Biologicals
(1991)
19, 67-70
tncremmd
Sensitivity of Antithymocyte GlobuHn-t Newborn Rat Assay for Metastasis
Evgenia Dragunsky,*t Dale Wierenga,$ lnessa Levenbook* *Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, U.S.A. and $-Pharmaceutical Manufacturers Association, Washington, DC, U.S.A.
Abstract. Tumorigenic and metastatic abilities of FL, transformed human amnion, Vero and LLC-MK2, continuous monkey kidney cell lines (CCLs), none of which produced lung metastases in nude mice, were studied in ATG-treated newborn Wistar rats. Effects of subcutaneous (s.c.) and intraperitoneal (i.p.) inoculation of lo6 and lo7 cells were compared. I.p. inoculation of the same number of FL cells produced larger primary tumors, increased incidence of lung metastasis and more metastases per rat than S.C. inoculation. The same was true of the higher inocula administered by either route as compared to the smaller inocula. The CCLs (Vero at passages 320 and 323, LLC-MK2 at passage 306) inoculated either S.C. or i.p. induced small tumors regressing in all animals. After S.C. inoculation of 10” Vero or LLC-MK2 cells lung metastases did not develop in any rats, but lo7 inoculum and i.p. administration of both size inocula caused single lung metastases. Thus, increased sensitivity of the rat system was achieved by larger size of the S.C. inoculum or the i.p. route of inoculation. Highly sensitive assay is especially important for evaluation of possible metastatic abilities of CCLs. Introduction
Materials
The most widely used model for studying the behaviour of tumors, the adult nude mouse, has a serious disadvantage. Xenografted malignant tumors rarely metastasize in these animals1~2 especially during relatively short-term observation.3 Van Steenis & van Weze14 recommended anti-rat thymocyte globulin (ATG))-treated newborn Wistar rats inoculated subcutaneously (s.c.) for testing the tumorigenicity of monkey continuous cell lines (CCL.9 being considered as substrates for vaccine production. Other authors, and our unpublished data, demonstrated the advantage of the rat over the adult nude mouse system in revealing metastatic potential of monkey CCLS.~,~ The objective of the present study was to increase the sensitivity of the rat system for testing the metastatic potential of cells. This is especially important for CCLs which are considered to be low or moderately malignant. We were able to demonstrate that this can be achieved by increasing the number of cells for the S.C. inoculum or using the intraperitoneal (i.p.) route of inoculation.
Cells A highly malignant, transformed human amnion cell line, FL, was obtained from ATCC, Rockville, Maryland, U.S.A, at passage 480 (CCL No. 621 and used as a positive control.’ Lines of special interest to this laboratory for their metastatic potential were monkey kidney CCLs, Vero, passages 320 and 323, and LLC-MK2, passage 306. Both monkey cell lines were acquired from J. Cleveland and J. Rodgers (Center for Biologics, Bethesda, Maryland, U.S.A.). Cells were grown in Eagle’s MEM supplemented with 10% fetal bovine serum. For cell passage the split ratio was 1:lO.
tTo whom requests for reprints should be sent. Address: CBER, Bldg. 29A, 8800 Rockville Pike, Bethesda, Maryland 20892, U.S.A. 1045--1056/91/020067+04
$03.00/O
and methods
Animals Pregnant Wistar rats were obtained from Charles River Breeding Labs, Inc., Wilmington, Massachusetts, U.S.A. ATG (kindly given by Dr van Steenis) was administered S.C. (O-1 ml per rat, on days 0 and 14) and lo6 or lo7 FL, Vero or LLC-MKa cells were injected either S.C.into the subscapular region or i.p. into newborn rats on day 0. Animals were necropsied on day 21, and the volume of the primary tumors was assessed. Inoculation sites, tumors and lungs examined histologically. Tissues were were embedded in paraffln, and sections were taken from @ 1991 The
International
Association
of Biological
Standardization
66
E. Dragunsky
Table
1. Frequency
of local tumors
et al.
and lung metastases,
number
and range of metastases
No. of rats Cell line passage No. FL
Inocul. route
lo6
8
i.p.
lo7 lo6 lo7
12 8 16
lo6
S.C.
lo7
320,323
i.p.
lo6 lo7
LLC-MKs
Inoculated
S.C.
486
Vero
No. of cells
S.C.
306
i.p.
With primary
tumors
7112t
With metastases
Median No. of metastases” (range)
16t
3 10 81 WI
2(1-2) 10(1-46)§ 4(3-8) 80(11-220)
8 9 19 10
6$ 9$ lO$ 6$
0 3 13 8
1(0-l) yJl--&? -
6t
lo6 lo7 lo6
7
31
0
16 13
12$
5
20-16)“”
lo7
10
it
2 3
l(l-2) l(l-2)
* Computed of the maximum No. of metastasesfound in one of the histological sectionsof the lungs (seeMaterial and Methods).
t Progressing tumors. $ Regressed tumors. $ The range of No. of metastasesin nine rats, whereas in the remaining rat the No. of metastasesexceeded 500. 7 Multiple metastases,many of them large. 11 The range of No. of metastasesin nine rats, whereas in four rats there were from 10 to 40 metastases. ** The range of No. of metastasesin four rats, whereas in one rat it reached 76.
two different levels of an inoculation site when a tumor was not detected macroscopically, or from one level of the tumor and from three levels of the lungs cut longitudinally. Sections were stained with hematoxylin and eosin. Results Our findings on the frequency of primary tumors and lung metastases in rats inoculated S.C. and i.p. with FL, Vero and LLC-MK2 cells are summarized in Table 1. Primary tumors
FL cell S.C. tumors were progressing in the majority of animals. These tumors were identified histologically as poorly differentiated and highly invasive adenocarcinomas. By the end of the observation period the average volume of the S.C. tumors which resulted from 10” cells was O-76 + 0.92 cm3 and those from lo7 cells 2.91 f 1.87 cm.3 None ofthe S.C.tumors which developed from the lo6 FL cell inoculum invaded the peritoneal cavity while almost half of the rats inoculated S.C. with lo7 cells had peritoneal invasion. I. p. tumor growth in all the rats inoculated with
lo7 cells was voluminous, and in some animals it filled the peritoneal cavity, All Vero and LLC-MK2 cell tumors, both S.C. and i.p., were regressing, so that by the end of the observation period only tiny nodules were detectable in some animals. After 3 weeks the average volume of the S.C. tumors resulting from a lo6 cell inoculum did not exceed 0.001 cm,3 and the average volume of those from lo7 cells was 0.058 + 0.05 cm.’ Histological examination revealed encapsulated remnants of adenocarcinomas. In rats inoculated i.p. with both size inocula of the CCLs, tiny nodules on the peritoneum were seen in half of the rats. Metastasis
Inoculation
of FL cells resulted in lung metastasis
in one third of the rats which received lo6 cells s.c., in
83% of those inoculated
S.C. with lo7 cells and in all
the rats inoculated i.p. with either lo6 or lo7 cells. After inoculation of lo7 FL cells S.C. the median number of lung metastases was five-times higher and, when inoculated i.p., 40-times higher than after
S.C. inoculation
of lo6 cells. In groups of rats which
received lo7 FL cell inocula, either S.C. or i.p., the numerical range of metastases was extremely broad.
Rat model for metastmio
studies
69
Figure 1. Lung metastasesin rats. Hematoxylin and eosin.(a) Metastasesin a rat inoculated S.C.with 10’ FL cells.Bar 500 .um. (b) The same animal as seen in (a). Metastases varied in size, some with necrotic centers (arrows). Bar 100 pm. (c) Metastasis in a rat inoculated i.p. with lo6 Vero cells.Bar 50 pm. (d) Metastasis with a mitosis(arrow) in a rat inoculated i.p. with lo6 LLC-MK2 cells. Bar 50 pm.
In rats inoculated S.C. with lo7 cells the number of metastases in nine individual animals ranged from one to 46 and in one rat it exceeded 500. This rat’s lungs are shown in Fig. l(a). In rats inoculated i.p. with 10’ cells the numerical range of metastases was between 11 and 220. Metastases varied in size, some had necrotic centers [Fig. l(b)]; many mitoses were seen. In four rats, additional metastases were found in the mediastinal lymph nodes. Vero cells in lo7 cell inocula administered S.C. caused metastases in three of nine rats and LLC-MKs in five of 16 rats, but not in any rats receiving lo6 cells. At the same time, i.p. 10’ inoculum of Vero cells resulted in metastases in 13 of 19 rats, and LLC-
MKc in two of 13. The CCLs metastases were detected histologically as single or very few foci consisting of a small number of cells (Figs. UC and d)] in majority of animals, although in some rats there were many and larger metastases. No signs of regression were seen in metastases. Only single mitoses could be found in some metastatic foci [(Fig. l(d)]. Discussion We found a direct correlation between the size of the FL cell inoculum and the size of primary S.C. tumors. The average volume of tumors 3 weeks post S.C. inoculation of lo7 FL cells was three-times larger
E. Oragunsky et al.
70
than the average volume of tumors formed after inoculation of lo6 cells. Since the widely-spread i.p. tumor growth could not be measured, similar calculation cannot be presented. However, tumor growth was more abundant in rats inoculated i.p. with lo7 FL cells. Higher or lower numbers of FL cells administered S.C. correlated with the number of rats which developed lung metastases as well as with the median number of metastases. The size of the i.p. inoculum did not effect the incidence of metastasis but directly correlated with the median number of metastases. When the CCLs were administered s.c., only lo7 cell inoculum was able to reveal their metastatic abilities, while there were no differences in effects of smaller and larger inocula for i.p. inoculation. The data presented here demonstrate further enhancement of the sensitivity of this valuable model for the study of tumorigenicity and metastasis by using a higher number of cells for the S.C. inoculum and the i.p. route of inoculation. In experiments with FL cells, the varying size of lung metastases with necrotic centers in some of them may indicate that metastases arose as a result of both initial inoculation of cells and cells released from invasively growing primary tumors. At the same time, in a few rats with lung metastases inoculated i.p. with the FL cells, primary tumors regressed, suggesting that metastases in these rats may have occurred early, most likely at the time of inoculation. As far as CCLs is concerned, in all metastasesbearing animals local tumors regressed, an indication that the metastases did occur at the time of inoculation. It has been shown that the formation of experimental metastases in lungs after intravenous injection of tumor cells, which can be defined as colonization, correlates well with metastatic potential of malignant
neoplasms
and can be used as an assay.7
Regression of local tumors caused by the CCLs can hardly be considered a reflection of their low malignancy but rather a feature of renal carcinomas. In our experiments in rats with the Caki-2 cell line of renal adenocarcinoma origin when 21 rats were inoculated S.C. and 19 i.p., local tumors also regressed in all of them (unpublished data). The lung tissue of the ATG-treated newborn rats is probably an appropriate ‘soil’ for metastasis. While primary tumors caused by the CCLs were always regressing, concurrently developed metastases survived though without remarkable progression.
References 1. Sharkey FE, Fogh J. Metastasis of human tumors in athymic nude mice. Int Cancer 1979; 24: 733-738. 2. Maguire H, Outzen HC, Custer P, Prehn RT. Invasion and metastasisof xenogeneic tumor in nude mice. J Natl Cancer Inst 1976; 57: 439-442. 3. Sordat B, Ueyama Y, Fogh J. Metastasis of tumor xenografts in the nude mouse.In: Fogh J and Giovanella BC, eds. The Nude Mouse in Experimental and Clinical Research.Academic Press, 1982: 95-147. 4. Van Steenis G, van Wezel Al. Use of the ATG-treated newborn rat for in vitro tumorigenicity testing of cell substrates. Devel Biol Stand, 1982; 50: 37-46. 5. Bather R, Becker BC, Contreras G, Furesz J. Heterotransplantation studies with tissue culture cell lines in various animal and in vitro systems.J. Biol Stand 1985; 13: 13-32. 6. Furesz J, Fanok A, Contreras G, Becker B. Tumorigenicity testing of various cell substrates for production of biologicals. Devel Biol Stand, 1989; 70: 233-243. 7. Talmage JR, Fidler IJ. Cancer metastasisis selective or random depending on the parent tumour population. Nature 1982; 27: 593-594. Received for publication 6 April 1990; accepted 14 January 1991.
(1991)
19, 67-70
tncremmd
Sensitivity of Antithymocyte GlobuHn-t Newborn Rat Assay for Metastasis
Evgenia Dragunsky,*t Dale Wierenga,$ lnessa Levenbook* *Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, U.S.A. and $-Pharmaceutical Manufacturers Association, Washington, DC, U.S.A.
Abstract. Tumorigenic and metastatic abilities of FL, transformed human amnion, Vero and LLC-MK2, continuous monkey kidney cell lines (CCLs), none of which produced lung metastases in nude mice, were studied in ATG-treated newborn Wistar rats. Effects of subcutaneous (s.c.) and intraperitoneal (i.p.) inoculation of lo6 and lo7 cells were compared. I.p. inoculation of the same number of FL cells produced larger primary tumors, increased incidence of lung metastasis and more metastases per rat than S.C. inoculation. The same was true of the higher inocula administered by either route as compared to the smaller inocula. The CCLs (Vero at passages 320 and 323, LLC-MK2 at passage 306) inoculated either S.C. or i.p. induced small tumors regressing in all animals. After S.C. inoculation of 10” Vero or LLC-MK2 cells lung metastases did not develop in any rats, but lo7 inoculum and i.p. administration of both size inocula caused single lung metastases. Thus, increased sensitivity of the rat system was achieved by larger size of the S.C. inoculum or the i.p. route of inoculation. Highly sensitive assay is especially important for evaluation of possible metastatic abilities of CCLs. Introduction
Materials
The most widely used model for studying the behaviour of tumors, the adult nude mouse, has a serious disadvantage. Xenografted malignant tumors rarely metastasize in these animals1~2 especially during relatively short-term observation.3 Van Steenis & van Weze14 recommended anti-rat thymocyte globulin (ATG))-treated newborn Wistar rats inoculated subcutaneously (s.c.) for testing the tumorigenicity of monkey continuous cell lines (CCL.9 being considered as substrates for vaccine production. Other authors, and our unpublished data, demonstrated the advantage of the rat over the adult nude mouse system in revealing metastatic potential of monkey CCLS.~,~ The objective of the present study was to increase the sensitivity of the rat system for testing the metastatic potential of cells. This is especially important for CCLs which are considered to be low or moderately malignant. We were able to demonstrate that this can be achieved by increasing the number of cells for the S.C. inoculum or using the intraperitoneal (i.p.) route of inoculation.
Cells A highly malignant, transformed human amnion cell line, FL, was obtained from ATCC, Rockville, Maryland, U.S.A, at passage 480 (CCL No. 621 and used as a positive control.’ Lines of special interest to this laboratory for their metastatic potential were monkey kidney CCLs, Vero, passages 320 and 323, and LLC-MK2, passage 306. Both monkey cell lines were acquired from J. Cleveland and J. Rodgers (Center for Biologics, Bethesda, Maryland, U.S.A.). Cells were grown in Eagle’s MEM supplemented with 10% fetal bovine serum. For cell passage the split ratio was 1:lO.
tTo whom requests for reprints should be sent. Address: CBER, Bldg. 29A, 8800 Rockville Pike, Bethesda, Maryland 20892, U.S.A. 1045--1056/91/020067+04
$03.00/O
and methods
Animals Pregnant Wistar rats were obtained from Charles River Breeding Labs, Inc., Wilmington, Massachusetts, U.S.A. ATG (kindly given by Dr van Steenis) was administered S.C. (O-1 ml per rat, on days 0 and 14) and lo6 or lo7 FL, Vero or LLC-MKa cells were injected either S.C.into the subscapular region or i.p. into newborn rats on day 0. Animals were necropsied on day 21, and the volume of the primary tumors was assessed. Inoculation sites, tumors and lungs examined histologically. Tissues were were embedded in paraffln, and sections were taken from @ 1991 The
International
Association
of Biological
Standardization
66
E. Dragunsky
Table
1. Frequency
of local tumors
et al.
and lung metastases,
number
and range of metastases
No. of rats Cell line passage No. FL
Inocul. route
lo6
8
i.p.
lo7 lo6 lo7
12 8 16
lo6
S.C.
lo7
320,323
i.p.
lo6 lo7
LLC-MKs
Inoculated
S.C.
486
Vero
No. of cells
S.C.
306
i.p.
With primary
tumors
7112t
With metastases
Median No. of metastases” (range)
16t
3 10 81 WI
2(1-2) 10(1-46)§ 4(3-8) 80(11-220)
8 9 19 10
6$ 9$ lO$ 6$
0 3 13 8
1(0-l) yJl--&? -
6t
lo6 lo7 lo6
7
31
0
16 13
12$
5
20-16)“”
lo7
10
it
2 3
l(l-2) l(l-2)
* Computed of the maximum No. of metastasesfound in one of the histological sectionsof the lungs (seeMaterial and Methods).
t Progressing tumors. $ Regressed tumors. $ The range of No. of metastasesin nine rats, whereas in the remaining rat the No. of metastasesexceeded 500. 7 Multiple metastases,many of them large. 11 The range of No. of metastasesin nine rats, whereas in four rats there were from 10 to 40 metastases. ** The range of No. of metastasesin four rats, whereas in one rat it reached 76.
two different levels of an inoculation site when a tumor was not detected macroscopically, or from one level of the tumor and from three levels of the lungs cut longitudinally. Sections were stained with hematoxylin and eosin. Results Our findings on the frequency of primary tumors and lung metastases in rats inoculated S.C. and i.p. with FL, Vero and LLC-MK2 cells are summarized in Table 1. Primary tumors
FL cell S.C. tumors were progressing in the majority of animals. These tumors were identified histologically as poorly differentiated and highly invasive adenocarcinomas. By the end of the observation period the average volume of the S.C. tumors which resulted from 10” cells was O-76 + 0.92 cm3 and those from lo7 cells 2.91 f 1.87 cm.3 None ofthe S.C.tumors which developed from the lo6 FL cell inoculum invaded the peritoneal cavity while almost half of the rats inoculated S.C. with lo7 cells had peritoneal invasion. I. p. tumor growth in all the rats inoculated with
lo7 cells was voluminous, and in some animals it filled the peritoneal cavity, All Vero and LLC-MK2 cell tumors, both S.C. and i.p., were regressing, so that by the end of the observation period only tiny nodules were detectable in some animals. After 3 weeks the average volume of the S.C. tumors resulting from a lo6 cell inoculum did not exceed 0.001 cm,3 and the average volume of those from lo7 cells was 0.058 + 0.05 cm.’ Histological examination revealed encapsulated remnants of adenocarcinomas. In rats inoculated i.p. with both size inocula of the CCLs, tiny nodules on the peritoneum were seen in half of the rats. Metastasis
Inoculation
of FL cells resulted in lung metastasis
in one third of the rats which received lo6 cells s.c., in
83% of those inoculated
S.C. with lo7 cells and in all
the rats inoculated i.p. with either lo6 or lo7 cells. After inoculation of lo7 FL cells S.C. the median number of lung metastases was five-times higher and, when inoculated i.p., 40-times higher than after
S.C. inoculation
of lo6 cells. In groups of rats which
received lo7 FL cell inocula, either S.C. or i.p., the numerical range of metastases was extremely broad.
Rat model for metastmio
studies
69
Figure 1. Lung metastasesin rats. Hematoxylin and eosin.(a) Metastasesin a rat inoculated S.C.with 10’ FL cells.Bar 500 .um. (b) The same animal as seen in (a). Metastases varied in size, some with necrotic centers (arrows). Bar 100 pm. (c) Metastasis in a rat inoculated i.p. with lo6 Vero cells.Bar 50 pm. (d) Metastasis with a mitosis(arrow) in a rat inoculated i.p. with lo6 LLC-MK2 cells. Bar 50 pm.
In rats inoculated S.C. with lo7 cells the number of metastases in nine individual animals ranged from one to 46 and in one rat it exceeded 500. This rat’s lungs are shown in Fig. l(a). In rats inoculated i.p. with 10’ cells the numerical range of metastases was between 11 and 220. Metastases varied in size, some had necrotic centers [Fig. l(b)]; many mitoses were seen. In four rats, additional metastases were found in the mediastinal lymph nodes. Vero cells in lo7 cell inocula administered S.C. caused metastases in three of nine rats and LLC-MKs in five of 16 rats, but not in any rats receiving lo6 cells. At the same time, i.p. 10’ inoculum of Vero cells resulted in metastases in 13 of 19 rats, and LLC-
MKc in two of 13. The CCLs metastases were detected histologically as single or very few foci consisting of a small number of cells (Figs. UC and d)] in majority of animals, although in some rats there were many and larger metastases. No signs of regression were seen in metastases. Only single mitoses could be found in some metastatic foci [(Fig. l(d)]. Discussion We found a direct correlation between the size of the FL cell inoculum and the size of primary S.C. tumors. The average volume of tumors 3 weeks post S.C. inoculation of lo7 FL cells was three-times larger
E. Oragunsky et al.
70
than the average volume of tumors formed after inoculation of lo6 cells. Since the widely-spread i.p. tumor growth could not be measured, similar calculation cannot be presented. However, tumor growth was more abundant in rats inoculated i.p. with lo7 FL cells. Higher or lower numbers of FL cells administered S.C. correlated with the number of rats which developed lung metastases as well as with the median number of metastases. The size of the i.p. inoculum did not effect the incidence of metastasis but directly correlated with the median number of metastases. When the CCLs were administered s.c., only lo7 cell inoculum was able to reveal their metastatic abilities, while there were no differences in effects of smaller and larger inocula for i.p. inoculation. The data presented here demonstrate further enhancement of the sensitivity of this valuable model for the study of tumorigenicity and metastasis by using a higher number of cells for the S.C. inoculum and the i.p. route of inoculation. In experiments with FL cells, the varying size of lung metastases with necrotic centers in some of them may indicate that metastases arose as a result of both initial inoculation of cells and cells released from invasively growing primary tumors. At the same time, in a few rats with lung metastases inoculated i.p. with the FL cells, primary tumors regressed, suggesting that metastases in these rats may have occurred early, most likely at the time of inoculation. As far as CCLs is concerned, in all metastasesbearing animals local tumors regressed, an indication that the metastases did occur at the time of inoculation. It has been shown that the formation of experimental metastases in lungs after intravenous injection of tumor cells, which can be defined as colonization, correlates well with metastatic potential of malignant
neoplasms
and can be used as an assay.7
Regression of local tumors caused by the CCLs can hardly be considered a reflection of their low malignancy but rather a feature of renal carcinomas. In our experiments in rats with the Caki-2 cell line of renal adenocarcinoma origin when 21 rats were inoculated S.C. and 19 i.p., local tumors also regressed in all of them (unpublished data). The lung tissue of the ATG-treated newborn rats is probably an appropriate ‘soil’ for metastasis. While primary tumors caused by the CCLs were always regressing, concurrently developed metastases survived though without remarkable progression.
References 1. Sharkey FE, Fogh J. Metastasis of human tumors in athymic nude mice. Int Cancer 1979; 24: 733-738. 2. Maguire H, Outzen HC, Custer P, Prehn RT. Invasion and metastasisof xenogeneic tumor in nude mice. J Natl Cancer Inst 1976; 57: 439-442. 3. Sordat B, Ueyama Y, Fogh J. Metastasis of tumor xenografts in the nude mouse.In: Fogh J and Giovanella BC, eds. The Nude Mouse in Experimental and Clinical Research.Academic Press, 1982: 95-147. 4. Van Steenis G, van Wezel Al. Use of the ATG-treated newborn rat for in vitro tumorigenicity testing of cell substrates. Devel Biol Stand, 1982; 50: 37-46. 5. Bather R, Becker BC, Contreras G, Furesz J. Heterotransplantation studies with tissue culture cell lines in various animal and in vitro systems.J. Biol Stand 1985; 13: 13-32. 6. Furesz J, Fanok A, Contreras G, Becker B. Tumorigenicity testing of various cell substrates for production of biologicals. Devel Biol Stand, 1989; 70: 233-243. 7. Talmage JR, Fidler IJ. Cancer metastasisis selective or random depending on the parent tumour population. Nature 1982; 27: 593-594. Received for publication 6 April 1990; accepted 14 January 1991.
