Neoplastic Cells. Uppsala, Almqvist & Wiksells Boktryckeri, 1951.

2 . Klein, E . : Transformation of Solid into Ascites Tumors. Uppsala, Almqvist & Wiksells Boktryckeri, 1955.

3. Yoshida, T . : Gann, 4 0 : 1,1949. 4. Makino, S., Tanaka, T . & Kano, K.: Gann, 4 2 : 199, 1961.

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5.

6. 7. 8. 9.

10.

11.

12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25. 26. 27. 28. 29. 30.

Takeda, K., Aizuwa, M., Zmamura, T., Sasage, S., Matsumoto , K. & Kanehira, S.:

Sato, H. & Aruj i , T.: Gann, 4 3 : 254, 1952. Tanaka, T. & Kano, K.: Cytologia, 17: 161,1952. Isaka, H.: Gann, 4 4 : 174,1953. Nakamura, K.: Gann, 4 5 : 374,1954. Teir, H., Voutilainen, A. & Kil junen , A.: Acta path. et microhiol. Srandinav. 3 4 :

Pyorbla, K., Teir, H., Voutilainen, A. & Kil junen , A.: Acta path. e t rnicrobiol.

Vincent Memorial Laboratory S t a f f : The Cytologic Diagnosis of Cancer. W. H.

Goldie, H. & Felix, M.D.: Cancer Research, 11: 73, 1951. Klein, G.: Exptl. Cell Res. 2: 518, 1951.

Yoshida, T.: J . Nat. Cancer Inst. 12: 947,1952. Tanaka, T., & Kano, K.: J . Fac. Sci., Hokkaido Univ. Ser. VI, Zool. 10: 289, 1951. Sato, H.: Gann, 4 2 : 132,1951, cited by Yoshida (16). Fogg, L. C.: Pub. Health Rep. 52 : 56,1936. Gnrdner, R. E., Ijailey, G. H. & Hyde, R. R.: Am. J. Hyg. (Sect. B.) 29: 1,1939. Shear, M . J . & Turner, F. C.: J . Nat. Cancer Inst. 4 : 81, 1943. Zahl , P. A., Hutner, S . H. & Cooper, F. S.: Proc. Sac. Exp. Biol. and Med. 5 4 : 187,

Shapiro, C . J.: Am. J. Hyg. (Sect. B) 3f: 114,1940. Algire, G. H., Legallais, F. Y . & Parks, H. D.: J . Nat. Cancer Inst. 8: 53,1947. Zahl, P. A. & Bjerknes, C.: Proc. Sac. Exp. Rial. and Med. 5 4 : 329,1943. Dillrr, 1. C.: Cancer Research, 7: 605,1947. Heilbrunn, L. V . & Wilson , W. L.: Science, 112: 56,1950. dizciwa, M., Oohashi, N. Kato, M., Matsumoto, K. & Arai, T . : Gann, 4 4 : 172,1953. Klein, G. & RCoPsz, L.: J . Nat. Cancer Inst. 1 4 : 229, 1953. RPodsz, L. & Klrin, G.: J. Nat. Cancer Inst. 1 5 : 253, 1954.

Gann, 4 3 : 132,1952.

417,1954.

Scandinav. 34 : 229,1954.

Saunders Camp., Philadelphia & London, 1950.

- Nature, 171: 398,1953.

1943.