World J. Surg. 3, 641-650, 1979

Workl Journal of Surgery

Heterotransplantation of Human Gastric Carcinomas into Nude Mice Katsunori Nakatani, M.D., Seiichi Takahashi, M.D., Tsuneo Shiratori, M.D., and Yoichi Konishi, M.D. First Department of Surgery and Department of Oncological Pathology, Cancer Center, Nara Medical University, Kashihara, Nara, Japan A total of 33 specimens of human gastric carcinoma were used for transplantation into nude mice. Initial tumor "take" was accomplished in 15 of the 33 tumors, and the transplantability rate was 45.5%. Transplantability correlated with histologic type, but not with clinical stage or Borrmann's classification. The transplantability rate of differentiated carcinomas, such as well-differentiated tubular adenocarcinoma, moderately differentiated tubular adenocarcinoma, and papillary adenocarcinoma was greater than that of poorly differentiated tumors, such as poorly differentiated adenocarcinoma and mucinous adenocarcinoma. The growth patterns of transplanted tumors were divided into 3 types: rapid, slow, and persistent. There were no specific relationships between growth pattern and histologic type. All histologic types, except signet ring cell carcinoma, could be transplanted serially. Tumor growth became rapid after serial transfer. However, the original histology of these tumors was unchanged. No invasion or metastases were encountered. Intraperitoneal injection of a tumor cell suspension, prepared from subcutaneous transplants of a poorly differentiated adenocarcinoma of Borrmann type III, grew in an ascites form, with invasion and metastasis. Ascitic fluid accumulated within 3-6 weeks after injection. Subsequently, intravenous injection of ascites fluid produced metastases in nude mice. The histology of the subcutaneous tumor was similar to that of the original tumor from the patient.

Since the original report of Rygaard and Povlsen [1], h u m a n cancers have b e e n successfully transplanted into nude mice by m a n y investigators [212]. Transplants of h u m a n malignant tumors in nude mice respond to c h e m o t h e r a p y , resulting in a potential model for the in vivo study of responses o f individual h u m a n tumors to different c h e m o l h e r a p e u t i c agents. H o w e v e r , at present, a satisfactory model for gastric c a r c i n o m a in the nude m o u s e does not exist, since invasion and metastases are not consistently e n c o u n t e r e d w h e n t u m o r s are grown in the s u b c u t a n e o u s tissues. We have been transplanting various histologic types o f h u m a n stomach c a n c e r into nude mice. The present e x p e r i m e n t was designed with two main purposes in mind: firstly, to establish a model for studying the c h e m o t h e r a p y of different histologic types of stomach cancer, and secondly, to develop a model in which the malignant potential of h u m a n gastric cancer could be studied. This p a p e r describes the results of the transplantation of various histologic types of h u m a n s t o m a c h cancer into nude mice and the successful adaptation of one t u m o r to grow in an ascites form in which invasion and metastasis occur.

Materials and Methods

Transplantation of Human Tumors Subcutaneously into Nude Mice Presented at the XXVIIth Congress of the Soci6t6 Internationale de Chirurgie, Kyoto, Japan, September 3-8, 1977. Supported by a grant from the Ministry of Education. Reprint requests: Katsunori Nakatani, M.D., The First Department of Surgery, Nara Medical University, Kashihara, Nara, Japan.

A t h y m i c male nude mice, 6-8 weeks old, with the

0364-2313/79/0003-0641 $02.00 ~) 1979 Soci6t6 International6 de Chirurgie 641

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Table 1. Heterotransplantation of human gastric tumors into nude mice. Transplanted tumor Cases Patient I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

YF MH SN HA KT KY TU NT E1 TT HK KK SN HN TM RK US AM TS YO SY SS MA GT YK NU SU KM GY NT KN KT TS TT SN embryo

Age (years)

Sex

70 52 59 58 38 41 75 59 73 71 59 42 53 61 55 28 61 44 57 64 66 47 48 70 62 64 19 36 79 74 65 72 69 65 59

M M M M F M M M M

F F F F F F M M F F M M F M M M F F M M M M M F F M

Clinical stage

Original tumor Borrmann

Histology

IV III pot IV IV por IV II por IV III muc III IV por II III muc IV I tub2 IV III pap IV IV sig IV II por IV IV por IV IV tuba IV IV pap III III por IV III tub~ III IV por IV IV por IV IV por IV IV por IV III por IV II pap II II por III IV por III II tub1 IV III por IV III muc III IV por IV IV por IV IV por III II tUbl IV IV por 1II III pap II III pap adenomatous polyp normal mucosa of stomach normal mucosa of stomach

g e n e t i c b a c k g r o u n d o f B A L B / c r e c e i v e d serial t r a n s p l a n t s o f t u m o r s a n d w e r e m a i n t a i n e d in flexible v i n y l i s o l a t o r s u n d e r specific p a t h o g e n - f r e e ( S P F ) c o n d i t i o n s . A t o t a l o f 33 h u m a n g a s t r i c c a n cers of various histologic types, classified by The G e n e r a l R u l e s f o r the G a s t r i c C a n c e r S t u d y in Surg e r y a n d P a t h o l o g y [13], w e r e utilized. T r a n s plantation was performed under aseptic conditions, a n d the t i m e b e t w e e n r e m o v a l o f t h e t u m o r t i s s u e and completion of the transplant was usually app r o x i m a t e l y 25 m i n u t e s . T u m o r t i s s u e w a s m i n c e d a n d p l a c e d in r e f r i g e r a t e d H A M F - 1 2 t i s s u e c u l t u r e m e d i u m . S e v e r a l t i s s u e f r a g m e n t s o f a b o u t 2 m m in d i a m e t e r w e r e t r a n s p l a n t e d w i t h a t r o c a r into t h e subcutaneous tissue of the back of nude mice.

Inoculation period (days)

No. of tumor takes/no, attempted

Size of tumor take (mm z)

Number of transfers

97-136 107-134 107-150 89-142 60 - 139 60 66-169 53-106 128 97-124 129 100 92 84 206 136 142

2/4 1/4 0/4 4/4 0/6 0/2 7/8 6/6 0/2 0/4 0/4 0/2 2/2 0/2 1/2 0/2 0/2 0/2 0/2 0/2 0/2 2/4 0/4 0/4 2/4 3/4 0/2 0/4 4/4 4/4 1/4 4/4 2/2 0/4 0/2 6/6

156,550 20

15

115

42-99 78 71 45-72 84-140 126 56-125 52-121 54 35 45-58 44-51 36 34-55 34 61 112 350-372

28-36 -56-124 56-225 ---120, 120 -135 --

6 10

4

---

--35, 49 -25, 25 25-50 --

3

-

25-48 25-36 36 30-144 30, 35 --35-96

2 2 2 2

T r a n s p l a n t a t i o n sites w e r e o b s e r v e d e v e r y d a y a n d the size o f i n d i v i d u a l t u m o r s w e r e m e a s u r e d w e e k ly. W h e n t u m o r s r e a c h e d 1 c m o r l a r g e r in size, serial t r a n s f e r s w e r e p e r f o r m e d b y the s a m e m e t h o d d e scribed above. T h e n u d e m i c e w e r e s a c r i f i c e d b y c e r v i c a l disl o c a t i o n , a n d a c o m p l e t e a u t o p s y w a s c a r r i e d out. T h e lung, h e a r t , liver, p a n c r e a s , s p l e e n , e s o p h a g u s , stomach, small and large intestine, kidney, adrenal, urinary bladder, testis, and tissues from the transp l a n t a t i o n sites w e r e fixed in 10% b u f f e r e d f o r m a l i n , e m b e d d e d in paraffin, s e c t i o n e d , a n d s t a i n e d w i t h hematoxylin and eosin, periodic acid-Schiff (PAS), a n d a l c i a n blue. T u m o r " t a k e s " w e r e d e t e r m i n e d by histologic examination.

K. Nakatani et al.: Animal Model for Human Gastric Carcinoma

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Table 2. Growth pattern of transplanted tumors in nude mice and survival of patients after operation.

Table 3. Conversion of human poorly differentiated adenocarcinoma (por) to ascites form in nude mice. Passage

Growth pattern of tumors in nude mice

Survival of patients after operation (month)

Rapid Slow Persistent Cases that did not "take"

1, 12 3, 3,9 a 12, 13, 13a'b, 7b, 6b, 6b, 5b, 4b, 4 b, 3b 17, 13, 11a, 7, 6 a, 6, 6, 18b, 17b, 15b 10a,b, 10b, 8b, 8b, 7b, 6b, 6b, 5b

I

aNonresectable cases. bStill alive on August 5, 1977.

Adaptation of Solid Tumors to Ascitic Growth

Number of mice with ascites/number of mice attempted Time ascites appeared (weeks) Number of mice with peritoneal carcinomatosis/number of mice attempted Number of mice with liver metastasis/number of mice attempted

Poorlydifferentiatedadenocarcinoma(por)

IV

V

3/3 2/2 2/2 2/2

2/2

3

6

4

4

4

3

2

2

2

2

0

1

1

0

1

Papillaryadenocarcinoma(pap)

mm2[

mm 2

500 [

500

/

III

tained f r o m the ascites t u m o r at the 3rd intraperitoneal passage in nude mice. Before injection, the n u m b e r of t u m o r cells was counted in a hemocytometer. The solid s u b c u t a n e o u s tumors were minced with scissors and passed through a cytosieve (100 mesh/ cm 2) with a rubber plug in Eagle M E M m e d i u m containing 10% fetal calf serum, s t r e p t o m y c i n (100 mg/ L), and penicillin (1 x 10~ units/L). The cell suspension, containing 1 x 107 cells per 0.5 ml, was in-

A t h y m i c male nude mice of the same strain and source were used. Mice receiving intraperitoneal or intravenous injections of t u m o r cells were maintained under conventional conditions, rather than in a specific pathogen-free environment. T u m o r cells used for intraperitoneal injection were obtained f r o m the 9th passage subcutaneous transplant o f a B o r r m a n n type III poorly differentiated adenocarcin o m a (por) obtained f r o m a 70-year-old male. Tum o r cells used for intravenous injection were ob-

L5 N~ 400 I

II

400

x

r xiv

I

so 09

VIII

200

,v

//,,, /,___ ,,,

I

I

0

Vl

IX

I

1

I

2

I

3

I

I

4

I

5

I

6

Weeksaftertransplantation

I

7

I

8

I

9

200

x

- IV

II

ix

l

I

I

I

I

I

!

I

I

1

2

3

4

5

6

7

8

9

Weeksafter transplantation

Fig. 1. Growth curves of transplant generations of two human gastric cancers serially transplanted in nude mice.

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Fig. 2. Photograph of transplanted poorly differentiated adenocarcinoma (por) io the 1st transplant generation.

jected intraperitoneally and, subsequently, ascites containing 1.5 • 106 cells per 0.5 ml was serially transplanted by intraperitoneal injection. F o r intravenous injection, ascitic fluid from the 3rd intraperitoneal passage, containing 6 x 105 cells per 0.2 ml, was injected into the tail veins o f nude mice. Mice were sacrificed by cervical dislocation at times indicated in Table 3. At the time of passage, smears of ascitic fluid were rapidly air dried, fixed with 100% methanol for 2 minutes, and stained with Giemsa stain. All animals were observed, and organs were examined grossly and microscopically in the same manner described above. Results

The tumors used for transplantation and the results o f the transplantation experiments are indicated in Table 1. The youngest patient was a 19-year-old fe-

World J. Surg. Vol. 3, No. 5, 1979

male, and the oldest one was a 79-year-old male. Clinically, all patients had advanced cancer, stages II to IV. All histologic types o f stomach cancer were included among these 33 cases. The shortest time period for tumor " t a k e " was 34 days, and all tumors which " t o o k " grew out within 100 days. At the present time, we are serially transplanting 2 cases of poorly differentiated adenocarcinoma (por), 1 case o f well-differentiated tubular carcinoma (tub1), 1 case o f moderately differentiated tubular adenocarcinoma (tub2), 4 cases o f papillary adenocarcinoma (pap), and 1 case of mucinous adenocarcinoma (muc). Embryonal gastric mucosa o f the stomach was grown successfully. H o w e v e r , an adenomatous polyp and a specimen of adult normal gastric mucosa were not successfully transplanted. Regarding the transplantability o f human gastric cancer in nude mice, tumors from 15 of 33 patients were successfully transplanted for a transplantability success rate of 45.5%. Considering the number of tumor specimens, 45 o f 114 tumors resulted in a " t a k e , " for a transplantability rate o f 39.5%. The transplantability rate was 66.7% in clinical stage II tumors, 25% in stage III tumors, and 50% in stage IV tumors. There was no specific correlation between transplantability and Borrmann's classification of tumors. All histologic types, except the signet ring cell cancer (sig), were successfully transplanted. The growth patterns o f transplanted tumors were divided into 3 categories, i.e., rapid, slow, and persistent, based on the tumor size and latency period after transplantation. Tub1 and tub2 tumors did not show rapid growth but showed slow or persistent growth. Pap tumors showed various growth patterns; 1 was rapid, 2 slow, and 1 persistent. Por tumors showed persistent growth except for 1 rapid

Fig. 3 A. Histology of the original por tumor from the patient. B. Histology of por tumor in the 4th transplant generation (x 100).

K. Nakatani et al.: Animal Model for Human Gastric Carcinoma

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Fig. 4 A. Histology of the original pap tumor from the patient. B. Histology of pap tumor in the 3rd transplant generation

(•

case. Muc tumors showed persistent growth in 2 o f 2 cases. The relationship between the growth patterns o f transplanted tumors and the survival o f patients after operation are shown in Table 2. Two patients whose tumors showed rapid growth in nude mice died at 1 and 12 months after operation. Three patients, 2 resectable and 1 nonresectable, whose tumors showed slow growth in nude mice died at 3 and 9 months after operation. Of 10 patients, 9 resectable and 1 nonresectable, whose tumors showed persistent growth in nude mice, 2 died at 12 and 13 months after operation, and the rest are still alive. T u m o r growth curves for individual transplant generations of por and pap are shown in Fig. 1. Both o f them showed rapid growth in the first transplant generation. Growth curves became more rapid with serial transplantation. By the 14th genera-

tion, the size of por transplants reached 310 mm 2 in 3 weeks and that of pap tumor transplants reached 180 mm 2 in 3 weeks. H o w e v e r , invasion and metastasis have never been encountered from these subcutaneous tumor transplants. Macroscopically, tumor transplants from gastric carcinomas of histologic types pap, t u b , tub2, and por grown in nude mice were whitish-grey and solid with an irregular surface (Fig. 2). The sizes of these tumors varied from 25 mm 2 to 550 mm 2. Muc transplants appeared partially cystic. The histologic appearance of the original tumors (obtained at operation) from which the por, pap, muc, and tub2 lines were established and transplants of these tumors are illustrated in Figs. 3-6. The histology o f the tumor transplants was, in e v e r y case, essentially identical to that of the original tumor. Invasion and metastases were not seen. The results o f the conversion of tumor por to the

Fig. 5 A. Histology of the original tub2 tumor from the patient. B. Histology of tub2 tumor in the 2nd transplant generation (• 100).

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Fig. 6 A. Histology of the original muc tumor from the patient. B. Histology ofmuc tumor in the 1st transplant generation (x 100).

ascites form and the results of serial transfers of this tumor in the ascites form are shown in Table 3. Growth in the ascitic form was accomplished in all 3 mice that received intraperitoneal injections of tumor cells. In each instance, ascitic fluid accumulated within 3 weeks. Serial transplantation in the ascites form has now reached the 5th passage generation, and ascites now develops within 4-6 weeks. Peritoneal carcinomatosis occurs consistently. Liver metastases were noted in 1 of 2 mice in the 2nd, 3rd, and 5th passages. Macroscopically, mice with peritoneal carcinomatosis showed multiple whitish-grey tumors invading the diaphragm, peritoneum, omentum, pancreas, and mesentery (Fig. 7A). In addition, metastases to the liver and spleen were seen. The Cytology of tumor cells in the ascites 3 weeks after intraperitoneal injection is shown in Fig. 7B. The cells are floating freely. Their nuclei are irregular in shape, and mitotic figures are seen. The cytoplasm does not stain with PAS or alcian blue.

The histologic appearance of invasive tumors in the mesentery and of metastatic deposits in the liver and spleen was similar to that of the original por tumor. Intravenous injection of ascitic fluid produced metastatic deposits in the lung and heart. The histologic appearances of the metastases in the lung and heart were similar to each other and similar to that of the original tumor specimen from which the por line was derived. Discussion Attempts to establish a model for the chemotherapy of human gastric carcinoma in nude mice have been reported previously by ourselves and others [ 10-12, 14]. However, results were disappointing since invasion and metastases were not consistently encountered when tumor transplants were grown sub-

Fig. 7 A. Photograph of peritoneal carcinomatosis produced by intraperitoneal injection of ascitic fluid. B. Cytology of the ascites produced by intraperitoneal injection of a tumor cell suspension derived from por tumor transplant (•

K. Nakatani et al.: Animal Model for Human Gastric Carcinoma

cutaneously [2, 3, 10, 12]. In the present experiments, 15 of 33 cases of stomach cancer were successfully transplanted subcutaneously in nude mice, and 9 lines of serially transplantable tumors were established. Transplantability rates were higher than previously reported [10, 11, 14]. Transplantability was found to be related to the histologic type of the tumor. Well-differentiated tumors showed greater transplantability than poorly differentiated tumors. However, transplantability did not depend on clinical stage or Borrmann's classification. In the present experiments, 7 of 10 (70%) differentiated tumors, such as tub1, tub2, and pap were successfully transplanted, whereas only 8 of 23 (34.8%) poorly differentiated tumors such as por and muc were successfully transplanted. Shimosato et al. [11] has stated that the low transplantability rate of desmoplastic or scirrhous carcinomas may be due to the presence of abundant human connective tissue elements which prevent contact of tumor cells with the tissues and body fluids of the mice. There was no specific relationship between the growth pattern and the histologic type of tumors. However, comparative studies of the relationship between tumor doubling time of various tumors and their histologic types are required. Tumor growth usually became more rapid following serial transplantation. This phenomenon is unexplained. Invasion and metastasis formation have been seen uncommonly when xenogenic human gastric cancers have been transplanted to nude mice [1012]. The present experiment has clearly demonstrated invasion and metastasis of human gastric cancer in nude mice following intraperitoneal injection of tumor cell suspensions or tumor cells in ascitic fluid. Ehrlich ascites tumor and the ascites form of Yoshida sarcoma are common and useful experimental models for cancer chemotherapy. However, they do not originate from human tumors. Therefore, the establishment of an ascites form of a human gastric carcinoma which results in invasion and metastases may be a very significant accomplishment. Shimosato et al. [11] reported only 1 " t a k e " in the ascitic form among 5 gastric cancers. Ueyama et al. [15] reported 1 case in which a human gastric cancer was adapted to grow in the ascitic form and was serially transplanted in nude mice. However, they did not clearly demonstrate metastasis formation in their experiments. It is possible that when tumor cells grow as a single cell suspension in ascitic fluid without adherence to other cells, attachment to the peritoneal surface and invasion into the blood stream may be facilitated, resulting in metastasis formation. Certainly, lung metastases were easily produced by the intravenous administration of tumor cells.

647

It has been suggested recently that the susceptibility of nude mice raised under conventional conditions to transplants of xenogeneic tumors is greater than that of germ-free nude mice [16]. Our experiments were performed under conventional conditions. These experimental conditions may also predispose to tumor invasion and metastasis formation. Additional research in these areas is required. Previous studies on the conversion of human tumor transplants in nude mice to the ascitic form have indicated that successful transplantation is dependent upon tumor type [15, 17]. Studies on the conversion of transplants of other histologic types of human gastric cancer to the ascitic form are in progress. As in previous reports [2, 3, 5, 7, 8, 10, 12, 18, 19], our experiments demonstrated that the histologic patterns of the original tumors were retained in nude mice. Nevertheless, it is evident from the present results that the malignant potential of the por tumor line, as evidenced by local invasion and metastasis formation, was expressed in nude mice when transplantation in the ascitic form was accomplished. Transplants of human gastric cancer in the ascitic form resulting in invasion and metastases may provide a useful model for studying the chemotherapy of these tumors.

R6sum6

Trente-trois sp6cimens de carcinome gastrique humain ont 6t6 transplant6s chez la souris nue. La "prise" initiale de la tumeur a 6t6 obtenue dans 15/ 33 cas et 45.5% des tumeurs sont transplantables. La possibilit6 de transplantation est en rapport avec le type histologique, mais pas avec le stade clinique ni avec la classification de Borrmann. Les carcinomes diff6renci6s (ad6nocarcinome tubulaire bien ou mod6r6ment diff6renci6, ad6nocarcinome papillaire) se transplantent mieux que les tumeurs peu diff6renci6es (ad6nocarcinome peu diff6renci6, ad6nocarcinome mucineux). Le mode de croissance des tumeurs transplantdes a 6t6 class6 en 3 groupes: rapide, lent et stationnaire. I1 n'y a pas de relation sp6cifique entre mode de croissance et type histologique. Tousles cancers, quel que soit le type histologique/t l'exception des tumeurs de Krukenberg, ont pu 6tre transplant6s en s6rie. Apr6s transplantation en s6rie, la croissance des tumeurs devient rapide. Pourtant l'aspect histologique de ddpart reste inchang6. Aucune invasion, aucune m6tastase n'ont 6t6 observ6es. L'injection intrap6riton6ale de suspensions de cellules tumorales pr6par6es ~t partir de transplants sous-cutan6s d'addnocarcinomes peu diff6renci6s de type Borrmann III donne une croissance avec

World J. Surg. Vol. 3, No. 5, 1979

648

production d'ascite, invasion r6gionale et m6tastases. L ' a s c i t e s ' a c c u m u l e en 3-6 semaines apr6s l'innoculation. L'injection intraveineuse de liquide d'ascite chez la souris nue p r o v o q u e le d6veloppement de m6tastases. L'histologie de la tumeur souscutan6e est c o m p a r a b l e aux aspects de la tumeur originale pr61ev6e chez le malade. References 1. Rygaard, J., Povlsen, C.O.: Heterotransplantation of a human malignant tumor to "nude" mice. Acta Pathol. Microbiol. Scand. 77:758, 1969 2. Povlsen, C.O., Rygaard, J.: Heterotransplantation of human adenocarcinomas of the colon and rectum to mouse mutant nude. A study of nine consecutive transplantations. Acta Pathol. Microbiol. Scand. Section A, 79:159, 1971 3. Povlsen, C.O., Rygaard, J.: Heterotransplantation of human epidermoid carcinomas to the mouse mutant nude. Acta Pathol. Microbiol. Scand. [A] 80:713, 1972 4. Visfeldt, J., Povlsen, C.O., Rygaard, J.: Chromosome analysis of human tumors following heterotransplantation to the mouse mutant nude. Acta Pathol. Microbiol. Scand. [A] 80:169, 1972 5. Povlsen, C.O., Rygaard, J.: Effects of cyclophosphamide (Endoxan) on a Burkitt's lymphoma serially grown in nude mice. In Proceedings of 1st International Workshop on Nude Mice. Stuttgart, Gusrave Fischer Verlag, 1974, pp. 285-292 6. Povlsen, C.O., Fialkow, P.J., Klein, E.: Growth and antigenic properties of a biopsy-derived Burkitt's lymphoma in thymus-less (nude) mice. Int. J. Cancer I1:30, 1973 7. Sordat, B., Fritsche, R., Mach, J.P.: Morphological and functional evaluation of human solid tumors serially transplanted in nude mice. In Proceeding of 1st International Workshop on Nude Mice. Stuttgart, Gustave Fischer Verlag, 1974, pp. 269-278 8. Giovanella, B.C., Stehlin, J.S., Williams, L.J., Jr.: Heterotransplantation of human malignant tumors in "nude" thymusless mice. II. Malignant tumors in-

Invited Commentary Beppino C. Giovanella, Ph.D. The Stehlin Foundation for Cancer Research, Houston, Texas, U.S.A. The use o f the nude m o u s e as a host for heterotransplanted h u m a n tumors has drastically altered the outlook in this field. The serial transplantation of h u m a n tumors has b e c o m e , for m a n y types o f

9.

10.

11.

12.

13.

14.

15.

16. 17.

18.

19.

duced by injection of cell cultures derived from human solid tumors. J. Natl. Cancer Inst. 52:921, 1974 Giovanella, B.C., Stehlin, J.S.: Influence of the host's sex on the growth of human tumors heterotransplanted in "nude" thymusless mice. Proc. Am. Assoc. Cancer Res. 15:23, 1974 Kuga, N., Yoshida, K., Seido, T., Oboshi, S., Koide, T., Shimosato, Y.: Heterotransplantation of cultured human cancer cells and cancer tissues into nude mice. Gann 66:547, 1975 Shimosato, Y., Kameya, T., Nagai, K., Hirohashi, S., Koide, T., Hayashi, H., Namura, T.: Transplantation of human tumors in nude mice. J. Natl. Cancer Inst. 56:51, 1976 Takahashi, S., Nakatani, K., Shiratori, T., Konishi, Y., Kojima, K.: Heterotransplantation of the human stomach carcinomas into nude mice. I. Studies on transplantability, growth patterns and histology (in Japanese). Jpn. J. Gastroenterol. 74:421, 1977 The General Rules For The Gastric Cancer Study In Surgery and Pathology, 9th edition, Japanese Research Society for Gastric Cancer. Tokyo, Kanehara, 1974, pp. 27-61 Schmidt, M., Deschner, E.E., Thaler, H.T., Clements, L., Good, R.A.: Gastrointestinal cancer studies in the human to nude mouse heterotransplantation system. Gastroenterology 72:829, 1977 Ueyama, Y., Kondo, Y., Tamaoki, N., Ohsawa, N.: Ascites form of a human cancer serially transplantable in nude mice. J. Natl. Cancer Inst. 57:965, 1976 Maguire, H., Outzen, H., Jr., Custer, R.P.: Invasion and metastasis of a xenogenic tumor in nude mice. J. Natl. Cancer Inst. 57:439, 1976 Hirohashi, S., Shimosato, Y., Nagai, K., Koide, T., Kameya, T.: Human breast cancer serially transplantable in nude mice in ascites form. Gann 67:431, 1976 Epstein, A.L., Kaplan, H.S.: Biology of the human malignant lymphomas. I. Establishment in continuous cell culture and heterotransplantation of diffuse histiocytic lymphomas. Cancer 34:1851, 1974 Shimosato, Y., Kameya, T., Koide, T.: Morphology of human lung cancers transplanted in nude mice (in Japanese). Proc. Jpn. Cancer Assoc. 32:23, 1974

tumors at least, a practical and easily accomplished procedure. F o r tumors of the gastrointestinal tract, the rate o f " t a k e s " is in general of the order of 50% [1, 2], as confirmed by the present paper. T u m o r s preserve well their m o r p h o l o g y through serial passages [3, 4], and their production of carcinoembryonic antigen (CEA) parallels the production of such an antigen in the t u m o r o f origin [ 1]. The m o s t interesting feature o f the heterotransplanted human tumors is that they p r e s e r v e the degree of differentiation of the t u m o r o f origin. This peculiarity