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Tumor transplantation in mice inoculated with polyoma virus
TUMOR
TRANSPLANTATION
IN
POLYOMA
MICE VIRUS
INOCULATED
WITH
l
L. SACHS Lubowtories
of I?ro/og~
and
Genetics. Received
lT’eirmann April
Institute
of Scienrr,
Kehoc~othT Isrnel
1, 1961
T sky recent note by Sjijgren et al. [6] pronlpts me to report the results of some experiments that originated during the course of studies on the possibility of transduction with pOlyOma Vii-US Using Cellular antigens as markers 141. It was noted during these studies that the transplantation of readily transplantable mouse tumors, induced by polyoma virus or benzpyrene, was considerably inhibited when the tumor cell suspension had been incubated in vitro with polyutna virus for 30 to 60 ruin before grafting into adult mice. These results ma\- be due to ((0 the itnmunzation of mice against new cellular antigens that may be produced as the result of transformation of normal cells (51 by the virus in uioo, (h) the death of transplanted cells as the result of lysis after virus infection [S, Xl, or (c) the possible cytotosic action of antiviral antibodies produced in the grafted mice. Further experiments were therefore undertaken including the transplantability of tumors in mice inoculated with polyoma virus either before or at about the same time as the tumor graft. The present report describes the results of these transplantation experiments with two tumors induced by polyoma virus, a parotid tumor originating in a l)E3;\:2 tnouse and a mammary tumor originating in a S\VR mouse, and with a subcutaneous sarcotna induced by benzpyrene in a C57BL/li mouse. ,411 three tutnors were readil!. transplantable into mice belonging to the strain of origin. The early transplantation behaviour of the DB.4/2 parotid (PT2) and the S\\‘R mammar>- (RITl) tumors have previously been described [S], and these tumors will be referred to as PT and XIT respectively. Neither tumor has spontaneously produced infective virus during the course of these esperitnents. The C5iBLjti sarcoma, which will be referred to as SU, was kindly supplied by Mrs. hmiella Globerson. Mice with this sarcoma had never previously been in a room with polyoma virus, and for each experiment fresh cells were taken from a mouse kept in a part of the building separated bg two floors from the polyoma room. Polyotua inoculations into adult mice were tnade with a virus stock producetl in Swiss (non-inbred) kidney cells containing 1 10” plaque forming units per nil, and for each inoculation 0.1 ml was injected intraperitoneally into 2 to 3-month-old animals. For the transplantation tests, suspensions of tumor cells were made lJ\ trypsinization, the cells suspended to the required number in Eagle’s medium with 10 per cent horse serum, and 0.1 ml of the cell suspension grafted subcutaneousI> into mice belonging to the strain of origin of the tumor.
186
Experimeniul
Cell
Nesetrrch
24
A summary of the results on the transplantability of the three tumors, when tested at various times after one or two inoculations of polyoma \-irus into adult mice, is given in Table I. These results show that Ihere was an inhibitory effect after inoculation with polyoma virus both in the polyoma induced tumors and in the tumor induced by benzpyrene. In all experiments the tumors in animals iuctculatetl with virus were smalkr than in the controls, and in experiment NT:%, and the rsperigroups a lower numlwi merits with 1’1‘ and SH. there were also in the esperimt~ntal or animals with successful tumor grafts. The inhibitory effect was generally brsl observed wit Ii a cell inoculum near the minimum required for a successful transplanl. ‘I’hr result of esperiment PTri indicates that an inhibitor!effect can be obtuinett C’VC‘II when the virus is inoculated itltraperitoneall~ and the cells are grafted subcutailt~ollsl~ at about the same time. The time of drvelopmenl of palpable tumors. in ant’ c\lwrinlerit with each of the three tumors. is given in Table Il.
In addition to these experiments with animals inoculated as adults, one group III S\VW mice (Experiment RITS), which were inoculated with polyorna when 4 days old and did not develop any palpable virus induced tumors, were grafted X19 days latc.1 with tumor LIT (Table I). These mice also showed a clear inhibitory effect as measured by tumor size, and the time of appearance of palpable MT tumors. It is of interest that attempts to transplant six different primary parotid tumors induced by ~I~~~OIIK~ Yirus in S\YII mice, by subcutaneous inoculation of tumor pieces back into the same mice lbearing the primary tumor, were all unsuccessful. The results obtained thus agree with those of Sjiigren et tr/. Iti] in showing that the transplantability of polyoma induced ~nouse tumors can be inhibited in mice itloculated when young or adult with polyoma virus. The present results also shov that an inhibitory effect can be obtained even when virus ant1 cells are incvxtlated
L. Sachs
188
at different sites and at about the same time, and that inoculation with polyoma virus can inhibit the transplantability of a tumor induced by benzpyrenc. The mechanism of the inhibitory effect, and whether it is the same in all the tumors, has still to be clarified. However the observation that the polyoma induced tumor PT used in these studies had shown sotne immunity to challenge infection with polyoma virus IS], suggests that the inhibition does not seem to be, at least in this tumor, merely the result of lysis of the tumor cells after virus infection in uioo. The explanation of inhibition due to the production of antibodies by virus inoculation is not incompatible with the results obtained when virus and cells were inoculated at about the same time. The production of antibodies, as measured by the polyoma hemagglutination inhibition test, can be detected between 4 to 5 days after virus inoculation into adult mice [3], and it takes at least this time and usually longer for the grafted cells to become established in the animal. However if the inhibitory effect is due to antibodies against all three tumors, one would have to postulate that inoculation with polyoma virus produces antibodies against antigens common to both the polyoma and benzpyrene induced tumors. The inhibitory effect would also seem to be relevant to the interpretation of data on the transplantability of polyoma induced primary tumors that can still produce virus [8], and to the use of in ho tests for the malignancy of in vitro transformed cultures [5] that produce virus. Since animals inoculated with polyoma when young generally produce high titers of antibodies [l, 21, the possibility exists that the inhibitory effect may furthermore be effective in preventing the development of primary tumors, and that differences in the strength of this effect may explain, either entirely or in part, the difference in tumor induction bg polgoma virus in various strains of mice [9]. REFERENCES 1. FoGEL, hr., and S.XHS, L.. Brit. J. Cancer 13, 2S6 (1959). 2. -~-~ J. .\‘at/. Cunccr Inst. 24, 839 (1960). 3. SACHS, L., FOGEL, BI., \VINOCOLJR, E., HELLER, E.. hIEt)IN.%, D. and 13. 251 11959). 4 SACHS. I,., and ~
9. --~
.I. AVcdl. Cnncer
E.xperimenlnl
Cell Research
Inst.
24
In press.
I
23, 204 (1961).
TRANSPLANTATION
IN
POLYOMA
MICE VIRUS
INOCULATED
WITH
l
L. SACHS Lubowtories
of I?ro/og~
and
Genetics. Received
lT’eirmann April
Institute
of Scienrr,
Kehoc~othT Isrnel
1, 1961
T sky recent note by Sjijgren et al. [6] pronlpts me to report the results of some experiments that originated during the course of studies on the possibility of transduction with pOlyOma Vii-US Using Cellular antigens as markers 141. It was noted during these studies that the transplantation of readily transplantable mouse tumors, induced by polyoma virus or benzpyrene, was considerably inhibited when the tumor cell suspension had been incubated in vitro with polyutna virus for 30 to 60 ruin before grafting into adult mice. These results ma\- be due to ((0 the itnmunzation of mice against new cellular antigens that may be produced as the result of transformation of normal cells (51 by the virus in uioo, (h) the death of transplanted cells as the result of lysis after virus infection [S, Xl, or (c) the possible cytotosic action of antiviral antibodies produced in the grafted mice. Further experiments were therefore undertaken including the transplantability of tumors in mice inoculated with polyoma virus either before or at about the same time as the tumor graft. The present report describes the results of these transplantation experiments with two tumors induced by polyoma virus, a parotid tumor originating in a l)E3;\:2 tnouse and a mammary tumor originating in a S\VR mouse, and with a subcutaneous sarcotna induced by benzpyrene in a C57BL/li mouse. ,411 three tutnors were readil!. transplantable into mice belonging to the strain of origin. The early transplantation behaviour of the DB.4/2 parotid (PT2) and the S\\‘R mammar>- (RITl) tumors have previously been described [S], and these tumors will be referred to as PT and XIT respectively. Neither tumor has spontaneously produced infective virus during the course of these esperitnents. The C5iBLjti sarcoma, which will be referred to as SU, was kindly supplied by Mrs. hmiella Globerson. Mice with this sarcoma had never previously been in a room with polyoma virus, and for each experiment fresh cells were taken from a mouse kept in a part of the building separated bg two floors from the polyoma room. Polyotua inoculations into adult mice were tnade with a virus stock producetl in Swiss (non-inbred) kidney cells containing 1 10” plaque forming units per nil, and for each inoculation 0.1 ml was injected intraperitoneally into 2 to 3-month-old animals. For the transplantation tests, suspensions of tumor cells were made lJ\ trypsinization, the cells suspended to the required number in Eagle’s medium with 10 per cent horse serum, and 0.1 ml of the cell suspension grafted subcutaneousI> into mice belonging to the strain of origin of the tumor.
186
Experimeniul
Cell
Nesetrrch
24
A summary of the results on the transplantability of the three tumors, when tested at various times after one or two inoculations of polyoma \-irus into adult mice, is given in Table I. These results show that Ihere was an inhibitory effect after inoculation with polyoma virus both in the polyoma induced tumors and in the tumor induced by benzpyrene. In all experiments the tumors in animals iuctculatetl with virus were smalkr than in the controls, and in experiment NT:%, and the rsperigroups a lower numlwi merits with 1’1‘ and SH. there were also in the esperimt~ntal or animals with successful tumor grafts. The inhibitory effect was generally brsl observed wit Ii a cell inoculum near the minimum required for a successful transplanl. ‘I’hr result of esperiment PTri indicates that an inhibitor!effect can be obtuinett C’VC‘II when the virus is inoculated itltraperitoneall~ and the cells are grafted subcutailt~ollsl~ at about the same time. The time of drvelopmenl of palpable tumors. in ant’ c\lwrinlerit with each of the three tumors. is given in Table Il.
In addition to these experiments with animals inoculated as adults, one group III S\VW mice (Experiment RITS), which were inoculated with polyorna when 4 days old and did not develop any palpable virus induced tumors, were grafted X19 days latc.1 with tumor LIT (Table I). These mice also showed a clear inhibitory effect as measured by tumor size, and the time of appearance of palpable MT tumors. It is of interest that attempts to transplant six different primary parotid tumors induced by ~I~~~OIIK~ Yirus in S\YII mice, by subcutaneous inoculation of tumor pieces back into the same mice lbearing the primary tumor, were all unsuccessful. The results obtained thus agree with those of Sjiigren et tr/. Iti] in showing that the transplantability of polyoma induced ~nouse tumors can be inhibited in mice itloculated when young or adult with polyoma virus. The present results also shov that an inhibitory effect can be obtained even when virus ant1 cells are incvxtlated
L. Sachs
188
at different sites and at about the same time, and that inoculation with polyoma virus can inhibit the transplantability of a tumor induced by benzpyrenc. The mechanism of the inhibitory effect, and whether it is the same in all the tumors, has still to be clarified. However the observation that the polyoma induced tumor PT used in these studies had shown sotne immunity to challenge infection with polyoma virus IS], suggests that the inhibition does not seem to be, at least in this tumor, merely the result of lysis of the tumor cells after virus infection in uioo. The explanation of inhibition due to the production of antibodies by virus inoculation is not incompatible with the results obtained when virus and cells were inoculated at about the same time. The production of antibodies, as measured by the polyoma hemagglutination inhibition test, can be detected between 4 to 5 days after virus inoculation into adult mice [3], and it takes at least this time and usually longer for the grafted cells to become established in the animal. However if the inhibitory effect is due to antibodies against all three tumors, one would have to postulate that inoculation with polyoma virus produces antibodies against antigens common to both the polyoma and benzpyrene induced tumors. The inhibitory effect would also seem to be relevant to the interpretation of data on the transplantability of polyoma induced primary tumors that can still produce virus [8], and to the use of in ho tests for the malignancy of in vitro transformed cultures [5] that produce virus. Since animals inoculated with polyoma when young generally produce high titers of antibodies [l, 21, the possibility exists that the inhibitory effect may furthermore be effective in preventing the development of primary tumors, and that differences in the strength of this effect may explain, either entirely or in part, the difference in tumor induction bg polgoma virus in various strains of mice [9]. REFERENCES 1. FoGEL, hr., and S.XHS, L.. Brit. J. Cancer 13, 2S6 (1959). 2. -~-~ J. .\‘at/. Cunccr Inst. 24, 839 (1960). 3. SACHS, L., FOGEL, BI., \VINOCOLJR, E., HELLER, E.. hIEt)IN.%, D. and 13. 251 11959). 4 SACHS. I,., and ~
9. --~
.I. AVcdl. Cnncer
E.xperimenlnl
Cell Research
Inst.
24
In press.
I
23, 204 (1961).