THE JOURNAL
Vol. 115, April
OF UROLOGY
Copyrii;ht © 1976 by The Williams & Wilkins Co.
Printed in U.S.A.
TRANSPLANTATION OF HUMAN RENAL CELL CARCINOMA TO THE NUDE MICE: AS AN INTERMEDIATE OF IN VIVO AND IN VITRO STUDIES YO,JI KATSUOKA, SHIRO BABA, MAKOTO HATA
AND
HIROSHI TAZAKI
From the Department of Urology, School of Medicine, Keio UniuNsity, Tokyo, Japan
ABSTRACT
Five human renal cell carcmomas were transplanted mice fed and grown condition. The of the tumor tissue was a close human cancer tissue in situ. The tumor tissue uv"°""'"' which was considered to be of host animal All from the nude mice. In this characteristic of features that indicated their Despite the recent accumulation of basic and clinical studies on human renal cell carcinoma as well as on other malignant tumors, its clinical and pathological features still remain obscure. Estabiishment of a cell line from human renal cell carcinoma, 1 of the future tools of biological studies for this clinical entity, has been extremely difficult. Herein we report on the successful transplantation of renal cell carcinoma to nude mice and the subsequent cell culture in vitro. MATERIALS A.'lD METHODS
Direct cell culture. The 24 cases of human renal cell carcinoma ( l from pulmonary metastasis and the others from primary lesions in the kidney) were submitted to primary cell culture, conventional histology and electron microscopy. Specimens for cell culture were minced with scissors, (0.25 per cent for 15 minutes) and then incubated at 37C. The number of cells at primary culture was 5 10' per ml. or more. The culture medium with 10 per cent calf serum and antibiotics (100 units penicillin and 100 mg. streptomycin per 100 ml.) was adjusted at pH 7 .3. Cultures were observed with a phase contrast microscope and some of them were sacrificed for histology and electron microscopy. Transplantation to the nude mice. Inbred 7 to 8-week-old male nude mice were used throughout the experiments. The surgical specimens, aseptically preserved on ice, were minced into 0.1 cm.3 in size (approximately 5 by 10 6 cells) and inoculated in subcutaneous space on the back and flank of the animal using a trocar needle. The tissue fragments, 2 to 3 pieces in 1 shot, were inoculated at ;3 to 4 areas. Three animals, not pre-treated with immunosuppression, were used in each experiment. The animals were fed and grown under specific pathogen-free conditions and we observed the fate of the inoculated tissue fragments. When new growths more than 1 cm. in diameter developed they were removed and aseptically transferred to another nude mouse in the same fashion as in the primary inoculation. Each specimen was examined by histology and electron microscopy. Cell culture from vowths in nude mice. The method of cell culture previously described was applied to the specimens transferred. If the culture ,vas successful chromosomal was performed along with conventional histology and electron Accernea for publication ,June 20. 19,.'i. at annual of American l:rolugical Association, l\iiian1i Beach. ?lorida, May 11 19,5.
RESULTS
Direct cell culture. Seven epithelial growths were maintained more than 4 weeks from 24 cases (fig. 1, A). Five were from clear cell carcinoma and 2 from granular cell carcinoma. All but 1 that altered the morphology from epithelial to fibrous were characterized a variety of degenerative changes, such as deformity, enlargement, flattening and fewer granules (fig. 1, B) and terminated their growth within :12 to 198 Transplantation to the nude mice. Results of successful transplantation are shown in the table. Five primary specimens formec! new growths visible under the hairless skin of the mice (fig. 2). Four cases were apparently absorbed from each inoculated site and designated as negative. Two of 5 successful cases were of a granular cell type and the remaining 3 were of a clear cell type. All 5 were from male patients, while 2 of 4 negative cases were from female patients. The time from surgical removal to transplantation ranged from 1 to 4.5 hours, while the successful cases were confined between 3 to 4.5 hours. The size of growths varied from 1.;3 to :3 cm. Most of them showed round multilobar protrusions, pinkish blue in color. Histology of the successful transplants in mice exactly reproduced the original patterns (fig. 3, A), for example 2 granular cell types and ;3 clear cell types. These tumor cells have a large cytoplasm with a round, relatively small nucleus and form cell sheets along with fine connective tissues with abundant capillaries. These capillaries are probably derived from animal blood vessels and increase in number as the transplanted tumor cells grow (fig. :3, H). There was no evidence of direct vascular invasion by the tumor cells. No distant metastases were recognized in sacrificed animals. Electron microscopic studies also confirmed that the transplanted tumor cells reproduced the original characteristics within the cytoplasm, including abundant mitochondria, glucose and lipid granules. The intercellular structure that resembled closely the brush borders of renal proximal tubules was recognized in some cases (fig. 4). T. N. was a man in whom pulmonary metastasis of renal cell carcinoma was resected surgically 2 1 , years after the primary renal tumor had been removed. The hemoglobin before than 18 gm. per dl. and A nude mouse was transand the autopsy shovved evidenc2 of 1n man.:,,; visceral organs and bone marrow.
374
KATSUOKA AND ASSOCIATES
Cell culture from growths in nude mice. Three of 5 growths in nude mice have been transferred successfully in cell culture. They form epithelial type colonies that are composed of large square-shaped cells in the outer zone (fig. 5, A), while cells in the middle tend to pile up (fig. 5, B). Each cell contains dense granules in cytoplasm and a relatively small nucleus. Electron microscopic observation revealed a large number of mitochondria and well developed endoplasmic reticulum in the cytoplasm. Also, characteristic intercellular junctions like the brush borders could be observed. Radial projections of microvilli to free space were seen in most outerzone cells (fig. 6). Chromosomal analysis of the 3 cell strains showed a variety of numbers with a suspected marker chromosome in 1 case (fig. 7). In 1 case the culture cells proliferated in a stable condition after surviving the fifth passage.
FIG. 1. A, successful direct cell culture from clear cell carcinoma after 30 days. Epithelial cells are growing as sheet. Cytoplasm contains large numbers of dense granules. B, direct cell culture after 152 days shows degenerating huge square cells.
FIG. 2. Nude mouse with successful transplants. New growths are easily visible under hairless skin. Measured 2.2 by 0.7 by 0.5 cm.
FIG. 3. A, histology of clear cell carcinoma from patient I. H. B, histology of new growth in nude mouse reproduced original patterns of clear cell carcinoma. H & E, reduced from x200.
Transplantation to nude mice Pt.-Age-Sex TF -:lS-M IH -65-M JY -72-M JY -31-F ST -54-F ST -69-M TN-67-M M0-71-M KY-54-M
Original Histology
Time Before Inoculation (hrs.)
Take
Number of Transfers
Histology of Transplants
Granular cell Clear cell Clear cell Clear cell Clear cell Granular cell Clear cellt Clear cell Clear cell
4.0 4.0 3.2
+ +
3 3
Granular*
;3 2
Granular Clear Clear
Clear
1.0
4.0
:3.5 4.5 3.0 4.5
+ +
1
* Sarcomatous in part. t Pulmonary metastasis.
,
__
TRANSPLANTATION OF RENAL CELL CARCINOMA TO MICE
375
FIG. 5. Cell culture from growth in nude mice. A, large squareshaped cells in outer zone after 15 days. B, central part of colony tends to pile up after 139 days.
Fi(;. 4. Electron microphotographs from new growth in nude mouse confirmed characteristic structure resembling brush borders of renal proximal tubules that had been transferred from human to animal. N. nucleus. mitochondrion. ER, endoplasmic reticulum. V, vacuole. BB, brush like structure.
DISCUSS101'
In recent years light has been shed on cell culture. 1 of the most feasible methods of disclosing the veil on human cancers. Cultures of human renal cancer cells have been attempted by various investigators. 1 · 5 They showed that prolonged in vitro culture of human renal cell carcinoma is possible. However, not included in the works of these authors was the study of established cell lines of human renal cell cancer. We have encountered extreme difficulties in trying to establish cell lines of tumor in vitro, although maintenance of primary culture alone for a sufficiently long period of time was generally feasible. In 1961 the discovery of nude mice was reported by Flanagan. 6 Little attention was paid to these hairless animals until the evidence of a congenital defect of the thymus in these animals was demonstrated by Pantelouris. 7 The latter discovery encouraged cancer researchers to make use of the animal in experimental A number of human malignant neoplasms have been successfully transplanted to nude mice and Povlsen 8 and others since 1969. At the first lntemational Workshop on Nude Mice held in 197:3 the general characteristics of the were recognized and confirmed: 0 1) original structures and functions are maintained of h ~nnan cancers t.o ~.he nud€ :tLice~ 2) if 1
FIG. 6. Electron microphotograph of cultured cells transferred in vitro from growth in nude mice. Radial projections of microvilli to free space appear as brush-border like structures at intercellular junction.
mice, even cultured cells restore their original structures and functions that have been lost during the in vitro processes, :3) the more differentiated the easier are epithelial tumors to grow in nude mice, 4) metastases from transplanted tumor are seen on rare occasions. Our results are essentially in agreement with these We have demonstrated that the structural charsct.eristics of hv,man rencd cell carcinnr.na is v-1ell rna:n-
376
KATSUOKA AND ASSOCIATES
ix1 tK11111 au
li~AIIIIII
B (4-5) l\UIIJ\l&R1'1AIJAU.lll,•Mlllflal\lli C ( 6- x-12)
1
2
.......,....
8(8-15)
E16
E(17-18)
................ F (19-20)
G (21-22)
"
V
Fm. 7. Chromosomal analysis from patient T. F.
tained after transplantation to nude mice. Furthermore, morphological findings indicated that the transplanted tumor tissue is supplied by the blood vessels that are probably of the host animal origin. The presence of blood vessels of host origin in the transplanted tumor tissue has been demonstrated also with choriocarcinoma, which is by nature a blood vessel-rich tumor. In the case of choriocarcinoma the inoculation of pure cultured cells into nude mice results in abundant neogenesis of blood vessels within the growths that are in fact of host animal origin. It is easily postulated that the presence of blood vessel supply plays a critical role in the maintenance of the transplanted tumor cell growth in nude mice. It is also easily understandable that in vitro cell culture is difficult because of lack of comparable blood vessel supply. We herein demonstrate the great advantage of using nude mice in human cancer research. In the present study electron microscopy confirmed the presence of the ultrafine structure, which resembled closely the brush borders of the renal proximal tubular cells, on the tumor cefls transplanted to nude mice. This, we believe, is proof that the original structural characteristics of renal cell carcinoma have been preserved throughout serial experimental manipulations, including transplantation to nude mice and subsequent passing of the strain to the in vitro cell culture system. 10 Erythrocytosis in human renal cell carcinoma is 1 of the most interesting clinical manifestations of the disease occurring in 3 to 5 per cent of the cases. Of great interest was that erythrocytosis was induced in the host animal by the successful transplantation of the surgical specimen of T. N., in whom pulmonary metastases oi renal cell carcinoma were resected surgically and whose hemoglobin before the prior nephrectomy
was more than 18 gm. per dl. Erythropoietin assay of the transplanted tumor tissue and of the medium of the subsequent cell culture is currently in progress. We are expecting that the result of the assay will not only provide direct proof that the transplanted and the cultured cells are of human renal cell carcinoma origin but also will demonstrate that the functional characteristics of the original tumor cells are preserved throughout transplantation to nude mice and culture in vitro later. REFERENCES
1. Richter, K. M. and Akin, R. H.: The cultivation of several genitourinary tract tumors. Trans. South Central Sect. Amer. Urol. Ass., p. 67, October 1957. 2. Bregman, R. V. and Bregman, E.T.: Tissue culture of benign and malignant human genitourinary tumors. J. Urol., 86: 642, 1961. 3. Jones, G. W.: Primary and metastatic epithelial tumors of the human kidney and bladder in tissue culture. Cancer, 20: 1893, 1967. 4. Shore, B., Wise, G. J., McBride, R. A. and Brendler, H.: Tissue culture of human renal cell carcinoma: effect of serum on monolayer growth. J. Urol., 103: 554, 1970. 5. Cummings, K. B., Peter, J. B. and Kaufman, J. J.: Cytotoxic serum factors in patients with renal cell carcinoma. J. Urol., 111: 330, 1974. 6. Flanagan, S. P.: "Nude" a new hairless gene with pleiotropic effects in the mouse. Genet. Res., 8: 295, 1966. 7. Pantelouris, E. M.: Absence of thymus in a mouse mutant. Nature, 217: 370, 1968. 8. Rygaard, J. and Povlsen, C. 0.: Heterotransplantation of a human malignant tumor to "nude" mice. Acta Path. Microbiol. Scand., 77: 758, 1969. 9. Rygaard, J. and Povlsen, J.: Proceedings of the First International Workshop on Nude Mice. Gustavfisher Verlag, Stuttgart, 1974. 10. Seljelid, R. and Ericsson, J. L. E.: Electron microscopic observations on specializations of the cell surface in renal clear cell carcinoma. Lab. Invest., 14: 435, 1965. COMMENT These authors have demonstrated that renal cancer, like many other human tumors, can be grown in nude mice. They imply that the technique may facilitate study of the cellular biology of this tumor, especially since it is difficult to maintain renal cancer in culture. Nude mice have a genetic defect that renders them hairless and athymic. Consequently, thEy often will accept heterologous grafts. However, the nude mice are difficult and expensive to maintain, and they are susceptible to generalized infections because of their immunodeficiency. Therefore, I suspect that extensive studies on renal cancer in this laboratory model may not be practical. Furthermore, recent reports suggest that it is now possible to establish human renal cancers in long-term monolayer cultures and, in fact, several such long-term cell lines are now available. E.E.F.
Vol. 115, April
OF UROLOGY
Copyrii;ht © 1976 by The Williams & Wilkins Co.
Printed in U.S.A.
TRANSPLANTATION OF HUMAN RENAL CELL CARCINOMA TO THE NUDE MICE: AS AN INTERMEDIATE OF IN VIVO AND IN VITRO STUDIES YO,JI KATSUOKA, SHIRO BABA, MAKOTO HATA
AND
HIROSHI TAZAKI
From the Department of Urology, School of Medicine, Keio UniuNsity, Tokyo, Japan
ABSTRACT
Five human renal cell carcmomas were transplanted mice fed and grown condition. The of the tumor tissue was a close human cancer tissue in situ. The tumor tissue uv"°""'"' which was considered to be of host animal All from the nude mice. In this characteristic of features that indicated their Despite the recent accumulation of basic and clinical studies on human renal cell carcinoma as well as on other malignant tumors, its clinical and pathological features still remain obscure. Estabiishment of a cell line from human renal cell carcinoma, 1 of the future tools of biological studies for this clinical entity, has been extremely difficult. Herein we report on the successful transplantation of renal cell carcinoma to nude mice and the subsequent cell culture in vitro. MATERIALS A.'lD METHODS
Direct cell culture. The 24 cases of human renal cell carcinoma ( l from pulmonary metastasis and the others from primary lesions in the kidney) were submitted to primary cell culture, conventional histology and electron microscopy. Specimens for cell culture were minced with scissors, (0.25 per cent for 15 minutes) and then incubated at 37C. The number of cells at primary culture was 5 10' per ml. or more. The culture medium with 10 per cent calf serum and antibiotics (100 units penicillin and 100 mg. streptomycin per 100 ml.) was adjusted at pH 7 .3. Cultures were observed with a phase contrast microscope and some of them were sacrificed for histology and electron microscopy. Transplantation to the nude mice. Inbred 7 to 8-week-old male nude mice were used throughout the experiments. The surgical specimens, aseptically preserved on ice, were minced into 0.1 cm.3 in size (approximately 5 by 10 6 cells) and inoculated in subcutaneous space on the back and flank of the animal using a trocar needle. The tissue fragments, 2 to 3 pieces in 1 shot, were inoculated at ;3 to 4 areas. Three animals, not pre-treated with immunosuppression, were used in each experiment. The animals were fed and grown under specific pathogen-free conditions and we observed the fate of the inoculated tissue fragments. When new growths more than 1 cm. in diameter developed they were removed and aseptically transferred to another nude mouse in the same fashion as in the primary inoculation. Each specimen was examined by histology and electron microscopy. Cell culture from vowths in nude mice. The method of cell culture previously described was applied to the specimens transferred. If the culture ,vas successful chromosomal was performed along with conventional histology and electron Accernea for publication ,June 20. 19,.'i. at annual of American l:rolugical Association, l\iiian1i Beach. ?lorida, May 11 19,5.
RESULTS
Direct cell culture. Seven epithelial growths were maintained more than 4 weeks from 24 cases (fig. 1, A). Five were from clear cell carcinoma and 2 from granular cell carcinoma. All but 1 that altered the morphology from epithelial to fibrous were characterized a variety of degenerative changes, such as deformity, enlargement, flattening and fewer granules (fig. 1, B) and terminated their growth within :12 to 198 Transplantation to the nude mice. Results of successful transplantation are shown in the table. Five primary specimens formec! new growths visible under the hairless skin of the mice (fig. 2). Four cases were apparently absorbed from each inoculated site and designated as negative. Two of 5 successful cases were of a granular cell type and the remaining 3 were of a clear cell type. All 5 were from male patients, while 2 of 4 negative cases were from female patients. The time from surgical removal to transplantation ranged from 1 to 4.5 hours, while the successful cases were confined between 3 to 4.5 hours. The size of growths varied from 1.;3 to :3 cm. Most of them showed round multilobar protrusions, pinkish blue in color. Histology of the successful transplants in mice exactly reproduced the original patterns (fig. 3, A), for example 2 granular cell types and ;3 clear cell types. These tumor cells have a large cytoplasm with a round, relatively small nucleus and form cell sheets along with fine connective tissues with abundant capillaries. These capillaries are probably derived from animal blood vessels and increase in number as the transplanted tumor cells grow (fig. :3, H). There was no evidence of direct vascular invasion by the tumor cells. No distant metastases were recognized in sacrificed animals. Electron microscopic studies also confirmed that the transplanted tumor cells reproduced the original characteristics within the cytoplasm, including abundant mitochondria, glucose and lipid granules. The intercellular structure that resembled closely the brush borders of renal proximal tubules was recognized in some cases (fig. 4). T. N. was a man in whom pulmonary metastasis of renal cell carcinoma was resected surgically 2 1 , years after the primary renal tumor had been removed. The hemoglobin before than 18 gm. per dl. and A nude mouse was transand the autopsy shovved evidenc2 of 1n man.:,,; visceral organs and bone marrow.
374
KATSUOKA AND ASSOCIATES
Cell culture from growths in nude mice. Three of 5 growths in nude mice have been transferred successfully in cell culture. They form epithelial type colonies that are composed of large square-shaped cells in the outer zone (fig. 5, A), while cells in the middle tend to pile up (fig. 5, B). Each cell contains dense granules in cytoplasm and a relatively small nucleus. Electron microscopic observation revealed a large number of mitochondria and well developed endoplasmic reticulum in the cytoplasm. Also, characteristic intercellular junctions like the brush borders could be observed. Radial projections of microvilli to free space were seen in most outerzone cells (fig. 6). Chromosomal analysis of the 3 cell strains showed a variety of numbers with a suspected marker chromosome in 1 case (fig. 7). In 1 case the culture cells proliferated in a stable condition after surviving the fifth passage.
FIG. 1. A, successful direct cell culture from clear cell carcinoma after 30 days. Epithelial cells are growing as sheet. Cytoplasm contains large numbers of dense granules. B, direct cell culture after 152 days shows degenerating huge square cells.
FIG. 2. Nude mouse with successful transplants. New growths are easily visible under hairless skin. Measured 2.2 by 0.7 by 0.5 cm.
FIG. 3. A, histology of clear cell carcinoma from patient I. H. B, histology of new growth in nude mouse reproduced original patterns of clear cell carcinoma. H & E, reduced from x200.
Transplantation to nude mice Pt.-Age-Sex TF -:lS-M IH -65-M JY -72-M JY -31-F ST -54-F ST -69-M TN-67-M M0-71-M KY-54-M
Original Histology
Time Before Inoculation (hrs.)
Take
Number of Transfers
Histology of Transplants
Granular cell Clear cell Clear cell Clear cell Clear cell Granular cell Clear cellt Clear cell Clear cell
4.0 4.0 3.2
+ +
3 3
Granular*
;3 2
Granular Clear Clear
Clear
1.0
4.0
:3.5 4.5 3.0 4.5
+ +
1
* Sarcomatous in part. t Pulmonary metastasis.
,
__
TRANSPLANTATION OF RENAL CELL CARCINOMA TO MICE
375
FIG. 5. Cell culture from growth in nude mice. A, large squareshaped cells in outer zone after 15 days. B, central part of colony tends to pile up after 139 days.
Fi(;. 4. Electron microphotographs from new growth in nude mouse confirmed characteristic structure resembling brush borders of renal proximal tubules that had been transferred from human to animal. N. nucleus. mitochondrion. ER, endoplasmic reticulum. V, vacuole. BB, brush like structure.
DISCUSS101'
In recent years light has been shed on cell culture. 1 of the most feasible methods of disclosing the veil on human cancers. Cultures of human renal cancer cells have been attempted by various investigators. 1 · 5 They showed that prolonged in vitro culture of human renal cell carcinoma is possible. However, not included in the works of these authors was the study of established cell lines of human renal cell cancer. We have encountered extreme difficulties in trying to establish cell lines of tumor in vitro, although maintenance of primary culture alone for a sufficiently long period of time was generally feasible. In 1961 the discovery of nude mice was reported by Flanagan. 6 Little attention was paid to these hairless animals until the evidence of a congenital defect of the thymus in these animals was demonstrated by Pantelouris. 7 The latter discovery encouraged cancer researchers to make use of the animal in experimental A number of human malignant neoplasms have been successfully transplanted to nude mice and Povlsen 8 and others since 1969. At the first lntemational Workshop on Nude Mice held in 197:3 the general characteristics of the were recognized and confirmed: 0 1) original structures and functions are maintained of h ~nnan cancers t.o ~.he nud€ :tLice~ 2) if 1
FIG. 6. Electron microphotograph of cultured cells transferred in vitro from growth in nude mice. Radial projections of microvilli to free space appear as brush-border like structures at intercellular junction.
mice, even cultured cells restore their original structures and functions that have been lost during the in vitro processes, :3) the more differentiated the easier are epithelial tumors to grow in nude mice, 4) metastases from transplanted tumor are seen on rare occasions. Our results are essentially in agreement with these We have demonstrated that the structural charsct.eristics of hv,man rencd cell carcinnr.na is v-1ell rna:n-
376
KATSUOKA AND ASSOCIATES
ix1 tK11111 au
li~AIIIIII
B (4-5) l\UIIJ\l&R1'1AIJAU.lll,•Mlllflal\lli C ( 6- x-12)
1
2
.......,....
8(8-15)
E16
E(17-18)
................ F (19-20)
G (21-22)
"
V
Fm. 7. Chromosomal analysis from patient T. F.
tained after transplantation to nude mice. Furthermore, morphological findings indicated that the transplanted tumor tissue is supplied by the blood vessels that are probably of the host animal origin. The presence of blood vessels of host origin in the transplanted tumor tissue has been demonstrated also with choriocarcinoma, which is by nature a blood vessel-rich tumor. In the case of choriocarcinoma the inoculation of pure cultured cells into nude mice results in abundant neogenesis of blood vessels within the growths that are in fact of host animal origin. It is easily postulated that the presence of blood vessel supply plays a critical role in the maintenance of the transplanted tumor cell growth in nude mice. It is also easily understandable that in vitro cell culture is difficult because of lack of comparable blood vessel supply. We herein demonstrate the great advantage of using nude mice in human cancer research. In the present study electron microscopy confirmed the presence of the ultrafine structure, which resembled closely the brush borders of the renal proximal tubular cells, on the tumor cefls transplanted to nude mice. This, we believe, is proof that the original structural characteristics of renal cell carcinoma have been preserved throughout serial experimental manipulations, including transplantation to nude mice and subsequent passing of the strain to the in vitro cell culture system. 10 Erythrocytosis in human renal cell carcinoma is 1 of the most interesting clinical manifestations of the disease occurring in 3 to 5 per cent of the cases. Of great interest was that erythrocytosis was induced in the host animal by the successful transplantation of the surgical specimen of T. N., in whom pulmonary metastases oi renal cell carcinoma were resected surgically and whose hemoglobin before the prior nephrectomy
was more than 18 gm. per dl. Erythropoietin assay of the transplanted tumor tissue and of the medium of the subsequent cell culture is currently in progress. We are expecting that the result of the assay will not only provide direct proof that the transplanted and the cultured cells are of human renal cell carcinoma origin but also will demonstrate that the functional characteristics of the original tumor cells are preserved throughout transplantation to nude mice and culture in vitro later. REFERENCES
1. Richter, K. M. and Akin, R. H.: The cultivation of several genitourinary tract tumors. Trans. South Central Sect. Amer. Urol. Ass., p. 67, October 1957. 2. Bregman, R. V. and Bregman, E.T.: Tissue culture of benign and malignant human genitourinary tumors. J. Urol., 86: 642, 1961. 3. Jones, G. W.: Primary and metastatic epithelial tumors of the human kidney and bladder in tissue culture. Cancer, 20: 1893, 1967. 4. Shore, B., Wise, G. J., McBride, R. A. and Brendler, H.: Tissue culture of human renal cell carcinoma: effect of serum on monolayer growth. J. Urol., 103: 554, 1970. 5. Cummings, K. B., Peter, J. B. and Kaufman, J. J.: Cytotoxic serum factors in patients with renal cell carcinoma. J. Urol., 111: 330, 1974. 6. Flanagan, S. P.: "Nude" a new hairless gene with pleiotropic effects in the mouse. Genet. Res., 8: 295, 1966. 7. Pantelouris, E. M.: Absence of thymus in a mouse mutant. Nature, 217: 370, 1968. 8. Rygaard, J. and Povlsen, C. 0.: Heterotransplantation of a human malignant tumor to "nude" mice. Acta Path. Microbiol. Scand., 77: 758, 1969. 9. Rygaard, J. and Povlsen, J.: Proceedings of the First International Workshop on Nude Mice. Gustavfisher Verlag, Stuttgart, 1974. 10. Seljelid, R. and Ericsson, J. L. E.: Electron microscopic observations on specializations of the cell surface in renal clear cell carcinoma. Lab. Invest., 14: 435, 1965. COMMENT These authors have demonstrated that renal cancer, like many other human tumors, can be grown in nude mice. They imply that the technique may facilitate study of the cellular biology of this tumor, especially since it is difficult to maintain renal cancer in culture. Nude mice have a genetic defect that renders them hairless and athymic. Consequently, thEy often will accept heterologous grafts. However, the nude mice are difficult and expensive to maintain, and they are susceptible to generalized infections because of their immunodeficiency. Therefore, I suspect that extensive studies on renal cancer in this laboratory model may not be practical. Furthermore, recent reports suggest that it is now possible to establish human renal cancers in long-term monolayer cultures and, in fact, several such long-term cell lines are now available. E.E.F.
