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Heterotransplantation of Human Transitional Cell Carcinoma in Athymic Mice
0022-534 7/79/ 1212-0159$02. 00/0 THE JOURNAL OF UROLOGY
Vol. 121, February
Copyright © 1979 by The Williams & Wilkins Co.
Printed in U.S.A.
HETEROTRANSPLANTATION OF HUMAN TRANSITIONAL CELL CARCINOMA IN ATHYMIC MICE GERALD SUFRIN,* MICHAEL P. MCGARRY, AVERY A. SANDBERG
AND
GERALD P. MURPHYt
From the Roswell Park Memorial Institute, New Yark State Department of Health, Buffalo, New Yark
ABSTRACT
Human bladder tumors were obtained and transplanted into nude mice or other control animals. Tumor measurements, growth rate and selected histological studies were completed. No correlation between the growth of the tumor in the nude mouse and the clinical course or histologic tumor appearance in the host was detected. Metastases were not found. This feature has been noted generally to be uncommon in the nude mice model with some other human tumors. Despite careful technique tumor growth was achieved in only 40 per cent of the appropriate experiments. The factors responsible for this variability and different growth rate in the nude mouse require further assessment before the results of other experimental treatments can be evaluated. The need for in vivo models for the study of human tumors and for the study of the action of anticancer agents on these human cancers is well acknowledged. 1- 3 In vivo models using human tumors offer an important change in the study of tumor biology and therapeutic systems. Tumor xenografts provide an invaluable source of material for the study of fundamental and practical aspects of human cancer. 2- 4 However, heterotransplantation of malignant human tumors, as explants, generally results in rejection of the true graft because of cellular immunity, that is thymic-dependent lymphocyte function. 5 Exception to this occurs in cases in which the animal host has been rendered immunologically unresponsive, as by thymectomy, sublethal radiation or use of antisera directed against recipient lymphocytes, or when tumors are transplanted into immunologically unresponsive sites. 4• 5 The mutant nude mouse (nu/nu), which is immunologically incompetent because of thymic aplasia, can be of use in such studies and, therefore, offers a unique model for the in vivo study of human xenografts. Indeed, a variety of solid and hematologic tumors has been reported to grow in the nude host. 4 - 6 We describe studies undertaken to explore the growth pattern of human transitional cell carcinoma of the bladder in such congenitally immunodeficient mice. Heterotransplant of this tumor is of special interest because of the current clinical need for more effective cytotoxic agents. It also is important to study the biologic behavior and characterize bladder-tumor growth with special consideration to observe possible differences among various malignant lesions. We, therefore, studied the growth of transitional cell carcinoma in the athymic nude host in an effort to define growth parameters, possibly to develop a model for chemotherapy and the further study of bladder tumor biology by obtaining successful implantation and growth of such tumors in these hosts. MATERIALS AND METHODS
Mice. We used 6 to 8-week-old athymic nude (nu/nu) and normal heterozygotic (+/nu) litter mates. The immunologically competent litter mates were used to demonstrate typical rejection reactions of the human tissues at the site of implantation. The congenitally athymic, nude mice were obtained Accepted for publication April 21, 1978. * Current address: Waldheim Department of Surgery, The Jewish Hospital of St. Louis, 216 S. Kingshighway, P.O. Box 14109, St. Louis, Missouri 63178. t Requests for reprints: Roswell Park Memorial Institute, 666 Elm St., Buffalo, New York 14263.
from the mating of heterozygous (+/nu) female mice with homozygous (nu/nu) male mice, resulting in 50 per cent nu/nu offspring. Stock mice were obtained initially from Giovanella of the Stehlin Foundation and were bred according to his and other published recommendations. 2• 3 The animals were kept according to germ-free techniques in plastic isolators. They were maintained as specific pathogen free with a single, detectable contaminant, Klebsiella species. 1- 9 Patients. Patient data and information regarding location, tumor type and histology are shown in table 1. Tumor tissue was obtained via transurethral forcep biopsy from 20 patients. A 1 cu. mm. portion of each tumor was directly implanted subcutaneously. Other specimens of tumor tissue also were concurrently fixed in formalin, embedded in paraffin, sectioned at 7 µ,. and stained with hematoxylin and eosin for histological examination by light microscopy. Inoculation. Surgical specimens, aseptically preserved on ice in RPMl-1640 media, were minced to 1 mm. 3 in size and inoculated subcutaneously into the flank of the animal using a trocar needle. One to several tumor tissue fragments were implanted subcutaneously along the lateral aspect of the abdomen at 3 to 4 sites within 20 to 45 minutes after surgical excision. Usually tissue from 1 tumor was inoculated into 3 to 6 mice. Transplantation sites were observed for 2 weeks for tumor growth. The average age of host mice was 50 days, with a range of 42 to 90 days. Tumor take. Tumor growth was considered to have occurred: 1) when there was an observed increase in the size of implant at the inoculation site and 2) when the tissues were removed and shown as histologically identical to the original implant and contained mitotic figures. The absence of such evidence or the occurrence of a necrotic residue generally was considered as indicating graft rejection. Evaluation and measurement of tumor growth. Animals were kept for observation for a mean of 165 days (range of 85 to 180 days). Autopsies were performed on all animals. The tumors, or remnants thereof, were dissected free and weighed. Tissue sections were made of all tumors and from any host organs showing evidence of macroscopic change. The size of subcutaneous tumors was observed daily and measured weekly in 2 dimensions by calipers. Data are expressed as the product of the 2 maximal perpendicular diameters. New growths were removed and studied when they exceeded 1 cm. in diameter. RESULTS
Transplantability of tumors - tumor growth. Transitional cell carcinoma from 20 patients was transplanted directly into 159
160
SUFRIN AND ASSOCIATES
104 athymic mice (table 2). The tumor from each patient was implanted in 4 to 6 animals, depending on the amount of tissue available. Successful takes were observed with 40 per cent of the tumors and in 41 of the 104 athymic murine recipients. If a grafted tumor grew in its nude host 75 to 100 per cent of the other animals implanted with the same tumor also exhibited tumor growth. No tumors grew in the normal, heterozygous immunologically competent recipients, indicating no loss of tumor antigenicity. The clinical features of patients with transitional cell carcinoma tumors that were implanted in nude mice are shown in table 1. The stage of tumor, its histological grade, the multicentricity and its tendency to recur had no apparent influence on the frequency of successful takes. Also, the subsequent clinical prognosis was not related to the success or failure of grafts to take in the nude host. Growth characteristics of human tumors in nude mice. The hairless condition of the congenitally athymic host mice facilitates observation of subcutaneously inoculated tissue. By 24 hours after tumor implantation a mass 1 to 2 mm. in diameter is visible. The subcutaneous nodules gradually regressed in mice with inoculate that failed to grow and they were visible only as pale areas less than 1 mm. in diameter by 12 days after implantation. Histologic examination revealed fibrotic nodules. There appeared to be another critical period between day 20 and day 25 after inoculation in the athymic host, during which heterotransplants underwent a period of selection as a result of which there was either an increase in size or regression. The transitional cell carcinoma that grew appeared initially as a minute discrete nodule, which slowly and progressively enlarged. In all cases the tumor showed only local growth without metastases to lymph nodes or other host organs. Fully developed xenografts were smooth with the overlying skin intact and not adherent. On section the tumors were well defined in the subcutaneous space as an apparent encapsulated solid mass. Relatively large blood vessels were seen crossing and penetrating the tumors. The tumors were generally from superficial arteries that dilate and ramify concurrently with the tumor growth. Transplants developing in the nude mouse maintained cytological and histological appearances in full accordance with those of the inoculated primary tumor. In all cases the TABLE
1. Summary of patient data from which transitional cell
carcinomas were derived
Pt. average age
Growth (No.)
No Growth (No.)
62
57
Sex: Male Female Tumor stage:
6 2
9 3
Low (I-II)
3
High (ill-IV) Tumor grade:
5
6 6
6 2
3 9
3
5
2 10
4 4
7 5
(yrs.)
Low (I-II) High (ill-IV) Subsequent clinical course: Stable Progression Distribution of lesions: Solitary Multiple
TABLE
2. Transplantation of human transitional cell carcinoma into
nude mice
Tumors transplanted Nude mice receiving xenografts Furred controls receiving xenografts
Implants (No.)
Tumor Growth
No Tumor Growth
(%)
(%)
20
40 40
60 60 100
104 43
0
grafts were the same as the initial tumor, especially regarding histological grade of the tumor. Mitotic figures were observed. No metastatic lesions were found. Chromosome study. Chromosome analysis was done in the xenografts from 2 patients. All of the mitoses observed were of a human karyotype and no mouse chromosomes were present, eliminating the possibility of host tissue being incorporated into the tumor. The modal chromosome number was 48. A marker chromosome was present frequently. These findings from tumor in male nude mice correspond to chromosomal analysis of the parent tumor. DISCUSSION
Heterotransplantation of malignant tumors to experimental animals normally results in rejection oftumor. 4 - 6 Well known exceptions from this general rule are cases in which the animal host has been made immunologically unresponsive (neonatal thymectomy, sublethal x-ray irradiation, steroid treatment or induction of immunological tolerance) or when implants are made into immunologically unresponsive locations (anterior eye chamber, hamster cheek pouch or outside the blood barrier). 6• 10 - 14 Tumor xenografting into nude mice provides a novel opportunity to study the biologic behavior of neoplastic tissue in the absence of normal host response. 6 Nude mice appear unable to reject such tumors because of their deficient thymus-dependent immune system. 6 The difficulties of using cell cultures in vitro for the purpose of developing effective drugs and chemotherapeutic regimens may be summarized as follows: 1) changes in metabolism imposed by growth in culture media may modify the effects of drugs, 2) in vitro experiments do not take into account in vivo disposition and metabolism of drugs and 3) rapid selection of particular tumor cell clones adapted to growth in vitro may occur and these may be unrepresentative of the tumor cell population in vivo. 10• 11 The relatively slow rate of growth of the transplanted bladder tumors observed in this study in the subcutaneous implants contrasts with the more typical rapidly proliferating anaplastic animal tumors. This behavior of the transplanted tumor is relevant since much tumor growth in the patient may be similar. 15 Our findings are in agreement with those of others who have reported that tumors implanted into athymic mice or immune-deprived mice do not always grow and certain tumors are more likely to take than others. 16 There appears to be a critical period between 20 and 25 days, during which the implanted bladder tumor undergoes a process of selection. The tumor growth rates observed in our study varied from active growth to mere persistence in an arrested survival state. This suggests, in our opinion, that the rate of human xenograft development is controlled by other unidentified intrinsic cellular properties of the tumor. Since we have used this technique for some time in our laboratory we do not believe that these variations are artifactual. The rate of tumor takes in this study did not seem related to tumor grade, stage, the response to treatment of the patient or frequency of such tumors. Mitchley and associates reported that many different types of human cancers have now been grown successfully in either mice that have been artificially deprived ofT-lymphocytes (B mice) or in nude athymic mice. 16• 17 Many of the tumors grown in this way have been transplanted for several generations but few metastasize. Transplantability usually has been found to be highest for carcinoma of the colon and lung and lowest for breast tumors. 3 Tumor growth rates in our study vary from exponential to persistence of tumor in an arrested survival state. Franks and associates, in a similar study of human bladder tumor material, noted that there appears to be a critical period between day +2 and day +25, during which the implanted bladder tumor undergoes a process of selection as a result of which there is either an increase in size or regres-
HETEROTRANSPLANTATION OF HUMAN TRANSITIONAL CELL CARCINOMA IN ATHYMIC MICE
161
sion. 18 Franks and associates observed that it is not clear what 8. Mirand, E. A., Gordon, A. S., Zanjani, E. D., Bennett, T. E. and Murphy, G. P.: Disappearance of exogenous erythropoietin determines the successful establishment of an implanted blad(ESF) from the blood of germfree mice. Proc. Soc. Exp. Biol. der tumor or why some tumor types preferentially survive in Med., 139: 161, 1972. a xenograft system. 18 In a previous study by Elhilali and 9. McGarry, M. P., Bahny, L., Jividen, J., Williams, P., Mirand, Nayak the success rate of growing bladder tumor tissue in E. A. and Murphy, G. P.: Particulate matter induced periortissue culture could not be correlated with the stage of the bital abscess in gnotobiotic athymic nu/nu (nude) mice. Lab. 19 tumor. Anim. Sci., 26: 956, 1976. This study has shown that the nude mouse can be used to 10. Sheard, C. E., Double, J. A. and Berenbaum, M. C.: The sensitivity to chemotherapeutic agents of a rat tumour grown demonstrate the variable growth rates of human bladder in immunosuppressed mice. Brit. J. Cancer, 25: 838, 1971. tumors. The rates are different from those reported for other tumors and appear to be near a mid range on a scale of O to 11. Editorial: Testing anti-cancer drugs. Lancet, 1: 827, 1972. 100 per cent. Factors for this difference require further study 12. Povlsen, C. 0. and Rygaard, J.: Heterotransplantation of human adenocarcinomas of the colon and rectum to the mouse mutant before this model can be used confidently to assay the response nude. A study of nine consecutive transplantations. Acta to chemotherapeutic agents. Path. Microbiol. Scand., 79: 159, 1971. REFERENCES
1. Shimosato, Y., Kameya, T., Nagai, K., Hirohashi, S., Koide,
2. 3.
4. 5. 6. 7.
T., Hayashi, H. and Nomura, T.: Transplantation of human tumors in nude mice. J. Natl. Cancer Inst. 56: 1251, 1976. Artzt, K.: Breeding and husbandry of mice. Transplantation, 13: 547, 1972. Giovanella, B. C. and Stehlin, J. S.: Heterotransplantation of human malignant tumors in "nude" thymus-less mice. I. Breeding and maintenance of "nude" mice. J. Natl. Cancer Inst., 51: 615, 1973. Rygaard, J. and Povlsen, C. 0.: Heterotransplantation of a human malignant tumor to "nude" mice. Acta Path. Microbiol. Scand., 77: 758, 1969. Povlsen, C. 0. and Rygaard, J.: Heterotransplantation of human epidermoid carcinomas to the mouse mutint nude. Acta Path. Microbiol. Scand., §0: 713, 1972. Gershwin, M. E., Ikeda, R. M., Kawakami, T. G. and Owens, R. B.: Irnmunobiology of heterotransplanted human tumors in nude mice. J. Natl. Cancer Inst., 58: 1455, 1977. Trexler, P. C.: The use of plastics in the design of isolator systems. Ann. N. Y. Acad. Sci., 78: 29, 1959.
13. Povlsen, C. 0., Fialkow, P. J., Klein, E., Klein, G., Rygaard, J. and Wiener, F.: Growth and antigenic properties of a biopsyderived Burkitt's lymphoma in thymus-less (nude) mice. Int. J. Cancer, 11: 1973. 14. Connors, T. A. Phillips, B. J.: Commentary: screening for anti-cancer agents; the relative merits c • in vitro and in vivo techniques. Biochem. Pharmacol., 24: 2217, 1975. 15. Fogh, J., Fogh, J. M. and Orfeo, T.: One hundred and twentyseven cultured human tumor cell lines producing tumors in nude mice. J. ·Natl. Cancer Inst., 59: 221, 1977. 16. Cobb, L. M. and Mitchley, B. C. V.: The growth of human tumours in immune deprived mice. Eur. J. Cancer, 10: 473, 1974. 17. Mitchley, B. C. V., Clarke, S. A., Connors, T. A., Carter, S. M. and Neville, A. M.: Chemotherapy of human tumors in Tlymphocyte-deficient mice. Cancer Treat. Rep., 61: 451, 1977. 18. Franks, C. R., Turner, D. R., Bishop, D. and Perkins, F. T.: Growth characteristics of a human bladder tumour subcutaneously implanted in immune deficient mice. Clin. Oncol., 2: 25, 1976. 19. Elhilali, M. M. and Nayak, S. K.: Immunologic evaluation of human bladder cancer: in vitro studies. Cancer, 35: 419, 1975.
Vol. 121, February
Copyright © 1979 by The Williams & Wilkins Co.
Printed in U.S.A.
HETEROTRANSPLANTATION OF HUMAN TRANSITIONAL CELL CARCINOMA IN ATHYMIC MICE GERALD SUFRIN,* MICHAEL P. MCGARRY, AVERY A. SANDBERG
AND
GERALD P. MURPHYt
From the Roswell Park Memorial Institute, New Yark State Department of Health, Buffalo, New Yark
ABSTRACT
Human bladder tumors were obtained and transplanted into nude mice or other control animals. Tumor measurements, growth rate and selected histological studies were completed. No correlation between the growth of the tumor in the nude mouse and the clinical course or histologic tumor appearance in the host was detected. Metastases were not found. This feature has been noted generally to be uncommon in the nude mice model with some other human tumors. Despite careful technique tumor growth was achieved in only 40 per cent of the appropriate experiments. The factors responsible for this variability and different growth rate in the nude mouse require further assessment before the results of other experimental treatments can be evaluated. The need for in vivo models for the study of human tumors and for the study of the action of anticancer agents on these human cancers is well acknowledged. 1- 3 In vivo models using human tumors offer an important change in the study of tumor biology and therapeutic systems. Tumor xenografts provide an invaluable source of material for the study of fundamental and practical aspects of human cancer. 2- 4 However, heterotransplantation of malignant human tumors, as explants, generally results in rejection of the true graft because of cellular immunity, that is thymic-dependent lymphocyte function. 5 Exception to this occurs in cases in which the animal host has been rendered immunologically unresponsive, as by thymectomy, sublethal radiation or use of antisera directed against recipient lymphocytes, or when tumors are transplanted into immunologically unresponsive sites. 4• 5 The mutant nude mouse (nu/nu), which is immunologically incompetent because of thymic aplasia, can be of use in such studies and, therefore, offers a unique model for the in vivo study of human xenografts. Indeed, a variety of solid and hematologic tumors has been reported to grow in the nude host. 4 - 6 We describe studies undertaken to explore the growth pattern of human transitional cell carcinoma of the bladder in such congenitally immunodeficient mice. Heterotransplant of this tumor is of special interest because of the current clinical need for more effective cytotoxic agents. It also is important to study the biologic behavior and characterize bladder-tumor growth with special consideration to observe possible differences among various malignant lesions. We, therefore, studied the growth of transitional cell carcinoma in the athymic nude host in an effort to define growth parameters, possibly to develop a model for chemotherapy and the further study of bladder tumor biology by obtaining successful implantation and growth of such tumors in these hosts. MATERIALS AND METHODS
Mice. We used 6 to 8-week-old athymic nude (nu/nu) and normal heterozygotic (+/nu) litter mates. The immunologically competent litter mates were used to demonstrate typical rejection reactions of the human tissues at the site of implantation. The congenitally athymic, nude mice were obtained Accepted for publication April 21, 1978. * Current address: Waldheim Department of Surgery, The Jewish Hospital of St. Louis, 216 S. Kingshighway, P.O. Box 14109, St. Louis, Missouri 63178. t Requests for reprints: Roswell Park Memorial Institute, 666 Elm St., Buffalo, New York 14263.
from the mating of heterozygous (+/nu) female mice with homozygous (nu/nu) male mice, resulting in 50 per cent nu/nu offspring. Stock mice were obtained initially from Giovanella of the Stehlin Foundation and were bred according to his and other published recommendations. 2• 3 The animals were kept according to germ-free techniques in plastic isolators. They were maintained as specific pathogen free with a single, detectable contaminant, Klebsiella species. 1- 9 Patients. Patient data and information regarding location, tumor type and histology are shown in table 1. Tumor tissue was obtained via transurethral forcep biopsy from 20 patients. A 1 cu. mm. portion of each tumor was directly implanted subcutaneously. Other specimens of tumor tissue also were concurrently fixed in formalin, embedded in paraffin, sectioned at 7 µ,. and stained with hematoxylin and eosin for histological examination by light microscopy. Inoculation. Surgical specimens, aseptically preserved on ice in RPMl-1640 media, were minced to 1 mm. 3 in size and inoculated subcutaneously into the flank of the animal using a trocar needle. One to several tumor tissue fragments were implanted subcutaneously along the lateral aspect of the abdomen at 3 to 4 sites within 20 to 45 minutes after surgical excision. Usually tissue from 1 tumor was inoculated into 3 to 6 mice. Transplantation sites were observed for 2 weeks for tumor growth. The average age of host mice was 50 days, with a range of 42 to 90 days. Tumor take. Tumor growth was considered to have occurred: 1) when there was an observed increase in the size of implant at the inoculation site and 2) when the tissues were removed and shown as histologically identical to the original implant and contained mitotic figures. The absence of such evidence or the occurrence of a necrotic residue generally was considered as indicating graft rejection. Evaluation and measurement of tumor growth. Animals were kept for observation for a mean of 165 days (range of 85 to 180 days). Autopsies were performed on all animals. The tumors, or remnants thereof, were dissected free and weighed. Tissue sections were made of all tumors and from any host organs showing evidence of macroscopic change. The size of subcutaneous tumors was observed daily and measured weekly in 2 dimensions by calipers. Data are expressed as the product of the 2 maximal perpendicular diameters. New growths were removed and studied when they exceeded 1 cm. in diameter. RESULTS
Transplantability of tumors - tumor growth. Transitional cell carcinoma from 20 patients was transplanted directly into 159
160
SUFRIN AND ASSOCIATES
104 athymic mice (table 2). The tumor from each patient was implanted in 4 to 6 animals, depending on the amount of tissue available. Successful takes were observed with 40 per cent of the tumors and in 41 of the 104 athymic murine recipients. If a grafted tumor grew in its nude host 75 to 100 per cent of the other animals implanted with the same tumor also exhibited tumor growth. No tumors grew in the normal, heterozygous immunologically competent recipients, indicating no loss of tumor antigenicity. The clinical features of patients with transitional cell carcinoma tumors that were implanted in nude mice are shown in table 1. The stage of tumor, its histological grade, the multicentricity and its tendency to recur had no apparent influence on the frequency of successful takes. Also, the subsequent clinical prognosis was not related to the success or failure of grafts to take in the nude host. Growth characteristics of human tumors in nude mice. The hairless condition of the congenitally athymic host mice facilitates observation of subcutaneously inoculated tissue. By 24 hours after tumor implantation a mass 1 to 2 mm. in diameter is visible. The subcutaneous nodules gradually regressed in mice with inoculate that failed to grow and they were visible only as pale areas less than 1 mm. in diameter by 12 days after implantation. Histologic examination revealed fibrotic nodules. There appeared to be another critical period between day 20 and day 25 after inoculation in the athymic host, during which heterotransplants underwent a period of selection as a result of which there was either an increase in size or regression. The transitional cell carcinoma that grew appeared initially as a minute discrete nodule, which slowly and progressively enlarged. In all cases the tumor showed only local growth without metastases to lymph nodes or other host organs. Fully developed xenografts were smooth with the overlying skin intact and not adherent. On section the tumors were well defined in the subcutaneous space as an apparent encapsulated solid mass. Relatively large blood vessels were seen crossing and penetrating the tumors. The tumors were generally from superficial arteries that dilate and ramify concurrently with the tumor growth. Transplants developing in the nude mouse maintained cytological and histological appearances in full accordance with those of the inoculated primary tumor. In all cases the TABLE
1. Summary of patient data from which transitional cell
carcinomas were derived
Pt. average age
Growth (No.)
No Growth (No.)
62
57
Sex: Male Female Tumor stage:
6 2
9 3
Low (I-II)
3
High (ill-IV) Tumor grade:
5
6 6
6 2
3 9
3
5
2 10
4 4
7 5
(yrs.)
Low (I-II) High (ill-IV) Subsequent clinical course: Stable Progression Distribution of lesions: Solitary Multiple
TABLE
2. Transplantation of human transitional cell carcinoma into
nude mice
Tumors transplanted Nude mice receiving xenografts Furred controls receiving xenografts
Implants (No.)
Tumor Growth
No Tumor Growth
(%)
(%)
20
40 40
60 60 100
104 43
0
grafts were the same as the initial tumor, especially regarding histological grade of the tumor. Mitotic figures were observed. No metastatic lesions were found. Chromosome study. Chromosome analysis was done in the xenografts from 2 patients. All of the mitoses observed were of a human karyotype and no mouse chromosomes were present, eliminating the possibility of host tissue being incorporated into the tumor. The modal chromosome number was 48. A marker chromosome was present frequently. These findings from tumor in male nude mice correspond to chromosomal analysis of the parent tumor. DISCUSSION
Heterotransplantation of malignant tumors to experimental animals normally results in rejection oftumor. 4 - 6 Well known exceptions from this general rule are cases in which the animal host has been made immunologically unresponsive (neonatal thymectomy, sublethal x-ray irradiation, steroid treatment or induction of immunological tolerance) or when implants are made into immunologically unresponsive locations (anterior eye chamber, hamster cheek pouch or outside the blood barrier). 6• 10 - 14 Tumor xenografting into nude mice provides a novel opportunity to study the biologic behavior of neoplastic tissue in the absence of normal host response. 6 Nude mice appear unable to reject such tumors because of their deficient thymus-dependent immune system. 6 The difficulties of using cell cultures in vitro for the purpose of developing effective drugs and chemotherapeutic regimens may be summarized as follows: 1) changes in metabolism imposed by growth in culture media may modify the effects of drugs, 2) in vitro experiments do not take into account in vivo disposition and metabolism of drugs and 3) rapid selection of particular tumor cell clones adapted to growth in vitro may occur and these may be unrepresentative of the tumor cell population in vivo. 10• 11 The relatively slow rate of growth of the transplanted bladder tumors observed in this study in the subcutaneous implants contrasts with the more typical rapidly proliferating anaplastic animal tumors. This behavior of the transplanted tumor is relevant since much tumor growth in the patient may be similar. 15 Our findings are in agreement with those of others who have reported that tumors implanted into athymic mice or immune-deprived mice do not always grow and certain tumors are more likely to take than others. 16 There appears to be a critical period between 20 and 25 days, during which the implanted bladder tumor undergoes a process of selection. The tumor growth rates observed in our study varied from active growth to mere persistence in an arrested survival state. This suggests, in our opinion, that the rate of human xenograft development is controlled by other unidentified intrinsic cellular properties of the tumor. Since we have used this technique for some time in our laboratory we do not believe that these variations are artifactual. The rate of tumor takes in this study did not seem related to tumor grade, stage, the response to treatment of the patient or frequency of such tumors. Mitchley and associates reported that many different types of human cancers have now been grown successfully in either mice that have been artificially deprived ofT-lymphocytes (B mice) or in nude athymic mice. 16• 17 Many of the tumors grown in this way have been transplanted for several generations but few metastasize. Transplantability usually has been found to be highest for carcinoma of the colon and lung and lowest for breast tumors. 3 Tumor growth rates in our study vary from exponential to persistence of tumor in an arrested survival state. Franks and associates, in a similar study of human bladder tumor material, noted that there appears to be a critical period between day +2 and day +25, during which the implanted bladder tumor undergoes a process of selection as a result of which there is either an increase in size or regres-
HETEROTRANSPLANTATION OF HUMAN TRANSITIONAL CELL CARCINOMA IN ATHYMIC MICE
161
sion. 18 Franks and associates observed that it is not clear what 8. Mirand, E. A., Gordon, A. S., Zanjani, E. D., Bennett, T. E. and Murphy, G. P.: Disappearance of exogenous erythropoietin determines the successful establishment of an implanted blad(ESF) from the blood of germfree mice. Proc. Soc. Exp. Biol. der tumor or why some tumor types preferentially survive in Med., 139: 161, 1972. a xenograft system. 18 In a previous study by Elhilali and 9. McGarry, M. P., Bahny, L., Jividen, J., Williams, P., Mirand, Nayak the success rate of growing bladder tumor tissue in E. A. and Murphy, G. P.: Particulate matter induced periortissue culture could not be correlated with the stage of the bital abscess in gnotobiotic athymic nu/nu (nude) mice. Lab. 19 tumor. Anim. Sci., 26: 956, 1976. This study has shown that the nude mouse can be used to 10. Sheard, C. E., Double, J. A. and Berenbaum, M. C.: The sensitivity to chemotherapeutic agents of a rat tumour grown demonstrate the variable growth rates of human bladder in immunosuppressed mice. Brit. J. Cancer, 25: 838, 1971. tumors. The rates are different from those reported for other tumors and appear to be near a mid range on a scale of O to 11. Editorial: Testing anti-cancer drugs. Lancet, 1: 827, 1972. 100 per cent. Factors for this difference require further study 12. Povlsen, C. 0. and Rygaard, J.: Heterotransplantation of human adenocarcinomas of the colon and rectum to the mouse mutant before this model can be used confidently to assay the response nude. A study of nine consecutive transplantations. Acta to chemotherapeutic agents. Path. Microbiol. Scand., 79: 159, 1971. REFERENCES
1. Shimosato, Y., Kameya, T., Nagai, K., Hirohashi, S., Koide,
2. 3.
4. 5. 6. 7.
T., Hayashi, H. and Nomura, T.: Transplantation of human tumors in nude mice. J. Natl. Cancer Inst. 56: 1251, 1976. Artzt, K.: Breeding and husbandry of mice. Transplantation, 13: 547, 1972. Giovanella, B. C. and Stehlin, J. S.: Heterotransplantation of human malignant tumors in "nude" thymus-less mice. I. Breeding and maintenance of "nude" mice. J. Natl. Cancer Inst., 51: 615, 1973. Rygaard, J. and Povlsen, C. 0.: Heterotransplantation of a human malignant tumor to "nude" mice. Acta Path. Microbiol. Scand., 77: 758, 1969. Povlsen, C. 0. and Rygaard, J.: Heterotransplantation of human epidermoid carcinomas to the mouse mutint nude. Acta Path. Microbiol. Scand., §0: 713, 1972. Gershwin, M. E., Ikeda, R. M., Kawakami, T. G. and Owens, R. B.: Irnmunobiology of heterotransplanted human tumors in nude mice. J. Natl. Cancer Inst., 58: 1455, 1977. Trexler, P. C.: The use of plastics in the design of isolator systems. Ann. N. Y. Acad. Sci., 78: 29, 1959.
13. Povlsen, C. 0., Fialkow, P. J., Klein, E., Klein, G., Rygaard, J. and Wiener, F.: Growth and antigenic properties of a biopsyderived Burkitt's lymphoma in thymus-less (nude) mice. Int. J. Cancer, 11: 1973. 14. Connors, T. A. Phillips, B. J.: Commentary: screening for anti-cancer agents; the relative merits c • in vitro and in vivo techniques. Biochem. Pharmacol., 24: 2217, 1975. 15. Fogh, J., Fogh, J. M. and Orfeo, T.: One hundred and twentyseven cultured human tumor cell lines producing tumors in nude mice. J. ·Natl. Cancer Inst., 59: 221, 1977. 16. Cobb, L. M. and Mitchley, B. C. V.: The growth of human tumours in immune deprived mice. Eur. J. Cancer, 10: 473, 1974. 17. Mitchley, B. C. V., Clarke, S. A., Connors, T. A., Carter, S. M. and Neville, A. M.: Chemotherapy of human tumors in Tlymphocyte-deficient mice. Cancer Treat. Rep., 61: 451, 1977. 18. Franks, C. R., Turner, D. R., Bishop, D. and Perkins, F. T.: Growth characteristics of a human bladder tumour subcutaneously implanted in immune deficient mice. Clin. Oncol., 2: 25, 1976. 19. Elhilali, M. M. and Nayak, S. K.: Immunologic evaluation of human bladder cancer: in vitro studies. Cancer, 35: 419, 1975.