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Growth of Primary Human Renal Cell Carcinoma on Embryonic Chick Skin in Organ Culture
0022-5347 /82/1285-1068$02.00/0 Vol. 128, November Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1982 by The Williams & Wilkins Co.
GROWTH OF PRIMARY HUMAN RENAL CELL CARCINOMA ON EMBRYONIC CHICK SKIN IN ORGAN CULTURE RUSSELL K. LAWSON,* STEPHEN C. JACOBS
AND
ELLIOT SILBAR
From the Department of Urology, The Medical College of Wisconsin, Milwaukee, Wisconsin
ABSTRACT
Embryonic chick skin was used as a biologic support medium for the growth of 2 human genital tract tumor cell lines and 3 primary human renal cell carcinomas. Three of 4 primary renal cell carcinomas invaded the dermal layer of the chick skin in organ culture. Additional studies are needed to determine if growth and invasion of the primary tumor correlates with the malignant potential of that tumor. The model may also be useful for studying the effect of various chemotherapeutic agents against individual human neoplasms. Animal models for the growth of human tumors are of great importance in many areas of cancer research. Transplantable or induced primary animal tumors have been used extensively to study the metabolic activity of these neoplasms and their response to various immunologic and chemotherapeutic manipulations. All of these studies suffer from the fact that animal tumors often do not behave as their counterparts in humans do. The discovery of the nude mouse as a biologic support system for the growth of human tumors was a major advance in the study of human cancers. 1- 3 Several other animal systems have been developed that depend on immunosuppression in the host animal for the growth of human tumors. 4 - 6 We reported such a tumor model several years ago that employed neonatal thymectomy and antilymphocyte serum in the newborn rat to provide the necessary degree of immunosuppression. 7 Human tumor cell lines grow rapidly in this model, deeply invade surrounding tissue and metastasize to multiple organs. Invasion of surrounding tissue is considered to be an important indicator of malignancy, as well as metastasis to other sites in the animal. Because invasion is one of the important characteristics of malignancy, tissue and organ culture systems have been developed to look at this parameter alone. Several papers have appeared describing the use of embryonic chick skin (ECS) as a biologic support medium for studying the invasive potential of various tumors and cell lines. 8- 10 In this study, we planned to determine if primary human renal cell carcinoma could be grown on embryonic chick skin and if the invasive potential of the tumor could be assessed. In order to determine if the model would work in our laboratory and also to serve as controls, we placed human cell lines H494 (benign prostatic hyperplasia) and DoT (cervical carcinoma) in this same organ culture system. 11 • 12
embryos were decapitated and the heads discarded. Approximately equal volumes of embryos and EBSS were placed in a Vertis tissue homogenizer and homogenized at medium to high speed, with care taken not to allow excessive foaming of the mixture. The extract was then allowed to stand overnight at 4C before centrifugation in the cold at 4,000 rpm for 30 minutes. The skin was removed from the dorsal surface of 9-day chick embryos and placed epidermal-side-down on the agar based medium. Cell suspensions of each primary renal tumor were prepared by trypsin collagenase digestion of small pieces of tumor tissue. 14 As cell separation progressed, the cells were decanted from the flask and fresh trypsin collagenase mixture added. The cells were immediately centrifuged at 4C and the trypsin collagenase supernatant was discarded. The cells were washed twice in EBSS, counted and prepared for inoculation on the embryonic chick skin. Monolayer cultures of cell lines H494 and DoT were trypsinized, washed and counted in the same manner. We inoculated the ECS by placing 1 drop of concentrated cell suspension (approximately 105 cells) directly onto the dermal surface of the skin. Each experiment consisted of 25 dishes inoculated with tumor cells and 5 dishes with ECS alone to serve as controls. The cultures were incubated at 37C for 3-5 days. They were then fixed in the Petri dishes by the addition of 10 per cent formalin directly to the culture dish. Following fixation, the skin was dehydrated and embedded in paraffin, and 8-µ, sections were cut in a sagittal plane to the skin. The sections were stained with hematoxylin and eosin and mounted for examination. RESULTS
Cell suspensions from cell lines H494 and DoT, both genitourinary tumors, showed deep invasion of the underlying dermis. At 3 days of incubation, clusters of cells could be seen invading MATERIALS AND METHODS almost through the dermal layer to the underlying epidermis. The embryonic chick skin was placed on a semi-solid agar After 5 days of incubation, in most cultures, the entire dermal based medium in small plastic Petri dishes. The Petri dishes layer was covered with tumor cells with moderate to severe were kept in a high humidity environment by incubation in a degenerative changes in the underlying dermis. In some areas, partially covered dish containing water in an atmosphere of 95 total necrosis of the supporting layer of skin was noted. Control per cent air, 5 per cent CO2 at 37C. A modified Wolff's medium cultures incubated for the same period of time demonstrated was used for these studies. 13 The medium consisted of 10 parts viable dermis and epidermis with no degenerative changes. of 1 per cent agar (Bacto agar) mixed in Earle's balanced salt Figure 1 demonstrates the early invasion of H494 seen at 3 solution (EBSS) with 4 parts of fetal calf serum and 4 parts of days. The malignant cells are easy to detect by their increased chick embryo extract. The medium was then poured into 3.5- size and darker staining quality. Figure 2 demonstrates the deep cm. plastic Petri dishes and allowed to cool. invasion and disruption of the underlying dermal support layer The chick embryo extract was prepared by the removal of 9- by DoT cells at 5 days of incubation. day chick embryos from their shells by sterile technique. The Four renal cell carcinomas were studied with this model. Three of the tumors grew well on the embryonic skin; 1 failed Accepted for publication March 11, 1982. to grow. The growth characteristics of the 3 tumors varied in * Requests for reprints: Department of Urology, The Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, Wisconsin the depth of invasion and the rate of growth. None of the 53226. primary tumors demonstrated the very rapid aggressive infil1068
1069
RENAL CELL CARCINOMA ON ECS
FIG. 1. Cell line H494 invading surface of ECS. H & E, reduced from X200.
FIG. 4. Invasion of ECS by renal cell carcinoma seen in figure 3. H & E, reduced from X200.
.,
FIG. 2. Cell line DoT sharing deep invasion of dermal layer of ECS. H & E, reduced from X200.
·l:.......
FIG. 5. Histopathology of renal cell carcinoma-poorly differentiated type. H & E, reduced from x200.
FIG. 3. Histopathology of renal cell carcinoma-clear cell type. H & E, reduced from X200.
tration noted with the cultured cell lines. However, invasion was found into the deep layers of the dermis at 5 days in all 3 primary renal cell carcinomas. Figure 3 is a hematoxylin and eosin section of one of the primary tumors. Figure 4 is the same tumor after 5 days incubation on EGS. Note the deep invasion of the dermal layer and the similarity of the histologic pattern
FIG. 6. Invasion of ECS by renal cell carcinoma seen in figure 5. H & E, reduced from X200.
of growth on the dermis when compared to the pathologic specimen. Figure 5 is a hematoxylin and eosin section of another primary tumor showing a more pleomorphic cell pattern. Figure 6 shows the same tumor invading the ECS and, again, the
1070
LAWSON, JACOBS AND SILBAR
histologic appearance is nearly identical with the primary tumor. DISCUSSION
Methods ofin vitro measurement of malignant potential have been investigated extensively for a number of years. Most of these studies have been carried out with the use of cultured tumor cells from both animals and man. Often the cells have been through many passages and resemble only slightly, if at all, the original tumor from which they were derived. Such cultured cells are considered to have undergone neoplastic transformation and exhibit certain characteristics in culture that define this transformation. Loss of density dependent inhibition, loss of anchorage dependent growth, infinite growth potential, decreased serum requirement, increased plating efficiency, ability to form colonies on contact inhibited normal fibroblasts, and growth on semi-solid agar medium are some of the characteristics ascribed to transformed cells in vitro. 15- 21 Animal models for the growth of xenogenic animal tumors and human tumors have been developed. 3- 7 The nude mouse and various immunosuppressed animals have been used to study malignant growth of primary and cultured tumors. Comparisons of in vitro criteria of malignancy with the ability of the tumor to grow in an immunologically deficient animal have shown that there is often a poor correlation between the in vitro and in vivo systems. 22 • 23 The use of various tissues from the embryonic chick as a biologic support medium for investigating the malignant potential of animal tumors has been studied extensively by Wolff. 24 He demonstrated that the embryonic chick mesonephros, when placed in culture with the test tumor and held in contact by a surrounding envelope of vitelline membrane, provides the best conditions for growth and invasion of the tumor. Wolff and Wolff also showed that embryonic chick tissue would support the growth of 2 different primary human tumors-carcinoma of the stomach and carcinoma of the cervix. 25 This early work was confirmed in 1975 when DeRidder and Mareel reported the growth of a primary human brain tumor on the stromal surface of ECS and stomach tissue. 26 Ikic and associates reported in 1977 that several tumor cell lines could be grown on the dermal surface of human fetal skin, mouse fetal skin and ECS and concluded that human fetal skin is the best support tissue of tumor growth. 10 Noguchi and associates compared the growth of a number of normal and neoplastic human cell lines in the ECS model and in the nude mouse and showed an excellent correlation of growth potential between the 2 systems. 23 No primary tumor growth was described in this study. Our results confirm the findings of Wolff and DeRidder that primary human tumor cells will grow on ECS in organ culture. The histologic pattern of the renal cell carcinomas is preserved in the organ culture system. The tumor cells invade deeply into the dermal support tissue, a finding which may correlate with the malignant potential of the tumors studied. These preliminary results have shown that at least some renal cell carcinomas will invade ECS in organ culture. Further study is needed to determine if this property correlates with the malignant potential of the neoplasm. The preparation of ECS organ cultures is simple and inexpensive when compared to the requirements for maintaining immunodeficient or immunosuppressed animals. The model may serve as a support system for the growth of primary human tumors to study their response to various chemotherapeutic agents, as well as being a possible indicator of the growth potential of any given human neoplasm. However, it appears that the degenerative changes seen in the ECS at 5 days when there is rapid aggressive invasion by the tumor cells may limit the usefulness of this model for long-term studies. REFERENCES
1. Rygaard, J. and Povlsen, C. 0.: Heterotransplantation of a human malignant tumour to "nude" mice. Acta. Pathol. Microbiol. Scand., 77: 758, 1969.
2. Povlsen, C. 0., Fialkow, P. J., Klein, E., Klein, G., Rygaard, J. and Wiener, F.: Growth and antigenic properties of a biopsy-derived Burkitt's lymphoma in thymus-less (nude) mice. Int. J. Cancer, 11: 30, 1973. 3. Giovanella, B. C., Stehlin, J. S. and Williams, L. J., Jr.: Heterotransplantation of human malignant tumors in "nude" thymusless mice. II. Malignant tumors induced by infection of cell cultures derived from human solid tumors. J. Natl. Cancer Inst., 52: 921, 1973. 4. Phillips, B. and Gazet, J.C.: Effect of antilymphocyte serum on the growth of Hep 2 and HeLa cells in mice. Nature, 220: 1140, 1968. 5. Aaronson, S. A. and Todaro, G. J.: Basis for the acquisition of malignant potential by mouse cells cultivated in vitro. Science, 162: 1024, 1968. 6. Wallace, R. and Vasington, P. J.: Heterotransplantation of cultured cell lines in newborn hamsters treated with antilymphocyte serum. Nature, 230: 454, 1971. 7. Oldroyd, R. I., Poole, R.R., Reed, R.R., Lawson, R. K. and Hodges, C. V.: An animal model for the growth of human tumor cell lines. Invest. Urol., 14: 434, 1977. 8. Wolff, E. and Schneider, N.: La culture d'un sarcome de souris sur des organes de poulet explantes in vitro. Arch. Anat. Microsc. Morphol. Exp., 46: 173, 1957. 9. DeRidder, L., Mareel, M. and Vaket, L.: Adhesion of malignant and nonmalignant cells to cultured embryonic substrates. Cancer Res., 35: 3164, 1975. 10. Ikic, D., Beck, M. and Turner, V.: The testing of cell invasiveness in embryonic tissue and in cell culture. Dev. Biol. Stand., 37: 235, 1977. 11. Hindsley, J.P., Jr., Fried, F. A., Auis, I. and Auis, F. P.: Transplantation of human prostate neoplasia into the nude mouse. Natl. Cancer Inst. Monogr., 49: 67, 1978. 12. Hussa, R. 0., Pattillo, R. A., Ruckert, A. C. F. and Scheuermann, K. W.: Effects of butyrate and dibutyryl cyclic AMP on hCGsecreting trophoblastic and non-trophoblastic cells. J. Clin. Endocrinol. Metab., 46: 69, 1978. 13. Wolff, E. and Haffen, K.: Sur une methode de culture d'organes embryonnaires "in vitro". Tex. Rep. Biol. Med., 10: 463, 1952. 14. Hilfer, S. R.: Collagenase treatment of chick heart and thyroid. In: Tissue Culture: Methods and Applications. Edited by P. F. Kruse, Jr. and M. K. Patterson, Jr. New York: Academic Press, sect. I, chapt. 3, p. 16, 1973. 15. Abercrombie, M., Heaysman, J. E. M. and Karthauser, H. M.: Social behavior of cells in tissue culture. Exp. Cell. Res., 13: 276, 1957. 16. Stroker, M. G. P. and Rubin, H.: Density dependent inhibition of cell growth in culture. Nature, 215: 171, 1967. 17. Shin, S. I., Freedman, V. H., Risser, R. and Pollack, R.: Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. Natl. Acad. Sci. USA, 72: 4435, 1975. 18. Barski, G. and Cassingena, R.: Malignant transformation in vitro of cells from C57BL mouse normal pulmonary tissue. J. Natl. Cancer Inst., 30: 865, 1963. 19. Dulbecco, R.: Topoinhibition and serum requirement of transformed and untransformed cells. Nature, 227: 802, 1970. 20. Aaronson, S. A., Todaro, G. J. and Freeman, A. E.: Human sarcoma cells in culture. Exp. Cell Res., 61: 1, 1970. 21. Marshall, C. J., Franks, L. M. and Carbonell, A. W.: Markers of neoplastic transformation in epithelial cell lines derived from human carcinomas. J. Natl. Cancer Inst., 58: 1743, 1977. 22. Freedman, V. H. and Shin, S. I.: Cellular tumorigenicity in nude mice: Correlation with cell growth in semi-solid medium. Cell, 3: 355, 1974. 23. Noguchi, P. D., Johnson, J.B., O'Donnell, R. and Petricciani, J.C.: Chick embryonic skin as a rapid organ culture assay for cellular neoplasia. Science, 199: 980, 1978. 24. Wolff, E.: Culture of tumors on embryonic organs explanted in vitro. In: Biological Interactions in Normal and Neoplastic Growth. Edited by M. J. Brennan and W. L. Simpson. Boston: Little, Brown, p. 413, 1962. 25. Wolff, E. and Wolff, E.: Culture de cancers humains sur du rein embryonnaire de poulet explante "in vitro". La Presse Medicale, 69: 1123, 1961. 26. DeRidder, L. and Maree!, M.: Characterization of malignancy in organotypical culture. In: Biological Characterization of Human Tumors. Edited by W. David and C. Maltoni. Sixth International Symposium, p. 370, 1975.
THE JOURNAL OF UROLOGY
Copyright© 1982 by The Williams & Wilkins Co.
GROWTH OF PRIMARY HUMAN RENAL CELL CARCINOMA ON EMBRYONIC CHICK SKIN IN ORGAN CULTURE RUSSELL K. LAWSON,* STEPHEN C. JACOBS
AND
ELLIOT SILBAR
From the Department of Urology, The Medical College of Wisconsin, Milwaukee, Wisconsin
ABSTRACT
Embryonic chick skin was used as a biologic support medium for the growth of 2 human genital tract tumor cell lines and 3 primary human renal cell carcinomas. Three of 4 primary renal cell carcinomas invaded the dermal layer of the chick skin in organ culture. Additional studies are needed to determine if growth and invasion of the primary tumor correlates with the malignant potential of that tumor. The model may also be useful for studying the effect of various chemotherapeutic agents against individual human neoplasms. Animal models for the growth of human tumors are of great importance in many areas of cancer research. Transplantable or induced primary animal tumors have been used extensively to study the metabolic activity of these neoplasms and their response to various immunologic and chemotherapeutic manipulations. All of these studies suffer from the fact that animal tumors often do not behave as their counterparts in humans do. The discovery of the nude mouse as a biologic support system for the growth of human tumors was a major advance in the study of human cancers. 1- 3 Several other animal systems have been developed that depend on immunosuppression in the host animal for the growth of human tumors. 4 - 6 We reported such a tumor model several years ago that employed neonatal thymectomy and antilymphocyte serum in the newborn rat to provide the necessary degree of immunosuppression. 7 Human tumor cell lines grow rapidly in this model, deeply invade surrounding tissue and metastasize to multiple organs. Invasion of surrounding tissue is considered to be an important indicator of malignancy, as well as metastasis to other sites in the animal. Because invasion is one of the important characteristics of malignancy, tissue and organ culture systems have been developed to look at this parameter alone. Several papers have appeared describing the use of embryonic chick skin (ECS) as a biologic support medium for studying the invasive potential of various tumors and cell lines. 8- 10 In this study, we planned to determine if primary human renal cell carcinoma could be grown on embryonic chick skin and if the invasive potential of the tumor could be assessed. In order to determine if the model would work in our laboratory and also to serve as controls, we placed human cell lines H494 (benign prostatic hyperplasia) and DoT (cervical carcinoma) in this same organ culture system. 11 • 12
embryos were decapitated and the heads discarded. Approximately equal volumes of embryos and EBSS were placed in a Vertis tissue homogenizer and homogenized at medium to high speed, with care taken not to allow excessive foaming of the mixture. The extract was then allowed to stand overnight at 4C before centrifugation in the cold at 4,000 rpm for 30 minutes. The skin was removed from the dorsal surface of 9-day chick embryos and placed epidermal-side-down on the agar based medium. Cell suspensions of each primary renal tumor were prepared by trypsin collagenase digestion of small pieces of tumor tissue. 14 As cell separation progressed, the cells were decanted from the flask and fresh trypsin collagenase mixture added. The cells were immediately centrifuged at 4C and the trypsin collagenase supernatant was discarded. The cells were washed twice in EBSS, counted and prepared for inoculation on the embryonic chick skin. Monolayer cultures of cell lines H494 and DoT were trypsinized, washed and counted in the same manner. We inoculated the ECS by placing 1 drop of concentrated cell suspension (approximately 105 cells) directly onto the dermal surface of the skin. Each experiment consisted of 25 dishes inoculated with tumor cells and 5 dishes with ECS alone to serve as controls. The cultures were incubated at 37C for 3-5 days. They were then fixed in the Petri dishes by the addition of 10 per cent formalin directly to the culture dish. Following fixation, the skin was dehydrated and embedded in paraffin, and 8-µ, sections were cut in a sagittal plane to the skin. The sections were stained with hematoxylin and eosin and mounted for examination. RESULTS
Cell suspensions from cell lines H494 and DoT, both genitourinary tumors, showed deep invasion of the underlying dermis. At 3 days of incubation, clusters of cells could be seen invading MATERIALS AND METHODS almost through the dermal layer to the underlying epidermis. The embryonic chick skin was placed on a semi-solid agar After 5 days of incubation, in most cultures, the entire dermal based medium in small plastic Petri dishes. The Petri dishes layer was covered with tumor cells with moderate to severe were kept in a high humidity environment by incubation in a degenerative changes in the underlying dermis. In some areas, partially covered dish containing water in an atmosphere of 95 total necrosis of the supporting layer of skin was noted. Control per cent air, 5 per cent CO2 at 37C. A modified Wolff's medium cultures incubated for the same period of time demonstrated was used for these studies. 13 The medium consisted of 10 parts viable dermis and epidermis with no degenerative changes. of 1 per cent agar (Bacto agar) mixed in Earle's balanced salt Figure 1 demonstrates the early invasion of H494 seen at 3 solution (EBSS) with 4 parts of fetal calf serum and 4 parts of days. The malignant cells are easy to detect by their increased chick embryo extract. The medium was then poured into 3.5- size and darker staining quality. Figure 2 demonstrates the deep cm. plastic Petri dishes and allowed to cool. invasion and disruption of the underlying dermal support layer The chick embryo extract was prepared by the removal of 9- by DoT cells at 5 days of incubation. day chick embryos from their shells by sterile technique. The Four renal cell carcinomas were studied with this model. Three of the tumors grew well on the embryonic skin; 1 failed Accepted for publication March 11, 1982. to grow. The growth characteristics of the 3 tumors varied in * Requests for reprints: Department of Urology, The Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, Wisconsin the depth of invasion and the rate of growth. None of the 53226. primary tumors demonstrated the very rapid aggressive infil1068
1069
RENAL CELL CARCINOMA ON ECS
FIG. 1. Cell line H494 invading surface of ECS. H & E, reduced from X200.
FIG. 4. Invasion of ECS by renal cell carcinoma seen in figure 3. H & E, reduced from X200.
.,
FIG. 2. Cell line DoT sharing deep invasion of dermal layer of ECS. H & E, reduced from X200.
·l:.......
FIG. 5. Histopathology of renal cell carcinoma-poorly differentiated type. H & E, reduced from x200.
FIG. 3. Histopathology of renal cell carcinoma-clear cell type. H & E, reduced from X200.
tration noted with the cultured cell lines. However, invasion was found into the deep layers of the dermis at 5 days in all 3 primary renal cell carcinomas. Figure 3 is a hematoxylin and eosin section of one of the primary tumors. Figure 4 is the same tumor after 5 days incubation on EGS. Note the deep invasion of the dermal layer and the similarity of the histologic pattern
FIG. 6. Invasion of ECS by renal cell carcinoma seen in figure 5. H & E, reduced from X200.
of growth on the dermis when compared to the pathologic specimen. Figure 5 is a hematoxylin and eosin section of another primary tumor showing a more pleomorphic cell pattern. Figure 6 shows the same tumor invading the ECS and, again, the
1070
LAWSON, JACOBS AND SILBAR
histologic appearance is nearly identical with the primary tumor. DISCUSSION
Methods ofin vitro measurement of malignant potential have been investigated extensively for a number of years. Most of these studies have been carried out with the use of cultured tumor cells from both animals and man. Often the cells have been through many passages and resemble only slightly, if at all, the original tumor from which they were derived. Such cultured cells are considered to have undergone neoplastic transformation and exhibit certain characteristics in culture that define this transformation. Loss of density dependent inhibition, loss of anchorage dependent growth, infinite growth potential, decreased serum requirement, increased plating efficiency, ability to form colonies on contact inhibited normal fibroblasts, and growth on semi-solid agar medium are some of the characteristics ascribed to transformed cells in vitro. 15- 21 Animal models for the growth of xenogenic animal tumors and human tumors have been developed. 3- 7 The nude mouse and various immunosuppressed animals have been used to study malignant growth of primary and cultured tumors. Comparisons of in vitro criteria of malignancy with the ability of the tumor to grow in an immunologically deficient animal have shown that there is often a poor correlation between the in vitro and in vivo systems. 22 • 23 The use of various tissues from the embryonic chick as a biologic support medium for investigating the malignant potential of animal tumors has been studied extensively by Wolff. 24 He demonstrated that the embryonic chick mesonephros, when placed in culture with the test tumor and held in contact by a surrounding envelope of vitelline membrane, provides the best conditions for growth and invasion of the tumor. Wolff and Wolff also showed that embryonic chick tissue would support the growth of 2 different primary human tumors-carcinoma of the stomach and carcinoma of the cervix. 25 This early work was confirmed in 1975 when DeRidder and Mareel reported the growth of a primary human brain tumor on the stromal surface of ECS and stomach tissue. 26 Ikic and associates reported in 1977 that several tumor cell lines could be grown on the dermal surface of human fetal skin, mouse fetal skin and ECS and concluded that human fetal skin is the best support tissue of tumor growth. 10 Noguchi and associates compared the growth of a number of normal and neoplastic human cell lines in the ECS model and in the nude mouse and showed an excellent correlation of growth potential between the 2 systems. 23 No primary tumor growth was described in this study. Our results confirm the findings of Wolff and DeRidder that primary human tumor cells will grow on ECS in organ culture. The histologic pattern of the renal cell carcinomas is preserved in the organ culture system. The tumor cells invade deeply into the dermal support tissue, a finding which may correlate with the malignant potential of the tumors studied. These preliminary results have shown that at least some renal cell carcinomas will invade ECS in organ culture. Further study is needed to determine if this property correlates with the malignant potential of the neoplasm. The preparation of ECS organ cultures is simple and inexpensive when compared to the requirements for maintaining immunodeficient or immunosuppressed animals. The model may serve as a support system for the growth of primary human tumors to study their response to various chemotherapeutic agents, as well as being a possible indicator of the growth potential of any given human neoplasm. However, it appears that the degenerative changes seen in the ECS at 5 days when there is rapid aggressive invasion by the tumor cells may limit the usefulness of this model for long-term studies. REFERENCES
1. Rygaard, J. and Povlsen, C. 0.: Heterotransplantation of a human malignant tumour to "nude" mice. Acta. Pathol. Microbiol. Scand., 77: 758, 1969.
2. Povlsen, C. 0., Fialkow, P. J., Klein, E., Klein, G., Rygaard, J. and Wiener, F.: Growth and antigenic properties of a biopsy-derived Burkitt's lymphoma in thymus-less (nude) mice. Int. J. Cancer, 11: 30, 1973. 3. Giovanella, B. C., Stehlin, J. S. and Williams, L. J., Jr.: Heterotransplantation of human malignant tumors in "nude" thymusless mice. II. Malignant tumors induced by infection of cell cultures derived from human solid tumors. J. Natl. Cancer Inst., 52: 921, 1973. 4. Phillips, B. and Gazet, J.C.: Effect of antilymphocyte serum on the growth of Hep 2 and HeLa cells in mice. Nature, 220: 1140, 1968. 5. Aaronson, S. A. and Todaro, G. J.: Basis for the acquisition of malignant potential by mouse cells cultivated in vitro. Science, 162: 1024, 1968. 6. Wallace, R. and Vasington, P. J.: Heterotransplantation of cultured cell lines in newborn hamsters treated with antilymphocyte serum. Nature, 230: 454, 1971. 7. Oldroyd, R. I., Poole, R.R., Reed, R.R., Lawson, R. K. and Hodges, C. V.: An animal model for the growth of human tumor cell lines. Invest. Urol., 14: 434, 1977. 8. Wolff, E. and Schneider, N.: La culture d'un sarcome de souris sur des organes de poulet explantes in vitro. Arch. Anat. Microsc. Morphol. Exp., 46: 173, 1957. 9. DeRidder, L., Mareel, M. and Vaket, L.: Adhesion of malignant and nonmalignant cells to cultured embryonic substrates. Cancer Res., 35: 3164, 1975. 10. Ikic, D., Beck, M. and Turner, V.: The testing of cell invasiveness in embryonic tissue and in cell culture. Dev. Biol. Stand., 37: 235, 1977. 11. Hindsley, J.P., Jr., Fried, F. A., Auis, I. and Auis, F. P.: Transplantation of human prostate neoplasia into the nude mouse. Natl. Cancer Inst. Monogr., 49: 67, 1978. 12. Hussa, R. 0., Pattillo, R. A., Ruckert, A. C. F. and Scheuermann, K. W.: Effects of butyrate and dibutyryl cyclic AMP on hCGsecreting trophoblastic and non-trophoblastic cells. J. Clin. Endocrinol. Metab., 46: 69, 1978. 13. Wolff, E. and Haffen, K.: Sur une methode de culture d'organes embryonnaires "in vitro". Tex. Rep. Biol. Med., 10: 463, 1952. 14. Hilfer, S. R.: Collagenase treatment of chick heart and thyroid. In: Tissue Culture: Methods and Applications. Edited by P. F. Kruse, Jr. and M. K. Patterson, Jr. New York: Academic Press, sect. I, chapt. 3, p. 16, 1973. 15. Abercrombie, M., Heaysman, J. E. M. and Karthauser, H. M.: Social behavior of cells in tissue culture. Exp. Cell. Res., 13: 276, 1957. 16. Stroker, M. G. P. and Rubin, H.: Density dependent inhibition of cell growth in culture. Nature, 215: 171, 1967. 17. Shin, S. I., Freedman, V. H., Risser, R. and Pollack, R.: Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. Natl. Acad. Sci. USA, 72: 4435, 1975. 18. Barski, G. and Cassingena, R.: Malignant transformation in vitro of cells from C57BL mouse normal pulmonary tissue. J. Natl. Cancer Inst., 30: 865, 1963. 19. Dulbecco, R.: Topoinhibition and serum requirement of transformed and untransformed cells. Nature, 227: 802, 1970. 20. Aaronson, S. A., Todaro, G. J. and Freeman, A. E.: Human sarcoma cells in culture. Exp. Cell Res., 61: 1, 1970. 21. Marshall, C. J., Franks, L. M. and Carbonell, A. W.: Markers of neoplastic transformation in epithelial cell lines derived from human carcinomas. J. Natl. Cancer Inst., 58: 1743, 1977. 22. Freedman, V. H. and Shin, S. I.: Cellular tumorigenicity in nude mice: Correlation with cell growth in semi-solid medium. Cell, 3: 355, 1974. 23. Noguchi, P. D., Johnson, J.B., O'Donnell, R. and Petricciani, J.C.: Chick embryonic skin as a rapid organ culture assay for cellular neoplasia. Science, 199: 980, 1978. 24. Wolff, E.: Culture of tumors on embryonic organs explanted in vitro. In: Biological Interactions in Normal and Neoplastic Growth. Edited by M. J. Brennan and W. L. Simpson. Boston: Little, Brown, p. 413, 1962. 25. Wolff, E. and Wolff, E.: Culture de cancers humains sur du rein embryonnaire de poulet explante "in vitro". La Presse Medicale, 69: 1123, 1961. 26. DeRidder, L. and Maree!, M.: Characterization of malignancy in organotypical culture. In: Biological Characterization of Human Tumors. Edited by W. David and C. Maltoni. Sixth International Symposium, p. 370, 1975.