[CANCER RESEARCH 46, 5687-5690, November 1986]

Cures and Partial Regression of Murine and Human Tumors by RecombinantHuman Tumor Necrosis FactorAbla A. Creasey,1 M. Theresa Reynolds, and Walter Laird

Departments of Cell Biology [A. A. C.. M. T. R.J and Toxicology [W. L.J, Cetus Corporation, Emeryville, California 94608

ABSTRACT

We tested the effect of recombinant human tumor necrosis factor(TNF) on the growth of the murine methylcholanthrene induced fibrosarcoma and the human ovarian carcinoma (NIH:OVCAR-3) in mice.The mice received multiple doses (25-250 jtg/kg) of TNF starting 7-10days after s.c. transplantation of tumors when they were easily palpable.TNF was administered i.v. every other day for a total of 6 injections permouse, or i.p. daily for 7 days. Complete tumor regression was observedin the methylcholanthrene induced tumor bearing mice in 90% of themice treated with TNF (100 Mg/kg), 67% treated with TNF (50 jig/kg),and 34% treated with TNF (25 fig/kg). Tumors which did not completelyregress were growth retarded during the course of TNF treatment. Allmice given the highest TNF dose are siili alive and tumor free (currentlyover 400 days), whereas the median survival of control mice was 28-39days. Partial regression was observed in 100% of mice bearing the ovariancarcinoma treated i.p. with 250 /ig/kg. Injections of TNF i.v. resulted inhigher percentage of cures than i.p. injections at similar dose levels.

These results suggest that tumor necrosis factor represents a likelypotent drug against solid tumors and that the method of administrationis critical in optimizing its use in cancer.

INTRODUCTION

TNF2 is a protein that was discovered in the serum of miceor rabbits given injections of Bacillus Calmette-Guérinor Co-rynebacterium parvum, followed by endotoxin (lipopolysaccha-ride), 1 to 2 weeks later (1-3). Serum containing TNF causesnecrosis of some tumors in vivo and is specifically cytotoxicand/or cytostatic to tumor cells in tissue culture (1-4). The i.v.administration of serum containing TNF into mice bearing theBALB/c sarcoma, Meth A, resulted in apparent destruction ofthe center of the tumor mass within 24-48 h, leaving a peripheral rim of viable tissue (1). Partially purified preparations ofmouse TNF were reported to exhibit antitumor activity againstboth mouse and human transplanted tumors in nude mice (5).Although it was noted that "sometimes" a complete cure was

observed in Meth A, Sarcoma 180, Ehrlich, and MM46 tumors(5), survival data with long-term follow-up on the cures has notbeen presented.

Partially purified native human TNF as isolated from thehuman promyelocytic leukemia cell line 111-60 was shown tocause significant necrosis of an established human breast carcinoma upon a single i.v. injection of TNF into nude mice (6)and the necrosis of the murine Meth A sarcoma when injectedi.v., i.p., or i.m. (7). Recently, recombinant human TNF produced in Escherlchia coli was also shown to cause hemorrhagicnecrosis of the Meth A tumor model (7) and of an undifferen-tiated human colon carcinoma when injected i.t. (8). It was alsoreported by the latter investigators that complete tumor regres-

Received 5/23/86; revised 7/29/86; accepted 8/5/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Department of CellBiology, Cetus Corp., 1400 Fifty-third Street, Emeryville, CA 94608.

2The abbreviations used are: TNF, tumor necrosis factor; Meth A, methyl

cholanthrene induced fibrosarcoma; PBS, phosphate buffered saline; i.t., minilumorally.

sion was observed in two of four mice receiving 5 x IO3 units

of TNF.Studies in our laboratories and those described above (6-8)

demonstrated that single injections of human recombinant TNFcause tumor necrosis. However, the tumors usually recover andkill mice. Long-term effects of multiple injections of TNF onsurvival of mice with tumors have not been reported. In thisstudy we demonstrate that repetitive systemic administrationof TNF (a) cured mice of s.c. murine methylcholanthreneinduced fibrosarcomas, prolonging their survival by >13months, and (h) caused partial regression of human ovariantumors in nude mice. In addition, we show that both tumor celllines were resistant to the cytotoxic action of TNF in vitro.

These results suggest that the mechanism of growth inhibition and regression of s.c. tumors in vivo by TNF may be dueto both the direct effect of TNF on the tumor cells and itsindirect effect on the tumor vasculature and/or the host immunesystem.

MATERIALS AND METHODS

Tumor Necrosis Factor. Highly purified recombinant human tumornecrosis factor expressed in /•:.coli (lot LYM12 022185B) was kindlyprovided by Dr. Leo Lin's laboratory at Cetus Corporation and was

used for all in vivo studies. The endotoxin level in the TNF preparation,was tíl.2Mg/ml as determined by the Limulus amebocyte lysate assay(Associates of Cape Cod, Inc., Woodshole, MA). Upon dilution of theconcentrated material in PBS with 2% bovine serum albumin, the micereceived «0.002Mgof endotoxin per injection. The endotoxin level inthe diluent bovine serum albumin was 0.03 ng/ml as determined by thequantitative chromogenic Limulus amebocyte lysate assay (WhittakerM. A. Bioproducts, Walkersville, MD).

Cells and Tumors. Murine (BALB/c) methylcholanthrene inducedfibrosarcoma cells (Meth A) were kindly supplied by Dr. Lloyd Old's

laboratory at Sloan Kettering, New York, NY. The cells were propagated as ascites in BALB/c mice. For s.c. tumors 5x10" cells were

suspended in 0.2 ml of serum free tissue culture medium and injectedin the suprascapular region on day 0. Tumors were evaluated by caliper(micrometer) measurements of 3 orthogonal directions (length x widthx height). Routinely the tumors reached about 100-200 mm3 in 10days. Human ovarian carcinoma cells NIH:OVCAR-3 (9) were suppliedto Cetus by Dr. T. Hamilton, National Cancer Institute, Bethesda,MD. This cell line is similar propagated as an ascites in BALB/c nudemice. Upon s.c. injection of 1 x 10*cells in 0.2 ml of serum free tissueculture medium, a 30-mm3 tumor is formed in about 10-12 days. For

both tumor models, TNF treatment began 10 days after tumors wereinitiated. Tumor volumes were recorded, and animals were weighedand randomized into groups. Tumor volumes and animal weight weremonitored every other day or every 3 days for the duration of theexperiments.

Mice. Female BALB/c inbred mice 7-10 weeks old, 18-20 g, wereobtained from Simonsen Labs, Gilroy, CA. Female nude athymicBALB/c mice, 7-10 weeks old, were obtained from Charles River,Wilmington, MA.

In Vitro Testing of Cell Lines for Response to TNF. Meth A andNIH:OVCAR-3 cells were suspended in growth media, seeded at 1 x10* cells/ml in 96-well microtiter plates, and incubated at 37*C in 5%CO2 for 18-24 h. Two-fold dilutions of a lO-^ig/ml TNF solution wereadded to the cells and the cultures were incubated for 5 days. Thecultures were evaluated for viability either microscopically (trypan blue

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TUMOR REGRESSION BY TUMOR NECROSIS FACTOR

dye exclusion) or by measurement of [3H]thymidine uptake (5 /¿Ci/ml,

78 Ci/m nini | for 18 h. The cells were harvested onto glass fiber filtersfor scintillation counting. Cell lines that routinely respond to TNF,such as murine fibrosarcoma L929 and human breast carcinoma MCF-7 (6), were used as positive controls for the assay.

RESULTS

The effect of six i.v. injections (given every other day) of TNFon the Meth A tumor starting 10 days after tumor transplantis shown in Fig. 1. An approximate 24-fold increase in tumorvolume was observed in control injected mice 18 days after thestart of treatment while no increase in tumor volume wasobserved in mice treated with TNF (50 Mg/kg) 18 days after thestart of treatment. Statistical analysis comparing the TNFtreated (50 Mg/kg) and diluent control treated mice 18 daysafter initiation of treatment showed significant differences intumor volume (x2 = 22.38, P = 0.0002) but no signficant

differences at the start of TNF treatment.Titration of the dose-response curve of the Meth A tumor to

TNF is shown in Table 1. Mice bearing solid tumors were giveni.v. TNF injections of 25, 50, and 100 tig/kg, respectively.Retardation of tumor growth as well as regression were observed in all groups of mice receiving TNF. Mice given injections of TNF (25 tig/kg) had tumors with an approximate sizeof one-half that of the controls 18 days after initiation oftreatment, while those receiving TNF (50 and 100 Mg/kg) hadtumors that were 0-3% of the control size. In this experiment,complete remission was observed in 38% of mice receiving TNF(25 /¿g/kg),88% in those mice receiving TNF (50 Mg/kg), and100% in those receiving TNF (100 /ig/kg). The survival pro-

Fig. I. Effect of systemic repetitive administration of recombinant humantumor necrosis factor on the growth of the murine s.c. Meth A sarcoma. TNFi.v. administration was initiated (arrows), using SOpg/kg/injection in 0.1 ml everyother day for a total of 6 injections, 10 days after the s.c. inoculation of S x 10sMeth A sarcoma cells. TNF (A); excipient control (PBS with 2% bovine serumaJbumin) (O). Bars, SD.

portions of the latter two groups are statistically distinguishablefrom the control groups using Fisher's (one-tailed) exact test

(P < 0.005). Although significant heterogeneity was observedin the response of the mice receiving TNF (25 /*g/kg), the meanlife span of all TNF treated groups was significantly higherthan that of the controls (Table 1). A repeat of this experimentusing 7 mice/group reproduced similar results. Cures wereobserved in 80, 43, and 29% of mice treated with 100, 50, and25 /ig/kg, respectively.

The 100-^g TNF/kg dose may be interpreted as the 25%lethal dose for the BALB/c mice bearing a Meth A solid tumorsince 2 of 8 mice died 1 day after the first injection. In a repeatexperiment 2 of 7 mice died 1 day after the first injection.

The effect of daily i.p. injections of TNF on the growth ofthe human ovarian carcinoma tumors NIH:OVCAR-3 in nudemice is shown in Fig. 2. After 7 days of TNF treatment with250 fig/kg/injection there was a 2.5-fold decrease in tumorvolume. The tumors, however, regained growth upon cessationof TNF treatment with a 4-fold increase in tumor volume overthe following 3 days (Fig. 2, day 10). The tumor volume of thediluent control animals increased (5-fold) over the 10 days.Statistical analysis indicated that the tumor volume for theTNF treated animals (250 ^g/kg/injection) was significantlysmaller than the diluent control animals on days 7 and 14 (P <0.005 and <0.02, respectively). Retardation of tumor growthbut not regression was observed in the groups of mice whichreceived 125 and 62.5 ^g/kg/injection.

In addition to decreasing tumor volume, i.v. TNF administration of 50 and 100 ¿tg/kg/injection resulted in a slightdecrease (3-5%) in body weight (P < 0.05) as evaluated on day28 (Table 1). However, the mice recovered after completion ofTNF administration and gained weight during further observation similar to results in controls (data not shown).

When injected i.v., the most pronounced effect of TNF in theMeth A model was to increase median survival time of the miceas shown in Fig. 3. The median survival times of the uninjectedand PBS control groups were 39 and 28 days, respectively,while those treated with TNF were 53, >400, and >400 for the3 doses, respectively. The survival times of the mice treatedwith TNF (50 Mg/kg) and with TNF (100 ng/kg) differedsignificantly from the survival of the excipient controls (P =0.001 and P = 0.002, respectively; Breslow test). All long-termsurvivors have been tumor free for >13 months.

When administered i.p., TNF was not as effective in theprolongation of survival time as compared to the i.v. route.This observation is demonstrated in Fig. 4. The median survivaltimes of uninjected and PBS control groups were 30 and 28days, respectively; results for those given TNF injections were46, 34, and 54 days. Both 25- and 75-Mg/kg doses of TNFsignificantly prolonged survival time (P = 0.003 and P = 0.016;Breslow test); however, the 50-jtg/kg dose did not (P = 0.082).The lack of a dose-response correlation is unclear but may berelated to the significant variability that was observed in mediansurvival times among controls in this model. Although the i.p.route of TNF administration was less effective in prolongingsurvival than the i.v. route, complete tumor regression wasobserved 8 days after the end of TNF treatment in approximately 10% of the mice given injections of 25 Mg/kg and 40%in those given injections of 75 Mg/kg. This finding suggests thatthe prophylactic efficacy of TNF is highly dependent on itsroute of administration. A significantly larger number of curesmight have been obtained using the i.p. route if TNF dosagewere increased to concentrations greater than 75 ¿ig/kg.

Administration of TNF i.p. into BALB/c mice bearing ascites5688

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Table 1 Efficacy testing of human recombinant TNF against mouse Meth AflbrosarcomaTumor volume (mm3 ±SD)Group12

345Treatment"Control,

no injectionsPBS control, 0.1 ml/injectionTNF, 25 Mg/kg/injectionTNF, 50 pg/kg/injectionTNF, 100 iig/kg/injectionDay

10Ì19±

142249 ±108254 ±132174 ±90307 ±201Day

284169

±21035443 ±30632646 ±3816

143 ±3690Change

intumor

volume*3850

51942392-31

-299No.

ofcures(%)°0/8

(0)0/8 (0)3/8 (38)7/8 (88)6/6 (100)'Median

survivaltime (days)39

2853

>400>400FBWVlBW1.12

1.151.050.950.98

" PBS control and TNF test animals were given i.v. injections every other day for a total of 6 injections, starting on day 10 after tumor cell injection.b Change in tumor size (day 28 —day 10) in mm3.' Number of animals with no tumor/number of animals per group at day 28. Survivors at day 28 were still alive at day 400." FBW/IBW, final body weight/initial body weight at day 28.' Two mice died after receiving the first injection of TNF.

12 13 14 15

DAYS

Fig. 2. Effect of systemic repetitive administration of recombinant tumornecrosis factor on the growth of the s.c. human ovarian carcinoma NIH:OVCAR-3 in nude mice. TNF i.p. administration was initiated (arrow*) every day for atotal of 7 injections. Treated animals received 0.5 ml containing 250 /ig/kg/injection (•),125 /¿gAg/injection (•),62.5 ng/kg/injection (A), or PBS with 2%bovine serum albumin (O). Bars, SD.

100

80

0 12 24 30 36 42 « 54

DAYS SINCE INITIATION OF TNF TREATMENT

Fig. 3. Survival curves for TNF i.v. administration [25 11%(•),50 ng (A), 100Mg(•)per kg per injection], excipient control [phosphate buffered saline with 2%bovine serum albumin (O)], and noninjected tumor bearing controls (A) are shown.The experimental protocol is described in "Materials and Methods" and in the

legend to Fig. 1.

of the Meth A fibrosarcoma or nude mice bearing ascites of theNIH:OVCAR-3 carcinoma proved ineffective in alleviating tumor burden or in prolonging survival (data not shown). Similarly, TNF (10 /ig/ml) had no effect on the growth of the murineMeth A fibrosarcoma cells and the human ovarian carcinomacells in vitro (Fig. 5), while it was cytotoxic to the human breastcarcinoma cells (MCF-7) at ng levels.

DISCUSSION

Tumor necrosis factor exhibits both cytotoxic and cytostaticactivities on a variety of tumor cells but not on many normal

DAYS SWCE INITIATION OF TNF TREATMENT78 IM

Fig. 4. Survival curves for i.p. administration of TNF [25 fig (•),50 ng (A),75 Mg(•)per kg per injection], excipient control [phosphate buffered saline with2% bovine serum albumin (O)], and untreated tumor bearing controls (A) areshown. The experimental protocol is described in "Materials and Methods" and

in the legend to Fig. 1 with the following modifications. Each group consisted of10 mice and all animals received TNF i.p.

160

140

120

100

80

s sS £ 60

£*40

20

« « » »

V

2.5 0.6 0.16 0.04

TNF (/iG/ML)

0.009 0.001

Fig. 5. ¡nvitro dose-response curves of murine methylcholanthrene inducedfibrosarcoma cells (Meth A) (O) and human ovarian carcinoma cells(NIHrOVCAR-3) (*) to recombinant tumor necrosis factor. Human breast carcinoma cell line (MCF-7) (Y) was used as a positive control. Representative meansof control [3H]thymidine uptake for the three cell lines ±SD (NIH:OVCAR-3,Meth A, and MCF-7) were 57,692 ±4,322, 141,232 ±10,123, and 310,000 ±22,453, respectively.

cell lines in culture (6, 10-12). The mechanism of tumorregression by tumor necrosis factor in vivo is still unknown. Itis possible that tumor necrosis factor acts both directly on thetumor cells and indirectly by activating certain arms of theimmune system. Recent reports show that TNF augments thephagocytic and cytotoxic activities of polymorphonuclear neu-

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trophils (13) and stimulates their adherence to umbilical veinendothelium (14), activation of eosinophils to kill schisiosomes(15), and enhancement of synthesis and expression of HLA-A,B antigens on endothelial cells and dermal fibroblasts in vitro(16).

The methylcholanthrene induced fibrosarcoma model isthought to be an unusually responsive tumor to various inumi-nomodulators (20). However, a stringent test of its usefulnessin predicting efficacy of anticancer agents is thought to be itsuse with slightly larger tumors at the onset of treatment, acondition which seems to minimize its nonselective response tovarious agents (17). We thus used established tumors thatranged in age from 10 to 15 days after cell transplantation (withan approximate size range of 125-600 mm3)for the assessment

of the therapeutic effect of TNF on this tumor model. Theabsence of spontaneous regression of tumors in the diluent(phosphate buffered saline) control animals supports the validity of this model and its nonresponsiveness to nonselectiveimmunostimulation. High doses of endotoxin (10-100 ng/ml)have been reported to produce hemorrhagic necrosis but nottumor regression in the Meth A tumor model (1). Endotoxinlevels in the TNF preparations used in this study are low, lessthan 2 ng/injection, and thus the tumor regression that wasobserved is highly unlikely to be due to endotoxin contamination of the TNF. One, however, cannot exclude the possibilitythat small quantities of endotoxin may have locally activatedthe host immune and/or inflammatory mechanisms whichmight have potentiated the effect of TNF.

The human ovarian carcinoma NIH.-OVCAR-3 model (9)was not as responsive to TNF as the murine methylcholanthrene induced fibrosarcoma. Complete tumor regression byTNF was not achieved in the ovarian tumors in the nude mice.The lack of cures in this model may be due to inherent differences in sensitivities of the tumors to TNF and the growthcharacteristics and vasculature patterns of the tumors. Bothtumor cell lines were found resistant to the cytotoxic action ofTNF i/i vitro and when subcultured as ascites in vivo. Thesefindings suggest that the mechanism of action of TNF in vivomay involve the activation of the host immune system and/ordirect action on the local vasculature of tumors. It is temptingto speculate that small quantities of TNF locally affect multiplecellular components of the tumor. TNF may activate macrophages to produce factors that potentiate TNF cytotoxic action;TNF may recruit neutrophils and lymphocytes into the tumorarea by activation of the endothelium or may directly affectneutrophils by altering their cell surface and enhancing theirability to adhere to the blood vessel endothelium. In light ofthe published reports on the interaction of TNF with cells ofthe immune system (13,14,16), any or all of the above scenariosmay lead to tumor regression by TNF. Hist(»pathologicalstudies on the kinetics of regression of tumors by TNF wouldundoubtedly shed some light on this area of research.

The putative receptor for TNF has been identified (18-22).Studies in our laboratories and those of other investigatorsshow that there is no correlation between receptor number andsensitivity to TNF. Thus, differences in receptor number between the murine Meth A tumor cells and the human ovariancarcinoma cells may not necessarily explain the differences weobserve in their response to TNF in vivo.

ACKNOWLEDGMENTS

We thank B. Richardson for the Meth A tumor cells; L. Lin, R.Yamamoto, and A. Lim for purified recombinant TNF; L. Doyle for

assisting in the development of this work; A. Carey and J. Kopplin forprotocols; J. Winkelhake for development of NIH:OVCAR-3 tumormodel at Cetus; C. Vitt, C. Franzini, T. Feng, G. White, E. Rosario,M. Tasch, E. Ladner, S. Nilson, S. Tickton, R. Bengelsdorf, J. Stelzner,and T. Gong for technical assistance; D. Carlin for helpful discussionson the statistical analyses of data; P. Ralph, M. Doyle, and F. C.Creasey, Jr., for critical review of the manuscript; and L. Lin, D. Mark,and T. White for their support and encouragement.

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14. Gamble, J. R., Harían,J. M., Klebanoff, S. J., and Vadas, M. A. Stimulationof the adherence of neutrophils to umbilical vein endothelium by humanrecombinant tumor necrosis factor. Proc. Nati. Acad. Sci. USA, 82: 8667-8671, 1985.

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1986;46:5687-5690. Cancer Res   Abla A. Creasey, M. Theresa Reynolds and Walter Laird  Recombinant Human Tumor Necrosis FactorCures and Partial Regression of Murine and Human Tumors by

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