(CANCERRESEARCH36,3584-3587,October1976] Effect of Defibrination on Tumor Growth and Response to Chemotherapy1 William D. DeWys, Hau C. Kwaan, and Sudha Bathina Department of Medicine (W. D. D. , H. C. K., 5. B.J and Cancer Center (W. D. D., S. B.J, Northwestern University 5chool of Medicine; Northwestern Memorial Hospital (W. D. D.J; and Veterans Administration Lakeside Hospital (H. C. K.J, Chicago, Illinois 60611 SUMMARY therapeutic manipulations were sufficiently effective to abolish completely the fibrin deposition within the tumor. Other investigators have demonstrated fibrin deposition Recently, the polypeptide Ancrod, extracted from the in tumors. Experiments were therefore designed to test venom of the Malayan pit viper (Agkistrodon rhodostoma) whether systemic defibrination would alter tumor growth or and known to convert fibrinogen to fibrin, has been used tumor response to chemotherapy with cyclophosphamide. i.v. to produce a complete defibrination in man and animals Defibrination with Ancrod, a venom extract of Agkistrodon (3, 4, 19, 21). This preliminary study utilizes this defibrinat rhodostoma, did not significantly affect tumor sensitivity to ing agent in association with cyclophosphamide in an at chemotherapy. Similarly, defibrination plus fibrinolytic tempt to prevent formation of fibrin deposits in tumors, therapy with streptokinase did not affect responsiveness to thereby enabling the assessment of the role of fibrin deposi cyclophosphamide. Long-term defibrination did not affect tion upon tumor sensitivity to chemotherapy. Additionally, a tumor growth. These results suggest three possible inter thrombolytic agent, streptokinase, is also used further to pretations: (a) the coagulation system may not be important decrease the amounts of tumor fibrin by fibrinolysis. in tumor growth and response to chemotherapy; (b) ade quate clearing of fibrin from the tumor was not accom MATERIALS AND METHODS plished in our experiments; or (c) other factors such as platelet deposition may be involved and platelet function Drugs. Ancrod (Venacil) was kindly supplied by Abbott was not inhibited by the therapies used in our experiments. Laboratories, North Chicago, III. Defibrination was initiated with a dose of 100 units/kg s.c. and followed by 200 units! INTRODUCTION kg aftera 12-hr interval. A maintenance dose of400 units/kg was given s.c. every 12 hr subsequently. Rapid death, pre Although an increased tendency to thrombosis in cancer sumably due to the production of disseminated intravascu patients has been recognized since Trousseau's classic ob lar coagulation, occurred when the defibrination was initi servation (28), the coagulation mechanism has only recently ated with the full 400 units/kg s.c. dose. been found to have a significant role in the growth of Streptokinase (Varidase, Lederle Labs., Pearl River, N. V.) primary tumors and their metastases (5, 6, 16, 24, 25). was given in doses of 800 lU s.c. every 8 hr. O'Meara and Jackson (25) first noted the deposition of fibrin Cyclophosphamide (NSC 26271) was kindly supplied by in a variety of tumors, especially at the invading periphery. Mead Johnson Laboratories, Evansville, Ind. The dose was He subsequently found various coagulants and fibrin in given s.c. as a single injection in a volume of 0.01 mg/g tumors at higher concentrations than in the surrounding body weight. tissues and suggested that a fibrin latticework facilitates Animals. Eight- to 12-week-old male C57BL x DBA/2 F1 vascularity and growth of the tumor tissue (24). This con mice, 20 to 26 g, were housed in groups of 5 in plastic cages cept is supported by other demonstrations of the presence (Chart 1). On Day 0, they were given injections of 10 Lewis of factors X (15), XIII (31), and other “thromboplastins.―lung carcinoma cells (6) in the right hind leg muscle by a Wood (30) observed the association between local fibrin previously described method (8). After the carcinoma cells formation and the successful experimental tumor emboliza were given on Day 0 and Ancrod was given from Days 6 tion in animals. He suggested that the development of mi through 10, mice of Groups I, II, and Ill received cyclophos crothrombi enveloping tumor cells is an important mecha phamide s.c. on Day 10 in a dose of 80, 160, and 240 mg/kg, nism by which tumor cells adhere to the vascular lining and respectively. migrate through vessel walls at metastatic sites. Accord In another series of experiments, mice of Group IV re ingly, various attempts to reduce the growth and spread of ceived 160 mg cyclophosphamide per kg on Day 17 with no tumors by anticoagulants and fibrinolysins have been made defibrination therapy. Mice of Group V received Ancrod in man and in experimental animals (11, 12, 18). The results from Days 13 through 17 and also 160 mg cyclophospha of these attempts are difficult to evaluate, since none of the mide per kg on Day 17. Mice of Group VI received Ancrod on Days 13 to 17, Ancrod plus streptokinase on Days 16 and 17, I Supported by Grant CA 5145 from the National Cancer Institute and by a and 160 mg cyclophosphamide per kg on Day 17. Mice of grant from the Wadsworth Memorial Fund of Northwestern University Medi calSchool. Group VII received Ancrod every 12 hr from the time of Received March 9, 1976; accepted June 14, 1976 tumor implant until death. 3584 CANCER RESEARCH VOL. 36 Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 1976 American Association for Cancer Research. Defibrination and Chemotherapy in Tumor Growth Blood Studies. Blood was collected by cardiac puncture. gen level was evident by 48 hr and, thereafter, fibrinogen The whole-blood clotting time, by the capillary method of was not detectable (Table 1). Creskoff (7), was determined at 24-hr intervals on animals The effect of Ancrod treatment from Days 6 to 10 on treated with Ancrod and on their controls. Fibrinogen levels cyclophosphamide treatment on Day 10 is shown in Chart were çletermined from platelet-poor plasma by Astrup's 2A. A dose of Ancrod was given just before cyclophospha modification (2) of the method of Ratnoff and Menzies, mide and another dose was given 12 hr later so that defibri using blood collected in 3.8% trisodium citrate; the lower nation would persist over the interval during which cyclo limit of sensitivity of this fibrinogen assay was found to be phosphamide was present in the blood. Ancrod in this ex 25 mg/100 ml. periment had no effect on tumor growth per Se. With in Tumor Measurements. Tumor growth was determined by creasing doses of cyclophosphamide, a greater antitumor measuring tumor diameter 3 times weekly; the tumor diam effect was seen as manifested by a greater degree of tumor eter was converted to tumor weight as previously described shrinkage. Pretreatment with Ancrod had no consistent ef (8). fect on sensitivity to cyclophosphamide, and there was a suggestion of greater shrinkage after the use of cyclophos phamide in the higher dose (240 mg/kg; Chart 2C). The RESULTS opposite effect seen after the use of cyclophosphamide, at With progressive defibrination, a significant prolongation a dose of 160 mg/kg (Chart 2B), may be explained by a of the whole-blood clotting time was produced so that, by 8 40% smaller tumor burden measurable initially, a ratio that hr, the blood was incoagulable. Twelve hr after the initial persisted for the duration of the observations. doses of 100 or 200 units/kg, however, the clotting time had These results led us to speculate that a period of 4 days of returned to pretreatment values. Effective and persistent defibrination might not have been sufficient to alter the defibrination was achieved, however, after doses of 400 fibrin content of the tumor. Thus, treatment regimens com units/kg every 12 hr. A significantly reduced plasma fibrino bining defibrinating and fibrinolytic agents were carried out. In experiments using this combination therapy, Lewis GROUP lung carcinoma of a more advanced stage was tested. More III‘6@,Ill4.40 advanced stages of this tumor are known to be less sensi tive to cyclophosphamide (8), and fibrin deposition might be a factor in this decreased sensitivity with increasing “0 ,IV•0 .‘@—;--VI@.. Table 1 Effect of Ancrodandfibrinogen treatment on whole-blood clotting times mice'Sampling levels in tumor-bearing (mg/100aftertime (hr Fibrinogen― initialml)0 dose) Clotting time (mm) @—z@VII 21724 3.5 10048 8.0 0 6 0 13 6 17 20 25 27 <2572 8.0 @ Tumor - Ancrod @ Implant •Streptokinase <2596 12.0 DAYSAFTER TUMOR IMPLANT CTX Cyclophosphamide 5.5 <25 Chart 1. Schematic diagram of different therapeutic regimens used in the a Mice received 400 units/kg s.c. every 12 hr. different groups of animals. b The lower limit of sensitivity of this assay in 25 mg/100 ml. 100 A 10.0 B 10.0 C 8.0 8.0 8.0 6.0 6.0 6.0 4.0 4.0 4.0 0) to E E E 0 (@ 2.0 @ (9. I E I @ 21.0 @l.O 1.0 [email protected] @;0.8 @ @O.6 @0.6 0.6 ‘a,, 0 I-.EO.4 I-E0.4 - 0.2 ‘5—.-.— Ancrod 0.2 I:: f Cyclophosphamide @-.———,Ancrod p—' Ancrod @ t Cyclophosphamide Cyclophosphamide 0.1 . ‘ ‘. ‘. 0.1 . ‘ 0.1 , , , , , 5 0 15 20 25 30 5 052025Daysafter 30 5 0 5 20 25 30 Days after Tumor Implant Tumor Implant Days after Tumor Implant Chart 2. Effect ofAncrod pretreatment (Days 6 to 10) on tumor sensitivity to cyclophosphamide given on Day 10. Cyclophosphamide dose was 80 mg/kg. A, 160 mg/kg; B, 240 mg/kg; C, 240 mg/kg.