"nude" mouse

4
256 Reports Invest. Ophthalmol. Visual Sci. March 1977 Heterotransplantation of retinoblastoma into the athymic "nude" mouse. BRENDA L. GALLIE, DANIEL M. ALBERT, JAMES J. Y. WONG, NED BUYUKMIHCI, AND CAR- MEN A. PULIAFITO. Fresh surgical specimens of retinoblastoma were successfully transplanted into the anterior cham- ber of the "nude" mouse (a homozygous mutant, nu/nu, with a severe defect in cellular immunity), filling the eyes but failing to grow subcutaneously. Retinoblastoma cells from an established cell line, Y-79, J spread from the intraocular injection site to invade the orbit, optic nerve, and brain and formed large tumors when implanted subcutaneous- ly. Tumor cells injected into the anterior chamber of immunologically normal litter-mates (heterozy- gotes, nu/+) survived for varying periods in the anterior chamber but showed little growth. The "nude" mouse is a hairless mutant (homozygous for the mutation nude, nu/nu), which is born without a thymus and possesses a severe defect in cellular immunity. 2 Recently, a variety of human malignant tumors have been transplanted subcutaneously and intraperitoneally into the nude mouse. Some human tumor cell lines, maintained in vitro prior to injection, have shown infiltrative and metastatic growth when so transplanted. 3 Fresh tumor specimens, on the other hand, typically grow slowly, if at all, when similarly implanted. 1 The experimental study of retinoblastoma, the most common intraocular tumor of childhood, has been limited by its low incidence and by the consequent scarcity of surgical specimens. Al- though short-term propagation of retinoblastoma is readily achieved, there are only two well- characterized, established cell lines of retino- blastoma in vitro. 1 ' 5 The results of heterologous transplantation into the anterior chamber of the eye has led to consideration of this site as an area of relative "immunologic privilege." 6 Recent studies have suggested that the mechanisms in- volved and the extent of "immunologic privilege" are not yet fully defined. 7 Our attempts at prop- agating numerous explants of retinoblastoma in the anterior chamber of rabbit, hamster, and monkey eyes and the cheek pouch of hamsters, over a 10 year period, have been unsuccessful. This is in marked contrast to the results with ocular melanomas and a variety of other tumors. In the present experiments, portions of fresh surgical specimens of retinoblastoma as well as cells from the established Y-79 cell line were transplanted into the anterior chamber of nude mice (nu/nu) and their heterozygote (nu/+) litter- mates, which have been shown to be immunologi- cally normal, 2 and into the subcutaneous site in nu/nu mice. Methods. Portions of 13 fresh retinoblastomas were obtained at the time of enucleation. Part of each specimen was placed in tissue culture with the use of methods previously described. 1 A single cell suspension of the fresh tumor was easily obtained by agitation in the tissue culture medium; trypsinization was unnecessary. Swiss background nu/nu and nu/+ mice were obtained from the animal facility at Memorial Sloan-Ketter- ing Cancer Center and were kept in a conven- tional environment in cages with filter tops. Fresh tumor cells or the cell line Y-79, in a single-cell suspension in RPMI 1640 with 1 percent penicillin and streptomycin, were implanted into the anterior chamber and subcutaneously into the nude mice and into the anterior chamber of their nu/+ litter- mates. The tumor cells were injected into the anterior chamber under sterile conditions, with a 30-gauge needle, under a dissecting microscope. The maximum volume of the anterior chamber injection was approximately 0.0001 ml., containing approximately 6,000 cells. As the needle was with- drawn, there was some slight reflux of fluid, so that the final number of cells remaining in the anterior chamber was less. The volume injected subcutaneously was approximately 0.20 ml. con- taining from 10" to 10" cells. Four nu/nu mice were treated with 300 mg./kg. of cyclophosphamide 4 days prior to subcutaneous implantation of fresh retinoblastoma, lowering their white cell counts from 10,000 to 400/mm 3 . Results. Growth of retinoblastoma in the various sites is shown in Table I. Fresh retinoblastoma heterotransplanted into nu/nu mice. Twelve of the 13 fresh retinoblastoma transplants filled the anterior chamber of the nu/nu mice. Spread into the vitreous and retina was typically seen. The fresh retinoblastomas failed to acquire a demonstrable stroma or vascular supply and remained as loosely clumped tumor cells floating inside the eye. Although infiltration of the iris and retina was seen, the tumor was avascular. No extraocular extension was seen. The histologic appearance of the tumor which grew within the eye remained similar to that in the original tumor. In two cases in which Flexner- Wintersteiner rosettes had been seen on original histopathologic study, similar structures were seen in the tumor which spread intraocularly in the nu/nu mice (Fig. 1). Tumor harvested from the eyes of nu/nu mice could be passed to the eyes of other nu/nu mice. Several tumors are now in the fifth and sixth passage. All tumors failed to grow subcutaneously in untreated nu/nu mice, but in three out of four nu/nu mice treated with cyclophosphamide, tu- mors grew subcutaneously to be as large as the whole animal. These large tumors spread along tissue planes but did not invade or destroy normal tissues. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933304/ on 02/03/2018

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Page 1: "nude" mouse

256 Reports Invest. Ophthalmol. Visual Sci.March 1977

Heterotransplantation of retinoblastomainto the athymic "nude" mouse. BRENDA

L. GALLIE, DANIEL M. ALBERT, JAMES

J. Y. WONG, NED BUYUKMIHCI, AND CAR-

MEN A. PULIAFITO.

Fresh surgical specimens of retinoblastoma weresuccessfully transplanted into the anterior cham-ber of the "nude" mouse (a homozygous mutant,nu/nu, with a severe defect in cellular immunity),filling the eyes but failing to grow subcutaneously.Retinoblastoma cells from an established cell line,Y-79,J spread from the intraocular injection site toinvade the orbit, optic nerve, and brain andformed large tumors when implanted subcutaneous-ly. Tumor cells injected into the anterior chamberof immunologically normal litter-mates (heterozy-gotes, nu/+) survived for varying periods in theanterior chamber but showed little growth.

The "nude" mouse is a hairless mutant(homozygous for the mutation nude, nu/nu),which is born without a thymus and possessesa severe defect in cellular immunity.2 Recently,a variety of human malignant tumors have beentransplanted subcutaneously and intraperitoneallyinto the nude mouse. Some human tumor celllines, maintained in vitro prior to injection, haveshown infiltrative and metastatic growth when sotransplanted.3 Fresh tumor specimens, on theother hand, typically grow slowly, if at all, whensimilarly implanted.1

The experimental study of retinoblastoma, themost common intraocular tumor of childhood, hasbeen limited by its low incidence and by theconsequent scarcity of surgical specimens. Al-though short-term propagation of retinoblastomais readily achieved, there are only two well-characterized, established cell lines of retino-blastoma in vitro.1' 5 The results of heterologoustransplantation into the anterior chamber of theeye has led to consideration of this site as an areaof relative "immunologic privilege."6 Recentstudies have suggested that the mechanisms in-volved and the extent of "immunologic privilege"are not yet fully defined.7 Our attempts at prop-agating numerous explants of retinoblastoma inthe anterior chamber of rabbit, hamster, andmonkey eyes and the cheek pouch of hamsters,over a 10 year period, have been unsuccessful.This is in marked contrast to the results withocular melanomas and a variety of other tumors.

In the present experiments, portions of freshsurgical specimens of retinoblastoma as well ascells from the established Y-79 cell line weretransplanted into the anterior chamber of nudemice (nu/nu) and their heterozygote (nu/+) litter-mates, which have been shown to be immunologi-cally normal,2 and into the subcutaneous site innu/nu mice.

Methods. Portions of 13 fresh retinoblastomaswere obtained at the time of enucleation. Part ofeach specimen was placed in tissue culture withthe use of methods previously described.1 Asingle cell suspension of the fresh tumor waseasily obtained by agitation in the tissue culturemedium; trypsinization was unnecessary. Swissbackground nu/nu and nu/+ mice were obtainedfrom the animal facility at Memorial Sloan-Ketter-ing Cancer Center and were kept in a conven-tional environment in cages with filter tops. Freshtumor cells or the cell line Y-79, in a single-cellsuspension in RPMI 1640 with 1 percent penicillinand streptomycin, were implanted into the anteriorchamber and subcutaneously into the nude miceand into the anterior chamber of their nu/+ litter-mates. The tumor cells were injected into theanterior chamber under sterile conditions, witha 30-gauge needle, under a dissecting microscope.The maximum volume of the anterior chamberinjection was approximately 0.0001 ml., containingapproximately 6,000 cells. As the needle was with-drawn, there was some slight reflux of fluid, sothat the final number of cells remaining in theanterior chamber was less. The volume injectedsubcutaneously was approximately 0.20 ml. con-taining from 10" to 10" cells.

Four nu/nu mice were treated with 300 mg./kg.of cyclophosphamide 4 days prior to subcutaneousimplantation of fresh retinoblastoma, loweringtheir white cell counts from 10,000 to 400/mm3.

Results. Growth of retinoblastoma in the varioussites is shown in Table I.

Fresh retinoblastoma heterotransplanted intonu/nu mice. Twelve of the 13 fresh retinoblastomatransplants filled the anterior chamber of thenu/nu mice. Spread into the vitreous and retinawas typically seen. The fresh retinoblastomasfailed to acquire a demonstrable stroma or vascularsupply and remained as loosely clumped tumorcells floating inside the eye. Although infiltrationof the iris and retina was seen, the tumor wasavascular. No extraocular extension was seen. Thehistologic appearance of the tumor which grewwithin the eye remained similar to that in theoriginal tumor. In two cases in which Flexner-Wintersteiner rosettes had been seen on originalhistopathologic study, similar structures were seenin the tumor which spread intraocularly in thenu/nu mice (Fig. 1). Tumor harvested fromthe eyes of nu/nu mice could be passed to theeyes of other nu/nu mice. Several tumors are nowin the fifth and sixth passage.

All tumors failed to grow subcutaneously inuntreated nu/nu mice, but in three out of fournu/nu mice treated with cyclophosphamide, tu-mors grew subcutaneously to be as large as thewhole animal. These large tumors spread alongtissue planes but did not invade or destroy normaltissues.

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Volume 16Number 3

Reports 257

Fig. 1. Flexner-Winteisteiner rosettes seen on intraocular growth of fresh retinoblastoma in thenude mouse. Similar structures were seen in the original tumor. (*600.)

Table I. Growth of retinoblastoma in nude mice0

Fresh retinoblastoma(13 tumors)

Retinoblastoma passed fromnu/nu anterior chamber

Y-79 cell line

nu/nuanteriorchamber

41/46

25/32

8/8

nu/nu subcutaneous

Untreated

0/26

0/6

0/4|7/9$

Cvchphosphamide-treated

3/4

nu/+anteriorchamber

3/6 §

2/2 §

3/7 §

"Expressed us number of animals showing tumor growth/number of animals implanted.f 10" tumor cells injected.1101 tumor cells injected,jMicroscopic growth only.

Y-79 retinohlastoma cell line heterotransplantedinto nu/nu mice. The established Y-79 cell linecompletely destroyed the eye in all instances andextended into the orbit, optic nerve, subarachnoidspace, and the brain (Fig. 2). This tumorcharacteristically showed a prominent fibrovascularstroma.

The Y-79 cells showed rapid subcutaneous

growth at the site of injection in the nu/nu micewhen the tumor inoculum contained 10T or morecells (Table I) . In four animals in which fewercells were injected, no subcutaneous growth wasseen.

Retinoblastoma heterotransplanted into the ante-rior chamber of nu/+ immunologically normalmice. Tumor growth was not clinically apparent

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258 Reports Invest. Ophthalmol. Visual Sci.March 1977

Fig. 2. Intracranial extension of tumor derived from Y-79 retinoblastoma cell line. (xl50.)

in the eyes of nu/+ mice injected with eitherfresh tumor or cells from the Y-79 line. Histo-pathologic examination, however, showed residual,viable tumor cells in the anterior segment, withsome infiltration of iris in one instance.

Attempts at tissue culture propagation. Explantsand single cell suspensions of all 13 fresh retino-blastoma specimens survived for only 1 or 2 weeksin vitro. Cells harvested from the mouse eyes alsosurvived only a short time in vitro.

Discussion. An experimental model of retino-blastoma using the immune-deficient nude mousehas been established. Two patterns of growthwere observed. Tumor from freshly enucleatedeyes spread diffusely throughout the ocularcavities when injected into the anterior chamberof nude mice. Cells from the established Y-79retinoblastoma cell line, which retain the mor-phologic characteristics of retinoblastoma,8 whensimilarly injected, showed a much more invasivegrowth pattern. The basis for the observed dif-ference in growth pattern between fresh and cul-tured tumor is not well understood but mayreflect the observation that tumor cells in cellculture become less differentiated and moreanaplastic than the cells of the original tumor.

The growth of fresh surgical specimens ofretinoblastoma in the eye of the nude mouse was

consistent; the only retinoblastoma tumor cellswhich did not grow intraocularly were derivedfrom a tumor that had received 3,500 rads ofradiation several months before enucleation. Rela-tively large amounts of fresh tumor could begrown in subcutaneous sites in the nude mousebut only when their immunologic defenses werefurther impaired by treatment with cyclophos-phamide.

The nude mouse provides an in vitro systemfor maintaining retinoblastoma for relatively longperiods through serial transplantation. This systemwill make available relatively large amounts oftumor, suitable for biochemical assays such asthose for lactic acid dehydrogenase (which someinvestigators have found to be elevated in theaqueous humor of eyes with retinoblastoma") orcatecholamines or their breakdown products.Moreover, this system provides a promising in vivomodel for studying tumor repsonse to chemo-therapy.

The authors wish to thank Doctors Robert M.Ellsworth, F. David Kitchin, and Robert A. Goodfor making this research possible.

From the Memorial Sloan-Kettering CancerCenter, New York; the Department of Ophthal-mology, Harvard Medical School and the Mas-sachusetts Eye and Ear Infirmary, Boston; and the

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Volume 16Nnviber 3

Reports 259

College of Veterinary Medicine, University ofTennessee, Knoxville. This work was supported bygrants from Fight for Sight, Incorporated, Grant-in-Aid G572; National Institutes of Health grants1 ROI EY CA01616-01 and CA-17404; and theOntario Ministry of Health. Submitted for publica-tion Oct. 14, 1976. Reprint requests: Dr. BrendaL. Gallie, Department of Ophthalmology, Wel-lesley Hospital, 160 Wellesley St. E., Toronto,Ontario, M4Y 1J3.

Key words: retinoblastoma, nude mouse, hetero-transplantation, intraocular tumor, animal models.

REFERENCES1. Albert, D. M., Rabson, A. S., and Dalton,

A. J.: Tissue culture study of human retino-blastoma, INVEST. OPHTHALMOL. 9: 64, 1970.

2. Wortis, H. H.: Immunological studies of nudemice, Contemp. Top. Immunobiol. 3: 243,1972.

3. Giovanella, B. C, Yim, S. O., Morgan, A. C,et al.: Metastases of human melanomas trans-

planted in "nude mice," J. Natl. Cancer Inst.50: 1051, 1973.

4. Schmidt, M., and Good, R. A.: Transplantationof human cancers to nude mice and the effectsof thymus grafts, J. Natl. Cancer Inst. 55: 1,1975.

5. Theodore Sery. Personal communication.6. Greene, H. S. N.: Heterologous transplantation

of human and other mammalian tumors,Science 88: 357, 1938.

7. Kaplan, H. J., Streilen, J. W., and Stevens,T. R.: Transplantation immunology of theanterior chamber of the eye. II. Immuneresponse to allogeneic cells, J. Immunol. 115:805, 1975.

8. Reid, T. W., Albert, D. M., Rabson, A. S.,et al.: Characteristics of an established line ofretinoblastoma, J. Natl. Cancer Inst. 53: 347,1974.

9. Swartz, M., Herbst, R. W., and Goldberg,M. F.: Aqueous humor lactic acid dehydro-genase in retinoblastoma, Am. J. Ophthalmol.78: 612, 1974.

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