The Effect of Human Fibrinolysin on Pulmonary MÃ©Tastases of Walker 256 Carcinosarcoma*

The Effect of Human Fibrinolysin on Pulmonary MÃ©tastases of Walker 256 Carcinosarcoma*

CARLOE. GROSSI,DOMENICOAGOSTINO,ANDEUGENEE. CLIFFTON

(Clotting Mechanisms Section, Din. Surgical Physiology, and Div. Biophysics and Experimental Pathology, Sloan-Keitering Institute; Memorial Centerfor Cancer and Allied Diseases; and Cornell University School of Medicine, New York, N.Y.)

SUMMARY 1. A significant in vitro fibrinolytic activity could be obtained in plasma of rats given human fibrinolysin intravenously. 2. Pulmonary mÃ©tastaseswere produced in a large percentage (81 per cent) of control rats given inoculations intravenously of 100,000 Walker 256 carcinosarcoma cells/ml. When cells were inoculated into animals with marked fibrinolytic activity produced by human fibrinolysin, the takes of tumor as measured by pulmonary mÃ©tas tases were markedly reduced to 25 per cent.

The demonstration of cancer cells in the blood tation of a tumor-bearing limb (7). The effect of in patients with resectable neoplasms has attract trauma has been confirmed by the demonstration ed a great deal of interest. Patients with primary that cancer cells can be dormant in the liver and colonie neoplasms have been studied most com apparently begin to grow after celiotomy and liver monly, and in these a higher percentage of mesen- trauma. Rats given injections intraportally of as teric vein blood samples showed tumor cells (5). few as 50 Walker 256 carcinosarcoma cells and ex This led to the use of chemotherapeutic agents at amined 3 months later showed no evidence of the time of surgery in an attempt to affect the hepatic tumor growth. If at this time the rats were showers of tumor cells in the systemic circulation subjected to repeated laparotomy and liver ex (8). Unfortunately, little is known of the mecha aminations at 7-day intervals, 100 per cent had a nism whereby tumor cells lodge in various organs. tumor within a few weeks (4). Some studies have been made with the Walker 256 Studies of the mechanisms of lodging of tumor carcinosarcoma of rats to ascertain the number of cells in organs have been limited. The VX2 carci cells needed for successful inoculation in the sys noma of rabbits was shown to have a thromboplas- temic and portal circulations. It has been found tic effect, severe enough to result in death of inocu that with this tumor the most successful route of lated animals by pulmonary embolization. Hepa- inoculation is the intraportal, with 96 per cent posi rin diminished the mortality caused by VX2 car tive takes in the liver. The intravenous inoculation cinoma (6), and later human fibrinolysin was yielded a lower percentage of takes (85 per cent). shown to have the same effect (2). In this same Nitrogen mustard has been shown to diminish the series of experiments human fibrinolysin was also inoculation rate in these animals (3). This has been highly effective in preventing the production of used as an additional reason for the use of this pulmonary mÃ©tastasesby the inoculation of the agent in humans during the surgery of malignant VX2 carcinoma (2). The fibrinolysin-treated ani neoplasms. mals had 21 per cent gross pulmonary mÃ©tastases, It has been shown that the intravenous injec whereas the control animals had 91 per cent. It tion of as few as 250 cells will result in mÃ©tastases was thought that the lysis of small fibrin deposits (4). Other investigators have shown that there is by fibrinolysis or the prevention of fibrin deposits an increase in pulmonary mÃ©tastasesafter ampu- by anticoagulants at the time of intravascular in * This work was supported by institutional funds and the oculation of cancer cells might be of value in di National Cancer Institute and U.S.P.H. Services. minishing the number of visceral mÃ©tastases. Received for publication November 27, 1959. More recently it was shown that VX2 carcinoma 605

cells aggregate in minute thrombi, and the fibrin posure of the femoral veins was then adopted as meshwork then adheres to the capillary wall and a the method by which intravascular inoculation nidus of tumor is formed (10). Nonthromboplastic could be ascertained beyond any doubt. Under tumors were sought to determine whether this satisfactory open drop ether anesthesia a 2-cm. phenomenon was a general one with tumors. With incision was made in the groin, and the femoral the Brown-Pearce carcinoma the results were not vessels were exposed. With a #24-gauge needle the quite so striking, but there was a definite decrease solutions and tumor suspensions were injected into in liver mÃ©tastaseswith fibrinolysin treatment the femoral vein under direct vision. (2). Since the Walker 256 carcinosarcoma produces In the control group 1 ml. of saline was injected mÃ©tastasesinboth liver and lung and has been the first, then 1 ml. of tumor cell suspension containing subject of extensive studies on mÃ©tastases, it about 100,000 cells/ml, and the wound was then seemed to be an excellent tumor with which to ex closed. Only saline rather than human plasma was pand these studies. used in the controls. In the treated group 1 ml. of fibrinolysin solution was injected, and 15 minutes MATERIALS AND METHODS later 1 ml. of tumor suspension (100,000 cells/ml) Adult, female, white Wistar rats weighing from was injected and the wound closed. The animals 180 to 200 gm. were used. The animals were fed a were sacrificed either when they seemed terminal stock diet of chow checkers and water. or at the end of 9 weeks. Gross autopsy was done The Walker 256 carcinosarcoma tumor is car in all animals, and the lungs, liver, heart, kidneys, ried in the ascitic form in the laboratory of radio- adrenals, and spleen were examined. biology and has been transplanted at weekly inter vals for over 2 years in more than 200 generations. RESULTS The ascitic fluid was aspirated under sterile pre In preliminary studies the infusion of 5,000 cautions, and a thin cell suspension was made by units of human fibrinolysin/kg in the rat was dilution with saline. An aliquot was taken and a shown to result in measurable fibrinolytic activity cell count carried out by a dilution and a direct in each animal, 15 minutes after the injection smear counting method (9). The ascitic fluid was (Table 1). After 30 minutes the activity was much found to have 5-10 million cells/cu mm. Dilutions less although still significant, and by 60 minutes were made with saline so that the tumor suspen the fibrinolytic activity had returned almost to sion used had about 100,000 cells/ml. Frequent control level. checks were made to assure a correct dilution of The effect of the human fibrinolysin on the anti cell suspension. To insure maximal viability of tu plasmin fibrinolytic titer was also evaluated. Rats mor cells, no suspension was used longer than 1 have a higher inhibitor level for human fibrinoly hour after preparation. Several rats were given sin, which may explain why a high concentration inoculations subcutaneously during each series of of fibrinolysin (5,000 units/kg) was necessary to experiments to have proof of viability at the be produce fibrinolytic activity in these animals. A ginning and end of the transfers. If tumors did not significant drop in antiplasmin fibrinolytic activity grow in these animals then the entire experimental was noted, and this effect lasted for at least 1 hour series was discarded. after injection. Human fibrinolysin.1â€”Twenty-five thousand No direct in vitro effect of human fibrinolysin (25,000) units were dissolved in 25 ml. of saline, could be detected on the viability of tumor cell and 1 ml. was given intravenously in 5 minutes to saline suspensions. Five thousand units of fibrino a 180-200-gm. animal. This was approximately lysin were added to 1 ml. of tumor cell saline sus 5,000-6,000 units of fibrinolysin/kg body weight. pension in vitro, and this was incubated at 37Â°C. The fibrinolytic activity of the plasma was de for 30 minutes. Two different concentrations of termined by the euglobulin fibrinolytic method cells were tested, i.e., 100,000 cells/ml and 5,000 (1). The normal value in the rat with the bovine cells/ml. There was no difference as to the number clot lysis time is over 24 hours. The antiplasmin of positive subcutaneous inoculations obtained in fibrinolytic method (1) was used to evaluate the rats between the control mixture and either of the inhibitory level of the plasma. tumor cell fibrinolysin mixtures. At the start it was felt that tail vein injections Two hundred and sixteen animals were inocu would provide a good means of inoculation. How lated intravenously with Walker 256 tumor sus ever, we could never be certain that the inocula pension. In the 103 control animals there was an tion was intravenous. The method of surgical ex- 81.5 per cent incidence of gross pulmonary mÃ©tas 1Human fibrinolysin, Thrombolysin, supplied by Merck, tases. This in the great majority of cases consisted Sharp & Dohme, West Point, Pa. of a large number of tumor nodules filling the

lungs. The tumor nodules measured 0.5-1 cm. in DISCUSSION diameter. Any lung containing even one tumor There is no obvious explanation for the above nodule was counted as positive. In the negative findings. Since little is known of the mechanism of animals the lungs appeared grossly normal, and no deposition of tumor cells in organs, a good deal of gross mÃ©tastaseswere detected (Fig. 1). In most supposition and additional work is necessary. The of the positive animals, the lungs were loaded with data suggest that the existence of fibrinolytic ac large tumor nodules replacing the parenchyma, tivity at the time of intravascular inoculation of and yet grossly the liver, spleen, and kidneys were tumor cells prevents the lodging of the tumor cells normal, without mÃ©tastases.On occasion meta- in the lungs, and as a result there are few mÃ©tas static deposits were found in the myocardium and tases. It is possible that clumping of cells is neces pericardium. These animals did not develop sary for lodging of cells and that fibrin formation pleural effusion. maintains them till growth can take place. Ap In the 113 animals given human fibrinolysin parently this phenomenon may be of significance prior to the tumor suspension only 28 animals (25 not only with the VX2 carcinoma of rabbit but

TABLE 1 PHYSIOLOGICALACTIONOFPLASMININRATSFOLLOWINGINJECTION OFsooo UNITSPLASMIN/KG

FlBHINOLTTIC ACTIVITY OF PLASMA

Timeafterinjection(min.)0 A>24 C>24 D>24

(Control)Iff3060Rathr.1 hr.2 hr.45 hr., 15min.2 hr., 10min.5 min.3 hr., 15min.22 hr., 57min.>24 hr., 10min.21 hr.Rat hr.Rat hr.

A.NTIPLA8MIN FlBBIJiOLYTIC ACTIVITY OF PLASMA

Timeafterinjection(min.)0 E>24hr.2 F17 H23

(Control)153060Rat hr., 7min.3 hr., 9min.2 hr., 40min.50 hr.1 hr., 55min.2 min.1 hr., 40min.2 hr., 41min.2 hr., 59 min.Rat hr., 30 min.Rat hr., 33 min.

per cent) were found to have gross evidence of also in other animal tumors, such as the Brown- metastatic deposits in the lungs. No deposits were Pearce carcinoma of the rabbit and the Walker found in the liver, spleen, or other organs inspect Carcinosarcoma of the rat. ed. The treated animals which had mÃ©tastaseswere The present findings pose more questions than similar to the control animals with mÃ©tastases. they answer. If the tumor cells do not lodge in the This 25 per cent incidence of positive takes in the lung, what becomes of them? Although there is lung is remarkable compared with that in the con transpulmonary passage of tumor cells, no liver trol group. The lungs of the treated animals which mÃ©tastaseswere found. It is not understood why were grossly negative were sectioned and found to the circulating cells do not lodge and produce be resilient and normal in appearance. In a few mÃ©tastasesafter the fibrinolytic enzyme activity animals tumor was found to grow in the subcuta ends in 2-3 hours. These data would suggest that neous tissues of the thigh near the site of femoral in the rat Walker 256 carcinosarcoma the cells vein injection, probably owing to leakage of would disappear from the circulation within 1 hour blood at this site. These animals were counted as and that fibrin deposition and microthrombi for negative if they did not have pulmonary mÃ©tas mation are significant mechanisms in the lodging tases, because the criterion being used was that of of blood-borne tumor cells in the Walker 256 car blood-borne mÃ©tastasesin the lung. cinosarcoma. However, no studies were done to

elucidate the problem of the dormant cell which Demonstration and Significance of Tumor Cells in the Mesenteric Venous Blood in Patients with Colorectal might begin to form gross mÃ©tastasesifmechanical Carcinoma. Surgery, Gynec. & Obst., 100:102-8, 1955. or chemical stimulation were applied. 6. LAWRENCE,E. A ; BOWMAN,D. E.; MOORE,D. B.; and BERNSTEIN,G. I. A Thromboplastic Property of Neo REFERENCES plasms. In: Surgical Forum; Proceedings of the Forum 1. CLIFFTON,E. E., and CANNAMELA,D.Fibrinolytic and Sessions, pp. 694-98, Thirty-eighth Clinical Congress of the Proteolytic Activity of a Human Plasminogen Prepared Am. College of Surgeons, New York City. Philadelphia: from Fraction III of Human Plasma. J. Appi. Physiol. W. B. Saunders & Co., 1953. 6:42-50, 1953. 7. LEWIS,M. R., and COLE,W. H. Experimental Increase of a. CLIFFTON,E.E., and GROSSI,C. E. Effect of Human Plas Lung MÃ©tastasesafter Operative Trauma (Amputation of min on the Toxic Effects and Growth of Blood-Borne Limb with Tumor). A.M.A. Arch. Surgery, 77:621-26, MÃ©tastasesof the Brown-Pearce Carcinoma and the V2 1958. Carcinoma of Rabbit. Cancer, 9:1147-52, 1956. 8. MIAZEK,R.; ECONOMON,S.;MCDONALD,G. O.; SLAUGH 3. CRUZ,E. P.; MCÃœONALD,G.O.; and COLE, W. H. Pro TER,D. P.; and COLE,W. H. Prophylactic and Adjuvant phylactic Treatment of Cancer; the Use of Chemothera- Use of Nitrogen Mustard in the Surgical Treatment of peutic Agents To Prevent Tumor Metastasis. Surgery, 40: Cancer. Ann. Surgery, 150:745-54, 1959. 291-96, 1956. 9. SEAL, S. H. Silicone Flotation, a Simple Quantitative 4. FISHER, B., and FISHER,E. R. Experimental Studies of Method for the Isolation of Free Floating Cancer Cells Factors Influencing Hepatic MÃ©tastases.III. Effect of from the Blood. Cancer, 12:590-95, 1959. Surgical Trauma with Special Reference to Liver Injury. 10. WOOD,S., JR. Pathogenesis of MÃ©tastasesFormation Ob Ann. Surgery, 150:731-43, 1959. served In Vivo in the Rabbit Ear Chamber. A.M.A. Arch. 5. FISHER,E. R., and TURNBULL,R. B., JR. The Cytologie Path., 66:550-68, 1958.

Fio. 1.â€”Autopsyspecimens. To the left, a lung of a rat that died of multiple pulmonary mÃ©tastasesafter 100,000 cells of Walker 256 carcinosarcoma by intravenous inoculation. Note the multiple tumor nodules. To the right, a lung of a rat that was sacrificed 9 weeks after inoculation of 100,000 cells of Walk er 256 carcinosarcoma intravenously preceded by plasmin, 5000 units/kg, intravenously. Note the absence of visible mÃ©tas tases. Both specimens were fixed in formalin for 2 weeks prior to photography, and this accounts for the apparent difference in size of the two lungs. The uninvolved lung tissue shrank when fixed.

Fio.l

Carlo E. Grossi, Domenico Agostino and Eugene E. Cliffton

Cancer Res 1960;20:605-608.

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