Transplantation of Human Tumors intoCortisone-treated Hamsters*

W. BRADFORDPATTERSON,!ROSANNAN. CHUTE,ANDSHELDONC. SOMMERSA

(Cancer Research Institute, New England Deaconess Hospital, Boston 15, Mass.)

The desirability of obtaining living humanmalignant tissue for experimental purposes continues to grow in proportion to our increasingknowledge of the genesis and treatment of cancer.Although several methods for growing humancancer outside of the primary host have been described and utilized (7-10, 13), human tumorswhich can be grown in predictable amounts, within a reasonable time, and by different investigatorsin laboratory animals, have not been reported.However, from the work of Toolan (14) it can besurmised that after 70 years of effort the tools andknowledge are at hand with which permanentand useful heterotransplantation may be accomplished.

The purpose of this paper is to report a humanepidermoid carcinoma, now established in cortisone-treated hamsters, which shows every evidence of possessing sufficient "growth potential"

to continue as a permanently transplantable tumor, growing with sufficient rapidity and certainty to make it of value for experimental use. Thistumor is one of a series of 37 human neoplasmstransplanted in the past 7 months. Histologicalsections are available in 33 of these cases, verifying "survival" in 23, or 70 per cent.

MATERIALS AND METHODSThe hamster cheek pouch technic described by

Lutz et al. (11) was selected for use because of itspromise as a simple method with easily observable results. Golden hamsters of both sexes, 3-5weeks old (40-70 gm.) were used. They were obtained from a local professional breeder. A number of older animals have been employed for particular experiments. They were fed Purina Laboratory Chow and water ad libitum, with lettuceprovided once a week. Cortisone acetate, 1 mg.

* This work was supported by funds from the AmericanCancer Society and United States Atomic Energy CommissionContract AT(SO-1)-901 with the New England Deaconess Hospital.

t Fellow in Cancer Research of the American Cancer Society. Present address: Peter Bent Brigham Hospital, Boston,Mass.

ÃŽPresent address : Massachusetts Memorial Hospitals,Boston, Mass.

Received for publication May 14, 1954.

for animals weighing less than 75 gm. and 2 mg.for those over this weight, was given subcutane-ously 3 times a week starting on the day of implantation. Tumors were obtained fresh from theoperating room and transplanted as rapidly aspossible. The total time required for this processvaried from 30 minutes to 3 hours. Frozen sectionswere prepared in most instances, and care wastaken to select fragments of tumor free of necrosisand gross infection. The tissue was not, as a rule,sterile on receipt, and no effort was made to sterilize it. However, sterile instruments were used,fragments from within the received tissue wereselected, and the recipient site was gently andbriefly cleansed with Zephiran (1:1000). The tissue was kept on a sponge wet with saline whilefragments roughly 1-2 mm. in diameter were cutand placed in No. 18 trocars. Transplantationwas performed with the animal under intraperi-toneal nembutal anesthesia, depositing the tumornear the tip of the everted cheek pouch. Thepouch was handled as little as possible in order tominimize trauma and reaction. Animals havingtumor transplants were anesthetized every 5—7days for inspection of the pouches. Transplantswere made to both cheek pouches. On a few occasions, two or three fragments rather than onehave been placed in each pouch. This has had noapparent effect on the growth or failure of thetransplant.

Transplants have been removed for study byeverting the cheek pouch and excising part or allof the tumor. Sterile technic was used if the animal was not to be sacrificed. Hemostasis was obtained by brief local pressure. Healing of the mucosa is definitely retarded by treatment of thehamster with cortisone, but a small mucosal rentis well tolerated in an otherwise unaffected pouch.Partial removal of the transplant is often followedby local infection, which may subside withouttreatment or become invasive.

A few transplants have been biopsied withinthe 1st week, but those utilized in calculating tumor survival statistics were all at least 12 daysold. Specimens for histological examination wereplaced in Zenker's fixative and stained withhematoxylin-eosin.

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PATTERSONet al.—Transplantation of Human Tumors into Hamsters 657

RESULTSTable 1 lists the results of this series. The usual

transplant shows a two- or threefold increase insize in the 1st week, with slight increase in vas-cularity. The enlargement may be due to any oneof several factors; there may clearly be growth ofthe tumor, with little host reaction except for anenlarging vascular supply; the existing tumorcells, if glandular in function, may produce asizable amount of secretion; a host reaction ofinflammatory exúdate, edema, and hemorrhagemay develop in spite of treatment with cortisone.We feel some confidence now in distinguishingthese reactions grossly. Growing tumors invariably have a healthy pink color, shading to blueaccording to their intrinsic vascularity. Theborders are sharp, and there is no gross evidenceof host reaction. Mucinous secretion has beenrecognizable as such in and around a few transplants. On the other hand, the presence of a muddy and variegated coloring with indistinct bordersindicative of edema is invariably associated witha considerable host reaction. In such transplantsthere may be a large portion of viable tumor centrally, but progressive growth does not occur. Inshort, if the host can react to an appreciable extent, it usually wins. Grossly infected transplantshave dead-white or yellow areas indicating abscess formation. Treatment with cortisone is apparently responsible for elimination of the markedgranulomatous reaction which in earlier experiments (1) was difficult to distinguish from viabletumor.

The growth period may last from 1 to 3 weeks,and in some cases is followed by partial or complete regression during the ensuing days or weeks.A few transplants show initial growth, then stabilization and persistence for long periods of time.One carcinoma of the breast (Fig. 1) has recentlybeen transplanted to a second generation of hamsters after surviving in the original animal for116 days. This tumor showed many of the characteristics of growth reported for anterior chambertransplants (3, 6); that is, it grew initially for aperiod of 4 weeks and then regressed. After aninterval of 6 weeks, during which time anotherbreast carcinoma was implanted and failed togrow in the same pouch, the original nodule slowlyenlarged tenfold. Biopsy at the time of the secondtransplantation showed a healthy, slowly growingcarcinoma (Fig. 2). Microscopically, it is difficultto differentiate this from a tumor which showsonly "persistence," but in view of the substantial

increase of the gross tumor mass, proliferativegrowth has very obviously taken place.

The tumor to be described below is one of theseries showing the striking growth which permits

repetitive, frequent transplantation. Two othertumors, to be discussed in a later paper, one epi-dermoid and one embryonal carcinoma, alsopromise to be permanently transplantable.

"Deac-l."—This tumor originated in the parotid gland of a 74-year-old man. The pathological diagnosis was muco-epidermoid carcinoma,grade II (Fig. 3). It was implanted in both cheekpouches of six hamsters within 30 minutes of itsremoval from the patient, and four of these animals were treated in the standard fashion with

TABLE1SURVIVALOFHUMANTUMORTRANSPLANTSIN

CORTISONE-TREATEDHAMSTERSCases Positive* Negative

1

01o0000

Adenocarcinomas:ParotidLungBreastPancreasSigmoidEndometriumProstate

Carcinoma simplex (breast)

Epidermoid carcinomas:MouthEsophagusLungRenal pelvisVulvaPenis

Mixed tumor of parotidUndifferentiated (scalp)Carcinoid (liver)Seminoma (testis)Small-cell carcinoma (lung)Embryonal carcinoma (testis)Fibrosarcoma (shoulder)LymphocytomaGlioblastoma (brain)Basal-cell carcinomaGiant-cell tumor (spine)

Totals:* Histologie evidence of cell survival and/or growth for a period of at

least lìdays. Microscopic sections demonstrating questionable survival ofonly a few cells were classed as negative.

cortisone. From the start the tumor showed exceptional growth, as evidenced by rapid andmarked increase in size and vascularity of fiveof the transplants. After 14 days in the first-generation animals, four tumors were large enoughto permit another transplantation. This processhas now been repeated to the 16th generation inthe course of 9 months. In view of the markedincrease in total mass of tumor now available,there seems little reason to anticipate a reversalof form and loss of the tumor.

These transplants have an initial latent periodof from 4 to 15 days, followed by an increase inthe vascular supply to the tumor and then steadyenlargement. The tumor maintains a healthypink color, a firm consistency, and a well-vascu-

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658 Cancer Research

larized surface. No detailed studies on growthrates have as yet been carried out, but in 3 weeksthe successful transplants of this tumor reach anaverage diameter of 0.4-0.7 cm., and at 6 weeksseveral tumors have weighed 0.3-0.5 gm. (A fewexperiments have been done which indicate that,with multiple initial fragments, resultant tumorsare proportionately larger.) After the tumors havegrown for 3-6 weeks, central liquefaction and degeneration are often evident. In several tumorswhich have been allowed to remain for periods upto 50 days, this degeneration has been followedby ulcérationthrough the pouch, with the formation of a lesion resembling many human carcinomas—a central ulcer with a rim of healthy tumor. Some of the transplants have regressed subsequent to the infection and inflammation whichhave followed the ulcération,but others are stillenlarging slowly at 60 days. No métastaseshavebeen observed, and no animal has died as a directresult of the tumor, although local recurrenceshave developed following what was regarded asan adequate excisional biopsy.

All transplants were verified microscopically.Sections of growing tumor showed a healthyepidermoid carcinoma, grade II, with numerousmitoses (Fig. 4). There was often evidence of localinvasion, especially of the overlying mucosa. Amoderate amount of vascular stroma was present,and host inflammatory response, if present at all,included varying degrees of peripheral vasodilatation, edema, and a scant lymphocytic infiltrate.Infected specimens excited a severe inflammatoryexúdate in spite of cortisone, with abscess formation and granulation tissue. Tumors that failed togrow showed degeneration, with fibrosis, mummification, and calcification. There have beenfew changes in the character of the neoplasm during the 9 months. No mucoid element has beenseen since the first transplant, and a number ofrecent transplants have shown an adeno-acan-thomatous character (Fig. 5).

Three animals in recent generations have beengiven no cortisone. Each implant has shown aninitial enlargement, with the edema and discoloration of inflammation, followed by rapid regressionwithin 10 days. Cortisone has also been stoppedon a number of animals bearing established transplants, with rapid résorptionof the tumor nodule.Studies are in progress relative to the optimum

cortisone treatment. Most animals have toleratedthe regimen of 6 mg/week fairly well. A few animals have died with post mortem findings suggestive of congestive heart failure. Animals treatedfor more than a month show generalized debilityand increased deposition of intrascapular brownfat.

Experiments have been carried out on the useof hydrocortone (Compound F), locally, to avoidthe systemic effects of cortisone. While this treatment will depress the host inflammatory responseand permit some tumor growth, it does not support growth adequately.

Transplants of the Deac-1 tumor have beenmade into 50 cortisone-treated hamsters. In thisgroup, microscopic survival has been noted in 84pouches of 45 animals, and active growth in 80of these. The tumor is at present growing in eighteen hamsters in this laboratory in varying stages.It is also being successfully grown and utilized inthree other laboratories in the Boston area. Transplantation from one animal to the next has beenaccomplished at varying times between 10 and52 days. Most transplants are made between 14and 21 days—when the tumors are sizable, anddegeneration is at a minimum.

DISCUSSIONThe use of cortisone to eliminate the initial

defense of an animal host to a foreign tumor, asfirst described by Toolan, has removed one of themost formidable obstacles to successful growthof tumors in animals. The epidermoid carcinomaand fibrosarcoma reported by Toolan and the onereported here are vigorous tumors, selected fortuitously, which seem to possess sufficient growthpotential to cross the species barrier, with the aidof cortisone, and grow progressively. In reviewingprevious attempts to transplant tumors heter-ologously, one is struck by the occasional widevariation in survival time reported by differentinvestigators and with different methods. Thisis suggestive of a "spectrum" of tumors, which

may be transplanted permanently to animals oneby one as our knowledge increases.

It is our conviction that there are many reasonsfor failures with heterologous transplantation ofmalignant tumors. Spontaneous tumors in humans and heterologous transplants in animalsboth excite varying responses, indicative of mul-

Fio. 1.—Carcinoma simplex of breast. X 250.Pio. t.—Same tumor growing in hamster 116 days after

transplantation. X 250.FIG. 3.—Muco-epidennoid carcinoma of parotid gland, the

Deac-1 tumor. X 250.

Fio. 4.—Deac-1, third-generation transplant in hamster.X250.

FIG. 5.—Deac-1, ninth-generation transplant showingadeno-acanthomatous tendency, with hamster cheek pouch onright. X 125.

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PATTERSONet al.—Transplantation of Human Tumors into Hamsters 659

tiple lines of host defense. The stromal responsedetermines the adequacy of the blood supply.Inflammatory exúdate and allergic responses mayregulate survival and spread. Endocrine factorshave been shown to be significant in the growthrate of both human and animal tumors. The nutritional state of the animal has long been knownto be related to the survival of transplanted tumors. Eichwald and others (5) have shown theimportance of a number of host and graft factorsand feel there are many others. It seems reasonable to compare our present-day technics for heter-ologous transplantation with the initial attemptsto culture bacteria. The most hardy varieties thengrew in the simplest media, but the culturing ofmore sensitive types of micro-organisms awaitedthe development of either more fortunate or morecarefully controlled substrates. There are severalways in which the less hardy tumors might beencouraged to grow; for example, the anteriorchamber transplants so intensively investigatedby Greene circumvent the species barrier becausethe particular medium allows the transplant aninitial period of recovery and adjustment beforevascularization brings host defenses to bear (4).These transplants have certainly been successfulin Greene's hands, but the numerous reports of

relative failures with this method (3, 12, 15) demonstrate that, while this technic may permitinitial transplantation of a wide variety of humanneoplasms, it will not provide investigators witheasily obtained, dependable sources of humantumors in animal hosts. It is pertinent to comparethe 3$ years, reported by Dobyns (2), requiredfor eight passages of a thyroid anterior chambertransplant, with the 3$ months required for eightpassages of the Deac-1 tumor reported here. It ispossible that certain tumors might respond well iftransplanted initially into the anterior chamber,with subsequent transfer to the hamster or rat,where technic may be less crucial and growth morerapid.

To us, the possibilities of early clinical application of this technic are as attractive as the searchfor other transplantable tumors. Although it mayseem overly optimistic to consider ways and meansof destroying the Deac-1 tumor in its animal hostsat this early date, we are initiating such studies,recognizing that the opportunity to test the correlation between tumor response in animal andhuman hosts will probably present itself in theforeseeable future. Should this patient, who is nowclinically free of disease, develop a recurrence,information on the susceptibility of his cancer toirradiation, endocrine changes, and antitumordrugs would be of immediate interest.

SUMMARY1. Transplantation of 33 human tumors into

the cheek pouch of cortisone-treated hamstershas resulted in the "survival" of 70 per cent of

the tumors for periods exceeding 12 days.2. One epidermoid carcinoma, now called

"Deac-1," holds promise of being permanently

transplantable. It has now been carried throughsixteen generations of hamsters in a 9-monthperiod.

3. Factors concerned in the eventual transplantation of other human neoplasms are brieflydiscussed, and possibilities for immediate clinicalapplication of experimental data are suggested.

REFERENCES1. CHUTE,R. N.; SOMMEES,S. C.; and WABREN,S. Hétéro-

transplantation of Human Cancer. II. Hamster CheekPouch. Cancer Research, 12:912-14, 1952.

2. DOBYNS,B. M., and LENNON,B. Changes in théHistologi-cal Pattern of Human Thyroid Cancer after Serial Transplantation in the Anterior Chamber of Eyes of GuineaPigs. Cancer, 6:45-51,1952.

3. EICHWALD,E. J. Heterologous Transplantation of Cancerof Childhood. Cancer Research, 8:273-77,1948.

4. EICHWALD,E. J., and CHANO,H. Y. The Significance ofthe Anterior Chamber in Tumor Transplantation. CancerResearch, 11:811-13, 1951.

5. EICHWALD,E. J.; CHANO,H. Y.; LANDA,M.; EVANS,R. G. Host and Extraneous Factors in Heterologous Tumor Transplantation. J. Nat. Cancer Inst., 2:17-81, 1950.

6. GREENE,H. S. N. Heterologous Transplantation of a Human Fibrosarcoma. Cancer Research, 2:649-54,1942.

7. . The Significance of the HeterotransplanUbility ofHuman Cancer. Cancer, 5:1952.

8. GREENE,H. S. N., and SAXTON,J. A., JR. Uterine Adenomata in the Rabbit. I. Clinical History, Pathology, andPreliminary Transplantation Experiments. J. Exper. Med.,67:691-707, 1938.

9. KAUFMAN,N.; PRIETO, L. C., JR.; MASON,E. J.; andKINNET,T. D. The Survival of Human Tumor Tissue onthe Chorio-allantoic Membrane of the Chick Embryo.Am. J. Path., 28:561-62, 1952.

10. LEMON,H. M.; Ltrrz, B. R.; POPE, R.; PARSONS,L.;HANDLER,A. H.; and PATT,D. I. Survival and Growth ofHuman Tissues Transplanted to Hamster Cheek Pouch.Science, 115:461-64, 1952.

11. LUTZ,B. R.; FULTON,G. P.; PATT,D. I.; and HANDLER,A. H. The Growth Rate of Tumor Transplants in theCheek Pouch of the Hamster. Cancer Research, 10:231-82, 1950.

12. Srn iLUM:. J. A.; SNELL,A. J.; and FAVATA,B. V. Heterologous Ocular Transplantation as a Practical Test for Cancer. Cancer, 2:480-90, 1949.

13. TOOLAN,H. W. Successful Subcutaneous Growth andTransplantation of Human Tumors in X-irradiated Laboratory Animals. Proc. Soc. Exper. Biol. & Med., 77:572-78, 1951.

14. . The Growth of Human Tumors in Cortisone-treated Laboratory Animals. The Possibility of Obtaining Permanently Transplantable Human Tumors. Cancer Research, 13:389-94, 1953.

15. TOWBIN,A. The Heterologous Transplantation of HumanTumors. Cancer Research, 11:716-22, 1051.

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1954;14:656-659. Cancer Res   W. Bradford Patterson, Rosanna N. Chute and Sheldon C. Sommers  HamstersTransplantation of Human Tumors into Cortisone-treated

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