Anticoagulants and Tumor Cell Lodgment1

[CANCER RESEARCH 27 Part 1, 421-425,February 1967] Anticoagulants and Tumor Cell Lodgment1

BERNARD FISHER AND EDWIN R. FISHER Departments of Surgery and Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Summary titative information relative to the effect of heparin, fibrinolysin (plasmin), and the antifibrinolytic agent EACA on deix>sition There is evidence that the coagulation mechanism plays a role and organ retention of 51Cr-labeledtumor cells in rats and rabbits. in the development of mÃ©tastasesand that anticoagulants, by interfering with the adherence of tumor cells to vascular endo- Materials and Methods thelium, may impair secondary tumor growth. The present study, employing 51Cr-labeled tumor cells in rats and rabbits, Sprague-Dawley (Holtzman, Madison, Wisconsin) female rats provided quantitative information relative to the effect of hep- weighing 180-200 gm and New Zealand female rabbits weighing arin, fibrinolysin, and the antifibrinolytic agent Â«-aminocaproic 2-3 kg, all housed in individual cages and fed a standard labora acid (EACA) on deposition and organ retention of such cells. It tory diet (Purina rat and rabbit chow) with water ad libitum were was observed: (a) that adequate continuous heparinization used in these studies. The Walker carcinoma, propagated in this failed to promote the residence of tumor cells in the blood stream laboratory continuously since 1957, was employed in rat experi or to affect the lodgment of tumor cells in lung, liver, kidney, and ments and the V2 tumor was utilized in experiments involving spleen of rats from 1 hr to 7 days following the jugular vein in rabbits. The technics for preparation and counting of tumor cell jection of Walker tumor cells; (6) that the intraportal inocula suspensions, as well as for labeling tumor cells with 51Cr,have tion of Walker tumor cells resulted in similar retention of tumor been described (12, 13). The latter was essentially the same as cells in livers of heparinized and unheparinized rats; (c) that that first described by Rajam et al. (25) for labeling Ehrlich findings similar to those in the rat were noted following the jug mouse ascites tumor cells, and later by Selecki (27). Preliminary ular vein injection of V2 carcinoma cells in heparinized and evaluation has confirmed the efficacy of 61Cr for this purpose. fibrinolysin-treated rabbits; and (a) that EACA failed to affect Tumor cells and heparin, fibrinolysin, and EACA were inoculated the organ retention of tumor cells in the rabbit. through polyethylene catheters (PE 50 in the rat and PE 90 in It is concluded that anticoagulants do not influence mÃ©tastases the rabbit) previously placed in the jugular vein and were flushed by preventing tumor cell lodgment and/or by promoting their with 0.25 ml of saline following injection. Catheters were in residence within blood vessels. Other mechanisms to explain serted in the rat 72 hr prior to anticoagulant injection to elimi their effects on secondary tumor growth must be sought. nate bleeding at the catheter site and in the rabbit 2-3 hr before heparin or fibrinolysin administration. For intraportal inocula Introduction tion of tumor cells, a 2nd catheter (PE 50) was placed in the portal vein 72 hr prior to tumor cell injection. Evidence that the coagulation mechanism plays a vital role in Rats received 5 X IO6 Walker tumor cells in 0.5 ml of saline the development of mÃ©tastasescomes from: (a) studies demon via the jugular catheter and 5 X IO5cells in a similar volume of strating that shortly following lodgment tumor cells become sur saline intraportally. Rabbits received 1, 3, or 10 X IO6tagged V2 rounded by a microcoagulum of fibrin and platelets (28, 30) ; (fe) carcinoma cells in 1 ml of saline via the jugular vein. Both rats the finding that some cancer cells may be rich in thromboplastin and rabbits were inoculated with 70 mg (7000 units) of heparin (10, 20, 21); (c) recognition of an increased incidence of mÃ©tas 30 min prior to tumor cell injection. This dose was repeated tases following administration of antifibrinolytic agents or during every 12 hr in the rat and every 9 hr in the rabbit until sacrifice. alimentary hyperlipemia when fibrinolytic activity is diminished Lee-White coagulation times were maintained over 30 min dur (9, 24); and (d) reports that heparin (1, 11, 31) and fibrinolysin ing the entire experiment. Control animals received injections of (5, 10, 11, 17, 29) reduce the incidence of experimental mÃ©tas equivalent volumes of saline. tases. It is generally believed that anticoagulants interfere with In experiments with fibrinolysin (thrombolysin, Merck Sharp the initial adherence of cancer cells to vascular endothelium, with and Dohme) rabbits were injected via jugular catheters over a their aggregation, and with enmeshment of tumor cells in fibrin period of 5 min with 100,000 units dissolved in 10 ml of 5% clot. Consequently, it has been speculated that the attainment glucose in water 20 min before tumor cell injection. Control rab of an extravascular position by such cells and their development bits received 10 ml of 5% glucose in the same manner. One ml of into overt mÃ©tastasesbecome hindered. V2 tumor cell suspensions containing 1 X IO6 tagged cells was This paper is concerned with studies performed to obtain quan- injected through the same catheter. Rabbits injected with EACA (Amicar, Lederle Laboratories, 1Supported by American Cancer Society Grant P-142 and Pearl River, New York) received 0.2 mg/gm diluted to 10 ml in USPHS Grants CA-05949 and CA-06670. saline slowly administered 10 min prior to tumor cell inoculation. Received July 6, I960; accepted September 30, 19G6. Control animals received 10 ml of saline.

FEBRUARY 1967 421

TABLE 1 EFFECTOFHEPABINONORGANANDBLOODRETENTIONFOLLOWINGJUGULARVEININJECTIONOFWALKERTUMORCELLSIN THERAT

TIMEorSACRIFICE

POSTTDMOK CELLINJECTION1

hr2 Â±2.8Â°6.0 Â±1.96.0 Â±8.021.1 Â±6.123.6 Â±1.54.0 Â±1.43.8 Â±0.31.3 Â±0.51.5 Â±5.08.6 Â±3.28.0 hr6hr24 Â±1.43.5 Â±2.23.1 Â±7.021.7 Â±7.920.4 Â±1.33.9 Â±1.33.5 Â±0.51.3 Â±0.61.4 Â±2.65.4 Â±3.16.1 Â±1.53.5 Â±1.03.7 Â±6.820.8 Â±8.816.2 Â±1.25.0 Â±1.23.2 Â±0.62.4 Â±0.61.6 Â±1.52.3 Â±1.52.6 hr48 Â±1.43.6 Â±1.33.1 Â±6.919.3 Â±6.017.2 Â±1.84.3 Â±0.63.4 Â±0.71.7 Â±0.81.3 Â±0.80.9 Â±1.00.9 hr7 Â±1.52.2 Â±2.11.7 Â±4.719.1 Â±3.915.0 Â±1.56.1 Â±0.74.5 Â±0.62.4 Â±0.51.5 Â±0.50.2 Â±0.20.2 daysLUNGControl8.4Â±0.8Heparin7.0Â±0.8LIVESControl22.6Â±7.5Heparin24.2Â±1.3KIDNEYControl4.1Â±2.4Heparin4.2Â±0.5SPLEENControl1.1Â±1.5Heparin1.3Â±0.2BLOODControl10.0Â±0.2Heparin10.9Â±0.1 All values are % of injected counts and/or cells. Each represents the mean and S.D. of 10 rats.

Rats were sacrificed 1 hr to 7 days and rabbits 1 hr and 24 hr TABLE 2 after tumor cell inoculation by exsanguination. A sample of vena EFFECTOFHEPARINONHEPATICRETENTIONOFWALKERTUMOR cava blood was removed from all rabbits for counting prior to CELLSFOLLOWINGTHEIRINTRAPORTALINJECTION sacrifice. Lungs, liver, kidneys, and spleen were removed, gently IN THE RAT washed free of blood, sectioned, and placed in glass tubes suitable for counting in a Baird-Atomic sodium iodide-thallium-activated TIMEor SACRIFICE LIVEE well scintillation counter. In all instances, the entire organ was INJECTION30 assayed for MCr activity. Results were calculated to provide the percent of injected radioactivity harbored in each sample at minIhr2hr4 Â±6.1Â»28.8 Â±9.624.5 time of sacrifice. Values for blood are expressed as percent of Â±6.829.5 Â±7.328.7 activity in the entire volume of blood which was estimated from Â±5.825.9 Â±10.829.2 body weight. Since it has been observed that there is a relation hr6 Â±5.928.8 Â±4.931.0 ship between numbers of cells and radioactivity, results are pre hr12 Â±6.629.2 Â±6.526.7 sented as percent of injected cells and/or percent of injected hr24 Â±9.823.1 Â±7.428.9 hr48 Â±7.325.9 Â±6.624.0 radioactivity. hrControl29.0 Â±8.4Heparin26.2 Â±7.2 Results " All values are % of injected counts and/or cells. Each repre sents the mean and S.D. of 10 rats. Organ and Blood Retention of Tumor Cells Following Their Jugular Vein Inoculation in Heparinized Rals (Table 1) by heparinization of such animals. Liver assays 1 to 48 hr after Animals were sacrificed 1 hr to 7 days following labeled tumor labeled cell injection demonstrated no significant difference in cell injection (5 X IO6 cells) and organs were assayed for 51Cr activity. In fact, whereas livers from control animals harbored activity. Half of the animals, selected at random, were heparin- 23.1 Â±7.3% of the cells, heparinized animals had 28.9 Â±6.6% in ized just prior to tumor cell inoculation and were anti- their livers 24 hr after injection. coagulated until sacrifice. The lungs of heparinized rats con tained a percentage of the injected cells and/or counts equivalent Organ and Blood Retention of Tumor Cells in Heparinized Rabbits to that observed in rats not anticoagulated. In both groups, (Table 3) with the passage of time the decrease in percent of retained 51Cr-labeled V2 carcinoma cells (1, 3, or 10 million) were in cells was of the same magnitude. Thus, for example, 48 hr after tumor cell injection there was 3.6 Â±1.5% of the cells in jected into the jugular veins of normal control or heparinized the lungs of control animals and 3.1 Â±2.1% in heparinized rabbits which were sacrificed 1 or 24 hr later. At that time the ])ercent of injected counts and/or cells present in various organs rats in contrast to 8.4 Â±2.8% and 7.0 Â±1.9% in the 2 groups 1 was determined. Onl3' heparinized rabbits injected with 1 X IO6 hr after injection. There was a slight but insignificant decrease in cells and sacrificed 1 hr later and those injected with 10 X IO6 the percent of cells in the liver, kidneys, and spleen of heparin ized rats only after 24 hr of anticoagulation. Likewise, blood cells and examined after 24 hr showed some reduction in tumor from control and heparinized animals sampled from 1 hr to 7 days cell retention in the lungs. Whereas in the former group control following tumor cell injection failed to reveal a difference in the rabbits harbored 19.3 Â±9.7% of the cells, anticoagulated ani number of cells in the 2 groups at any time. mals had 12.4 Â±6.7%. Of the latter group, control animals had 13.2 Â±4.6% of cells and heparinized animals 7.8 Â±3.7%. Com parison of control and heparinized rabbits injected with 1 X IO6 Hepatic Retention of Tumor Cells Following Their Intraportal In cells and sacrificed 24 hr later, 3 X IO6cells and sacrificed after 1 jection in Heparinized Rats (Table 3) and 24 hr, or those injected with 10 X IO6 cells and examined The hepatic retention of injected Walker tumor cells following after 1 hr demonstrated no significant difference in the number their intraportal inoculation (5 X IO5cells) was not influenced of cells in the lungs.

422 CANCER RESEARCH VOL. 27

TABLE 3 EFFECTOFHEPARINONORGANRETENTIONOFV2 CARCINOMACELLSIN RABBITSFOLLOWINGJUGULARVEININJECTION

CELLSControl19.3 X 10' CELLSControl23.7 X 10Â« CELLSControl28.2 X 10Â«

CEIL INJECTION(hr)1241241

LungLiverKidneySpleenBloodTIMEPOSTTt'MOÂ»Â±9.7Â« Â±6.7 Â±8.4 Â± 6.6 Â± 10.6 Â±11.67.8 4.5 Â±2.62.3 3.4 Â±1.53.3 10.9 Â±4.12.8 8.9 Â±2.53.9 13.2 Â±4.62.4 Â±3.72.9 Â±0.94.5 Â±1.75.0 Â±1.56.1 Â±1.67.4 Â±1.35.9 Â±0.77.5 Â±1.41.1 Â±1.51.0 Â±3.11.6 Â±2.61.6 Â±1.91.8 Â±3.21.9 Â±0.5 Â±0.3 Â±1.0 Â±0.8 Â±0.7 Â±0.62.6 241 1.6 Â±0.30.2 1.5 Â±0.30.2 2.2 Â±1.10.1 2.3 Â±1.10.2 2.6 Â±0.60.2 Â±1.00.3 Â±0.1 Â±0.2 Â±0.1 Â±0.2 Â±0.1 Â±0.1 241241 0.7 Â±0.57.4 0.7 Â±0.66.1 0.6 Â±0.36.8 0.8 Â±0.47.6 0.6 Â±0.38.6 0.8 Â±0.310.5 Â±4.33.2 Â±2.52.2 Â±3.13.7 Â±2.95.2 Â±5.23.1 Â±2.73.3 Â±3.1Heparin12.4 Â±1.73 Â±2.1Heparin21.6Â±2.410 Â±1.3Heparin29.1 Â±2.1 Â«Allvalues are % of injected counts and/or cells. Each represents the mean and S.D. of at least 10 rabbits.

TABLE 4 Organ Retention of Tumor Cells in Fibrinolysin-treated Rabbits EFFECTOFFIBRINOLYSINONORGANRETENTIONOFV2 CARCINOMA (Table 4) CELLSIN RABBITSFOLLOWINGJUGULARVEIN INJECTION Rabbits that received fibrinolysin were injected intravenously CELLSControl13.9 X 10" with 1 X IO6V2 carcinoma cells and sacrificed 1 or 24 hr later.

IN No significant difference in the percentage of counts and/or cells JECTION(hr)1241241241 injected was found in the lungs or livers of control animals or those receiving the fibrinolysin. While the blood of fibrinolysin- Â±6.2Â«5.0 LungLiverBloodTIMEPOSTTUMOHCELL Â±7.93.5 treated animals demonstrated slightly less counts and/or cells Â±3.52.4 Â±2.52.5 Â±0.94.7 Â±1.04.4 than controls (3.9 Â±3.4% versus 5.6 Â±3.6% at 1 hr, and 2.3 Â± Â±2.25.6 Â±1.73.9 2.7% versus 4.0 Â±2.5% at 24 hr), these differences were not Â±3.64.0 Â±3.42.3 statistically significant. Â±2.5Heparin13.3 Â±2.7 Organ Retention of Tumor Cells in t-Aminocaproic Add (EACA)- " All values are % of injected counts and/or cells. Each is the treated Rabbits (Table 5) mean and S.D. of 10 rabbits. EACA administration prior to tumor cell inoculation failed to alter the retention of such cells in lungs or liver 1 and 24 hr later. TABLE 5 Likewise, activity and/or cell numbers in blood was no different EFFECTOFEACA ONORGANRETENTIONOFV2 CARCINOMACELLS in treated or control animals. IN RABBITSFOLLOWINGJUGULARVEININJECTION

CELLSControl18.8 Discussion

IN Cliffton and associates have reported that heparin and fibrino JECTION(hr)1241241241X10Â« lysin administration prior to intravenous inoculation of irradiated or nonirradiated tumor cells reduced the incidence of lung mÃ©tas LungLiverBloodTIMEPOSTTUMORCELLÂ±6.7"4.8 Â±5.64.1 Â±1.44.0 Â±1.83.9 tases in rats (1, 2, 17) and rabbits (7,10) and also augmented the Â±1.16.3 Â±0.66.2 salutory effect of nitrogen mustard in this regard (3). Moreover, Â±2.07.9 Â±1.37.0 they observed a decreased number of mÃ©tastasesat sites of Â±2.53.6 Â±3.42.7 trauma (4) and in the liver following local massage of bowel Â±2.9EACAÂ«17.6 Â±1.9 tumors when animals were anticoagulated (5). Conversely, ad ministration of the antifibrinolytic agent EACA increased lung Â«EACA,e-aminocaproic acid. mÃ©tastasesinrats from 33 to 85% (8). Their findings are in keep 6All values are % of injected counts and/or cells. Each is the ing with those of Wood (30, 31), who had noted a decreased in mean and S.D. of 8 rabbits. cidence of lung mÃ©tastasesinmice and, as observed in the rabbit ear chamber, a reduction in endothelial attachment of V2 carci It was of interest that livers of anticoagulated animals con noma cells following heparinization. Our own observations (11), tained a slightly greater, but not significant, percent of cells both reported in 1961, of the effect of anticoagulants on experimental 1 and 24 hr after injection of each of the 3 tumor doses. No signifi hepatic mÃ©tastasesdiffers from those of Cliffton et al. in that cant difference was found in the kidneys or spleens of such ani while these investigators observed that 1 injection of heparin mals. Likewise, the blood did not harbor a significantly different (40 units) prior to tumor cell injection was adequate to inhibit number of cells whether animals were or were not heparinized. mÃ©tastases,wenoted a striking inhibition of metastatic develop-

FEBRUARY 1967 423

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1967 American Association for Cancer Research. Bernard Fisher and Edwin R. Fisher ment only with prolonged anticoagulation (4-7 days) and with following endothelial permeation tumor cells do not necessarily larger doses of heparin. Moreover, while we did note a reduction remain in the extravascular tissue but may traverse the inter in the incidence of mÃ©tastasesfollowing plasmin administration, stitial space and enter lymphatics. It might be conjectured that results were not as striking as those recorded by them. Having anticoagulants promote the transorgan passage of cells via this observed that heparin and plasmin, when given hours after in route, thus preventing their extravascular lodgment and stromat jection of tumor cells, were less effective than when administered ization. The results of this study fail to substantiate such a possi prior to their inoculation, it was conjectured by us that anti bility, since it would have been anticipated that if such occurred coagulants interfered with the initial sticking of cancer cells to a clear difference in radioactivity of the organs from treated and vascular endothelium and/or the formation of intravascular untreated animals would have resulted. thrombi, thus hindering the attainment of an extravascular position by tumor cells with prolongation of their residence in Acknowledgments blood vessels. Since, however, mÃ©tastasesreadily occurred follow The technical assistance of Mrs. Edith Melczer, Miss Joyce ing withdrawal of heparin after 48 hr of administration, and since Mitchell, and Miss Betty Hitchey is gratefully acknowledged. no greater numbers of such cells traversed livers so as to be found in hepatic vein blood of heparinized rats, it was concluded that References the effect of heparin was probably not achieved by the interfer 1. Agostino, D.( and Cliffton, E. E. Anticoagulants and the De ence with lodgment of tumor cells in the liver. velopment of Pulmonary MÃ©tastases. Arch. Surg., 84: 449-53, The results reported here support this conclusion. For, the 1962. organ retention of MCr-labeled Walker tumor cells was essentially 2. . Decrease of MÃ©tastases of Carcinosarcoma Walker 256 no different in adequately heparinized rats following the jugular with Irradiation and Heparin or Fibrinolytic Agents. Radi vein or intraportal injection of such cells than in unheparinized ology, 79: 848-55, 1962. control animals. Moreover, heparin or plasmin failed to effect the 3. . Decrease in Pulmonary MÃ©tastases. Potentiation of Nitrogen Mustard Effect by Heparin and Fibrinolysis. Ann. lodgment of V2 tumor cells in rabbit organs. The failure to ob Surg., 157: 400-8, 1963. serve an alteration in the number of tumor cells in the circulating 4. â€” . Trauma as a Cause of Localization of Blood-borne blood of heparinized rats and rabbits was in keeping with the MÃ©tastases: Preventive Effect of Heparin and Fibrinolysis. findings in the organs and suggests that heparin does not promote Ibid., 161: 97-102, 1965. the retention of tumor cells in the blood stream, as has been con 5. Agostino, D., Grossi, C. E., and Cliffton, E. E. Effect of Hu jectured. Madden and Malmgren (23) have also shown that addi man Fibrinolysis on Hepatic MÃ©tastases in Simulated Colon tion of heparin to injected tumor cell suspensions failed to alter Carcinoma in Rats. Ibid., 153: 365-68, 1961. the rate of cell disappearance from the blood of mice. Recently, 6. Balazs, H., and Holmgren, H. Effect of Sulfomucopolysac- we have rei>orted (14) that adequate heparinization of rats like charides on Growth of Tumor Tissues. Proc. Soc. Exptl. Biol. wise fails to impair the localization of labeled tumor cells at sites Med., U: 142-45, 1949. of mechanical, chemical, or surgical trauma. The failure of EACA 7. Cliffton, E. E., and Agostino, D. Irradiation and Anticoagu lant Therapy to Prevent Pulmonary MÃ©tastases of the V2 to enhance cell retention and to reduce the number of circulating Carcinoma in Rabbits. Radiology, 80: 236-43, 1963. tumor cells would lend further discredit to the importance of the 8. â€¢ â€”.Effect of Inhibitors of Fibrinolytic Enzymes on Devel coagulation mechanism insofar as cell lodgment is concerned. In opment of Pulmonary MÃ©tastases. J. Nati. Cancer Inst., SS: this regard, it should be mentioned that Greene and Harvey (16) 753-62, 1964. have failed to confirm the assumption that aggregation or en- 9. Cliffton, E. E., Agostino, D., and Minde, K. The Effect of meshment in a fibrin clot are of primary significance in the organ Hyperlipemia on Pulmonary MÃ©tastases of the Walker 256 retention of tumor cells. Carcinosarcoma in the Rat. Cancer Res., el: 1062-67, 1961. Since anticoagulants fail to inhibit deposition of tumor cells, 10. Cliffton,E. E., and Grossi, C. E. Effect of Human Plasmin on other mechanisms whereby they impair mÃ©tastasesmust be con the Toxic Effects and Growth of Blood-borne Metastasis of the Brown-Pearce Carcinoma and the V2 Carcinoma of Rabbit. sidered. While failure of tumor cells to clump as a result of these Cancer, 9: 1147-52, 1956. agents has been considered as possible explanation, some investi 11. Fisher, B., and Fisher, E. R. Experimental Studies of Factors gators (26) feel that this very action may increase the number of Influencing Hepatic MÃ©tastases. VIII. Effect of Anticoagu cells free to form mÃ©tastases.Retik and associates (26) employed lants. Surgery, 50: 240-47, 1961. this explanation for their observation that heparinization of mice 12. . The Interrelationship of Hematogenous and Lym failed to reduce metastasis formation and, in fact, in 1 tumor- phatic Tumor Cell Dissemination: An Experimental Study. host system actually enhanced them. Surg. Gynecol. Obstet., Hi: 791-98, 1966. There is evidence that heparin may act as a mitotic inhibitor 13. . The Organ Distribution of Disseminated 61Cr-labeled (6, 15, 18, 19, 22, 32) and thus exert its tumor-inhibitory effect. Tumor Cells. Cancer Res., #7: 412-20, 1967. We (11), however, did not observe tumor growth to be delayed 14. Fisher, B., Fisher, E. R., and Feduska, N. Trauma and the or prevented following subcutaneous or intraportal injection of Localization of Tumor Cells. Cancer, in press. tumor cell suspensions prepared in heparin-saline solution, and 15. Goerner, A. The Influence of Anticlotting Agents on Trans plantation and Growth of Tumor Tissvie. J. Lab. Clin. Med., subcutaneous tumor growth was similar in animals systemically 16: 369-72, 1931. heparinized for 7 days as in untreated animals. 16. Greene, H. S. N., and Harvey, E. K. The Relationship Be Tumor cell permeation of vascular endothelium and subsequent tween the Dissemination of Tumor Cells and the Distribution stromatization by interstitial tissues is an essential requisite of of MÃ©tastases. Cancer Res., H: 799-811, 1964. secondary growth. Recently, we (12) have demonstrated that 17. Grossi, C. E., Agostino, D., and Cliffton, E. E. The Effect of

424 CANCER RESEARCH VOL. 27

Human Fibrinolysin on Pulmonary MÃ©tastases of Walker 256 26. Retik, A. B., Arons, M. S., Ketcham, A. S., and Mantel, N. Carcinosarcoma. Ibid., 20: 605-8, 1900. The Effect of Heparin on Primary Tumors and MÃ©tastases. 18. Heilbrunn, L. V., and Wilson, W. L. The Effect of Heparin on J. Surg. Res., 2: 49-51, 1962. Cell Division. Proc. Soc. Exptl. Biol. Med., 70: 179-82, 1949. 27. Selecki, E. E. A Study of the Metastatic Distribution 19. Jolies, B., and Greening, S. G. Effect of Heparin Upon Tumor of Ehrlich Ascites Tumour Cells in Mice. Australian J. Exptl. Growth. Acta UniÃ³ Intern. Contra Cancrum, 16: 682-85, 1960. Biol. Med. Sci., 37: 489-98, 1959. 20. Lawrence, E. A., Bowman, 1)., Moore, D. B., and Bernstein, 28. Wood, S., Jr. Pathogenesis of Metastasis Formation Observed G. I. A Thromboplastic Property of Neoplasms. Surg. Forum, in vivo in the Rabbit Ear Chamber. A. M. A. Arch. Pathol., 3: 694-98, 1953. 66: 550-68, 1958. 21. Lawrence, E. A., Dugan, M. J., and Overley, T. M. Further 29. . Experimental Studies of the Intravascular Dissemina Observations on the Thromboplastic Property of Neoplastic tion of Ascitic V2 Carcinoma Cells in the Rabbit, with Spe Tissue and the Effect of Its Control on the Distribution of cial Reference to Fibrinogen and Fibrinolytic Agents. Bull. Intravenous Implants. Proc. Am. Assoc. Cancer Res., 1: 32, Schweiz. Akad. Med. Wiss., 20: 92-121, 1964. 1953. 30. Wood, S., Jr., Holyoke, E. D., and Yardley, J. H. Mechanisms 22. Lippman, M. The Growth Inhibitory Action of Heparin on the of Metastasis Production by Blood-borne Cancer Cells. Proc. Ehrlich Ascites Tumor in Mice. Cancer Res., 17: 11-14, 1957. 4th CaÃ±ad. Cancer Conf., pp. 167-223. New York: Academic 23. Madden, R. E., and Malmgren, R. A. Quantitative Studies on Press, Inc., 1961. Circulating Cancer Cells in the Mouse. Ibid., 22: 62-66, 1962. 31. Wood, S., Jr., Yardley, J. H., and Holyoke, E. D. The Rela 24. O'Neal, R. M., and Hartroft, W. S. Cited as Personal Com tionship Between Intravascular Coagulation and the Forma munication (1960) to Wood, S., Holyoke, E. D., and Yardley, tion of Pulmonary MÃ©tastasesin Mice Injected Intravenously J. H. (See Ref. 30). Proc. 4th CaÃ±ad. Cancer Conf., pp. 167- with Tumor Suspension. Proc. Am. Assoc. Cancer Res., 2: 260, 223. New York: Academic Press, Inc., 1961. 1957. 25. Rajam, P. C., Jackson, A. L., and Black, S. H. The Intracel- 32. Zahl, P. A. The Action of Bacterial Toxins on Tumors. VIII. lular Labeling of Ehrlich Mouse Ascites Tumor Cells with Factors in Their Use for Cancer Therapy. J. Nati. Cancer Radiochromate. J. Lab. Clin. Med., 51: 767-72,1958. Inst., 11: 279-88, 1950.

FEBRUARY 1967 425

Bernard Fisher and Edwin R. Fisher

Cancer Res 1967;27:421-425.

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