Znt. J. Cancer: 20, 120-128 (1977)
THE EFFECT OF AZATHlOPRINE ON HOST CELL INFILTRATION A N D GROWTH OF A MURINE FIBROSARCOMA R. EVANS Department of Turnour Immunology, Chester Beatty Research Institute, Institute of Cancer Research, Clijton Avenue, Belmont, Sutton, Surrey, S M 2 5PX, England
Azathioprine (AZ) was injected into C57 Black mice before or after IM implantation of the syngeneic fibrosarcoma FS6, and observations were made on tumour growth and on the cellular composition of the fibrosarcomas. When multiple injections were given t o the tumour-bearing host the tumours regressed. The percentage of tumour-associated macrophages remained essentially stable as regression occurred whereas the percentage of B-antlgenpositive cells increased. In the case of single I P injections of AZ, changes i n the cellular composition of tumours were seen only when A 2 was given 1-72 h before implantation but not when it was given after implantation. Compared w i t h controls there was a 2-t o 3-week delay in the appearance of large numbers of tumour-associated macrophages. The percentage of 0-antigen-positive cells within the turnours mass was elevated 7 days post A Z treatment but the level declined t o control values between days 10 and 13. The significance of the results i s discussed i n relation t o the dependency of the macrophage content of t h i s tumour upon the development o f an immune response towards the tumour.
multiple injections of this antimetabolite or its derivative, 6 mercaptopurine (6 M-P), a monocytopenia (Van Furth et al., 1975; Gassmann and Van Furth, 1975; Hurd and Ziff, 1968) and in some cases tumour regression (Schlossberg and Hollander, 1973) were induced; and (2) that a single intraperitoneal (IP) injection of AZ resulted in the death of a subpopulation of thymic lymphocytes, as well as reducing B antigenicity of residual thymic lymphocytes (Poulter et al., 1974). A summary of part of this work has been reported elsewhere (Evans, 1976).
In a recent report (Evans, 1977) it was demonstrated that whole-body irradiation of mice before or after implantation of the syngeneic C57 Black fibrosarcoma, FS6, affected the growth and cellular composition of the tumour. The macrophage content of the tumours was particularly low during the initial 2-3 weeks of growth and the evidence suggested a possible relationship between a reduced rate of tumour growth, a lack of infiltration of host macrophages and restricted vascularization of the tumours. Tn the present report, the effect of the immunosuppressive agent, azathioprine (AZ), on the cellular composition and growth of the C57 Black fibrosarcoma, FS6, was studied using either a regimen of multiple injections into tumour-bearing mice or a single intraperitoneal injection before or after tumour implantation. In view of our interest in the interrelationships of tumour growth, tumour macrophage content, blood monocyte levels and immunogenicity (Evans, 1972; Eccles and Alexander, 1974; Eccles et al., 1976), it was hoped that use of this antimetabolite might provide further insight into the mechanisms controlling or determining the level of host cell infiltration. The rationale was based on reports demonstrating: (1) that after
Azathioprine
MATERIAL A N D METHODS
Mice
Eight- to 10-week-old C57 Black male mice (18-21 g) were used for growth of the syngeneic FS6 tumour.
Azathioprine (Imuran, Burroughs Wellcome, Beckenham, Kent, England) was in a relatively insoluble form as tablets for clinical use each containing 50 mg azathioprine and was brought into suspension by vigorous agitation in isotonic saline. Mice were injected intraperitoneally in all experiments, each mouse receiving 0.1 ml containing approximately 150 mg azathioprine/kg. Controls received 0. I ml of saline. Turnour The FS6 fibrosarcoma, a highly immunogenic benzpyrene-induced tumour, was transplanted at regular intervals with frequent returns to the tumour bank (after 20-30 animal passages). Cell suspensions from solid tumours were prepared as fully described elsewhere (Evans, 1977). In all experiments 1 Os tumour cells were injected intramuscularly into the right hind leg. An inoculum of this size gave a palpable tumour in 6 days. Tumour size was expressed as average tumour diameter after
Received: January 20, 1977 and in rev,sed for,,,
April 5 , 1977.
121
AZ AND HOST CELL INFILTRATION IN TUMOUR
measuring and averaging the largest and smallest diameters with calipers. Identification of ceN types
The full procedure for identification of cells associated with the FS6 tumour mass was described in detail elsewhere (Evans, 1977). Briefly, macrophages were identified by their capacity to adhere and spread in culture, and by the presence of Fc receptors, as measured by their capacity to bind and ultimately to phagocytose antibody-coated sheep red blood cells (EA): neoplastic cells by morphology under phase contrast microscopy and by smears; polymorphonuclear cells (PMNs) by nuclear morphology; and 0-antigen-positive cells were identified in preliminary studies by incubating the whole tumour cell population with anti-8 serum dilutions followed by incubation with complement. Because of the low percentage of T-cells associated with this tumour and the difficulty of accurate enumeration, this procedure was replaced by separation of lymphocytes on a cell separator by velocity sedimentation at unit gravity (Miller and Phillips, 1969). They were then identified by lysis with AKR anti-C,H anti-theta serum (Searle Laboratories, High Wycombe, England) using guinea-pig serum diluted to 1/20 (Burroughs Wellcome) as the source of complement. Lysis was assessed after addition of trypan blue to cells. Specificity of the anti-0 serum for lymphocytes bearing the 0 antigen was tested against thymus and spleen cells, and also tumour-associated and peritoneal macrophages. Serum dilutions of 1 / 6 4 lysed 90% thymus cells, 1/32 lysed 20-30% spleen cells, whereas 1/5 dilution did not lyse macrophages after the addition of complement. No attempt was made to identify other cell types possibly present, such as B lymphocytes or ‘‘ null ” cells.
after four injections of AZ) tumour diameters had stopped increasing at the same rate as controls and thereafter tumours began to regress. By 10 days regression was clearly occurring, resulting ultimately in complete disappearance of tumours without recurrence over a period of 6 months. Mice reinjected with lo6 FS6 cells after regression of the first inoculum showed complete resistance. If only five injections of AZ were given it was seen that
20
zE
15
-lx w
IW
II
3
10-
2I
3
I-
5-
b
4
Histology
Tumour tissue was fixed in Bouin’s solution for 1 h and then transferred to 70% alcohol. Sections were stained with haematoxylin and eosin. RESULTS
Effect of multiple injections of A Z on growth of FS6 tumours Mice were injected IM with lo6cells. After 10 days, when the tumours measured approximately 1 cm in diameter, mice received eight IP daily injections of AZ. This course of treatment was not lethal to the mice although they lost weight and spleens became very small. Figure 1 compares tumour diameters at various times in control and AZtreated groups. It is seen that by the 4th day (that is,
8
12
16 20 24
DAYS FIGURE 1 Effect of multiple injections of AZ on growth of the FS6 fibrosarcoma. Treatment started on day 10 (arrow).
controls: 0-0,
AZ-treated: o----o.
tumour growth was stopped for about 7 days, and thereafter the tumours began to grow again. The histological appearance of the FS6 tumour (Fig. 2 A) showed large neoplastic cells and smaller mononuclear cells. Non-specific esterase staining (not shown) revealed a widespread distribution of positively stained small mononuclear cells (presumably macrophages), while the obvious large tumour cells were negative. In controls the
122
EVANS
histological appearance of the tumour was similar for the duration of the experiments except that the amount of necrotic material increased. There was no obvious infiltration by PMNs of viable tumour tissue, while necrotic areas contained predominantly PMNs. After AZ treatment of tumour-bearing mice the tumours assumed a pale yellow appearance compared with controls which showed rich vascu-
culture. The macrophage percentage remained essentially stable as the tumour mass decreased in size although a slight increase was apparent after the course of injections was completed. When placed in culture the macrophages showed an exaggerated capacity to spread compared with controls. PMNs were at a low level throughout (less than 5 %) and very few were detected in the small regressing
FIGURE 2 (A) Histological section of FS6 fibrosarcomaexcised I8 days post implantation. Small mononuclear cells are visible amongst the large neoplastic cells. Haernatoxylin and eosin, x 240. (B) Histological section of FS6 fibrosarcoma after mice had received 8 IP injections. There appear to be fewer neoplastic cells, and mononuclear cells are more prominent. Haematoxylin and eosin, x 240.
larization. The architecture of the tumours appeared to change and sections appeared less dense after staining. Mononuclear cells became more prominent (Fig. 2 B). During regression (from the 4th day onwards) the neoplastic cells showed vacuolation of cytoplasm. At the time when tumours had been reduced to a small size the cellular composition became sparse and cell types were difficult to identify among the fibrotic material. Cellular composition
This is summarized in Table I. The percentage of neoplastic cells declined as regression proceeded. Moreover, they became more difficult to grow in
tumours. Lymphocytes with 0 antigen showed a dramatic increase in percentage from an average of 4 % at the start of experiments to as much as 45 % in the regressing tumours. This rise occurred concomitantly with the decrease in the percentage of neoplastic cells and with decreasing tumour diameter. Compared with thymus cells from AZpretreated or control tumour-bearing mice, or control non-tumour-bearing mice, those &positive lymphocytes derived from the fibrosarcomas required higher concentrations of anti- 0 serum to induce complement-mediated lysis (a minimum dilution of 1/20 of anti-serum compared with 1/3201/640 for thymus cells). Those small cells not lysed
123
AZ AND HOST CELL INFILTRATION IN TUMOUR
TABLE I THE EFFECT OF MULTIPLE INJECTIONS OF AZ ON THE CELLULAR COMPOSITION OF THE FS6 FIBROSARCOMA ~~
Mean percentage of total cell population after start of AZ injection '
Cell types
Neoplastic Macrophages PMNs
&antigenpositive lymphocytes
4 days
8 days
12 days
45 (39-47) 45 (38-47) 5 (2-7)
20 (16-22) 43 (37-47) 4 (1-4)
8 (5-10) 50 (45-55) 3 (1-4)
52 (48-56) 42 (38-46) 7 (5-12)
4 (3-8)
31 (25-41)
35 (28-45)
4 (3-6)
Controls
a
Mice received eight IP injection AZ (150 mg/kg) starting 10 days after implantation of lo1 FS6 fihrosarcoma cells. - a Average values on day 0 ( = 10 days post implantation) when treatment was started. Control fibrosarcomas showed little variation in percentages over the period of study. Ranges in variation from six different experiments given in parentheses.
following anti-0 serum and C treatment did not form EA rosettes nor did they adhere to culture dishes. After accounting for the above categories of cells there was usually a small percentage (
THE EFFECT OF AZATHlOPRINE ON HOST CELL INFILTRATION A N D GROWTH OF A MURINE FIBROSARCOMA R. EVANS Department of Turnour Immunology, Chester Beatty Research Institute, Institute of Cancer Research, Clijton Avenue, Belmont, Sutton, Surrey, S M 2 5PX, England
Azathioprine (AZ) was injected into C57 Black mice before or after IM implantation of the syngeneic fibrosarcoma FS6, and observations were made on tumour growth and on the cellular composition of the fibrosarcomas. When multiple injections were given t o the tumour-bearing host the tumours regressed. The percentage of tumour-associated macrophages remained essentially stable as regression occurred whereas the percentage of B-antlgenpositive cells increased. In the case of single I P injections of AZ, changes i n the cellular composition of tumours were seen only when A 2 was given 1-72 h before implantation but not when it was given after implantation. Compared w i t h controls there was a 2-t o 3-week delay in the appearance of large numbers of tumour-associated macrophages. The percentage of 0-antigen-positive cells within the turnours mass was elevated 7 days post A Z treatment but the level declined t o control values between days 10 and 13. The significance of the results i s discussed i n relation t o the dependency of the macrophage content of t h i s tumour upon the development o f an immune response towards the tumour.
multiple injections of this antimetabolite or its derivative, 6 mercaptopurine (6 M-P), a monocytopenia (Van Furth et al., 1975; Gassmann and Van Furth, 1975; Hurd and Ziff, 1968) and in some cases tumour regression (Schlossberg and Hollander, 1973) were induced; and (2) that a single intraperitoneal (IP) injection of AZ resulted in the death of a subpopulation of thymic lymphocytes, as well as reducing B antigenicity of residual thymic lymphocytes (Poulter et al., 1974). A summary of part of this work has been reported elsewhere (Evans, 1976).
In a recent report (Evans, 1977) it was demonstrated that whole-body irradiation of mice before or after implantation of the syngeneic C57 Black fibrosarcoma, FS6, affected the growth and cellular composition of the tumour. The macrophage content of the tumours was particularly low during the initial 2-3 weeks of growth and the evidence suggested a possible relationship between a reduced rate of tumour growth, a lack of infiltration of host macrophages and restricted vascularization of the tumours. Tn the present report, the effect of the immunosuppressive agent, azathioprine (AZ), on the cellular composition and growth of the C57 Black fibrosarcoma, FS6, was studied using either a regimen of multiple injections into tumour-bearing mice or a single intraperitoneal injection before or after tumour implantation. In view of our interest in the interrelationships of tumour growth, tumour macrophage content, blood monocyte levels and immunogenicity (Evans, 1972; Eccles and Alexander, 1974; Eccles et al., 1976), it was hoped that use of this antimetabolite might provide further insight into the mechanisms controlling or determining the level of host cell infiltration. The rationale was based on reports demonstrating: (1) that after
Azathioprine
MATERIAL A N D METHODS
Mice
Eight- to 10-week-old C57 Black male mice (18-21 g) were used for growth of the syngeneic FS6 tumour.
Azathioprine (Imuran, Burroughs Wellcome, Beckenham, Kent, England) was in a relatively insoluble form as tablets for clinical use each containing 50 mg azathioprine and was brought into suspension by vigorous agitation in isotonic saline. Mice were injected intraperitoneally in all experiments, each mouse receiving 0.1 ml containing approximately 150 mg azathioprine/kg. Controls received 0. I ml of saline. Turnour The FS6 fibrosarcoma, a highly immunogenic benzpyrene-induced tumour, was transplanted at regular intervals with frequent returns to the tumour bank (after 20-30 animal passages). Cell suspensions from solid tumours were prepared as fully described elsewhere (Evans, 1977). In all experiments 1 Os tumour cells were injected intramuscularly into the right hind leg. An inoculum of this size gave a palpable tumour in 6 days. Tumour size was expressed as average tumour diameter after
Received: January 20, 1977 and in rev,sed for,,,
April 5 , 1977.
121
AZ AND HOST CELL INFILTRATION IN TUMOUR
measuring and averaging the largest and smallest diameters with calipers. Identification of ceN types
The full procedure for identification of cells associated with the FS6 tumour mass was described in detail elsewhere (Evans, 1977). Briefly, macrophages were identified by their capacity to adhere and spread in culture, and by the presence of Fc receptors, as measured by their capacity to bind and ultimately to phagocytose antibody-coated sheep red blood cells (EA): neoplastic cells by morphology under phase contrast microscopy and by smears; polymorphonuclear cells (PMNs) by nuclear morphology; and 0-antigen-positive cells were identified in preliminary studies by incubating the whole tumour cell population with anti-8 serum dilutions followed by incubation with complement. Because of the low percentage of T-cells associated with this tumour and the difficulty of accurate enumeration, this procedure was replaced by separation of lymphocytes on a cell separator by velocity sedimentation at unit gravity (Miller and Phillips, 1969). They were then identified by lysis with AKR anti-C,H anti-theta serum (Searle Laboratories, High Wycombe, England) using guinea-pig serum diluted to 1/20 (Burroughs Wellcome) as the source of complement. Lysis was assessed after addition of trypan blue to cells. Specificity of the anti-0 serum for lymphocytes bearing the 0 antigen was tested against thymus and spleen cells, and also tumour-associated and peritoneal macrophages. Serum dilutions of 1 / 6 4 lysed 90% thymus cells, 1/32 lysed 20-30% spleen cells, whereas 1/5 dilution did not lyse macrophages after the addition of complement. No attempt was made to identify other cell types possibly present, such as B lymphocytes or ‘‘ null ” cells.
after four injections of AZ) tumour diameters had stopped increasing at the same rate as controls and thereafter tumours began to regress. By 10 days regression was clearly occurring, resulting ultimately in complete disappearance of tumours without recurrence over a period of 6 months. Mice reinjected with lo6 FS6 cells after regression of the first inoculum showed complete resistance. If only five injections of AZ were given it was seen that
20
zE
15
-lx w
IW
II
3
10-
2I
3
I-
5-
b
4
Histology
Tumour tissue was fixed in Bouin’s solution for 1 h and then transferred to 70% alcohol. Sections were stained with haematoxylin and eosin. RESULTS
Effect of multiple injections of A Z on growth of FS6 tumours Mice were injected IM with lo6cells. After 10 days, when the tumours measured approximately 1 cm in diameter, mice received eight IP daily injections of AZ. This course of treatment was not lethal to the mice although they lost weight and spleens became very small. Figure 1 compares tumour diameters at various times in control and AZtreated groups. It is seen that by the 4th day (that is,
8
12
16 20 24
DAYS FIGURE 1 Effect of multiple injections of AZ on growth of the FS6 fibrosarcoma. Treatment started on day 10 (arrow).
controls: 0-0,
AZ-treated: o----o.
tumour growth was stopped for about 7 days, and thereafter the tumours began to grow again. The histological appearance of the FS6 tumour (Fig. 2 A) showed large neoplastic cells and smaller mononuclear cells. Non-specific esterase staining (not shown) revealed a widespread distribution of positively stained small mononuclear cells (presumably macrophages), while the obvious large tumour cells were negative. In controls the
122
EVANS
histological appearance of the tumour was similar for the duration of the experiments except that the amount of necrotic material increased. There was no obvious infiltration by PMNs of viable tumour tissue, while necrotic areas contained predominantly PMNs. After AZ treatment of tumour-bearing mice the tumours assumed a pale yellow appearance compared with controls which showed rich vascu-
culture. The macrophage percentage remained essentially stable as the tumour mass decreased in size although a slight increase was apparent after the course of injections was completed. When placed in culture the macrophages showed an exaggerated capacity to spread compared with controls. PMNs were at a low level throughout (less than 5 %) and very few were detected in the small regressing
FIGURE 2 (A) Histological section of FS6 fibrosarcomaexcised I8 days post implantation. Small mononuclear cells are visible amongst the large neoplastic cells. Haernatoxylin and eosin, x 240. (B) Histological section of FS6 fibrosarcoma after mice had received 8 IP injections. There appear to be fewer neoplastic cells, and mononuclear cells are more prominent. Haematoxylin and eosin, x 240.
larization. The architecture of the tumours appeared to change and sections appeared less dense after staining. Mononuclear cells became more prominent (Fig. 2 B). During regression (from the 4th day onwards) the neoplastic cells showed vacuolation of cytoplasm. At the time when tumours had been reduced to a small size the cellular composition became sparse and cell types were difficult to identify among the fibrotic material. Cellular composition
This is summarized in Table I. The percentage of neoplastic cells declined as regression proceeded. Moreover, they became more difficult to grow in
tumours. Lymphocytes with 0 antigen showed a dramatic increase in percentage from an average of 4 % at the start of experiments to as much as 45 % in the regressing tumours. This rise occurred concomitantly with the decrease in the percentage of neoplastic cells and with decreasing tumour diameter. Compared with thymus cells from AZpretreated or control tumour-bearing mice, or control non-tumour-bearing mice, those &positive lymphocytes derived from the fibrosarcomas required higher concentrations of anti- 0 serum to induce complement-mediated lysis (a minimum dilution of 1/20 of anti-serum compared with 1/3201/640 for thymus cells). Those small cells not lysed
123
AZ AND HOST CELL INFILTRATION IN TUMOUR
TABLE I THE EFFECT OF MULTIPLE INJECTIONS OF AZ ON THE CELLULAR COMPOSITION OF THE FS6 FIBROSARCOMA ~~
Mean percentage of total cell population after start of AZ injection '
Cell types
Neoplastic Macrophages PMNs
&antigenpositive lymphocytes
4 days
8 days
12 days
45 (39-47) 45 (38-47) 5 (2-7)
20 (16-22) 43 (37-47) 4 (1-4)
8 (5-10) 50 (45-55) 3 (1-4)
52 (48-56) 42 (38-46) 7 (5-12)
4 (3-8)
31 (25-41)
35 (28-45)
4 (3-6)
Controls
a
Mice received eight IP injection AZ (150 mg/kg) starting 10 days after implantation of lo1 FS6 fihrosarcoma cells. - a Average values on day 0 ( = 10 days post implantation) when treatment was started. Control fibrosarcomas showed little variation in percentages over the period of study. Ranges in variation from six different experiments given in parentheses.
following anti-0 serum and C treatment did not form EA rosettes nor did they adhere to culture dishes. After accounting for the above categories of cells there was usually a small percentage (
