Yuji KUROKAWA (Department of Oncology, Research Institute for Tuberculosis, Leprosy and Cancer, Tohoku University*)

[GANN, 61, 461-471; October, 1970] UDC 616-006-033.2: 616.428 EXPERIMENTS ON LYMPH NODE METASTASIS BY INTRALYMPHATIC INOCULATION OF RAT ASCITES TUMOR CELLS, WITH SPECIAL REFERENCE TO LODGEMENT, PASSAGE, AND GROWTH OF TUMOR CELLS IN LYMPH NODES (Plates LXXVI-LXXVIII) Yuji KUROKAWA (Department of Oncology, Research Institute for Tuberculosis, Leprosy and Cancer, Tohoku University*) Synopsis The mechanism of lymph node metastasis, especially lodgement, passage, and growth of tumor cells in lymph nodes, was investigated serially by direct inoculation of various strains of rat ascites tumor cells into the testicular lymphatics of Donryu rats. It was observed that passage of tumor cells through lymph nodes did not occur until the nodes were almost invaded by growing tumor cells. Successful inoculation of a small number of tumor cells and lack of considerable reaction of lymph node cells to tumor cells indicated that lymph nodes may be in a sense a suitable soil for proliferation. Based on the findings obtained, the functions of lymph nodes were discussed as a barrier in metastasis formation. INTRODUCTION In the experimental reports on lymphatic metastasis, there are many investigators describing data on lymph node metastasis by inoculating tumor cells into either subcutis, muscle, or various organs.4,11,12,19,20,22,32) However, direct inoculation of tumor cells into the lymphatics seems to be a better technique to analyse the behavior of tumor cells such as lodgement, passage, and growth in the lymph nodes. The intralymphatic inoculation of tumor cells was first reported by Zeidman in 1954 using lymph vessels of a rabbit.34,35) In the same year, Engeset also described the possibility of the injection into testicular lymphatics of a rat.5) Recently, several papers have been published on experimental lymph node metastasis utilizing the intralymphatic injections,6,7,8,13,15,16, 24,25,28,36) In the present paper, the behavior of tumor cells such as the lodgement, passage, and growth in the lymph nodes, is dealt with, considering the morphological and biological characteristics of various strains of ascites tumors of rats which have been developed and studied in Japan. MATERIALS AND METHODS Animals The animals used were male Donryu rats weighing 200-300g, obtained from Nippon Rat Co. Ltd. (Tokyo). Tumors Tumors used were nine strains of ascites hepatoma (AH-13, AH-66F, AH-130, AH-109A, AH-225A, AH-131A, AH-84A, AH-136B, AH-108A), and

Y. KUROKAWA Yoshida sarcoma. These ascites tumors are individually different from each other in various characteristics, such as the survival time, transplantability, invasiveness, metastasizability, chromosome constitution, drug sensitivity, and ascitic picture (the ratio of free cells and island cells).33) They have been serially transplanted by intraperitoneal inoculation into Donryu rats at Sasaki Institute (Tokyo) and our laboratory (Sendai). Topography of the Lymphatics Lymphangiography using Micropaque was performed before the experiments to determine the lymphatic pathways from the left testicle. It was found that in 98% of the rats examined (50/51), the lymph vessel passed through at least one lymph node (either left lumbar node or left renal node) before it reached the blood stream (Fig. 1). Only in one case, the lymph vessel emptied directly into the thoracic duct without passing through any lymph node. From the findings obtained from lymphangiography, it was concluded that the lymph vessel of the left testicle was a suitable site to cause lymph node metastasis in Donryu rats. Fig. 1. Topography of lymphatic system from left testicle A total of 51 rats were examined. The lymph vessel passed through both lumbar and renal node in 29 cases (Type I), passed through only renal node in 15 cases (Type II). Six cases were variations of type I and II in which branching lymph vessels were found. Only in one case, the lymph vessel emptied directly into thoracic duct. Intralymphatic Inoculation Tumor cells were harvested from the peritoneal cavity of the rat by a glass pipette, diluted in Eagle's minimum essential medium (MEM) with heparin (500units/ml), and counted in hemocytometer so as to contain 100-100,000 cells in 0.02-0.05ml. Viability of tumor cells was estimated by Trypan Blue staining in every case. For injection of tumor cells, the rats were anesthetised with pentobarbital solution administered intraperitoneally in dose of 4mg/100g body weight. The left testicle with its tunica albuginea was exposed through incision in the scrotum. Physiological saline (0.1-0.3ml) was injected blindly into the left testicle to cause swelling and enlargement of the lymph vessels and to expand the testicle, so that the later injection procedure would be easier. Then the tunica albuginea was incised and 0.01-0.02ml of 10% aqueous solution of sky blue dye was injected at the upper pole of the testicle under caput epidydimis. At that time the lymph vessels were most readily visualised, 462 GANN

METASTASIS BY INTRALYMPHATIC INOCULATION the course, length, and thickness of which could be easily recognized (Photo 1). The tumor cells were inoculated with a 0.5-ml glass tuberculin syringe and a 1/5 hypodermic needle (diameter 0.35mm) without using a dissecting microscope (Photo 2). The injected volume ranged from 0.02 to 0.05ml. This injection procedure was performed within 30-60 sec so as not to burst the lymph vessels, but the pressure of injection was not estimated in this series of experiments. After the inoculation, the spermatic cord was ligated as proximal as possible and then the testicle was removed. The scrotum was closed with usual suture. These operations were done under aseptic condition as much as possible. Penicillin was used to prevent further infection. The rats which developed local tumors at the ligated site of the spermatic cord were discarded. Detection of Tumor Cells in the Thoracic Duct Lymph and Heart Blood Thoracic duct lymph was collected according to the methods of Yamada31) or Saldeen.21) Tumor cells in the collected lymph was detected by making 5-10 smear preparations stained with Wright-Giemsa after 800rpm centrifugation for 10min. In some cases in which amount of the lymph collected was not enough, a number of smears were prepared without centrifugation. Tumor cells in the blood stream were detected by bioassay by sampling 3-5ml of blood with puncture of the heart shortly after sacrifice and injecting it into the peritoneal cavity of two recipient rats. The ascites of these rats were examined 4-5 days after the injection. Thereafter, the rats were autopsied following tumor death or by sacrifice one month after injection. Methods of Observation The rats were sacrificed under chloroform anesthesia and fixed in 4% formaldehyde solution. Histological sections were made of lymph nodes (left and right lumbar, renal, and mediastinal nodes) and other organs. The specimens were usually stained with Hematoxylin and Eosin, and sometimes with Gomori's reticulum stain. RESULTS Growth of Tumor Cells in the Lymph Node after Intralymphatic Inoculation In this series, the rats were all sacrificed 7 days after inoculation of 100-100,000 tumor cells. The total number of 202 rats were used in this experiment in which metastases were found in 56 cases (28%). As shown in Table I, metastasis formation was highest in 2/9 animals (22%) inoculated with 100 tumor cells, 6/10 (60%) with 1,000 cells of AH-66F, and in 7/7 (100%) of 10,000 tumor cells of AH-13. It is also shown in Table I that metastasis formation in the lymph node correlated well with the cell constitution of each tumor strain. In completely free cell-type tumors, such as Yoshida sarcoma, AH-13, and AH-66F, metastasis was far higher than in island cell-type tumors. Metastasis formation described above does not mean the grade of invasion by tumor cells in each lymph node. The grade of lymph node metastasis was divided into three classes and is shown schematically in Fig. 2. The relationship between the number of tumor cells inoculated and the grade of lymph node metastasis is shown in Table II. These four strains of tumors (Yoshida sarcoma, AH-13, AH-66F, AH-130) grew only in marginal sinuses 7 days after inoculation of 100 tumor cells. When 1,000 tumor cells were inoculated, AH-66F and Yoshida sarcoma invaded as far as intermediary sinus. When 10,000 tumor cells were inoculated, they replaced almost all the tissues of the 61(5) 1970 463

Y. KUROKAWA Table I. Metastasis Formation in Lymph Nodes 7 Days after Intralymphatic Inoculation of Tumor Cells * Main cell component of each tumor strain. Figures in parentheses are percentages. Grade I. Tumor cells proliferate in marginal sinus. Grade II. Tumor cells proliferate as far as intermediary sinus and invade into parenchyma. Fig. 2. Schematic representation of grade of lymph node metastasis Grade III. Tumor cells occupy almost all the lymph node. lymph node in the case of AH-66F, while AH-130 cells, which are the island cell type, were found in marginal sinus yet. This grade of lymph node metastasis shows the speed of invasiveness of each tumor strain in the lymph node. Free cell type tumors were far more faster than island cell type. It is also suggested from these data that the grade of lymph node metastasis at the same period after inoculation was well related to the number of each tumor cell first reaching the lymph node. 464 GANN

METASTASIS BY INTRALYMPHATIC INOCULATION Table II. Grade of Lymph Node Metastasis 7 Days after Intralymphatic Inoculation of Tumor Cells Histology of the Early Phase of Lymph Node Metastasis Three strains of tumor, i.e., AH-66F, a completely free cell type, AH-84A, large island cell type, and AH- 225A, an intermediate type, were used. The total of 106 rats were inoculated, and sacrificed immediately, or 3, 6, 12, 24, 72, or 120hr after inoculation of 100,000 tumor cells into the lymph vessels. (1) Incidence of Lymph Node Metastasis According to Elapsed Time: Histological observation for embolism of tumor cells or metastatic foci revealed that these phenomena did not increase according to the time elapsed after inoculation. It is apparent as shown in Table III that the percentage of positive lymph nodes decreased twice, at 6 and 24hr. This phenomenon will be discussed later with regard to the sinus histiocytosis. Table III. Incidence of Lymph Node Metastasis According to the Time Elapsed after Intralymphatic Inoculation of 100,000 Tumor Cells (2) Morphology of Tumor Cells in the Lymph Node during the First 5 Days after Inoculation: AH-66F (This series was observed from 24hr after inoculation): Invasion of these tumor cells was seen from marginal sinus to intermediary sinus rather than to cortical pulp after 24hr. These places were more highly invaded by tumor cells 3 days later. It was found that cells of AH-66F moved deeply into cortical pulp singly and freely as they are in ascitic form, accompanying the proliferation of reticulum fibers (Photos 3 and 4). In some instances, the lymph nodes were fully occupied by tumor cells without any normal architecture already around 5 days after inoculation. AH-225A: Tumor cells were found embolic as clusters consisting of 5-50 cells in marginal sinus or in afferent lymph vessels immediately after inoculation (Photos 5 and 6). They seemed to continue embolic state for 3-12hr, until they began to grow in marginal sinus 24hr after inoculation. In some instances, the first sign of invasion was 61(5) 1970 465

Y. KUROKAWA seen more often into cortical pulp directly than to the intermediary sinus. Simultaneously, Gomori's reticulum stain revealed outgrowth of reticulum fibers at the invaded site. After 3 to 5 days, the growth of tumor cells advanced diffusely into both cortical pulp and intermediary sinus (Photo 7). AH-84A: Until 12hr after inoculation, only a few clusters of large island cells were seen to be embolic in marginal sinus (Photo 8). Then 24hr after inoculation, tumor cells began to grow and invade into cortical pulp. Characteristic spreading pattern was observed during 3-5 days after inoculation; that is, the tumor cells advanced through the marginal sinus around the lymph node from where they started growing. When the tumor cells had completely occupied these marginal sinuses, they began to invade into cortical pulp for the first time as if they could find no other place to live (Photo 9). These findings could be summarized as follows. For the first 24hr, tumor cells were simply embolic in marginal sinus. The growth and invasion occurred thereafter, the usual route of which was at first more directly to cortical pulp rather than to intermediary sinus. When tumor cells proliferated and spread to some extent in marginal sinus, intermediary sinus seemed to be secondarily invaded. This mode of spread, however, was somewhat different among these three kinds of tumors. Such differences might be initiated by the speed of invasiveness based on the cell constitution and mobility of tumor cells of each tumor strain.23) (3) Sinus Histiocytosis and Tumor Growth in Lymph Node: The occurrence of the so-called sinus-histiocytosis in metastasized lymph nodes was observed from 6hr to 3 days, the highest grade of which was observed at 12hr. In the lymph nodes which were almost replaced by tumor cells, no sign of sinus-histiocytosis was found. The distribution of sinus-histiocytosis in each lymph node seemed to be related to that of tumor cells, e.g., when the metastasis formation was limited to marginal sinus, sinus-histiocytosis was also localized around it (Photo 10). If the invasion expanded into intermediary sinus, sinus-histiocytosis went with it and spread widely. These findings might be an evidence that lymph node may react with tumor cells in the early phase of metastasis formation. Spread of Tumor Cells into the Thoracic Duct Lymph or Heart Blood from Lymph Node After intralymphatic inoculation of 100,000 tumor cells (AH-66F, AH-225A, AH-84A), the presence of tumor cells in the thoracic duct lymph and heart blood was examined. Each rat was sacrificed and autopsied at various time intervals after inoculation. The results from 45 cases are summarized in Table IV. The first appearance of tumor cells in the thoracic duct lymph was after 3 days in AH-66F, 5 days in AH-225A, and 18 days in AH-84A. Tumor cells in the heart blood were detected later than in the thoracic duct lymph, namely, after 9 days in AH-66F and AH-225A, and 35 days in AH-84A. This speed of spreading of each tumor correlated well with that of invasion in the early phase of lymph node metastasis as described above. Distant metastases were found in the lungs only in 2 cases of AH-84A series. In Table V, the cases in which tumor cells were positive in thoracic duct lymph and/or heart blood are classified according to the grade of lymph node metastasis. The cases were divided into one each of Grade I and II, and 9 of Grade III. It was noteworthy that tumor cells were negative in 21 out of 30 cases of Grade III. In addition, the survival time of the 466 GANN

METASTASIS BY INTRALYMPHATIC INOCULATION Table IV. Detection of Tumor Cells in Thoracic Duct Lymph, Heart Blood, and Distant Organs with regard to the Grade of Lymph Node Metastasis

Y. KUROKAWA Table V. Cases in which Tumor Cells were Positive in Heart Blood and/or Thoracic Duct Lymph with regard to its Grade of Lymph Node Metastasis recipient rats which were injected with the heart blood was as long as when a small number of tumor cells inoculated intraperitoneally. The above data might indicate that although tumor cells could be detected in 24% of cases examined, their number was considerably small. DISCUSSION Among the many problems on lymph node metastasis, the most widely discussed and argued problem is whether the lymph node has a function of a "barrier" to tumor cells or not.14,26,30) This term "barrier", however, should have two phases in itself, as Straeuli pointed out.26) The first is the function of preventing passage of tumor cells through the lymph node as a filter (mechanical barrier) and the second is that of reacting with entering tumor cells and inhibiting further growth in the lymph node (biological barrier). Therefore, it would be better to take into account the dual meaning of the word "barrier" when discussing lymph node metastasis. As to the problem of mechanical barrier in the lymph node, Zeidman demonstrated his experimental results stressing that the tumors did not spread to the next node for at least 3 weeks after initial arrest of the emboli, and in cases when lymph node metastasis did occur, the incidence of lung metastasis was relatively few and delayed.6,34) Other workers concluded, in their reports, that successful detection of tumor cells in the efferent lymphatics shortly after intralymphatic inoculation might be the evidence that lymph node functions little as a mechanical barrier.8,16) In the present experiments, the first appearance of tumor cells in the thoracic duct lymph varied in each tumor strain (between 3 and 18 days). These results demonstrated clearly that the free cell-type tumor (AH-66F) passed through lymph nodes earlier than the island cell-type (AH-84A), but the histological observation of these lymph nodes revealed they were almost fully occupied by tumor cells, as far as hilus of the lymph node in most cases. Results of an experiment show that passage of tumor cells through lymph node may not occur until tumor cells have invaded and proliferated in the lymph node to a great extent.27) In our experiments, detection of tumor cells in the thoracic duct lymph and blood stream was made from 24hr after inoculation. Some investigators point out that translymph nodal passage of tumor cells mostly occur within 24hr after inoculation.16) Although we did not try the detection of tumor cells during that interval, it could be 468 GANN

METASTASIS BY INTRALYMPHATIC INOCULATION speculated that only a few cells or almost nonviable cells passed through the lymph node, because lung metastases were found only in 2 out of 45 cases by histological observation. It is also noteworthy that in cases of Grade III lymph node metastasis, tumor cells were negative both in the lymph and blood in 70% of the cases (21/30) examined. This fact may indicate that, besides the function of a lymph node as a mechanical barrier, the main route for hematogenous dissemination of tumor cells from lymph node is through thoracic duct and not through the post-capillary venule of the lymph node itself.9) This hypothesis was also drawn from data indicating that when tumor cells were positive in blood, they were all simultaneously positive in the lymph, i.e., there were no cases in which tumor cells were positive only in the blood. Until Willis' review, despite a good deal of speculation, there had been no direct evidence about the function of the lymph node as a biological barrier.30) Recently, however, the problem has been studied from the point of transplantability and cellular reaction. Transplantability of tumor cells into the lymph node was analysed by direct inoculation into lymph nodes and intralymphatic inoculation, comparing with that of other organs, and the result showed that lymphoid tissue including lymph nodes had higher transplantability than other organs.1,17,18,28.36) The results obtained in the present experiment, the successful inoculation of 100 tumor cells of Yoshida sarcoma and AH-66F, and high transplantability of 10,000 tumor cells of AH-13 (100%) and AH-66F (91%), may be understood along the same line as those of the above-mentioned workers. The reaction which seems to occur between tumor cells and lymph node cells falls into two groups; one is the phenomenon of sinus-histiocytosis, and the other is the lymphocyte as cellular antibody. The question of whether sinus-histiocytosis is related to the prognosis of cancer patient has been debated among many clinical investigators.2, 3,10,29) In the animals in which 100,000 tumor cells were inoculated and sacrificed at various time intervals, it was found that low grade of sinus-histiocytosis existed with metastatic foci in lymph node around 24hr after inoculation. This behavior of histiocytes seems interesting in order to know the early stage of sinus-histiocytosis. Neither a direct cellular reaction between tumor cells and lymph node cells nor necrosis and degeneration of tumor cells were observed at any times. Although metastatic rate decreased twice between 6 and 24hr after inoculation and sinus-histiocytosis occurred to some extent, it will be suggested that the function of a lymph node as a biological barrier may be very weak and act only in early stage of metastasis. It is concluded from our findings that lymph node functions considerably as a mechanical barrier but little as a biological barrier, which may be enhanced by some method of host conditioning in further researches. The author's grateful thanks are due to Prof. Haruo Sato for his helpful guidance and valuable suggestions. (Received April 28, 1970) 61(5) 1970 469

Y. KUROKAWA REFERENCES 1) Asahina, A., Fukushima Igaku Zasshi, 17, 65 (1967). 2) Berg, J. W., Cancer, 9, 935 (1956). 3) Black, M. M., Speer, F. D., Surg. Gynec. Obstet., 110, 477 (1960). 4) Costachel, O., Rivenson, A., Comisel, V., Oncologia, 19, 273 (1965). 5) Engeset, A., Cancer Res., 14, 277 (1954). 6) Idem, Acta Unio Intern. Contra Cancrum, 15, 879 (1959). 7) Engzell, U., Rubio, C., Tjernberg, B., Zajicek, J., Europ. J. Cancer, 4, 305 (1969). 8) Fisher, B., Science, 152, 1397 (1966). 9) Fisher, B., Fisher, E., Surg. Gynec. Obstet., 122, 791 (1966). 10) Gnirs, L., Z. Krebsforsch., 60, 94 (1954). 11) Hashimoto, K., Sci. Rept. Res. Inst. Tohoku Univ. Ser. C., 11, 406 (1964). 12) Kanno, K., Fukushima Igaku Zasshi, 10, 473 (1960). 13) Kurokawa, Y., Suzuki, M., Sato, H., Proc. Japan Cancer Assoc., 27th General Meeting, 225 (1968). 14) Leighton, J., "The Spread of Cancer," (1967). Academic Press, Inc., New York and London. 15) Ludwig, J., Titus, J. L., Arch. Pathol., 84, 304 (1967). 16) Madden, R. E., Gyure, L., Oncology, 22, 281 (1968). 17) Miyawaki, H., Proc. Japan Cancer Assoc., 27th General Meeting, 182 (1968). 18) Idem, ibid., 28th General Meeting, 161 (1969). 19) Plenk, H. P., Sorensen, F. M., Eichwald, E. J., Cancer Res., 14, 580 (1954). 20) Rivenson, A., Comisel, V., Popp, I., Experientia, 25, 70 (1969). 21) Saldeen, T., Linder, E., Acta Pathol., 49, 433 (1960). 22) Sato, H., Cancer Chemotherapy Rept., 13, 33 (1961). 23) Sato, H., Goto, M., Kuroki, T., Gann Monograph, 2, 127 (1967). 24) Stoker, T. A. M., Brit. J. Cancer, 23, 132 (1969). 25) Idem, ibid., 23, 136 (1969). 26) Straeuli, P., "Endogenous Factors Influencing Host-Tumor Balance," p. 249 (1967). The University of Chicago Press, Chicago. 27) Tjernberg, B., Zajecek, J., Acta Cytol., 9, 197 (1965). 28) Wallace, A. C., Hollenberg, N. K., Brit. J. Cancer, 19, 338 (1965). 29) Wartman, W. B., ibid., 13, 389 (1960). 30) Willis, R. A., "The Spread of Tumours in the Human Body," (1952). Butterworth & Co. Ltd., London. 31) Yamada, T., Igaku-no-Ayumi, 56, 234 (1966). 32) Yamaguchi, I., Takahashi, T., Narisawa, T., Tohoku J. Exptl. Med., 87, 338 (1965). 33) Yoshida, T., Sato, H., Natl. Cancer Inst. Monograph, 16 (1960). 34) Zeidman, I., Buss, J. M., Cancer Res., 14, 403 (1954). 35) Zeidman, I., ibid., 15, 719 (1955). 36) Idem, ibid., 25, 324 (1965). EXPLANATION OF PLATES LXXVI-LXXVIII Photo 1. The left testicle of a rat after injection of physiological saline and sky blue dye. The lymph vessel can be easily recognized. Photo 2. Intralymphatic inoculation of tumor cells into testicular lymph vessel. This injection can be carried out without dissecting microscope. Photo 3. AH-66F cells were highly invasive into cortical pulp with outgrowth of reticulum fibers 3 days after inoculation (left lumber node). Gomori's reticulum stain. Photo 4. Higher magnification of the cortical pulp invaded by AH-66F cells 3 days after inoculation (left lumbar node). H-E. 470 GANN

METASTASIS BY INTRALYMPHATIC INOCULATION Photo 5. Embolic state of AH-225A cells in marginal sinus immediately after inoculation (left lumbar node). H-E. Photo 6. Embolism by AH-225A cells in the afferent lymphatic immediately after inoculation. H-E. Photo 7. AH-225A cells which began to invade into cortical pulp 3 days after inoculation (left lumber node). H-E. Photo 8. AH-84A cells which were yet in embolic state in marginal sinus 12 hours after inoculation (left renal node). H-E. Photo 9. AH-84A cells in marginal sinus 3 days after inoculation. Note that invasion went through within the marginal sinus more than to cortical pulp (left renal node). H-E. Photo 10. Sinus-histiocytosis of the metastasized lymph node (AH-84A) 6 hours after inoculation (left lumbar node). H-E. H-E=Hematoxylin and Eosin stain 61(5) 1970 471

GANN, Vol. 61 PLATE LXXVI

GANN, Vol. 61 PLATE LXXVII

GANN, Vol. 61 PLATE LXXVIII