[CANCER RESEARCH 44, 5286-5290, November 1984]

Characterization of a Xenograft Model of Human Ovarian Carcinoma Which

Produces Ascites and Intraabdominal Carcinomatosis in Mice

Thomas C. Hamilton, Robert C. Young, Karen G. Louie, Brent C. Behrens, Wilma M. McKoy, Karen R. Grotzinger,and Robert F. Ozols1

Medicine Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20205

ABSTRACT

We have used in vivo and in vitro procedures to select asubpopulation of cells from the human ovarian carcinoma cellline, NIH:OVCAR-3, with the capacity to grow i.p. in female nudeathymic mice. After i.p. injection of these cells, animals developmetastatic spread similar to that of clinical ovarian cancer. Disease progression is characterized by the development of massiveascites, extensive invasive i.p. tumors, and pulmonary métastases. The malignant ascites cells are transplantable, manifestcytoplasmic androgen and estrogen receptors, and express theovarian cancer associated antigen CA125 (116,000 units/ml ofascites supernatant). The cells also have the same chromosomemarkers which were present in the original cell line, NIH:OVCAR-

3. Survival following i.p. passage of ascites is dependent ontumor cell inoculum ranging from a median survival of 39 dayswith 40 million cells to 84 days for 11.5 million transplanted cells.The characteristics of this unique in vivo model make it wellsuited for the evaluation of new drugs and novel experimentaltherapies in ovarian cancer. In addition, this in vivo model,together with ovarian cancer cell lines, may prove particularlyuseful for the study of pharmacological ways to specificallyincrease the cytotoxicity of anticancer agents in tumor cells whilenot increasing toxicity in normal tissues. The presence of hormone receptors should facilitate the experimental evaluation ofhormonal therapy in ovarian cancer.

INTRODUCTION

A variety of experimental models of ovarian cancer have beenexamined to identify an optimum system for the study of thebiology of the human disease and to evaluate the potential roleof new experimental therapeutic modalities. Both in vivo and invitro systems have been studied, and these include ovariancancer cell lines (3, 5, 8, 9, 12, 14, 19, 23, 29), s.c. humanovarian tumor xenografts in immunodeficient or ¡mmunodeprivedhosts (4,16, 26), growth of xenografts at immunoprivileged sites(22), direct cloning of human ovarian cancer cells in soft agar,i.e., the human tumor stem cell assay (10, 20, 24), and atransplantable murine teratocarcinoma (18). None of thesemodels exhibits all the following features of an ideal modelsystem: (a) histopathology and embryology consistent with human ovarian cancer; (£>)a reproducible pattern of métastaseswhich parallels human disease (30); (c) drug sensitivity profilessimilar to that of human ovarian cancer; (d) immunological andbiological properties consistent with human ovarian cancer; and(e) suitable markers for following response to treatment. In

1To whom requests for reprints should be addressed, at Medicine Branch,

National Cancer Institute, Building 10, Room 12N226, 9000 Rockville Pike, Bethesda, MD 20205.

Received April 2, 1984; accepted August 13,1984.

addition, an experimentally useful model system must also provide adequate numbers of malignant cells for detailed biochemical and biological analyses.

We have used in vivo and in vitro selection procedures toobtain a subpopulation of cells from the well-characterized human ovarian carcinoma cell line, NIH:OVCAR-3 (12)[NIH:OVCAR-3 refers to NIH ovarian carcinoma cell line 3;NIH:OVCAR-3nu(A<>+)refers to a variant of OVCAR-3 selected for

growth in nude mice (nu) and substrate-independent growth in

agarose (Ag+)|, which grows i.p. in female nude athymic mice.This new in vitro-in vivo model system fulfills many of the

requirements for a suitable experimental system of ovarian cancer as set forth above. The tumor is transplantable, and progression closely parallels the human disease with animals developingintraabdominal carcinomatosis and ascites which leads to deathfrom respiratory compromise and bowel obstruction. BecauseNIH:OVCAR-3 was derived from a patient refractory to combination chemotherapy with Adriamycin, cisplatin, and cyclophos-

phamide and the cell line retained resistance to these drugs invitro (12), this model is an appropriate system in which to studydrug resistance and its pharmacological manipulation. In addition,NIH:OVCAR-3 contains steroid hormone receptors making the

model potentially useful for the evaluation of hormonal regulationof malignant cell growth and function and the potential significance of hormonal therapy in ovarian cancer.

MATERIALS AND METHODS

Chemicalsand Reagents

[2,4,6,7-3H]Estradk>l(90 to 115 Ci/mmol) and [17a-mei/7y/-3H]R1881(70 to 87 Ci/mmol; 17/3-hydroxy-17a-methylestra-4,9,11-trien-3-one)were purchasedfrom NewEnglandNuclear,Boston, MA. Hydroxyflutam-¡de(SCH 16423) was a gift from Schering Corp., Bloomfield, NJ. Unla-beled 5«-dihydrotestosterone,diethylstilbestrol, 9«-fluoro-110,21-dihy-droxy-16«,17rt-isopropyledenedioxy-1,4-pregnadiene-3,20-dione(triam-cinolone acetonide), and agarose (type VII) were acquired from SigmaChemical Co., St. Louis, MO. Lymphocyte separation medium (Ficoll)was from Bionetics Laboratory Products, Kensington, MD.

Selectionfor i.p. Growth

The initiation of NIH:OVCAR-3from the malignantascites of a patientwith common epithelialovarian cancer and the characteristicsof the cellline have been described in detail previously (12).Subsequently,this cellline has been subjected sequentially to selection procedures whichfavored acquisition of a subpopulation of cells with the capacity for i.p.growth in heterologous hosts. NIH:OVCAR-3was first selected for invivo growth by s.c. injectionof the cell line(10ecells) into the subscapular

areas of nude athymic mice (BALB/c genetic background). After in vivopassage, the s.c. tumors were selected for anchorage-independentgrowth. This was accomplished by mechanical dissociation of tumors(10) when they were 1 cm in diameter (at approximately 6 weeks) and

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Intraperitoneal Model of Human Ovarian Cancer

plating the cell suspension in agarose in a double-layer agar system as

previously described (12, 20). The colonies of cells which developed inagarose were harvested in mass and plated in tissue culture T-flasks (75

sq cm) in RPM11640 as used for the parental cell line (12). These cellsattached, grew in monolayer, and were passaged in vitro as a subline,NlttOVCAR-S"**«*', of the parental NIH:OVCAR-3 cell line.

NIH:OVCAR-3nu|A9't')was selected, a second time, for in vivo prolifer-

ative capacity by s.c. injection (as above), and the s.c. tumors thusformed were the source of cells for i.p. injection in female nude athymicmice. Prior to injection, these tumors were dissociated with collagenase(12), and tumor cells were separated from host cells on discontinuousgradients of FicoH (28). The tumor cell fraction was suspended in sodiumchloride solution (0.9%, w/v), and 107 cells were injected i.p. into 3 female

8-week-old nude athymic mice.

In Vivo Passage of Tumor Cells

All animals given injections of the tumor cell preparation describedabove developed abdominal distention due to asertes. The peritonealcavity was irrigated with sodium chloride solution (0.9%, w/v), and thewashings were combined with the ascites. The cell suspension thusobtained was centrifuged (300 x g at 4°for 10 min), and the cell pellet

was resuspended in sodium chloride solution (0.9%, w/v), after whichaliquots were counted, as nuclei, on an electronic cell counter (ModelZBI; Coulter Electronics, Hialeah, FL). Between 10" and 10" cells were

injected i.p. into subsequent hosts. Ascites cells were tested periodicallyby the mouse antibody production test and by this criterion were deemedto be negative for the 13 mouse-associated viruses assayed by thisprocedure.2

Assessment of Steroid Hormone Receptor Status

Buffer. The buffer used in the following procedures consisted of Tris-

HCI (10 rtiM), pH 7.4, containing EDTA (1 mw), dithiothreitol (1 mw),phenylmethylsulfonyl fluoride (1 HIM), and sodium molybdate (10 mu).

Protein Estimation. Cytosol protein was measured by the modifiedmethod (13) of Lowry e?al. (17) with bovine serum albumin as standard.

Preparation of Cytosol. Ascites harvested from animals (as above)was maintained on ice, and cells were obtained by centrifugation (asabove). Cells were suspended in 3 volumes of buffer and homogenizedwith a motor-driven glass-Teflon homogenizer. The homogenate wascentrifuged at 100,000 x g for 1 hr at 3°to obtain the cell cytosol (12).

Sedimentation Analysis. Cytosol labeled with [3H]estradiol or |3H|-R1881 in the absence or presence of potential competitors (3 hr at 4°)

and then treated with charcoal to remove free steroid was analyzed on4-ml linear (5 to 20%, w/v) gradients of sucrose prepared in buffer as

described previously (11) in detail. After centrifugation (150,000 x g for16 hr at 3°),gradients were fractionated into scintillation vials.

Measurement of Radioactivity. Scintillation fluid (10 ml; Aquassure;New England Nuclear) was added to vials and mixed. Samples werecounted at an efficiency of approximately 40%.

Survival Studies

The effect of cell inoculum on duration of survival was determined byi.p. passage of either 11.5 million or 40 million malignant ascites cells togroups of 9 female nude mice.

Detection Of CA125 Antigen

The presence of an antigen, CA125, which is associated with commonepithelial tumors of the human ovary (1), was kindly determined by Dr.Robert Bast (Boston, MA) and Dr. Mark Zweig (Bethesda, MD) on thesupernatant from the NIH:OVCAR-3-derived malignant ascites of animals

and on their serum using a radioimmunoassay procedure based on amonoclonal antibody, OC125, with specificity for the antigen (2).

2Animal Health Diagnostic Laboratory, Frederick Cancer Research Facility,NationalCancer Institute, personal communication.

Table 1Effect of i.p. cell inoculum size on host survival

No. of cellsinjected11.5x10"(n = 9)

40 x10«(n= 9)Days

ofsurvivalMedian84(67-112)'

39 (31-46)Mean

±S.O.89

±1638± 5

* Numbers in parentheses, range

Cytogenetics

Cytogenetic analysis on short-term cultures of NIH:OVCAR-3-derivedmalignant ascites was kindly performed by Dr. J. Whang-Peng as de

scribed previously (12).

RESULTS

General Observations. The i.p. injection of female nudeathymic mice with 10 million NIH:OVCAR-3 cells selected for thecapacity for substrate-independent and in vivo growth was followed in 40 to 50 days by visible abdominal distention (Fig. ~\A).

Examination of the peritoneal cavity at this stage revealed diffusestudding of tumor deposits on all peritoneal surfaces, the viscera,and the diaphragm (Fig. 1C), as well as copious ascites (Fig. Mi).The size of the tumors varied from 0.1-mm implants on thediaphragm to 1- to 2-cm masses in the bowel mesentery. Cytc-

logical examination of the ascitic fluid (Fig. 1D) revealed groupsand sheets of adenocarcinoma cells which in some cases showacinar and papillary-like structures. The volume of ascites usuallywas 2 to 5 ml per animal, and 10e cells were frequently obtainable

by aspiration of the ascites combined with peritoneal lavage.These cells could be grown and passaged in vitro. Histologicalexamination of tumor implants revealed invasive malignant cellswith a growth pattern which varied from solid tumor to glandularand papillary with numerous mitotic figures (Fig. 1£).Late in thecourse of the disease, pulmonary métastasesfrequently (-80%)

developed (Fig. 1F).Animals died from complications of extensive i.p. disease

(massive ascites and carcinomatosis) within 1 to 3 weeks afterabdominal distention was noted. Malignant cells in the asciticfluid were readily passaged to subsequent hosts and to date hasbeen serially transplanted 6 times. A dose-response relationship

was found to exist between size of the cell inoculum, abdominaldistention, and time of death. Injection of 40 million cells i.p.resulted in a mean survival for 9 animals of 38 ±5 (S.D.) days,while survival increased to 89 days ± 16 (S.D.) days on i.p.inoculation of 11.5 million cells (Table 1). It is feasible to inject30 to 50 million cells per animal, for experiments requiring a largenumber of animals, since between 300 million and 1 billion cellscan routinely be harvested from a single mouse. An inoculum ofthis size will produce the degree of reproducibility in mediansurvival required for comparative survival studies with this model.It should be noted, however, that animals inoculated with as fewas 1 million cells will ultimately succumb to the disease, althoughthe duration of survival is variable (data not shown).

Cytogenetics. Cytogenetic analysis of malignant ascites subjected to short-term culture revealed the cells to be derived from

a human female and to manifest those chromosomal markersdescribed previously for the cell line NIH:OVCAR-3 (12).3

3J. Whang-Peng (Medicine Branch, National Cancer Institute), personal com

munication.

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T. C. Hamilton et al.

Chart 1. Cytosol (protein, 14 mg/ml) prepared fromOVCAR-3-induced malignant ascites cells was sub

jected to sucrose density gradient analysis followingincubation (3 hr, 4°)with 4 nu [3H]R1881 alone (O) orin the presence of unlabeled Sa-dihydrotestosterone(•)at 400 nw, triamcinolone acetonide (D) at 400 HM,or hydroxyflutamide (A) at 2 pM (A); and 4 RM |3H|^

estradici alone (O) or in the presence of unlabeleddiethylstilbestrol (•)at 400 nw (B). After centrifugation,gradient fractionation was bottom to top (shown left toright), and markers of comparative sedimentation arebovine catalase (a, sx_w = 11.35), human igG (ó,sM,= 6.9S), and bovine serum albumin (c, SM.„= 4.6S).

2000

1500

1000

' 500

250

200

100

3000

2000

1000.

I 300•o

200

100

10 20 TOPFRACTION

10 20 TOPFRACTION

CA125 Antigen. The supernatant derived from centrifugationof malignant ascites was found to contain 100,000 to 400,000units of the CA125 antigen per ml, which is expressed by ovariancarcinoma cells from a majority of patients with nonmucinousovarian cancer (1, 2). The serum level of CA125 in mice withestablished tumors was in the range of 12,500 units/ml.4 The

level of antigen expressed in the ascites supernatant is 6 timesgreater than the highest serum level in any ovarian cancerpatients thus far studied.5

Steroid Hormone Receptors. Cytosol prepared from ascitescells, incubated with labeled estradiol or labeled R1881 alone orin the presence of potential competitors and analyzed on low-

salt sucrose density gradients (Chart 1), was found to containdistinct androgen- and estrogen-binding components with sedi

mentation coefficients of 7 to 9S and with specificities consistentwith the requisite receptors (11,12). Whole-cell estradiol-binding

assays of ascites cells further revealed approximately 5000estrogen-specific sites (diethylstilbestrol competible) per cell,

assuming equivalent binding in all cells.

DISCUSSION

This in vivo model of human ovarian cancer which mimics thehuman disease in growth pattern and progression was successfully developed through the use of in vivo and in vitro selectionprocedures to obtain a subpopulation of cells enriched for thecombined capacities of substrate-independent and in vivo

growth. The use of the approach was successful where previousattempts by us to establish such a model by direct i.p. implantation of human ovarian carcinoma cell lines, fresh ascites, andsolid tumor failed.

In an effort to develop more specific therapies for ovariancancer, we utilized experimental models including the humantumor stem cell assay (20), a murine teratoma (18), and humanovarian cancer cell lines (3, 9, 12, 19-23) for the preclinical

' M. Zweig (Clinical Pathology, Clinical Center, NIH), personal communication.5 R. Bast (Dana-Farber Cancer Center), personal communication.

evaluation of potential new therapeutic modalities. Studies inthese experimental systems have formed, in part, the rationalefor clinical trials of i.p. Adriamycin therapy (21) and enhancementof Adriamycin cytotoxicity by its use in combination with vera-

pamil (19, 23). The ability of these model systems to accuratelypredict the clinical efficacy of new treatment modalities may belinked by their inability to (a) differentiate between normal tissuetoxicity and tumor cytotoxicity and (b) accurately reflect important determinants of in vivo response such as bioavailability anddrug metabolism. The model system reported here has thepotential to be more predictive for clinical utility of experimentaltherapies than were previously described models of ovariancancer. The NIH:OVCAR-3 human ovarian cancer i.p. xenograft

model parallels the human disease by producing ascites, intraabdominal carcinomatosis, pulmonary métastases,and death fromdisease. In vitro findings of potential therapeutic significance canbe evaluated in this in vivo system where results can be monitored by the clear-cut parameter of increased survival.

The pattern of métastasesand the human derivation suggestthat the model may be useful in the evaluation of the therapeuticeffect of natural killer cells (25, 27), müllerianregression substance (6), bacterial toxins (15), and monoclonal antibodies aloneor conjugated to toxic agents (7). Likewise, the continued presence of available androgen and estrogen receptors in the ascitescells obtained from intact animals (ovaries not removed) suggeststhe relevance of this model for the evaluation of hormonal andantihormonal therapy in a well-controlled in vivo setting. The

evaluation of therapy with conjugates of steroids and cytotoxiceffectors should also be feasible in the model. The presence ofthe CA125 antigen also offers the opportunity to correlate antigen levels with experimental therapies in living animals.

The rapid development of drug resistance frequently limits theeffectiveness of chemotherapy in the treatment of patients withadvanced ovarian cancer. We have recently demonstrated inhuman ovarian cancer cell lines that resistance to Adriamycinand melphalan can be reversed by calcium channel blockers (19,23) and depletion of glutathione (9), respectively. The i.p. model

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of ovarian cancer described here appears to be well suited todetermine whether these in vitro manipulations can also besuccessfully performed in vivo. The demonstration that survivalcan be improved by using pharmacological means to reversedrug resistance or increase chemotherapeutic agent efficacywould indicate that clinically exploitable differential effects existbetween normal tissues and tumor cells. Studies with Adriamycinplus verapamil and of the effects of glutathione depletion uponmelphalan cytotoxicity are currently in progress in this model.

REFERENCES

1. Bast, R. C., Freeney, M., Lazarus, H., Nadler, L. M., Colvin, R. B., and Knapp,R. C. Reactivity of a monoclonal antibody with human ovarian carcinoma. J.Clin. Invest., 68:1331 -1337,1981.

2. Bast, R. C., Klug, T. L, St. John, E., Jenison, E., Mito«,J. M., Lazarus, H.,Berkolwitz, R. S., Leavitt, T., Griffiths, T., Parker, L., Zurawski, V. R., Jr., andKnapp, R. C. Radioimmunoassay using a monoclonal antibody to monitor thecourse of epithelial ovarian cancer. N. Engl. J. Med., 309: 883-887,1983.

3. Behrens, B. C., Louie, K. G., Hamilton, T. C., Curt, G., Kinsella, T., Young, R.C., and Ozols, R. F. Resistance (R) and cross-resistance (XR) of human

ovarian cancer (OC) cell lines to Adriamycin (AD), melphalan (ME) and irradiation (XRT). Proc. Am. Assoc. Cancer Res., 25: 336, 1984.

4. Davy, M., Mossiage, J., and Johannessen, J. V. Heterologous growth ofhuman ovarian cancer. Acta Obstet Gynecol. Scand., 56: 55-59,1977.

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7. FitzGerald, D. J. P., Trowbridge, I. S., Pastan, I., and Willingham, M. C.Enhancement of toxicity of antitransferrin receptor antibody-Pseuctomonasexotoxin conjugates by adenovirus. Proc. Nati. Acad. Sci. USA, 80: 4134-

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10. Hamburger, A. W., Salmon, S. E., Kim, M. B., Trent, J. M., Soehnten, B. J.,Alberts, D. S., and Schmidt, H. J. Direct cloning of human ovarian cancer cellsin agar. Cancer Res., 38: 3438-3444,1978.

11. Hamilton, T. C., Davies, P., and Griffiths, K. Androgen and oestrogen bindingin cytosds of human ovarian tumors. J. Endocrino!., 90:421-431,1981.

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Intraperitoneal Model of Human Ovarian Cancer

13. Hartree. E. F. Determination of protein: a modification of the Lowry methodthat gives a linear photometric response. Anal. Biochem., 48: 422-427,1972.

14. loachim, H. L., Dorsett, B. H., Sabbath, M., and Barber, H. K. Electronmicroscopy, tissue cultures, and immunology of ovarian carcinoma. Nati.Cancer Inst. Monogr., 42: 45-62, 1975.

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16. Kullander, S., Reusing, A., and Trope, C. Human ovarian tumors neterotrans-planted to 'nude' mice. Acta Obstet. Gynecol. Scand., 57:149-159,1978.

17. Lowry, O. H., Rosebrough, N. J., Fan-, A. L., and Randall, R. J. Protein

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18. Ozols, R. F., Locker, G. Y., Doroshow, J. H., Grotzinger, K. R., Myers, C. E.,and Young, R. C. Pharmacokinetìcsof Adriamycin and tissue penetration inmurine ovarian cancer. Cancer Res., 39:3209-3214,1979.

19. Ozols, R. F., Rogan, A. M., Hamilton, T. C., Klecker, R., and Young, R. C.Verapamil (V) plus Adriamycin (ADR) in refractory ovarian cancer (OC): designof a clinical trial on basis of reversal of ADR resistance (R) in human OC celllines (CL). Proc. Am. Assoc. Cancer Res., 25: 300, 1984.

20. Ozols, R. F., Willson, J. K. V., Weitz, M. D., Grotzinger, K. R., Myers, C. E.,and Young, R. C. Inhibition of human ovarian cancer colony formation byAdriamycin and its major metabolites. Cancer Res., 40: 4109-4112,1980.

21. Ozols, R. F., Young, R. C., Speyer, J. L., Sugarbaker, P. H., Greene, R.,Jenkins, J., and Myers, C. E. Phase I and pharmacological studies of Adriamycin administered intaperitoneally to patients with ovarian cancer. CancerRes., 42: 4265-4269,1982.

22. Reich, S. D., Griffin, T. W., Antonelli, D. M., Costanza, M. E., and Bogden, A.E. Activity of drugs against human tumors as determined by the 6-day subrenalcapsule assay. Proc. Am. Assoc. Cancer Res., 23: 222, 1982.

23. Rogan, A. M., Hamilton, T. C., Young, R. C., Klecker, R. W., and Ozols, R. F.Reversal of Adriamycin resistance by Verapamil in human ovarian cancer.Science (Wash. DC), 224; 994-996, 1984.

24. Selby, P., Butek, R. N., and Tannock, I. A critical appraisal of the 'humantumor stem cell assay.' N. Engl. J. Med., 308:129-134,1983.

25. Serrate, S. A., Vose, B. M., Timonen, T., Ortaldo, J. R., and Herberman, R. B.Association of human natural killer cell activity against human primary tumorswith large granular lymphocytes. In: R. B. Herberman (ed.), N. K. Cells andOther Natural Effector Cells, pp. 1055-1060. New York: Academic Press, Inc.,1982.

26. Taette, R., Howell, S. B., Giuliani, F. C., Koziol, J., and Koessler, A. Comparisonof the activity of doxorubicin analogues using colony forming assays andhuman xenografts. Cancer (Phila.), 50:1455-1461,1982.

27. Uchida, A., and Mtaksche, M. Lysis of fresh human tumor cells by autologouslarge granular lymphocytes from peripheral blood and pleural effusions. Int. J.Cancer, 32: 37-44,1983.

28. Vose, B. M. Quantitation of proliferative and cytotoxic precursor cells directedagainst human tumors: limiting dilution analysis in peripheral blood and at thetumor site. Int. J. Cancer, 30: 135-142,1982.

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7. C. Hamilton et al.

mm

Fig. 1. A, female nude athymic mouse with abdominaldisientan apparent 45 days after injectionof 10 millioncells prepared as described in "Materials and Methods."8, presenceof milky ascites (arrow) in animal shown \nA.C, presenceof bulky tumor and small foci of tumor throughout peritonealcavity. D, malignant-appearingsheetsof cells in the ascites. Papanicolaou,x 220. E, surface invasion of the liver; note numerous mitotic figures. H & E, x 54. F, metastatic lesion in lung of animal shown inAH&E, X130.

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1984;44:5286-5290. Cancer Res   Thomas C. Hamilton, Robert C. Young, Karen G. Louie, et al.   Carcinomatosis in MiceCarcinoma Which Produces Ascites and Intraabdominal Characterization of a Xenograft Model of Human Ovarian

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