1
Cancer Letters, 51 (1990) l-5 Elsevier Scientific Publishers Ireland Ltd.
Tumor cell implantation sponge disc
with the use of Gelaspon@ gelatin
E. Uzvolgyi, A. Katona and P. Kertai Department of Hygiene and Epidemiology, Debrecen, DOTE (Hungary)
University Medical School of Debrecen, Nagyerdei
krt. 98.
H-4012
84012
(Received 15 May 1989) (Revision received 3 November 1989) (Accepted 18 December 1989)
Summary
Myelomonocytic leukemia (My) mesoblastic nephroma (Ne) and hepatocellular carcinoma (He) cells were implanted under the renal capsule of F344, Long-Evans (LE) and BDZX rats. Gelaspon@ sponge discs were used in the implantation procedure, which were resorbed within a few days. The tumor cells, which were located on the surface of these discs could then attach themselves to the renal capsule and thus grow. There was a correlation between the number of tumor cells and the difference between the two kidney masses. The correlation was linear between 104 and 106 cells, thus the method proved to be a simple, fast and quantitative model in experimental cancer prevention and therapy.
Keywords: Gelaspon@ ; subrenal capsule; tumor growth assay; tumor implantation.
immunological properties, along with previously tested chemotherapeutical drugs. The principle of chemotherapeutic drug testing is that growth delay or cure of transplanted tumors is proportional to the dosage. The advantages or disadvantages in using rodent tumors are discussed in special papers and books [8, lo]. Recently, a new method has been found for testing the chemosensitivity of human tumor sections, in which immundeficient and immuncompetent mice are used [1,2,9]. In our laboratories we examined the effect of chemopreventive compounds. We needed to find a quantitative method, in which the transplanted tumor cell growth could be measured and recorded. For a carrier, we used a sterile, fine porous type gelatin sponge, which in a few days could be reabsorbed. The tumor cells attached to the capsule grew in all directions. In our paper the use of Gelaspon@ gelatin sponge disc in implantation of tumor cells will be discussed.
Introduction
Materials and methods
The transplantation of tumor cells has been a well known and used procedure in cancer research. The tumor cells and their host organisms are tested for biochemical and
Experimental animals Studies were carried out on female and male F344, Long-Evans and BDIX inbred, or FBIX F, hybrid rats, weighing 150-200 g.
0304-3835/90/$03.50
0
1990 Elsevier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
2
They were kept in a conventional laboratory environment and fed on a semi-synthetic diet (LATI, Godollo, Hungary) and tap water ad libitum . Experimental tumors My cells were obtained from Long-Evens rats pretreated with DMBA, using the Huggins and Sugiyama method [7]. Ne and He were induced in newborn F344 rats with 125 pg of NDMA-intraperitoneally [ 121. The tumor cells were maintened by serial passage or stored in liquid nitrogen. The cells were suspended in MI99 medium and thus diluted so that 1 ~1 of medium contained 1O1- lo6 cells, respectively . Preparation of Gelaspon@ discs In order to transplant tumor cells Gelaspon@ (Germed, G.D.R.) gelatin sponge were used 4 mm in diameter and 1 mm thick discs were prepared, sterilized by ethyleneoxide and used within 1 week. Before impiantation 1~1 of cell suspension was dropped on each disc.
Results
On examining the validity of the method Gelaspon@ discs containing 1 ~1 cell-free MI99 medium, or lo6 myelomonocytes were used which were then implanted under the left renal capsule. After 5, 10, 15, 20 and 25 days the animals were killed. Histological examinations revealed, that discs containing the MI99 medium were resorbed within 5 days, and only a few disc fibers remained visible under the renal capsule. Discs containing myelomonocytes were also resorbed, but preceeding this, the cells attached to the renal capsule first formed a monolayer, then expanded in all directions. This process is similar to the in vitro cell cultures, with one A9
Surgical procedure The rats were anesthetized by ether, and the lumbal area, previously shaved was prepared. The left kidney was isolated, the disc containing the tumor cells was placed under the renal capsule, and the wound was closed. Every 5 days one group of rats was anesthetized, both kidneys were removed, weighed and the difference between the intact and tumorous kidneys was recorded. Histological examination Histological sections were made by the conventional technique, stained by haematoxylin-eosin, Gieson and silver van impregnation methods. Statistical eoaluation Statistical calculations were made by the 4 point assay method [3].
days The difference between the two kidney’s mass after the implantation of 102, lo4 and lo6 myelomonocytic cells in LBF, female rats at different time intervals. The reason the graph starts under 0 point is that the rat right kidney mass is larger than the left kidney used for transplantation.
Fig. 1.
3
difference, that the renal capsule substitutes the wall of the culture dish. After lo-15 days the tumor cells infiltrated the kidney parenchyma and the interstitial area among the tubules. Metastases could be seen in the parathymical lymph nodes 20-25 days later. In the following experiments 102, lo4 and lo6 My, Ne and He cells were implanted under the renal capsule. Following the proce-
lob
5
lo
15
20
25
30 days
2
104
1
VI2
5
10
is
ti
25
30
dure the animals were killed on days 5, 10, 15, 20, 25 and 30. The two kidneys were weighed, and the difference in mass between the two kidneys gave the growth curve. Each point on the curve represents the average of 6 independent results. Of the three tumor the My cells showed accelerated types, growth, while the Ne and He cells showed a slower growth rate (Figs 1, 2a, 2b). The differences found in tumor growth agree with our earlier observation, when My, Ne and He cells in solid tumor forms were transplanted subcutaneously. After the implantation of lo’, 102, 103, 104, lo5 and lo6 tumor cells, LBF, or FBF, hybrids were killed on the 15th day. The two kidney masses were weighed and by taking the difference between the two kidney’s mass a dose-effect curve was constructed. The curves show that there is a correlation between the number of implanted cells and the difference between the two kidney’s masses and the correlation is linear between lo4 and lo6 cells (Fig. 3). On the basis of these findings it seems possible to apply the 4 point assay method to compare tumor growth rates [3]. In our experiments male LE, BDIX and LBF, rats were used and 104-106 I\;ry cells were implanted under the renal capsule. After exactly 15 days, the animals were killed. Each of the 6 groups contained 10 animals, respectively. The curves clearly show that the tumor cells did not grow at all in BDIX rats, while in LE and LBF, rats, no difference was observed. We can conclude, that genetic factor(s) regulating tumor growth are inherited dominantly. A similar result is found with Ne and He cells, with one difference, that F344 and FBF, hybrid rats were used (Fig. 4). :
days
Fig. 2.
(a) The difference between the two kidney’s mass after implanting lo*, lo4 and lo6 mesoblastic nephroma cells in female FBF, rats at different time intervals. (b) The difference between the two kidney’s mass after implanting lo*, 10” and 10” hepatocellular carcinoma cells in FBF, male rats at different time intervals.
Discussion
The implantation of tumor pieces under the renal capsule is a widely used method. In evaluating the results, the tumor pieces contain not only the tumor cells, but also interstitial parenchyma and non-tumor cells.
The difference between the two kidney’s mass Fig. 4. after the implantation of 104-W myelomonocytic (My) mesoblastic nephroma (Ne) and hepatocellular carcinoma (He)-cells in Le, F344 and BDIX inbred rats, and FBF,, LBF, hybrids after 15 days.
The difference between the two kidney’s mass Fis. 3. after the implantation of lo*- 106 myelomonocytic (My), mesoblastic nephroma (Ne) and hepatocellular carcinoma cells (He) in LBF, and FBF, hybrid rats, after 15 days.
Another known method, where given amounts of tumor cell containing suspension is used, was injected under the renal capsule. This procedure is also debatable because, after injecting the suspension, cell loss occured through the puncture channel. To correct this problem, Fingert [6] used fibrinclots in the implantation of human tumor cells under the renal capsule. Another possibility is the use of collagen. Ehrmann and Gey [4] and Elsdale and Bard [5) showed that collagen fibers could be used in cell cultures after
which this procedure was also used in tumor cell cultures (131. Recently, collagen has been used in in vitro experimentation of tumor cell invasion [ll]. The advantages of using Gelaspone are (1) that the gelatin is resorbed and the cells attached themselves to the kidney’s collagen fibers; (2) the cell loss is available and (3) suitable blood- flow is provided for the growing tumor cells. Further advantages are that this experimental system is inexpensive and is also useful for quantitative evaluation of chemotherapeutic and chemopreventive drugs. Acknowledgements
This research was supported by grants from the Ministry of Health (ETT l/011) and Academy of Sciences (AKA 715) of Hungary. Mrs. Judith Cs. Nagy is acknowledged for excellent technical assistance.
References Bogden, A.E., Kelton, D.E., Cobb, W.R. and E&r, H.J. (1978) A rapid screening method for testing chemotherapeutic agents against human tumor xenografts. In: Proceedings of the use of athymic (nude) mice in cancer research, pp. 231-250. Editors: D.P. Houchens and A.A. Ovejera, Gustav Fischer, New York. Bogden, A.E. (1985) The subrenal capsule assay/SRCA/ and its predictive value in oncology. Ann. ChIr. Gynecol., 74, Suppl. 199,12-27. Bum, J.H., Finney, D.J. and Goodwin, L.G. (1952) Biological Standardization, pp. 75-86. Oxford University Press, London-New York-Toronto. Ehrmann, R.L. and Gey, G.O. (1956) The growth of cells on a transparent gel of reconstituted rattail collagen. J. Natl. Cancer Inst., 16, 1375-1390. Elsdale, T. and Bard, J. (1972) Collagen substrate for studies on cell behavior. J. Cell. Biol., 54, 626-637. Fingert, H.J., Chen, Z., Mizrahi, N., Gajewski, W.H., Bamberg, M.P. and Kradin, R.L. (1987) Rapid growth of human cancer cells in a mouse model with fibrin clot subrenal capsule assay. Cancer Res., 47,3824-3829. Huggtns, C.B. and Sugiyama, T. (1966) Induction of leukemia in rat by pulse doses of 7,lBdimethylbenz[a]anthracene. Proc. Natl. Acad. Sci., 55, 74.
8
9
10
11 12
13
14
Kallman, R.F. (1987) Rodent Tumor Models in Experimental Cancer Therapy, Pergamon Press, New York, pp. l-310. Kangas, I. and Perila, M. (1985) Clinical praxis and laboratory procedures in subrenal capsule assay (SCRA). Ann. Chir. Gynaecol., 74, Supp. 1990, 7- 11. Metcalf, D. (1975) Transplantation of Tumors. In: A training manual for cancer research workers. UICC Technical Report Series Vol. 20, pp. 40-43, Editor: D. Metcalf, Geneva. Schor, S.L., Schor, A.M., Wfnn, B. and Rushton, G. (1982) The use of three-dimensional collagen gels for the study of tumour cell invasion in vitro: experimental parameters influencing cell migration into the gel matrix. Int. J. Cancer, 29, 57-62. Terracini, B. and Magee, P.N. (1964) Renal tumours in rats following injection of dimethylnitrosamine at birth. Nature, 202,502. Yang, J., Richards, J., Bowman, P., Guxman, R., Enami, J., McCormick, K., Hamamoto, S., Pitelka, D. and Nandi, S. (1979) Sustained growth and threedimensional organization of primary mammary tumor epithelial cells embedded in collagen gels. Proc. Natl. Acad. Sci., 76,3401-3405.
Cancer Letters, 51 (1990) l-5 Elsevier Scientific Publishers Ireland Ltd.
Tumor cell implantation sponge disc
with the use of Gelaspon@ gelatin
E. Uzvolgyi, A. Katona and P. Kertai Department of Hygiene and Epidemiology, Debrecen, DOTE (Hungary)
University Medical School of Debrecen, Nagyerdei
krt. 98.
H-4012
84012
(Received 15 May 1989) (Revision received 3 November 1989) (Accepted 18 December 1989)
Summary
Myelomonocytic leukemia (My) mesoblastic nephroma (Ne) and hepatocellular carcinoma (He) cells were implanted under the renal capsule of F344, Long-Evans (LE) and BDZX rats. Gelaspon@ sponge discs were used in the implantation procedure, which were resorbed within a few days. The tumor cells, which were located on the surface of these discs could then attach themselves to the renal capsule and thus grow. There was a correlation between the number of tumor cells and the difference between the two kidney masses. The correlation was linear between 104 and 106 cells, thus the method proved to be a simple, fast and quantitative model in experimental cancer prevention and therapy.
Keywords: Gelaspon@ ; subrenal capsule; tumor growth assay; tumor implantation.
immunological properties, along with previously tested chemotherapeutical drugs. The principle of chemotherapeutic drug testing is that growth delay or cure of transplanted tumors is proportional to the dosage. The advantages or disadvantages in using rodent tumors are discussed in special papers and books [8, lo]. Recently, a new method has been found for testing the chemosensitivity of human tumor sections, in which immundeficient and immuncompetent mice are used [1,2,9]. In our laboratories we examined the effect of chemopreventive compounds. We needed to find a quantitative method, in which the transplanted tumor cell growth could be measured and recorded. For a carrier, we used a sterile, fine porous type gelatin sponge, which in a few days could be reabsorbed. The tumor cells attached to the capsule grew in all directions. In our paper the use of Gelaspon@ gelatin sponge disc in implantation of tumor cells will be discussed.
Introduction
Materials and methods
The transplantation of tumor cells has been a well known and used procedure in cancer research. The tumor cells and their host organisms are tested for biochemical and
Experimental animals Studies were carried out on female and male F344, Long-Evans and BDIX inbred, or FBIX F, hybrid rats, weighing 150-200 g.
0304-3835/90/$03.50
0
1990 Elsevier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
2
They were kept in a conventional laboratory environment and fed on a semi-synthetic diet (LATI, Godollo, Hungary) and tap water ad libitum . Experimental tumors My cells were obtained from Long-Evens rats pretreated with DMBA, using the Huggins and Sugiyama method [7]. Ne and He were induced in newborn F344 rats with 125 pg of NDMA-intraperitoneally [ 121. The tumor cells were maintened by serial passage or stored in liquid nitrogen. The cells were suspended in MI99 medium and thus diluted so that 1 ~1 of medium contained 1O1- lo6 cells, respectively . Preparation of Gelaspon@ discs In order to transplant tumor cells Gelaspon@ (Germed, G.D.R.) gelatin sponge were used 4 mm in diameter and 1 mm thick discs were prepared, sterilized by ethyleneoxide and used within 1 week. Before impiantation 1~1 of cell suspension was dropped on each disc.
Results
On examining the validity of the method Gelaspon@ discs containing 1 ~1 cell-free MI99 medium, or lo6 myelomonocytes were used which were then implanted under the left renal capsule. After 5, 10, 15, 20 and 25 days the animals were killed. Histological examinations revealed, that discs containing the MI99 medium were resorbed within 5 days, and only a few disc fibers remained visible under the renal capsule. Discs containing myelomonocytes were also resorbed, but preceeding this, the cells attached to the renal capsule first formed a monolayer, then expanded in all directions. This process is similar to the in vitro cell cultures, with one A9
Surgical procedure The rats were anesthetized by ether, and the lumbal area, previously shaved was prepared. The left kidney was isolated, the disc containing the tumor cells was placed under the renal capsule, and the wound was closed. Every 5 days one group of rats was anesthetized, both kidneys were removed, weighed and the difference between the intact and tumorous kidneys was recorded. Histological examination Histological sections were made by the conventional technique, stained by haematoxylin-eosin, Gieson and silver van impregnation methods. Statistical eoaluation Statistical calculations were made by the 4 point assay method [3].
days The difference between the two kidney’s mass after the implantation of 102, lo4 and lo6 myelomonocytic cells in LBF, female rats at different time intervals. The reason the graph starts under 0 point is that the rat right kidney mass is larger than the left kidney used for transplantation.
Fig. 1.
3
difference, that the renal capsule substitutes the wall of the culture dish. After lo-15 days the tumor cells infiltrated the kidney parenchyma and the interstitial area among the tubules. Metastases could be seen in the parathymical lymph nodes 20-25 days later. In the following experiments 102, lo4 and lo6 My, Ne and He cells were implanted under the renal capsule. Following the proce-
lob
5
lo
15
20
25
30 days
2
104
1
VI2
5
10
is
ti
25
30
dure the animals were killed on days 5, 10, 15, 20, 25 and 30. The two kidneys were weighed, and the difference in mass between the two kidneys gave the growth curve. Each point on the curve represents the average of 6 independent results. Of the three tumor the My cells showed accelerated types, growth, while the Ne and He cells showed a slower growth rate (Figs 1, 2a, 2b). The differences found in tumor growth agree with our earlier observation, when My, Ne and He cells in solid tumor forms were transplanted subcutaneously. After the implantation of lo’, 102, 103, 104, lo5 and lo6 tumor cells, LBF, or FBF, hybrids were killed on the 15th day. The two kidney masses were weighed and by taking the difference between the two kidney’s mass a dose-effect curve was constructed. The curves show that there is a correlation between the number of implanted cells and the difference between the two kidney’s masses and the correlation is linear between lo4 and lo6 cells (Fig. 3). On the basis of these findings it seems possible to apply the 4 point assay method to compare tumor growth rates [3]. In our experiments male LE, BDIX and LBF, rats were used and 104-106 I\;ry cells were implanted under the renal capsule. After exactly 15 days, the animals were killed. Each of the 6 groups contained 10 animals, respectively. The curves clearly show that the tumor cells did not grow at all in BDIX rats, while in LE and LBF, rats, no difference was observed. We can conclude, that genetic factor(s) regulating tumor growth are inherited dominantly. A similar result is found with Ne and He cells, with one difference, that F344 and FBF, hybrid rats were used (Fig. 4). :
days
Fig. 2.
(a) The difference between the two kidney’s mass after implanting lo*, lo4 and lo6 mesoblastic nephroma cells in female FBF, rats at different time intervals. (b) The difference between the two kidney’s mass after implanting lo*, 10” and 10” hepatocellular carcinoma cells in FBF, male rats at different time intervals.
Discussion
The implantation of tumor pieces under the renal capsule is a widely used method. In evaluating the results, the tumor pieces contain not only the tumor cells, but also interstitial parenchyma and non-tumor cells.
The difference between the two kidney’s mass Fig. 4. after the implantation of 104-W myelomonocytic (My) mesoblastic nephroma (Ne) and hepatocellular carcinoma (He)-cells in Le, F344 and BDIX inbred rats, and FBF,, LBF, hybrids after 15 days.
The difference between the two kidney’s mass Fis. 3. after the implantation of lo*- 106 myelomonocytic (My), mesoblastic nephroma (Ne) and hepatocellular carcinoma cells (He) in LBF, and FBF, hybrid rats, after 15 days.
Another known method, where given amounts of tumor cell containing suspension is used, was injected under the renal capsule. This procedure is also debatable because, after injecting the suspension, cell loss occured through the puncture channel. To correct this problem, Fingert [6] used fibrinclots in the implantation of human tumor cells under the renal capsule. Another possibility is the use of collagen. Ehrmann and Gey [4] and Elsdale and Bard [5) showed that collagen fibers could be used in cell cultures after
which this procedure was also used in tumor cell cultures (131. Recently, collagen has been used in in vitro experimentation of tumor cell invasion [ll]. The advantages of using Gelaspone are (1) that the gelatin is resorbed and the cells attached themselves to the kidney’s collagen fibers; (2) the cell loss is available and (3) suitable blood- flow is provided for the growing tumor cells. Further advantages are that this experimental system is inexpensive and is also useful for quantitative evaluation of chemotherapeutic and chemopreventive drugs. Acknowledgements
This research was supported by grants from the Ministry of Health (ETT l/011) and Academy of Sciences (AKA 715) of Hungary. Mrs. Judith Cs. Nagy is acknowledged for excellent technical assistance.
References Bogden, A.E., Kelton, D.E., Cobb, W.R. and E&r, H.J. (1978) A rapid screening method for testing chemotherapeutic agents against human tumor xenografts. In: Proceedings of the use of athymic (nude) mice in cancer research, pp. 231-250. Editors: D.P. Houchens and A.A. Ovejera, Gustav Fischer, New York. Bogden, A.E. (1985) The subrenal capsule assay/SRCA/ and its predictive value in oncology. Ann. ChIr. Gynecol., 74, Suppl. 199,12-27. Bum, J.H., Finney, D.J. and Goodwin, L.G. (1952) Biological Standardization, pp. 75-86. Oxford University Press, London-New York-Toronto. Ehrmann, R.L. and Gey, G.O. (1956) The growth of cells on a transparent gel of reconstituted rattail collagen. J. Natl. Cancer Inst., 16, 1375-1390. Elsdale, T. and Bard, J. (1972) Collagen substrate for studies on cell behavior. J. Cell. Biol., 54, 626-637. Fingert, H.J., Chen, Z., Mizrahi, N., Gajewski, W.H., Bamberg, M.P. and Kradin, R.L. (1987) Rapid growth of human cancer cells in a mouse model with fibrin clot subrenal capsule assay. Cancer Res., 47,3824-3829. Huggtns, C.B. and Sugiyama, T. (1966) Induction of leukemia in rat by pulse doses of 7,lBdimethylbenz[a]anthracene. Proc. Natl. Acad. Sci., 55, 74.
8
9
10
11 12
13
14
Kallman, R.F. (1987) Rodent Tumor Models in Experimental Cancer Therapy, Pergamon Press, New York, pp. l-310. Kangas, I. and Perila, M. (1985) Clinical praxis and laboratory procedures in subrenal capsule assay (SCRA). Ann. Chir. Gynaecol., 74, Supp. 1990, 7- 11. Metcalf, D. (1975) Transplantation of Tumors. In: A training manual for cancer research workers. UICC Technical Report Series Vol. 20, pp. 40-43, Editor: D. Metcalf, Geneva. Schor, S.L., Schor, A.M., Wfnn, B. and Rushton, G. (1982) The use of three-dimensional collagen gels for the study of tumour cell invasion in vitro: experimental parameters influencing cell migration into the gel matrix. Int. J. Cancer, 29, 57-62. Terracini, B. and Magee, P.N. (1964) Renal tumours in rats following injection of dimethylnitrosamine at birth. Nature, 202,502. Yang, J., Richards, J., Bowman, P., Guxman, R., Enami, J., McCormick, K., Hamamoto, S., Pitelka, D. and Nandi, S. (1979) Sustained growth and threedimensional organization of primary mammary tumor epithelial cells embedded in collagen gels. Proc. Natl. Acad. Sci., 76,3401-3405.
