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Metastatic growth of human tumour xenografts in thymectomised irradiated mice reconstituted with syngeneic bone marrow cells
Europ. J. CancerVol. 11, pp. 619-622. Pergamon Press 1975. Printed in Great Britain
Metastatic Growth of Human Tumour Xenografts in Thymectomised Irradiated Mice Reconstituted with Syngeneic Bone Marrow Cells C. R. FRANKS*, L. R. BOULGER, A. J. GARRETT, DIANA BISHOP, DIANE REESON and F. T. PERKINSt Division of Viral Products, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London N W 3 6RB. Abstra©t--Human tumour xenografls were grown by s.c. implantation zn thymectomised irradiated mice reconstituted with syngeneic bone marrow cells. Sixty-three tumour specimens were implanted into 462 mice, and tumour survivals of 1 to 20 weeks were recorded. At autopsy, a comprehensive macroscopic and microscopic examination was made. Eighteen cases of metastasis were observedfrom 5 different tumour types.
MATERIAL AND M E T H O D S
INTRODUCTION
HeLa tumours
THE OROWTH of h u m a n tumours in thymectomised irradiated mice reconstituted with syngeneic bone marrow cells ( T - B + ) has been studied extensively [1-4]. In all these investigations using the T - B + mouse, the tumours were implanted either in the renal capsule [1, 2], or subcutaneously [3, 4]. There has not been a single recorded case of metastatic growth in any of these studies. In 1974, Franks et al. reported that a h u m a n melanoma implanted in a mouse had metastasised to a para-aortic node [5]. Since then, metastatic growth from 3 other turnout types has been reported [6]. This report includes another 14 cases of metastases, making a total of 18 cases. The analysis is based on autopsy examination of 462 mice, into which 63 tumour specimens were implanted.
HeLa cells were originally obtained from the Central Public Health Laboratories at Colindale. Thereafter, the cells were passaged in the laboratory; they were shown to be free from mycoplasma. Each mouse received 2"5 x 10 5 cells s.c., when the H e L a cells were given as a suspension, or, at least 3 m m 3, when transplanted from a mouse carrying a solid HeLa tumour. One hundred and sixty implantations were made from 12 H e L a samples. Human tumours
T u m o u r specimens were obtained at operation from patients with primary cancer. These were transported to the laboratory in a medium containing Eagle's minimum essential medium, 2% calf serum, 200 units m1-1 of penicillin, and 100 pg ml - 1of streptomycin sulphate. At least 3 m m 3 pieces of tumour were implanted s.c. in the iliac region of the mouse. Three hundred and two implantations were made from 51 tumour specimens.
Accepted 27 May 1975. *ICRF Breast Cancer Unit, Guy's Hospital, London, SEI 9RT. tChief, Biological Standardization, W.H.O., 1211 Geneva 27, Switzerland.
Mice
Female CBA mice bred in the specific 619
620
C. R. Franks, L. R. Boulger, A. J. Garrett, Diana Bishop, Diane Reeson and F. T. Perkins
pathogen-free colony at the National Institute for Medical Research were used.
Immune deprivation A method similar to that of Miller et al. (1963) [7] was used. The mice were thymectomised at 4 weeks of age, and 3 weeks later were given a lethal dose of 900R whole body irradiation from a 6°Co source. Within 5 hr of irradiation, each mouse received 10 7 syngeneic bone marrow cells i.v.
Measurement of tumours The vertical and transverse diameters of the solid tumour implants were measured immediately after implantation, and thereafter once a week, using vernier calipers. As soon as a palpable tumour appeared after the inoculation of a cell suspension, the vertical and transverse diameters were also measured. Where possible, the same person took all the measurements for a particular tumour specimen.
Autopsy examination When the implanted tumours threatened the life of the mouse, or showed signs of regression, the mouse was killed. Histological sections were made of the following: (1) (3) (5) (7) (8)
T u m o u r implant (2) Lungs Liver (4) Spleen Kidneys (6) Ovaries L y m p h nodes Vertebral column and long bones
H e L a tumour specimens, only 94 mice showed evidence of a palpable tumour at one week (Table 1). By eight weeks, 40 mice had palpable tumours, and, at 20 weeks, only 7 of the original 160 mice had palpable turnouts. Thirteen cases of metastatic growth were found at autopsy. O f the 302 mice implanted with 51 tumour specimens from patients with primary cancer, 13 mice with 2 caecal, 3 rectal, and 5 anal carcinoma implants were still palpable at 20 weeks. Three mice had palpable melanomas (Table 1). At 16 weeks, just under half of the mice, implanted with 17 specimens from patients with breast cancer, had palpable tumours, and 3 of the 18 mice implanted with colonic carcinomas also had palpable tumours. O f the remaining 6 tumour types investigated, only the mice implanted with squamous cell carcinomas had palpable tumours beyond 8 weeks. Mice with gastric and basal cell carcinomas had detectable tumours at 8 weeks, and the remaining 3 tumour types, mainly carcinomas of the tongue, pancreas, and bronchus did not grow beyond the first week following implantation. In all the specimens in Table 1, the histological analysis at autopsy, and the comparison of the implant with the original tumour specimen removed from the patient, confirmed the presence of the specific tumour specimen being studied. It also demonstrated 5 cases of metastatic growth.
Metastatic growth of tumours
RESULTS
Survival of tumour implants O f the 160 mice implanted with the 12
Metastases were found at autopsy in mice into which 2 melanomas, a colon, a rectal, and a squamous cell carcinoma had been implanted. In addition, 13 cases of metastases, following implantation with HeLa, were also found (Table 2). Nine out of 18 cases were to lymph nodes; 8 out 18 to lung, and 1 out 18 to spleen.
Table 1. Survival of s.c. implanted tumours in T - B + mice i
Tumour Tumour type implants HeLa Melanoma Colon Breast Caecum Rectum Anus Sq cell ca Tongue Pancreas Bronchus Stomach Basal cell ca
Mice implants
12 17 3 3 6 5 7 2 2
160 84 18 17 61 27 43 12 7
1 1
Mice with Mice takes metastases 1 wk 4 wk 8 wk 12 wk 16wk 13 2 1 0 0 1 0 1 0
94 67 12 11 42 25 37 11 2
59 40 8 15 28 13 21 2
8 8
0 0
8 7
1
3
15
0
15
7
4
1
2
0
1
1
1
ill
i
40 21 4 8 13 6 16 2
24 6 3 7 7 5 8 1
21 5 3 6 5 3 5
20 wk
Fig. 1.
Metastatic growth of carcinoma of rectum to mouse lung. x 80.
Fig. 2.
Metastatic growth of carcinoma of colon to mouse lung. × 80.
(to face page 620)
Fig. 3.
Metastatic growth of melanoma to mouse para-aortic node. x 200.
Fig. 4.
Metastatic growth
of tteLa
tumour
to mouse lung. x 4 0 .
Metastatic Growth of Huron Tumour Xenografts Table 2. Tumour type
621
Metastaticgrowth of s.c. implanted tumours in T - B+ mice Day killed after implantation
Melanoma Melanoma Colon Rectum Sc Ca Pinna HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa
Passage
19 69 146 47 98 38 46 64 66 84 84 91 106 124 154 161 161 199
These occurred from day 19 to day 199 after implantation. Fig. 1-4 show metastatic growth of the rectum, colon, a melanoma, and HeLa tumours.
DISCUSSION
It is not entirely clear why metastatic growth of tumour xenografts is now occurring. Although this is a recent finding in T - B + mice, Cobb has observed metastatic spread in immune deficient hamsters [8]. In these studies, 3.9% of the mice demonstrated metastases, and 2-3% occurred from HeLa tumours. In mice treated with antilymphocyte serum, HeLa tumours never metastasise [9]. Why HeLa should demonstrate such radically different behaviour in two similar experimental groups of mice, both of which were immune incompetent to a greater or lesser extent, is not possible to say. O f the 12 primary carcinomas investigated, metastases were found only in 1.1% of implanted mice. Clearly, in the T - B+ mouse, these tumours do not retain their h u m a n metastatic characteristics. Were this the case, metastases would be found in a high percentage of mice, because m a n y of the tumours studied have a high tendency to metastasise in humans. Metastases do not appear to be related to any particular growth period of a tumour in the mouse, nor are they governed by the
1st 1st 1st 1st 1st 1st 1st 1st 1st 1st 1st 1st 2nd 6th 1st 1st 1st 3rd
Site of metastases Lymph node (no type) Para-aortic node Lung Lung Para-aortic node and mesentery Lung Inguinal node Axillary node Lung Lung Inguinal node Lung Inguinal node Lung Para-aortic node Spleen Lung Para-aortic node
number of passages of an individual tumour. Most of the metastatic growth observed occurred during the first passage, and varied from day 19 to day 199, these being the days the mice were killed. It is of course possible that metastases may have developed long before the mice were withdrawn from the experiment. Five of the tumours included in this study were in mice killed after the 20 week study period. The only significant change that has been made recently is the use of mice from the specific pathogen-free unit at the National Institute of Medical Research. This was done to guarantee pathogen-free mice unlike the outbred stock of mice which often died following thymectomy. In view of the rarity of metastatic growth of tumour xenografts in T B+ mice, it is possible that the process of transplantation of primary tumours to mice represents essentially the process of metastasis. As such, when carcinomas are removed from their primary h u m a n environment and transplanted to mice, they no longer behave as primary tumours, and therefore lose the h u m a n characteiistic of being able to metastasise, being metastases themselves now. This hypothesis, however, does not account for the rare finding of mouse metastases from implanted human tumours, nor does it explain why HeLa tumours metastasise in T - B + mice, and not in mice treated with antilymphocyte serum. It is an interesting finding, which needs further study.
622
C. R. Franks, L. R. Boulger, A. J. Garrett, Diana Bishop, Diane Reeson and F. T. Perkins REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9.
J . E . CASTRO, Human tumours grown in mice. Nature (New Biol.) 239, 83 (1972). J . E . CASTRO,A method of in vivo maintenance of human prostatic tissue in immunosuppressed mice. Brit. J. Urol. 45, 163 (1973). C.R. FRANKS,F. T. PERKINSand J. THORNTONHOLMES,Subcutaneous growth of human tumours in mice. Nature (Lond.) 243, 91 (1973). L . M . COBB and B. C. V. MITeHLE'Z,Growth of human tumours in immune deprived mice. Europ. J. Cancer 10, 473 (1974). C . R . FRANKS,F. T. PERKINSand J. THORNTON HOLMES,Growth of human tumours in immune suppressed mice. Proc. roy. Soc. Med. ~ , 287 (1975). C . R . FRANKS, F. T. PERKINS, L. R. BOULGER and J. THORNTON HOLMES, Maintenance of h u m a n tumours in i m m u n e deficient mice. Brit. J. Cancer 31~
260 (1975). J . F . A . P . MILLER,SHEILAM. A. DOAKand A. MARJORIECROSS,Role of the thymus in recovery of the immune mechanism in the irradiated adult mouse. Proc. Soc. exp. Biol. Med. 112, 785 (1963). L . M . COBB, Metastatic spread of human tumour implanted into thymeetomised antilymphocyte serum treated hamsters. Brit. J. Cancer 26, 183 (1972). C . R . FRANKS,KRYSTYNACURTIS and F. T. PERKINS, Long-term survival of HeLa tumours in mice treated with antilymphocyte serum. Brit. J. Cancer 27, 390 (1973).
Metastatic Growth of Human Tumour Xenografts in Thymectomised Irradiated Mice Reconstituted with Syngeneic Bone Marrow Cells C. R. FRANKS*, L. R. BOULGER, A. J. GARRETT, DIANA BISHOP, DIANE REESON and F. T. PERKINSt Division of Viral Products, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London N W 3 6RB. Abstra©t--Human tumour xenografls were grown by s.c. implantation zn thymectomised irradiated mice reconstituted with syngeneic bone marrow cells. Sixty-three tumour specimens were implanted into 462 mice, and tumour survivals of 1 to 20 weeks were recorded. At autopsy, a comprehensive macroscopic and microscopic examination was made. Eighteen cases of metastasis were observedfrom 5 different tumour types.
MATERIAL AND M E T H O D S
INTRODUCTION
HeLa tumours
THE OROWTH of h u m a n tumours in thymectomised irradiated mice reconstituted with syngeneic bone marrow cells ( T - B + ) has been studied extensively [1-4]. In all these investigations using the T - B + mouse, the tumours were implanted either in the renal capsule [1, 2], or subcutaneously [3, 4]. There has not been a single recorded case of metastatic growth in any of these studies. In 1974, Franks et al. reported that a h u m a n melanoma implanted in a mouse had metastasised to a para-aortic node [5]. Since then, metastatic growth from 3 other turnout types has been reported [6]. This report includes another 14 cases of metastases, making a total of 18 cases. The analysis is based on autopsy examination of 462 mice, into which 63 tumour specimens were implanted.
HeLa cells were originally obtained from the Central Public Health Laboratories at Colindale. Thereafter, the cells were passaged in the laboratory; they were shown to be free from mycoplasma. Each mouse received 2"5 x 10 5 cells s.c., when the H e L a cells were given as a suspension, or, at least 3 m m 3, when transplanted from a mouse carrying a solid HeLa tumour. One hundred and sixty implantations were made from 12 H e L a samples. Human tumours
T u m o u r specimens were obtained at operation from patients with primary cancer. These were transported to the laboratory in a medium containing Eagle's minimum essential medium, 2% calf serum, 200 units m1-1 of penicillin, and 100 pg ml - 1of streptomycin sulphate. At least 3 m m 3 pieces of tumour were implanted s.c. in the iliac region of the mouse. Three hundred and two implantations were made from 51 tumour specimens.
Accepted 27 May 1975. *ICRF Breast Cancer Unit, Guy's Hospital, London, SEI 9RT. tChief, Biological Standardization, W.H.O., 1211 Geneva 27, Switzerland.
Mice
Female CBA mice bred in the specific 619
620
C. R. Franks, L. R. Boulger, A. J. Garrett, Diana Bishop, Diane Reeson and F. T. Perkins
pathogen-free colony at the National Institute for Medical Research were used.
Immune deprivation A method similar to that of Miller et al. (1963) [7] was used. The mice were thymectomised at 4 weeks of age, and 3 weeks later were given a lethal dose of 900R whole body irradiation from a 6°Co source. Within 5 hr of irradiation, each mouse received 10 7 syngeneic bone marrow cells i.v.
Measurement of tumours The vertical and transverse diameters of the solid tumour implants were measured immediately after implantation, and thereafter once a week, using vernier calipers. As soon as a palpable tumour appeared after the inoculation of a cell suspension, the vertical and transverse diameters were also measured. Where possible, the same person took all the measurements for a particular tumour specimen.
Autopsy examination When the implanted tumours threatened the life of the mouse, or showed signs of regression, the mouse was killed. Histological sections were made of the following: (1) (3) (5) (7) (8)
T u m o u r implant (2) Lungs Liver (4) Spleen Kidneys (6) Ovaries L y m p h nodes Vertebral column and long bones
H e L a tumour specimens, only 94 mice showed evidence of a palpable tumour at one week (Table 1). By eight weeks, 40 mice had palpable tumours, and, at 20 weeks, only 7 of the original 160 mice had palpable turnouts. Thirteen cases of metastatic growth were found at autopsy. O f the 302 mice implanted with 51 tumour specimens from patients with primary cancer, 13 mice with 2 caecal, 3 rectal, and 5 anal carcinoma implants were still palpable at 20 weeks. Three mice had palpable melanomas (Table 1). At 16 weeks, just under half of the mice, implanted with 17 specimens from patients with breast cancer, had palpable tumours, and 3 of the 18 mice implanted with colonic carcinomas also had palpable tumours. O f the remaining 6 tumour types investigated, only the mice implanted with squamous cell carcinomas had palpable tumours beyond 8 weeks. Mice with gastric and basal cell carcinomas had detectable tumours at 8 weeks, and the remaining 3 tumour types, mainly carcinomas of the tongue, pancreas, and bronchus did not grow beyond the first week following implantation. In all the specimens in Table 1, the histological analysis at autopsy, and the comparison of the implant with the original tumour specimen removed from the patient, confirmed the presence of the specific tumour specimen being studied. It also demonstrated 5 cases of metastatic growth.
Metastatic growth of tumours
RESULTS
Survival of tumour implants O f the 160 mice implanted with the 12
Metastases were found at autopsy in mice into which 2 melanomas, a colon, a rectal, and a squamous cell carcinoma had been implanted. In addition, 13 cases of metastases, following implantation with HeLa, were also found (Table 2). Nine out of 18 cases were to lymph nodes; 8 out 18 to lung, and 1 out 18 to spleen.
Table 1. Survival of s.c. implanted tumours in T - B + mice i
Tumour Tumour type implants HeLa Melanoma Colon Breast Caecum Rectum Anus Sq cell ca Tongue Pancreas Bronchus Stomach Basal cell ca
Mice implants
12 17 3 3 6 5 7 2 2
160 84 18 17 61 27 43 12 7
1 1
Mice with Mice takes metastases 1 wk 4 wk 8 wk 12 wk 16wk 13 2 1 0 0 1 0 1 0
94 67 12 11 42 25 37 11 2
59 40 8 15 28 13 21 2
8 8
0 0
8 7
1
3
15
0
15
7
4
1
2
0
1
1
1
ill
i
40 21 4 8 13 6 16 2
24 6 3 7 7 5 8 1
21 5 3 6 5 3 5
20 wk
Fig. 1.
Metastatic growth of carcinoma of rectum to mouse lung. x 80.
Fig. 2.
Metastatic growth of carcinoma of colon to mouse lung. × 80.
(to face page 620)
Fig. 3.
Metastatic growth of melanoma to mouse para-aortic node. x 200.
Fig. 4.
Metastatic growth
of tteLa
tumour
to mouse lung. x 4 0 .
Metastatic Growth of Huron Tumour Xenografts Table 2. Tumour type
621
Metastaticgrowth of s.c. implanted tumours in T - B+ mice Day killed after implantation
Melanoma Melanoma Colon Rectum Sc Ca Pinna HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa
Passage
19 69 146 47 98 38 46 64 66 84 84 91 106 124 154 161 161 199
These occurred from day 19 to day 199 after implantation. Fig. 1-4 show metastatic growth of the rectum, colon, a melanoma, and HeLa tumours.
DISCUSSION
It is not entirely clear why metastatic growth of tumour xenografts is now occurring. Although this is a recent finding in T - B + mice, Cobb has observed metastatic spread in immune deficient hamsters [8]. In these studies, 3.9% of the mice demonstrated metastases, and 2-3% occurred from HeLa tumours. In mice treated with antilymphocyte serum, HeLa tumours never metastasise [9]. Why HeLa should demonstrate such radically different behaviour in two similar experimental groups of mice, both of which were immune incompetent to a greater or lesser extent, is not possible to say. O f the 12 primary carcinomas investigated, metastases were found only in 1.1% of implanted mice. Clearly, in the T - B+ mouse, these tumours do not retain their h u m a n metastatic characteristics. Were this the case, metastases would be found in a high percentage of mice, because m a n y of the tumours studied have a high tendency to metastasise in humans. Metastases do not appear to be related to any particular growth period of a tumour in the mouse, nor are they governed by the
1st 1st 1st 1st 1st 1st 1st 1st 1st 1st 1st 1st 2nd 6th 1st 1st 1st 3rd
Site of metastases Lymph node (no type) Para-aortic node Lung Lung Para-aortic node and mesentery Lung Inguinal node Axillary node Lung Lung Inguinal node Lung Inguinal node Lung Para-aortic node Spleen Lung Para-aortic node
number of passages of an individual tumour. Most of the metastatic growth observed occurred during the first passage, and varied from day 19 to day 199, these being the days the mice were killed. It is of course possible that metastases may have developed long before the mice were withdrawn from the experiment. Five of the tumours included in this study were in mice killed after the 20 week study period. The only significant change that has been made recently is the use of mice from the specific pathogen-free unit at the National Institute of Medical Research. This was done to guarantee pathogen-free mice unlike the outbred stock of mice which often died following thymectomy. In view of the rarity of metastatic growth of tumour xenografts in T B+ mice, it is possible that the process of transplantation of primary tumours to mice represents essentially the process of metastasis. As such, when carcinomas are removed from their primary h u m a n environment and transplanted to mice, they no longer behave as primary tumours, and therefore lose the h u m a n characteiistic of being able to metastasise, being metastases themselves now. This hypothesis, however, does not account for the rare finding of mouse metastases from implanted human tumours, nor does it explain why HeLa tumours metastasise in T - B + mice, and not in mice treated with antilymphocyte serum. It is an interesting finding, which needs further study.
622
C. R. Franks, L. R. Boulger, A. J. Garrett, Diana Bishop, Diane Reeson and F. T. Perkins REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9.
J . E . CASTRO, Human tumours grown in mice. Nature (New Biol.) 239, 83 (1972). J . E . CASTRO,A method of in vivo maintenance of human prostatic tissue in immunosuppressed mice. Brit. J. Urol. 45, 163 (1973). C.R. FRANKS,F. T. PERKINSand J. THORNTONHOLMES,Subcutaneous growth of human tumours in mice. Nature (Lond.) 243, 91 (1973). L . M . COBB and B. C. V. MITeHLE'Z,Growth of human tumours in immune deprived mice. Europ. J. Cancer 10, 473 (1974). C . R . FRANKS,F. T. PERKINSand J. THORNTON HOLMES,Growth of human tumours in immune suppressed mice. Proc. roy. Soc. Med. ~ , 287 (1975). C . R . FRANKS, F. T. PERKINS, L. R. BOULGER and J. THORNTON HOLMES, Maintenance of h u m a n tumours in i m m u n e deficient mice. Brit. J. Cancer 31~
260 (1975). J . F . A . P . MILLER,SHEILAM. A. DOAKand A. MARJORIECROSS,Role of the thymus in recovery of the immune mechanism in the irradiated adult mouse. Proc. Soc. exp. Biol. Med. 112, 785 (1963). L . M . COBB, Metastatic spread of human tumour implanted into thymeetomised antilymphocyte serum treated hamsters. Brit. J. Cancer 26, 183 (1972). C . R . FRANKS,KRYSTYNACURTIS and F. T. PERKINS, Long-term survival of HeLa tumours in mice treated with antilymphocyte serum. Brit. J. Cancer 27, 390 (1973).