Propagation of Human Tumors in Antithymocyte Serum-Treated Mice 1,2

Paul Arnstein,3 Dee O. N. Taylor,4 Waiter A. Nelson-Rees,5 Robert J. Huebner,3 and Edwin H. Lennette 4,6

SUMMARY-NIH Swiss mice were treated with antithymocyte serum (ATS) to diminish their cell-mediated immune response; subse­quently, these mice proved satisfactory sub­strates for the propagation of a variety of human malignant tumors. Among 18 cultured tumor lines tested which exhibited malignant in vitro growth patterns, 13 were transplant­able and produced appropriate neoplasms. Carcinomas and sarcomas were most con­sistently initiated by subcutaneous implanta­tion of 2X106 trypsin-dispersed cells from cell cultures. Lymphomatous neoplasms were most consistently transplantable intracere­brally, with 2X105 suspension culture cells. Human cell cultures, indistinguishable from input cells, were reestablished from all tumors propagated on the ATS-treated (ATST) mice. Of 18 other cultures with benign in vitro growth patterns, none were transplantable. Direct transplantation of surgically excised human tumors received from the operating room proved less successful and only 4 takes were accomplished in 19 attempts. The ATST mouse system should be adaptable for testing chemi­cal, immunologic, and physical influences directly on specified human tumors, without actually involving human subjects, since transplants of representative tumor types (carcinomas, sarcomas, lymphomas) can be consistently produced by inoculation of ap­propriate malignant culture cells. The replica­tion of human neoplasms in a mouse host may also provide an environment for activa­tion and detection of C-type tumor viruses.­J Natl Cancer Inst 52: 71-84, 1974.

NUMEROUS PUBLISHED REPORTS describe the transplantation of human neoplasms to newborn and older animals of many species (1-28). Hamsters, monkeys, mice, cats, guinea pigs, and rats have been tested by various investigators.

We wished to test a simple method of hetero­transplantation to immunologic ally deficient hosts, using laboratory animals that are easily bred and cared for. We modified the technique described by Stanbridge and Perkins (16) for the transplantation of HeLa cells into laboratory mice treated with anti­lymphocyte serum (ALS), but elected to use anti­thymocyte serum (ATS) which reportedly was more uniform in potency when purchased commercially (29).

The study, designed to amplify the data secured by earlier investigators and to use their techniques with some modifications, had the following aims:

1) Determine the consistency with which malignant cells or tumors can be transplanted in antithymocyte serum-treated (ATST) mice and their litter mates receiving no ATS.

2) Study the characteristics of human tumors transplanted to the mice. Compare progression and transplantability of representative tumor types (carcinomas, sarcomas, lymphomas).

3) Demonstrate reestablishment in vitro of the original "input" human cells, noting morphologic or karyologic altera­tion post transplant (if any).

Subsequent to the completion of these steps, we planned to examine transplanted tumors and re­established cultures for tumor virus activity, as was described by McAllister et al. (30) who, after trans­plantation of human sarcoma to fetal kittens, isolated a C-type virus (RDl14) in the reestablished cell line. Results of our viral studies will be reported separately.

MATERIALS AND METHODS Mice.-Breeding stock of NIH Swiss albino mice

(NIH SW) supplied by the National Institutes of Health, Laboratory Aids Branch, was used to establish a closed, random breeding colony in one of our facilities. NIH SW mice were chosen because they have a very low incidence of spontaneous neoplasia and have not yet yielded endogenous, C-type, RNA tumor viruses (31). The mice used to propagate tumors were 5-21 days old when inoculated. For each test, whole litters were usually used, thus resulting in approximately equal distribution between the sexes.

A TS.-The same lot of ATS (Microbiological As­sociates, Inc., #57-110. Courtesy Dennis Reeder), produced in rabbits by a modified Levey-Medawar method (32), was used throughout. Activity of the serum was tested by the manufacturer and the speci­fications furnished were as follows:

Specific cytotoxicity titer (vs. mouse thymocyte) 1: 12,800 Skin transplant (unrelated) survival:

ATS-treated 30.3 days average Controls lOA days average

1 Received April 30, 1973; accepted September 7, 1973. 2 Supported in part by Public Health Service contract

NOI-CP-43209 and E73-20l-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, Bethesda, Md. 20014.

3 Viral Carcinogenesis Branch, National Cancer Institute, National Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. Dr. Arnstein was on PHS assign men t to the California State Department of Health, Berkeley, Calif. 94704.

4 Viral and Rickettsial Disease Laboratory, California State Department of Health, Berkeley, Calif. 94704.

5 University of California, School of Public Health, Naval Biomedical Research Laboratory, Oakland, Calif. 94625.

6 We thank the following for their excellent assistance: Miss Jean Chin and Mrs. Maureen Hanahoe of the Viral and Rickettsial Disease Laboratory, State of California Department of Health, who propagated, standardized, and evaluated cultures before and after tumor induction; Mrs. Paula K. Hawthorne and Mr. Jack F. Weaver for karyology, and Mr. Robert B. Owens for the selection and preparation of many of the successfully implanted cell cultures from the collection at Naval Biomedical Research Laboratory.

JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 52, NO. I, JANUARY 1974 71

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72 ARNSTEIN ET AL.

Intraperitoneal doses of 0.1-0.25 m1/mouse were administered in proportion to age: 0.1 ml, up to 1 week of age; 0.15 ml, 1-2 weeks; 0.2 m1, 2-3 weeks; and 0.25 ml when over 3 weeks. The appropriate dose of ATS was injected on days -1, 0, + 1, +3, and subsequently twice weekly, terminating day 21-40 (day O=injection of tumor material or cells).

Fresh tumors.-Fresh human tumor tissue was ob­tained after operations in local hospitals and from collaborating colleagues in Los Angeles.7 For rapid collection of suitable tumor specimens, containers with culture medium [Eagle's minimal essential me­dium (EMEM) with 5% fetal bovine serum, penicillin, streptomycin, chlortetracycline, and fungizoneJ were supplied to the pathology staff of each hospital. Tumors were trimmed aseptically and immersed into refrigerated culture medium immediately after sur­gery; subsequently, they were processed within 48 hours (usually <24 hours) after surgery as follows: A 5-10% suspension of finely minced tissue in the medium was prepared for cultivation in vitro and simultaneous mouse inoculation. Six mice pretreated with ATS were usually inoculated/tumor specimen; each received 0.5 ml of the suspension subcutaneously (sc). Some tumor suspensions were also inoculated intracerebrally (ic), 0.03 ml/mouse. An equal number of untreated controls was inoculated if sufficient tumor material was available. At the start of these experi­ments, preoperative or surgical diagnosis was con­sidered sufficient to evaluate results. Subsequently, however, a portion of the specimen received in the laboratory was reexamined histologically by one of us (D.T.) to be certain that the tissue included the neoplasm.

Cell cultures.-Vigorous cultures derived from a variety of neoplasms and a small number of normal fetal cells were used in this series. 8 Most cell cultures were adapted to and grown in EMEM (with Earle's salts), supplemented with 10% fetal bovine serum and antibiotics (penicillin, streptomycin, fungizone), at 37° C, either in 8-ounce prescription flasks or in 250-cc plastic culture flasks. Cell morphology in vitro was re­corded while cultures were being expanded to produce sufficient cells to inoculate 6-12 mice and subsequently maintain the culture in vitro.

The optimal sc dose was determined in preliminary tests using HeLa cells and nOl mal diploid cells in graded doses on our mice and with the specific lot of ATS to be subsequently used for all transplants. When fewer than 1 million cells were given, HeLa tumor growth was slow and ceased relatively early. If over 5 million cells were given, normal diploid lines produced large inflammatory nodules difficult to distinguish in vivo from tumors during the first 10 days post transplantation. The groups receiving 2 and 2.5 million cells seemed most suitable. For convenience and technical reasons (doses in excess of 500,000 cells ic frequently resulted in immediate postinoculation death), the ic cell inoculum used throughout was one-tenth the number of cells (and one-tenth the volume) of the sc inoculum, i.e., 2-2.5 X 105 viable cells. 9

For inoculation, monolayer cells were dispersed in trypsin-Versene, resuspended in EMEM, and counted as well as checked for viability (by trypan blue exclusion) in a hemocytometer. Cells growing in suspension were concentrated by light centri­fugation; subsequent vigorous pipetting in a reduced amount of medium re suspended single cells, which could be accurately counted and checked for viability as above.

The number of cells was then adjusted to 8-10 million/ml and mice were given injections of appro­priate volumes for sc or ic implantation. Each mouse was incoculated with 2-2.5 million cells sc and/or 200,000-250,000 ic. Identical doses of each specimen were injected into nonimmunosuppressed controls, in parallel with the ATST mice.

All cell cultures were initially inoculated into 4-6 ATST mice and an equal number of untreated litter­mates. Most cultures were inoculated twice, with 1-2 months between tests.

A few tests were performed with 3 parallel cohorts: the ATST, the untreated controls, and normal rabbit serum (NRS)-treated controls. NRS was administered in the same manner as ATS.

Tumors propagated in mice.-Inoculated mice were examined at least twice weekly and daily after measurable tumors or neurologic abnormalities ap­peared. Tumors and brains of inoculated mice were usually harvested by day 21 post inoculation. After chloroform euthanasia and dissection of the mouse, gross observations were noted, tissues excised asepti­cally, and portions fixed in formalin for histopatho­logic diagnosis. Tissues that appeared neoplastic were immersed in EMEM with 2 times the usual concentration of antibiotics and allowed to infuse overnight in the refrigerator. The following day they were prepared for direct transplantation to addi tional mice and for in vi tro cuI tu re : Each neoplasm was finely minced until a 5-10% suspension could readily be taken up with a hypodermic syringe fitted with a 20- or 25-gauge needle. For direct transplantation, ATST and untreated control mice

7 We thank the following physicians for their generous collaboration in supplying fresh tumor material: Dr. Bernard Horn, Kaiser Hospital, Vallejo, Calif.; Dr. William J. Wede­meyer, Herrick Hospital, Berkeley, Calif.; Dr. Barbara von Schmidt, Children's Hospital, Oakland, Calif.; Dr. Murray Gardner and Dr. Brian Henderson, University of Southern California Medical Center, Los Angeles, Calif.

S We gratefully acknowledge the contribution of cell lines for these studies by: Dr. John Riggs, California State Depart­ment of Health, Berkeley, Calif.; Dr. Robert McAllister, Children's Hospital of Los Angeles, Calif.; Dr. Donald Morton, University of California at Los Angeles; Dr. Richard Lerner, Scripps Research Foundation, La Jolla, Calif.; Dr. Etienne Lasfargues, Institute for Medical Research, Camden, N.J.; Dr. John Sykes, Southern California Cancer Center, Los Angeles; Dr. Michael Brennan, Michigan Cancer Foundation, Detroit, Mich.; Dr. Roland Patillo, Marquette School of Medicine, Milwaukee, Wis.; Dr. Lloyd Old, Sloan-Kettering Institute for Cancer Research, New York, N.Y.; Dr. Ward Peterson, Jr., Child Research Center of Michigan, Detroit; Dr. Nelly Auersperg, Cancer Research Centre, University of British Columbia, Vancouver, B.C., Canada.

9 In a current series of transplants, inoculation of 3.5 million viable cells sc and/or 350,000 ic is producing slightly more consistent results.

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HUMAN TUMORS PROPAGATED IN ATS-TREATED MICE 73

were inoculated with 0.3 ml of the suspension sc (20-gauge needle aspirate) and/or 0.03 ml ic (25-gauge needle aspirate). In each transplant generation, litters were divided into 4-6 ATST and a similar number of control mice.

On the day of the transplant, in vitro cultures of each tumor were also established from the minced fragments and observed daily for viable cell outgrowth.

Medium was partly replaced 1-2 times weekly, as needed. When adequate outgrowth resembling the "input" cells was observed, cultures were dis­persed by trypsin-Verse ne and then propagated by usual tissue culture methods. All cells derived from the tumors were examined karyologically and results were compared to those of the input cells.

These procedures were repeated for 3 transplant generations with each transplantable tumor line listed in tables 3, 4, and 5.

Karyology.-Chromosome observations were made on early passage cultures of tumors recovered from ATST mice and results were compared with those on cells of cultures before inoculation. Cells in metaphase were prepared for study by the air-dry method (33), with a slight modification, and stained in Giemsa. Presence or absence of Y chromosomes was studied after fluorescent staining (34).

Isozyme.-The mobility pattern of glucose-6-phos­phate dehydrogenase (G6PD) was determined by Dr. W. D. Peterson, Jr. (35).

Histopathology.-All tissue samples were fixed in 10% formalin buffered with 2% sodium acetate. After fixation, they were embedded in paraffin, sectioned at 5fJ., stained with hematoxylin and eosin, and examined microscopically.

RESULTS Tumors Received From the Operating Room

The surgical specimens reexamined histopatho­logically in this laboratory were surprisingly often devoid of neoplastic tissues. This may have been due to wide excisions necessary in cancer surgery and the consequent difficult task of recognizing the margins of malignancy. About half the specimens contained no neoplastic cells.

Results of direct transplantation are presented in table 1. Only 1 tumor, a transitional cell carcinoma of the bladder (HTI051), was successfully transplant­able by direct serial passage in the ATST juvenile mice. This tumor produced slow-growing carcinomas resembling the original human neoplasm (figs. 1, 2) for 7 transplant generations; 4-6 ATST mice were used per generation and at least three-fourths devel­oped tumors. Untreated controls receiving identical inocula developed no tumors or persistent nodules. After removal from tumor-bearing animals, the carcinoma cells could not be grown in vitro; instead mouse fibroblasts were repeatedly isolated [the culture

TABLE 1.-Direct transplants, tissues received from tumor surgery

Number Preoperative diagnosis Results of inoculation into ATS-treated mice*

(A) Positive (turn or growth)

HT873t HT887t H51

HT1051 t

Gross observations

Carcinoma, colon ___ - - - - - - - - -lvery slow-growing, encapsulated Carcinoma, colon___ _ _ _ _ _ _ _ _ _ tumors persisting 21 days. t Carcinoma, colon ___________ _

Carcinoma, bladder _ _ _ _ _ _ _ _ _ _ Slow-growing, progressi ve tumors transplanted serially 7 passages. §

Histopathology

Carcinomas composed of columnar epithelial cells containing lipid vac­uoles. Acinar structures observed (fig. 3).

Thick layers of pleomorphic epithelial cells; some areas suggestive of squamous metaplasia as seen in transitional cell carcinoma (fig. 2).

CB) Negative (no growth) HT889t HT776 HT829 HT775 HT738t HT874 H49 H50 H58 H53 H54 HTI045 HT1049 HTlO71 H59

Fi brosarcoma. Carcinoma, bronchus. CarCinoma, breast. Carcinoma, endometrium. Carcinoma, gastric. Carcinoma, colon. Carcinoma, colon. Carcinoma, colon. Carcinoma, rectum. Carcinoma, metastatic. Carcinoma, breast. Osteosarcoma. Hodgkin's disease. Hodgkin's disease. Lymphoma.

*Tumor dose: approximately 0.3 ml of 5% tumor suspension, mechanically dispersed, sc. tIn vitro cultures of these tumors were also inoculated and are listed in table 2, group A. tNot transplantable serially and would not grow in vitro. §No growth of tumor cells in vitro.

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74 ARNSTEIN ET AL.

established from the original tumor in vitro resembled a fibroblastoid cell monolayer; 2 X 106 of these cells did not produce tumors (table 2)]. Three other neoplasms, colon carcinomas HT873, HT887, and H51, reproduced small tumors composed of mucus­secreting colon carcinoma cells in the first transplant passage (fig. 3). The tumors grew very slowly for about 2 weeks, reaching 3--4 mm. These, however, failed to transplant to additional mice or to gr ow tumor cells in vitro. Cells from these tumors, which were initially established in vitro, also failed to pro­duce tumors upon inoculation (table 2).

Cell Cultures Derived From Tumors Many human tumor-derived cultures were avail­

able in our laboratories; more were obtained through the generous assistance of other cancer research groups. Although these cultures were derived from neoplastic specimens with widely ranging diagnoses, their appearance in vitro (morphology) could be classified into 3 general categories: a) "henign­appearing"-flat, contact-inhibited, fibroblastoid monolayers, with parallel alignment of cells (fig. 5); b) "malignant-appearing"-pleomorphic, piled-up non-contact-inhibited cultures with consistent pecu­liarities (vide infra) (figs. 7, 8); and c) "suspension­cells"-floating, unattached clusters of spherical cells (fig. 6, 9f) often containing 10-30% dead cells.

"Benign-appearing" monolayers None of the cultures in this group produced tumors

in ATST mice. Small reactive nodules were often present for a few days after inoculation in ATST and control mice, but disappeared in 5-10 days. Cells

TABLE 2.-Human tumor-derived cells: Monolayers, in vitro morphology "benign"

Number Derivation

Group (A)t

HT738 Carcinoma, gastric ___ HT889 Fibrosarcoma _______ HT1051 Carcinoma, bladder __ HT874 Carcinoma, colon ____ HT873 Carcinoma, colon ____ HT887 Carcinoma, colon ____

Group (BH HT680 Carcinoma, kidney __ HS293T Adenocarcinoma,

cecum. HT871 Carcinoma, prostate_ HS432 Malignant melanoma_ HS444 Metastatic seminoma_ MT Osteosarcoma _______ TE417 Osteosarcoma _______ MBA8387 Fibrosarcoma ______

·Injected with 2XI06 viable cells sc.

Results Passage in A TS-

No. tested treated mice*

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tested are listed in table 2. The first 6 (Group A) represent in vitro outgrowths from tumors in table 1. The other 8 cultures (Group B) were derived from neoplasms received during the past 4 years, dimethyl sulfoxide (DMSO) preserved and frozen at early passages. They were reestablished in culture and their in vitro appearance was recorded; they were then harvested and inoculated in ATST and control mice.

"Malignant-appearing" attached cells Cells were classified as morphologically malignant

if they exhibited one or more of the following traits while actively replicating in vitro: a) polymorphism of cells, including variations of shape, size, and density even though well-adapted to growth in vitro (figs. 7, 8), b) tendency to heap, cord, form multi­layered sheets, or form dense masses but remain viable and undetached from flask surface (fig. 7), c) bizarre cell population, including vacuolated, multinucleated and giant cells (fig. 8), d) highly refractile, "glassy" cells. Additional criteria better observed in stained preparations were: e) high nuclear to cytoplasmic ratio (fig. 9a vs. 9b-9f), j) prominent and multiple nucleoli (figs. 9b-9f) , g) bizarre division figures (figs. 9c, ge), h) irregularly shaped nuclei (figs. 9c-9f), i) multinucleate cells (figs. 9d, ge).

The cultures meeting at least one of these criteria are listed in tables 3 and 4, with results of inoculation into ATST mice. Actually all traits listed above were found to some extent in most of the cell cultures judged "malignant." As shown in tables 3 and 4, 10 of 12 such cultures transplanted successfully to ATST mice, and produced tumors having an architecture similar to the original human tumor from which each had been derived. Transplanted sc tumors, harvested and reestablished in vitro, resulted in cultures con­taining 90-100% human tumor cells in the first set of flasks planted. The mouse fibroblasts, if present in the first re cultured passage, disappeared by the second, so that only human karyotypes were seen after subculture.

The observed sequence after sc implantation of tumorigenic cell cultures or minced tumor suspension was a transitory postinocula tion "bleb," followed by a 1- to 3-mm nodule which sometimes persisted 5-10 days. The actual tumor growth became detect­able about days 5-7, reaching maximum size by day 25; subsequently, most tumors remained stationary in size despite continued ATS administration. The only culture behaving differently was BT20, a breast carcinoma line which grew more slowly; small neoplasms were detected first on the 22d day after inoculation; reisolation of the "input" human cells was accomplished from carcinomas reaching peak size on the 48th day.

The ATST members of each litter inoculated sc with the transplantable cells or tumors almost always developed palpable lesions simultaneously or within 1-2 days of each other; very rarely, 1-2 mice in a cohort of 6 failed to develop tumors. The consistency of "takes" and the rate oftumor growth were highest

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in mice 5-10 days old. In older mice, tumors often grew slowly and sometimes became necrotic, ap­parently due to scar formation. In mice <5 days old, the delicacy of skin and other body structures caused either excessive loss of tumor implant or trau­matic deaths. Cannibalism by the dam was rare in the NIH SW. Untreated mice or those treated with NRS and receiving tumor inoculations identical to the ATST did not develop significant lesions (except the postinoculation reactions noted previously, dis­appearing in 5-10 days).

The gross appearance of sc carcinomas and sar­comas on the ATST mice was quite similar: They were glistening white, very firm, and usually covered with a vascular capsule. There was no tendency to invade the abdominal cavity. The central area of each neoplasm looked firm and viable for the first 25 days; the sarcomas remained so as long as ATS treatment was continued. Carcinomas tended to undergo central necrosis after 25 days and also to ulcerate through the skin, possibly as a result of the very rapid increase in tissue mass which could not be adequately nourished by the available blood supply. The input tumor cells could, however, be recovered from the peripheral, firm areas of such centrally cavitated lesions. Interestingly, the 3 sarcoma cultures (RD, T174, and SA4TxSl) grew luxuriantly when injected sc or ic; the latter route resulted in death of ATST mice in 10-14 days post inoculation. Subcutaneous sarcomas reached a max­imum of approximately 10 mm in diameter. By the same routes, carcinomas attained a larger size in the subcutis (up to 25 mm in diameter if undisturbed) but with 2 exceptions failed to grow or produce neoplasms ic.10 Five carcinoma and 3 sarcoma cultures assembled tumors of sufficiently large size for detection and subsequent reculture. All 8 yielded tissue cultures morphologically identical to the input cultures described in tables 3 and 4.

Subcutaneous neoplasms were harvested from mice 20-40 days after cell inoculation; ic neoplasms were recultured when mice showed spastic or paralytic signs, usually by day 15. Results are recorded in tables 3 and 4.

To summate, none (0/12) of the attached cell cultures exhibiting malignant traits in vitro induced neoplasms in untreated mice; 10 of 12 reproduced tumors in ATST mice, histologic ally compatible with the diagnosis of the original human neoplasm from which the culture was initiated; 5 of 10 were trans­plantable in the ATST mice by either the ic or the sc route, the remainder by the sc only .

"Suspension cells" All cultures in this group had been adapted at least

6 months to stationary suspension growth in vitro. Included in this series were 5 derived from lymphoid

10 BeWo/cc and C4 Cl I: The former is derived from chorio­carcinoma in a newborn and produced massive brain invasion and rapid death when inoculated ic, and large cystic tumors sc. C4 Cl I is the more undifferentiated of 2 distinct clones established from a specimen of cervical carcinoma; it formed carcinomas in the brain and the subcutis.

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HUMAN TUMORS PROPAGATED IN ATS-TREATED MICE 77

neoplasms and 1, the LEVIn, reportedly from pleural effusion of a mammary carcinoma patient. When viewed microscopically, they resembled each other and can best be described as clustering lymphoblastoid cell suspensions. Clusters of the LEVIII cells were the largest, where undisturbed flasks contained spheres up to 2 mm in diameter. The other suspensions grew in smaller clusters, about one-quarter to one-half mm in diameter. Results of the inoculation of these 6 suspension cultures in the ATST NIH SW mice are summarized in table 5. The LEVIII culture was the most consistently tumorigenic. All 3 neoplasm­producing suspension cultures (including LEVIII) replicated much more rapidly and in greater propor­tion of inoculated animals ic than in the subcutis. Brains of mice which exhibited spastic or paralytic behavior were harvested, usually about 15 days after ic inoculation. Pure suspension cultures morpho­logically identical to input cells described in table 5 were reestablished in vitro from brains inoculated with the 3 transplantable lines. No sc or ic takes occurred in mice not receiving ATS. Thus, of the 6 suspension cultures transplanted: N one produced neoplasms in untreated mice. Half (3 of 6) consistently produced ic neoplasms (and occasionally sc neo­plasms) in ATST mice.

Cell Cultures From Non-Neoplastic Tissues Two human embryo-derived diploid cultures (at

passages 14 and 20) inoculated sc into ATST mice, using 2 million viable cells per mouse, produced small 1- to 3-mm nodules that persisted for 6-10 days and could not be located thereafter. This agrees with results reported by Stanbridge (16) that such cells did not produce neoplasms.

Two million high-passage (60-100) feline embryo cells and canine (beagle) embryo cells were also inoculated, with results identical to those obtained with the human diploid lines.

Histopathology Most transplanted tumors in mice had recognizable

similarities to the types of human neoplasms from which they were derived (tables 1, 3, 4, and 5). Exceptions were tumors derived from rhabdomyo­sarcoma T174 and mammary carcinoma LEVIII.

The tumor transplants of rhabdomyosarcoma T174 consisted of pleomorphic spindle cells; strap cells or large bizarre cells resembling myoblasts, as seen in typical rhabdomyosarcomas, were virtually non­existent. Thus, rather than rhabdomyosarcomas, these neoplasms resembled fibrosarcomas.

The LEVIII suspension culture, reportedly derived from pleural effusion of a woman with mammary carcinoma, had an in vitro growth pattern resembling lymphoblastoid cells; carcinoma-like cells are not apparent in the culture. Stained sections of intra­cranial neoplasms resulting from transplantation of LEVIII cells resembled those of the other 2 trans­plantable suspension cell lines. The most noteworthy features in all were invasive infiltrates along meningeal surfaces and filling ventricular spaces; infiltration of the brain parenchyma along perivascular spaces

was also observed (fig. 4). The tumor cells were rounded, with a moderate amount of acidophylic cytoplasm. The nuclei were usually round or oval, but sometimes indented. In most nuclei a single prominent nucleolus was evident. These lesions are best summarized as resembling lymphomatous infiltrates.

Identity of and Chromosome Observations on Tumor Cells

Karyologic and isozyme determinations served 2 purposes. Since most cell lines employed had been grown for long periods in culture, we sought, first, to determine whether each cell line was of human origin and whether chromosomal sex and G 6PD mobility pattern conformed to the sex and racial origin of the donor. Second, where tumors were in­duced in ATST mice, we determined whether they consisted of mouse or human cells, and in the case of the latter, whether they varied grossly from the cells inoculated (table 6).

With 3 exceptions, the Y-chromosome fluorescence pattern and G 6PD mobility coincided with that expected for the sex and race of the donor. The exceptions were T174, SA4TxSI and LEVIII. The lack of a Y chromosome in the first 2 cultures, although derived from males, could be explained by the loss of this chromosome during long-term cultiva­tion and conforms to other examples of such "epiphenomena" as cited by Peterson et al. (50). The appearance of an A-band mobility pattern in SA4TxSI cells, although derived from a Caucasian, is more difficult to explain, but has precedent in cell line Ma160 (51) where long-term cultivated cells of a Caucasian male whose own blood pattern is B (52) showed an A-type pattern and did not reveal Y -chromosome fluorescence (52, 53). The existence of a Y chromosome in LEVIII (53- 55) cells, which were derived from a female, could be the result of a mosaic or mixoploid condition in the donor (56).

In every instance where transplanted tumors yielded analyzable cultures, the recovered population resembled the inoculated as to presence or absence of Y -chromosome fluorescence, median chromosome number, and presence or absence of other marker chromosomes. In addition, as mentioned earlier, all recovered cultures were also morphologically identical to the input cells as described in tables 3, 4, and 5.

DISCUSSION The low proportion of original human tumors

forming recognizable neoplastic structures on trans­plantation to ATST mice may be attributable to causes too numerous to list in their entirety, but among them are: the low prevalence of tumors rich in actual neoplastic (as opposed to stromal) cellular population; lapse of time between surgery and implantation of the tumors; unknown optimal method of suspending neoplastic cells before implantation; possible previous antitumor treatment in the patient; relatively slow proliferation after transplantation.

Also, the excised tumor mass may contain immune lymphocytes that are ineffective in the patient due to

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HUMAN TUMORS PROPAGATED IN ATS-TREATED MICE 79

TABLE 6.-Chromosomal and G6PD isozyme identity of transplantable human tumor cell lines before implantation and after explantation from A TS T mouse-borne tumors *

Cell line Sex of donor

Racet Y -chromosome fluorescence

Median No.t Marker G6PD§ chromosome

Carcinoma BT20_______________ F Carcinoma ME 180 (Sy-180) _ _ _ _ _ F Carcinoma Det562 _ _ _ _ _ _ _ _ _ _ _ _ _ F Carcinoma C4# L _ _ _ _ _ _ _ _ _ _ _ _ _ _ F Choriocarcinoma Be W 0 _ _ _ _ _ _ _ _ _ M Sarcoma RD#2________________ F Sarcoma TI7L________________ M Sarcoma SA4TxSl (HuSal)_______ M Suspension H37________________ F Suspension LEVIIL_ _ _ _ _ _ _ _ _ _ _ _ F Suspension Raji (SCRF-2) _ _ _ _ _ _ M

C C C C C C C C C C N

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Yes B Yes B Yes B Yes B Yes B No B Yes B Yes A No B Yes B No B

* All parameters listed refer to and were identical in implanted and explanted cells of each line. tC=Caucasian, N=Negro. tThe diploid chromosome number of NIH SW host mouse is 40, and metaphases are clearly distinguishable from those of human tumor cells at low

magnification. §Isozyme mobility patterns determined through courteous assistance of Dr. W. D. Peterson, Jr.

"blocking" antibody, described by Hellstrom et al. (57,58). Upon in vitro explanation or transplantation to ATST mice, these lymphocytes could become cytotoxic for the tumOl cells, resulting in fibroblastic overgrowth in vitro and failure to take in vivo.

A new series of direct transplants from more nearly optimal neoplastic surgical specimens to mice is in progress and should present fewer technical problems. Also, a modification in the maintenance of the tumor transplant growth beyond I month is being tried; it involves substitution of prednisone for ATS on day 25 after transplantation. This procedure looks promising, but has not been tried often enough for adequate evaluation.

In vitro cultured tumor cells were far more pre­dictable as to their transplantability. Inoculation of either "benign" or "malignant" rated cultures into untreated or NRS-treated 5- to 10-day-old NIH SW mice never resulted in sc or ic tumor takes.

In contrast, when transplanted to ATST groups, the fate of most attached cell cultures correlated very well with their derivation and their in vitro culture morphology: Those rated "benign" in vitro included 4 embryonic lines and 14 lines explanted from human tumors, but exhibiting the "benign" growth pattern described before. Of these 18, none produced tumors or even palpable nodules by day 21 post inoculation of 2 X 106 cells sc. Among those rated "malignant" 10 of 12 proved transplantable; the concurrence was particularly impressive as to the tumor type grown in the experimental animal. In each instance, sarcoma­derived cells proliferated and formed a recognizable sarcomatous neoplasm; similarly, carcinoma cultures produced characteristic carcinomatous structures.

These dissociated tumor cells confined in the sc space of A TST mice reconstructed a tumor mass closely resembling spontaneous tumors, including a fibroblastoid stroma, a vascular supply, and an integral tumor mass. The stroma and blood vessels are almost certainly mouse tissue, stimulated to grow into the proliferating human tumor. Since the entire process can be completed by dissociated cells, in a

525-344 0 - 74 - 6

foreign host, and in a time period during which de novo transformation of host cells is unlikely to cause massive tumors, it is additional evidence for a concept often taken for granted-that colonies of tumor cells possess all the genetic and somatic requirements for completion of a cancerous structure. Subsequent reestablishment of human karyotype cell cultures from the transplanted tumors with either none or very few mouse cells strengthens this view, as do the observa­tions and deductions of other investigators (1- 28).

In this series, sarcomas could be transplanted either sc or ic, whereas most carcinomas transplanted sc only.

The failure of 2 "malignant"-looking cultures (neuroglioma H4 and breast carcinoma 734B) to form tumors may be due to imperfections in the immuno­suppression procedure or due to the inability of the cells themselves to adapt to mouse subcutis and brain environment. Of course, it is also possible that the cells are not really descendants of the malignant com­ponent of the tumor, despite their in vitro growth pattern. We cannot at this point state whether trans­plantation to ATST mice (or other immunodeficient hosts) or in vitro morphology should be given greater weight in judgment of a culture as containing tumor­ous versus nontumorous cells. Where the 2 criteria coincide, i.e., "benign" morphology with no tumor take or "malignant" morphology with positive take (especially if the take, histopathologically, resembles the tumor type from which the cells had been cultured), we believe the judgment may be rendered with confi­dence.

Cells capable of continued suspension growth may by this very characteristic be rated "malignant" or "transformed," whether originating from neoplastic or non-neoplastic tissue. Only 6 lines tested are inade­quate for proper evaluation; half failed to induce neo­plasms. The 3 positive takes were in each instance reproducible ic only, in marked contrast to the at­tached cells. This may reflect the intrinsic preference of suspension cells for brain (including the cerebro­spinal fluid) versus sc environment or the peculiarity of partial immunosuppression induced by ATS. Addi-

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tional attempts to transplant suspension cultures, either neoplasm-derived or other, are needed and are in preparation; they should demonstrate whether the low rate of take is consistent or is accidental to the first 6 attemptsY It is interesting that Southam et al. (J 3) found 9 of 22 tested suspension lines to be trans­plantable to newborn rats, resulting in lymphomatous infiltrates, the remainder being nontransplantable. These results are not vastly different from ours.

The suspension line LEVIII, although derived from a carcinomatous pleural effusion, gave rise to lympho­matous lesions in mouse brains, which closely resem­bled those produced by B37 (malignant lymphoma) and Raji (Burkitt's lymphoma) cells; this result, in conjunction with its in vitro suspension growth in stationary vessels, suggests a lymphoreticular 01 igin for culture LEVIII.

Our data indicate that established tumor cell cul­tures can be evaluated for tumorigenic potential in the ATST mouse by careful observation of morpho­logic characteristics and their correspondence with the neoplasm from which they were derived. Before use of anyone of such cultures for extensive experimentation, confirmation by a simple transplant to a small gl oup of ATST mice would be indicated. Since, in each in­stance in which prompt tumor formation took place in this series, morphologically and karyologically unal­tered human cells could easily be reestablished after 15-48 days of tumor growth, the ATST mouse seems suitable as a host in which to test for activation of C-type RNA tumor virus genomes by heterospecies transplantation. During the period of solid tumor growth in the ATST mice, the humoral and cellular environment may favor virogene activation without interference by antiviral controls (repressors, anti­bodies) directed against the sought after human tumor viruses or human-murine viral hybrids.

One confirmed C-type virus, ATSI24, has been isolated from rhabdomyosarcoma RD cells reestab­lished in vitro after 3 transplant generations in the ATST NIH Swiss mice. It has been partially charac­terized (61) and contains detectable murine C-virus components including GS-I antigen and RNA­dependent DNA polymerase enzyme closely related to those of murine leukemia viruses. Its infectivity in vitro, however, is greatest for human and primate cell cultures. Such a virus has not been reported before in any mouse strain; the NIB SW mouse strain in partic­ular had been studied intensively with consistently negative virus isolation results. It thus appears that ATS-mediated transplantation of human tumor cells had provided the necessary conditions for its emergence.

The propagation of various human tumors in the ATST mouse system may have other applications in cancer research. A particularly timely use may be in the evaluation of anticancer measures, either thera­peutic or preventive. In a recent study, Adams et al. (24) studied selected chemotherapeutic and immuno­therapeutic procedures against human lymphomas transplanted to newborn hamsters. They successfully demonstrated differences in efficacy among antitumor drugs tested, as well as a saving effect with immuno-

therapy. Their ingenious design can serve as a model for studies of other human tumor types transplantable to tolerant hosts, including ATST mice. The responses of these tumor cells that have grown into a solid tumor mass may be closer to reactions of spontaneous cancers than the responses obtained with tumor cells in a more restrictive tissue culture system.

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FIGURE I.-Transitional cell carcinoma, HT1051, excised from an 87-year-old man. Hematoxy1in and eosin. X 198

FIGURE 2.-Sixth direct transplant passage of HT1051 in subcutis of ATS-treated mouse 36 days post inoculation. Neoplastic epithelial cells are similar to but larger and more rounded than those in figure 1. Also, nucleus to cytoplasm ratio appears greater, indicating these cells are less differentiated. H & E. X 198

FIGURE 3.-Human colon carcinoma, HT873, direct tumor transplant in subcutis of an ATS-treated mouse 30 days post inoculation. Columnar mucus-producing acinus-like structures. H & E. X 127

FIGURE 4.-Suspension culture LEVIII in brain of an ATS-treated mouse 15 days post inoculation. Undifferentiated lymphoid infiltrate has obliterated lateral ventricle and is invading hippocampus at left. H & E. X 198

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FIGURE 5.-Normal human fibroblast cells, monolayer, high passage. X 45.

FIGURE 6.-Malignant lymphoma H37 cells, suspension, high passage. X 45

FIGURE 7.-Rhabdomyosarcoma, RD cells, monolayer, high passage. X 45

FIGURE 8.-Pharyngeal carcinoma Det562 cells, monolayer, high passage. X 45

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FIGURE g.-Comparison of morphologic characteristics of stained nonconfluent cells: a) Nonmalignant early passage foreskin; b) neuroglioma H4; c) choriocarcinoma, BeWo; d) rhabdomyosarcoma, RD#2; e) rhabdomyosarcoma, T174; f) lymphoma CCRF-CEM. May-Griinwald. X 450

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