Immunologic Parameters of Ultraviolet Carcinogenesis"
2
Margaret L. Kripke 3 and Michael S. Fisher 4.5.6
Chronic exposure of mice to UV radiation induces a high incidence of skin tumors with long latent periods. In at least two inbred strains of mice, tumors induced in this way are extremely antigenic. In fact, most UVinduced tumors are immunologically rejected when transplanted to normal syngeneic recipients (1). These neoplasms resemble those induced by chemical carcinogens, in that each UV-induced tumor is antigenically unique, as judged by in vivo cross-reactivity tests (1,2). The finding that most UV-induced tumors are rejected by normal syngeneic mice raises the question why these tumors are able to persist and grow progressively in the autochthonous host. Further, it suggests that the autochthonous host may be immunologically altered in a way that favors, rather than retards, tumor development and/or that there is some mechanism that permits these tumors to overcome immunologic destruction in situ. The question why primary hosts fail to reject their developing tumors has been studied after tumor induction by chemical carcinogens (3, 4). Primary hosts are frequently more susceptible to rechallenge with their own tumors than are normal syngeneic recipients. However, the chemically induced tumors usually grow progressively in both primary hosts and normal syngeneic mice. Thus one must rely on tumor growth rates to assess the reactivity of the primary host relative to that of normal mice. In contrast, with the UV-induced tumor system, there is a clear distinction between the fate of a tumor in the primary host (progressive growth) and its fate in a normal animal (immunologic rejection). This permits us to examine the immunologic response of the primary host against its own tumor and against other non-cross-reacting tumors induced by UV and to investigate the mechanisms underlying the failure of the immune system to deal with extremely antigenic tumors. MATERIALS AND METHODS
Mice.-Most animals in these experiments were male VOL. 57, NO. I, JULY 1976
211
or female C3Hf/Sm mice from the Inbred Rodent Colony of the University of Utah. In the one experiment involving skin allograft rejection the graft recipients were C3H/Hej mice from The jackson Laboratory (Bar Harbor, Maine). Strain A/j mice were also obtained from The jackson Laboratory. UV irradiation.-The UV light source was an intermediate pressure 100-W quartz-mercury arc lamp (Hanovia) that delivered a dose rate of approximately 3 x 105 ergs/cm 2 /second over the wavelength range of 280320 nm. The backs of the irradiated mice were shaved once a week; the animals were exposed to UV light for 60 seconds three times a week (Mon., Wed., Fri.) for the duration of the experiment, unless otherwise indicated. The primary UV-induced tumors used in these studies arose on the backs of the irradiated mice and were classified histologically from fixed sections stained with hematoxylin and eosin. Tumor transplantation.-Primary tumors were removed from anesthetized donors, cut into l- mm 3 fragments, and transplanted sc with a trocar to syngeneic male recipients. The tumors were maintained by serial passage in mice immunosuppressed by adult thymectomy and whole-body (450 R) X-irradiation (ATX recipients). The recipients were inspected once a week for tumors which were measured in three perpendicular diameters. The product of the three diameters is used as an approximation of tumor volume. Skin grafting. -Full-thickness, circular, abdominal skin grafts (15 mm in diameter) were transplanted to the thorax by the method of Billingham and Medawar (5). Plaster casts were removed under light ether anesthesia after 8 days. Grafts were inspected daily for gross signs of rejection and scored in double-blind fashion. Complete destruction of the epidermis was judged the rejection end point. RESULTS Comparison of Growth of Primary Tumors as Autografts Versus Isografts to Normal Mice
This experiment was designed to determine whether ABBREVIATIONS USED: A TX=adult thymectomy and X·irradiation (450 R); MCA=3-methylcholanthrene. Received December 4, 1975; accepted January 23, 1976. Supported by Public Health Service contract NOI CP33263 from the Division of Cancer Cause and Prevention, National Cancer Institute, to the Department of Pathology, University of Utah College of Medicine. 3 Basic Research Program, Frederick Cancer Research Center, P. O. Box B, Frederick, Md. 21701. 4 Department of Pathology, University of Utah College of Medicine, Salt Lake City, Utah 84132. 5 Study performed by M.S.F. in partial fulfillment of the requirements for the Ph.D. degree in Experimental Pathology. 6 The contributions of Dr. Ernst J. Eichwald and Dr. John D. Spikes to this work are acknowledged with thanks. J
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ABSTRACT-Skin tumors induced in mice by UY light are usually immunologically rejected by normal syngeneic recipients. We evaluated the immune status of primary hosts against these highly antigenic tumors immediately after surgical removal of the primary tumor. All primary hosts were susceptible to challenge with their autochthonous tumors, though most of these were reo jected by untreated control mice. Primary hosts were also susceptible to challenge with isografts of antigenically dissimilar UY·induced neoplasms. The susceptibility of the primary hosts to tumor challenge was probably induced by chronic exposure to UY light, since UY·irradiated non·tumor·bearing mice were also susceptible to challenge with these tumors. Although UY·treated mice were unable to reject these syngeneic tumors, they could reject skin and tumor allografts. Further, UY irradiation did not interfere with the second·set rejection of syngeneic UY·induced tumors in mice that were specifically immunized before UY treat· ment.-J Natl Cancer Inst 57: 211-215, 1976.
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KRIPKE AND FISHER
the failure of a primary host to reject its own tumor is a local phenomenon, restricted to the site of tumor development, or a phenomenon that is expressed systemically. Primary tumors were surgically excised and immediately returned sc to the ventral (nonirradiated) side of the autochthonous host. At the same time, tumor fragments were implanted sc in groups of normal and immunosuppressed (ATX) syngeneic recipients. Since the primary hosts were often more than 12 months old when tested, normal controls of the same age as the primary tumor donors were usually used as recipients. Table 1 shows that all 13 of the tumors tested grew progressively upon retransplantation to their autochthonous hosts. Only 2 of these tumors grew in normal recipients (#91, 1 of 5; #81, 3 of 5); 12 of the 13 tumors grew in I or more ATX recipients. From this, we conclude that the primary host is systemically altered in a way that permits tumor growth.
We then asked whether the susceptibility of the primary host to tumor growth was restricted to autochthonous tumors or extended to other, non-cross-reacting tumors induced with UV. In the first experiment, primary tumors were excised and immediately transplanted to groups of normal and A TX mice, to the autochthonous host, and to a different primary host. Thus each primary host received an autograft and an isograft. In all 7 isografted mice (syngeneic primary hosts) the tumors grew progressi"ely, which indicated that a primary host is susceptible to the growth of its autochthonous tumor and to other UV-induced tumors (table 1). To determine whether a primary host is more suscepTABLE I.-Transplantation of primary UV-induced tumors into various syngeneic recipients Number of mice with progressively growing tumor/No. challenged Tumor No.
41 91 53 45 126 913 115
Histologic type
Spindle cell tumor "
Squamous cell carcinoma Myxosarcoma Spindle cell tumor
230 231 81 " 82 71 102 Total number of progressively growing implants/No. ofrecipients Total number of progressively growing tumors/No. of tumors tested a
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NT=not tested.
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Autoch- Normal thonous . mIce host
ATX mice
Syngeneic primary host a
1/1 1/1 1/1 1/1 1/1 1/1 1/1
0/5 1/5 0/5 0/5 0/5 0/5 0/5
2/4 3/5 1/4 0/5 3/5 1/5 1/5
NT NT NT NT NT NT NT
1/1 1/1 1/1 1/1 1/1 1/1 13/13
0/5 0/5 3/5 0/5 0/5 0/3 4/63
2/5 3/5 5/5 5/5 1/5 1/3 28/61
1/1 1/1 1/1 1/1 2/2 1/1 7/7
13/13
2/13
12/13
6/6
Tumor No.
Number of weeks between implantation and development of a palpable tumor Autograft
82 102 230 231 81 71 a
2 2 4
2 2 2
Isograft 4
5 8 5 2 2,2a
This tumor was tested in two syngeneic primary hosts.
tible to the growth of its own tumor than to a different primary one, we compared the growth of the 6 tumors as autografts versus isografts to a different primary host. Table 2 shows that, after transplantation, 4 of the 6 tumors were detectable earlier in the autochthonous hosts than in the syngeneic recipients. In these 4 cases, the tumors grew more rapidly in their autochthonous hosts than in the syngeneic hosts, but in the remaining 2, no differences were detectable in the time of appearance or in the growth rates of the tumors transplanted into autologous or syngeneic hosts. No tumor grew better as an isograft than an autograft. Although only a few comparisons were made, the data suggest that, in general, primary hosts may be more susceptible to the growth of their own than to syngeneic tumors. Similar experiments were performed with transplanted, rather than primary, UV-induced tumors. Autochthonous tumors were excised from primary hosts, and these animals were challenged with another (UV-induced) tumor that had been passaged in vivo in ATX mice. Normal mice the same age as the primary hosts were used as controls. The results, depicted in table 3 (expts 1,2), again indicate that primary hosts are more susceptible to the growth of UV-induced tumors than are normal mice. Growth of Syngeneic UV-Induced Tumors in UV-Irradiated Mice
We next asked whether the state of susceptibility to tumor transplantation in the primary host was brought about by the presence of the primary tumor or by the chronic exposures to UV light. Groups of C3Hf mice were UV-irradiated for various periods before challenge with a syngeneic UV-induced tumor. After tumor challenge, the UV treatment was continued until the experiment ended. Groups of age-matched normal mice and A TX mice were included at each testing. The results are summarized in table 3. In experiments 3-16, all UV-treated recipients were free of macroscopic primary tumors. 7 Nevertheless, in all mice treated with UV light for 17 or more weeks, the transplanted tumors continued to grow. Most animals treated for less than 17 weeks also developed progressively growing implants, and even in mice treated for only 2 weeks before chal7 The mean time of tumor appearance for this regimen of UV treatment is approximately 45 weeks, with a range of 27-60 weeks (SpikesJD. Kripke ML. Eichwald EJ: Unpublished data).
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Growth of Syngeneic UV-Induced Tumors in Primary Hosts
TABLE 2.-Time of appearance of transplanted tumors in autochthonous or syngeneic primary hosts
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IMMUNOLOGIC PARAMETERS OF ULTRAVIOLET CARCINOGENESIS
Expt No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Totals
TABLE 3.--Growth of transplanted UV-induced tumors in UV-irradiated, normal, and ATX recipients Weeks ofUV Recipients b Transplant gen- treatment a before Challenge tumor eration challenge UV-treated Normal" 231 82 45 231 231 231 231 154 231 231 861 231 231 861 231 231
50 d 45 d 51 50 30 26 23 21 17 15 15 8 8 8 4 2
2 2 1 2 5 4 2 1 5 4 2 4 4 4 3 3
3/3 4/4 4/4 2/2 5/5 5/5 4/4 5/5 10/10 6/9 9/10 3/5 9/10 7/10 5/5 3/5 84/96(88%)
0/5 2/5 0/5 0/5 0/5 0/5 0/5 1/4 1/10 0/10 1/10 0/5 0/10 0/10 0/5 0/5 5/104(5%)
ATX 2/5 5/5 0/5 2/5 5/5 4/5 2/5 5/5 5/5 3/5 5/5 6/10 10/10 9/12 3/7 2/10 68/104(65%)
b
TABLE 4.-Duration of susceptibility to tumor challenge after 3 months of UV treatment Time of tumor challenge, mob Group
UV treatment"
I II III
3 mo Continuous None
6
7
8
5/5" 5/5 0/5
5/5 5/5 0/5
5/5 5/5 2/5
" 60 sec three times/week. b Mice tested at 6 and 7 mo received implants of tumor #231 from the 4th passage in ATX mice; those tested at 8 mo received implants of tumor #231 from the 5th passage. C Number of mice with progressively growing tumor/No. challenged.
lenge (expt 16), 3 of 5 recipients were susceptible to tumor challenge. Since chronic treatment with UV light rendered mice susceptible to challenge with UV-induced tumors, we tested the duration of this effect after cessation of UV ir~adi.ation. In the following preliminary experiment, mICe m groups I and II were irradiated for 60 seconds three times/week. After 3 months, group I received no f~rther U,:, treatment. UV treatment of group II contmued untIl the end of the experiment. Group III received no UV irradiation (age-matched controls). At 3, 4, and 5 months after the end of UV treatment in group I (months 6, 7, and 8, respectively), mice from each group were tested for their ability to reject a transplanted UV-induced tumor. As shown in table 4 all mice treated continuously with UV developed tu~ors when challenged (group II). Mice treated with UV for 3 months and given implants at 6,7, or 8 months were also susceptible to tumor challenge (group I). Animals receiving no UV treatment were resistant to tumor challenge at the 6- and 7-month testing, but 2 animals developed tumors after the 8-month testing (group III). However, these 2 tumors in group III appeared at 6 weeks after challenge, whereas those in groups I and II were apparent at 2 weeks after the 8-month challenge. VOL. 57, NO.1, JULY 1976
TABLE 5.--Effect of UV irradiation on skin allograft rejection Months ofUV treatment" before grafting 2 4 6 8 10
Sham-treated
UV-treated
Numberb
Median (range)"
Number
Median (range)
16 15 16 16 12
11(9-18) 11(10-14) 13(8-21) 11(9-18) 14(9-20)
15 16 13 11 9
10(8-20) 12(8-17) 13(11-19) 12(9-18) 13(9-16)
" 20 sec three times/week. b Number of mice given grafts. C Median and range of graft survival times, in days. Graft donors were female A/J (H-2 incompatible) mice. Effect of UV Irradiation on In Vivo Cell-Mediated Immunity
Since UV irradiation rendered mice susceptible to with highly antigenic syngeneic tumors, we next mvestigated the possibility that these animals had a general immunologic impairment. First we tested the in vivo rejection response against skin allografts. In this experiment, C3H/He] males and females were exposed to UV irradiation for 20 seconds three times/ weekS for 2,4,6,8, or 10 months before ~rafting. Agematched controls were shaved once a week and sham treated to subject them to the same amount of stress and handling as the irradiated mice. Strain A/] females served as H-2-incompatible graft donors. The results are .given in table 5. Analysis of the data by the MannWhItney U test detected no significant differences in graft survival times between UV-treated and control mIce. chall~nge
8 This UV treatment produced tumors in 100% of the mice with an average latent period of 60 weeks and a range in time of appearance from 48 to 74 weeks (Spikes jD, Kripke ML, Eichwald EJ: Unpublished data).
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60 sec three times/week. Number of mice with progressively growing implants/No. challenged. : Norma~ ~ecipi~nts were the same age as the ~V-treated mice in each experiment. All recIpIents In the UV-treated group had prImary tumors that were excised just before challenge. a
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KRIPKE AND FISHER
TABLE 6.-Effect of UV irradiation on tumor allograft rejection Recipients
UV treatment, wk a
C3Hf UV-treated C3Hfnormal C3Hf ATX A/J normal C3Hf UV-treated C3Hfnormal C3Hf ATX A/J normal
UV-induced challenge tumorb C3Hf
A/J
8
3/5 0/5 7/10
0/5 0/5
17
10/10 1/10 5/5
5/5 0/10 0/10 5/5
60 sec three times/week. Number of mice with progressively growing tumors/No. challenged. a b
TABLE 7.-Effect of UV irradiation on preimmunized mice Challenge tumor a Recipients Normal ATX UV-treated b 231-immunized + UV -treated c
#231
#861
0/10 3/5 6/9 0/10
1/10 5/5 8/10 6/10
a Number of mice with progressively growing implants/No. challenged. b 60 sec of UV three times/week x 15 weeks. C Mice immunized against tumor #231, then given 60 sec of UV three times/week x 15 weeks.
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tion response against the syngeneic UV -induced tumors. DISCUSSION
These experiments are directed to the question of why highly antigenic UV-induced tumors are not immunologically rejected by the autochthonous host. Although the answer to this question is still incomplete, our studies have established several points: 1) The primary host is systemically altered in such a way that it is unable to immunologically eliminate transplanted UV -induced tumors. It is possible that this systemic alteration is responsible for, or at least contributes to, the inability of the primary host to deal with its autochthonous tumor in situ. 2) The failure of immunologic rejection in a primary host is not limited to the autochthonous tumor but extends to others induced with UV as well. Since these tumors do not exhibit any detectable cross-reactivity in vivo (1,2), it is unlikely that the progressive growth of an isograft in a primary host results from immunologic tolerance to tumor-specific transplantation antigens or from the presence of specific serum blocking substances. These factors may account for our finding that UV-induced tumors tend to grow better as autografts than as isografts in a different primary host (table 2); however, the failure of the primary host to reject all transplanted UV-induced tumors suggests a lack of immunologic specificity in the major mechanism responsible for this phenomenon. 3) A systemic alteration that results in an inability to reject transplanted syngeneic UV-induced tumors can be detected in UV -irradiated mice free of detectable primary tumors (table 3). This demonstrates that the carcinogen itself can alter host reactivity against a tumor in addition to initiating the neoplastic transformation. It seems reasonable to assume that this UV-induced alteration is responsible for the progressive growth of autochthonous tumors as well as transplanted UV tumors but, as yet, we have no direct proof of this. 4) UV irradiation does not alter the host immune response against strong histocompatibility antigens, nor does it abrogate the secondary response against syngeneic UV-induced tumors in preimmunized mice (tables 5-7). This demonstrates that UV -irradiated mice are capable of mounting an in vivo rejection response to certain antigens, even though they do not reject syngeneic UV-induced tumors upon first challenge. Thus their failure to reject the latter cannot be attributed to total immunologic suppression. Several explanations are possible for our findings. First, the systemic alteration induced by UV irradiation may not be an immunologic one. Conceivably, UV irradiation alters the host environment (i.e., hormonally or by the endogenous production of growth-promoting substances) in a way that permits UV-induced tumors to outgrow the immune response. This possibility was raised by Potter et al. (6, 7) who, in their studies with mineral oil-induced plasmacytomas, found that primary plasmacytomas grow poorly in normal syngeneic recipiVoL. 57, NO. I,JULY 1976
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We then tested the ability of UV -treated mice to reject a tumor allograft, which was induced in an A/] mouse by chronic UV treatment and was selected because of its relatively low antigenicity. This tumor exhibited progressive growth when transplanted into normal A/] recipients. Groups of C3Hf/Sm mice were treated with 60 seconds of UV three times/week for 8 or 17 weeks before challenge with the allogeneic or a syngeneic UV-induced tumor. Even though the UV-treated mice were susceptible to the growth of a syngeneic UV-induced tumor, they uniformly rejected a tumor allograft (table 6). We also examined the effect of UV treatment on established immunity to a syngeneic UV -induced tumor. We immunized the mice by implanting tumor fragments and then allowing these to regress. Two weeks after implantation, these animals and a group of unimmunized mice were started on a course of UV irradiation for 60 seconds three times/week. After 15 weeks of treatment, these mice and groups of normal and ATX mice were challenged with the immunizing tumor (#231) or with a different UV-induced tumor (#861). As indicated in table 7, UV irradiation did not abrogate tumor rejection in specifically immunized mice, but most of the unimmunized animals and those given tumor #231 and challenged with tumor #861 were susceptible to challenge. The preceding experiments indicated that the first-set rejection of allografts and the second-set rejection of syngeneic UV tumors were unaffected by UV treatment. The only alteration that we detected in the UVirradiated mice (aside from eventual carcinogenesis) was their inability to mount an effective first-set rejec-
IMMUNOLOGIC PARAMETERS OF ULTRAVIOLET CARCINOGENESIS
VOL. 57, NO. I, JULY 1976
nous and UV-irradiated hosts are highly susceptible to challenge with syngeneic UV-induced tumors rejected by normal animals. In the UV and MeA tumor systems, the primary host clearly is not equivalent to a normal animal that has received a tumor transplant. In fact, a normal animal will cure itself of a transplanted syngeneic UV-induced tumor by immunologic means, whereas the autochthonous host will succumb to progressive tumor growth. These studies also show that a relatively short course of UV irradiation makes mice susceptible to challenge with UV-induced tumors which, in addition to its carcinogenic action, induces a systemic alteration that changes the outcome of this particular host-tumor interaction. Further, it raises the possibility that this systemic alteration might also affect the balance between host and tumor during viral or chemical carcinogenesis as well as during UV carcinogenesis. Since UV light is an important environmental carcinogen, this possibility warrants further investigation. REFERENCES (1) KRIPKE ML: Antigenicity of murine skin tumors induced by
ultraviolet light. J Nat! Cancer Inst 53:1333-1336, 1974 (2) PASTERNAK G, GRAFFI A, HORN K-H: Der Nachweis individualspezifischer Antigenitiit bei UV-induzierten Sarkomen der Maus. Acta BioI Med Ger 13:276-279, 1964 (3) ST]ERNSWARD J: Immune status of the primary host toward its own methylcholanthrene-induced sarcomas. J Natl Cancer Inst 40: 13-22, 1968 (4) BASOMBRIO MA, PREHN RT: Immune status of autochthonous and adoptively protected mice toward spontaneous and chemically induced tumors. Cancer Res 32:2545-2550, 1972 (5) BILLINGHAM RE, MEDAWAR PG: The technique of free skin grafting in mammals. J Exp BioI 28:385-402, 1951 (6) POTTER M, PUMPHREY JG, WALTERSJL: Growth of primary plasmacytomas in the mineral oil-conditioned peritoneal environment. J Nat! Cancer Inst 49:305-308, 1972 (7) POTTER M, WALTERS JL: Effect of i"ntraperitoneal pristane on established immunity to the Adj-PC-5 plasmacytoma. J Nat! Cancer Inst 51:875-881,1973 (8) HEPPNER GH: Blocking antibodies and enhancement. Ser Haematol 4:41-66, 1972
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ents but can be successfully transplanted in carcinogen (mineral oil)-pretreated mice. Potter et al. suggest that these findings might be due to an environmental change in the carcinogen-treated animals, rather than an immunologic change. A second possibility is that UV irradiation causes only a moderate reduction in the number of immunologically competent cells. This reduction might be insufficient to alter the rejection of skin and tumor tissue bearing strong histocompatibility antigens, or to eliminate all the reactive cells generated by immunization against a syngeneic UV-induced tumor before UV irradiation. However, it could still be sufficient to prevent the first-set rejection of relatively less antigenic syngeneic tumors. Third, specific serum blocking factors [tumor antigens, antitumor antibodies, or tumor antigen-antibody complexes; see (8) for review] or immunologic tolerance to tumor antigens could account for our findings. However, this seems unlikely, since the UV -irradiated animals would have to be made tolerant to or have blocking factors against all possible tumor antigens that could be expressed on UV-transformed cells. The fourth hypothesis is that UV irradiation selectively suppresses a subpopulation of cells that plays a decisive role in the primary response to syngeneic tumors, but not in the secondary response to syngeneic tumors or in first-set allograft rejection. Although our findings in the UV-induced tumor system are quite striking, they do not differ qualitatively from the results of others who studied the response of the autochthonous host after chemical carcinogenesis. Stjernsward (3) and Bosombrfo and Prehn (4) also reported that the autochthonous host was more susceptible to the growth of its own MeA-induced tumor than were normal mice. In addition, Stjernsward showed that primary hosts were more susceptible to autochthonous tumors than to isografts of primary MeA tumors and, further, that MeA-treated mice showed increased susceptibility to tumor challenge compared with untreated controls. Our experiments indicated that autochtho-
215
2
Margaret L. Kripke 3 and Michael S. Fisher 4.5.6
Chronic exposure of mice to UV radiation induces a high incidence of skin tumors with long latent periods. In at least two inbred strains of mice, tumors induced in this way are extremely antigenic. In fact, most UVinduced tumors are immunologically rejected when transplanted to normal syngeneic recipients (1). These neoplasms resemble those induced by chemical carcinogens, in that each UV-induced tumor is antigenically unique, as judged by in vivo cross-reactivity tests (1,2). The finding that most UV-induced tumors are rejected by normal syngeneic mice raises the question why these tumors are able to persist and grow progressively in the autochthonous host. Further, it suggests that the autochthonous host may be immunologically altered in a way that favors, rather than retards, tumor development and/or that there is some mechanism that permits these tumors to overcome immunologic destruction in situ. The question why primary hosts fail to reject their developing tumors has been studied after tumor induction by chemical carcinogens (3, 4). Primary hosts are frequently more susceptible to rechallenge with their own tumors than are normal syngeneic recipients. However, the chemically induced tumors usually grow progressively in both primary hosts and normal syngeneic mice. Thus one must rely on tumor growth rates to assess the reactivity of the primary host relative to that of normal mice. In contrast, with the UV-induced tumor system, there is a clear distinction between the fate of a tumor in the primary host (progressive growth) and its fate in a normal animal (immunologic rejection). This permits us to examine the immunologic response of the primary host against its own tumor and against other non-cross-reacting tumors induced by UV and to investigate the mechanisms underlying the failure of the immune system to deal with extremely antigenic tumors. MATERIALS AND METHODS
Mice.-Most animals in these experiments were male VOL. 57, NO. I, JULY 1976
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or female C3Hf/Sm mice from the Inbred Rodent Colony of the University of Utah. In the one experiment involving skin allograft rejection the graft recipients were C3H/Hej mice from The jackson Laboratory (Bar Harbor, Maine). Strain A/j mice were also obtained from The jackson Laboratory. UV irradiation.-The UV light source was an intermediate pressure 100-W quartz-mercury arc lamp (Hanovia) that delivered a dose rate of approximately 3 x 105 ergs/cm 2 /second over the wavelength range of 280320 nm. The backs of the irradiated mice were shaved once a week; the animals were exposed to UV light for 60 seconds three times a week (Mon., Wed., Fri.) for the duration of the experiment, unless otherwise indicated. The primary UV-induced tumors used in these studies arose on the backs of the irradiated mice and were classified histologically from fixed sections stained with hematoxylin and eosin. Tumor transplantation.-Primary tumors were removed from anesthetized donors, cut into l- mm 3 fragments, and transplanted sc with a trocar to syngeneic male recipients. The tumors were maintained by serial passage in mice immunosuppressed by adult thymectomy and whole-body (450 R) X-irradiation (ATX recipients). The recipients were inspected once a week for tumors which were measured in three perpendicular diameters. The product of the three diameters is used as an approximation of tumor volume. Skin grafting. -Full-thickness, circular, abdominal skin grafts (15 mm in diameter) were transplanted to the thorax by the method of Billingham and Medawar (5). Plaster casts were removed under light ether anesthesia after 8 days. Grafts were inspected daily for gross signs of rejection and scored in double-blind fashion. Complete destruction of the epidermis was judged the rejection end point. RESULTS Comparison of Growth of Primary Tumors as Autografts Versus Isografts to Normal Mice
This experiment was designed to determine whether ABBREVIATIONS USED: A TX=adult thymectomy and X·irradiation (450 R); MCA=3-methylcholanthrene. Received December 4, 1975; accepted January 23, 1976. Supported by Public Health Service contract NOI CP33263 from the Division of Cancer Cause and Prevention, National Cancer Institute, to the Department of Pathology, University of Utah College of Medicine. 3 Basic Research Program, Frederick Cancer Research Center, P. O. Box B, Frederick, Md. 21701. 4 Department of Pathology, University of Utah College of Medicine, Salt Lake City, Utah 84132. 5 Study performed by M.S.F. in partial fulfillment of the requirements for the Ph.D. degree in Experimental Pathology. 6 The contributions of Dr. Ernst J. Eichwald and Dr. John D. Spikes to this work are acknowledged with thanks. J
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ABSTRACT-Skin tumors induced in mice by UY light are usually immunologically rejected by normal syngeneic recipients. We evaluated the immune status of primary hosts against these highly antigenic tumors immediately after surgical removal of the primary tumor. All primary hosts were susceptible to challenge with their autochthonous tumors, though most of these were reo jected by untreated control mice. Primary hosts were also susceptible to challenge with isografts of antigenically dissimilar UY·induced neoplasms. The susceptibility of the primary hosts to tumor challenge was probably induced by chronic exposure to UY light, since UY·irradiated non·tumor·bearing mice were also susceptible to challenge with these tumors. Although UY·treated mice were unable to reject these syngeneic tumors, they could reject skin and tumor allografts. Further, UY irradiation did not interfere with the second·set rejection of syngeneic UY·induced tumors in mice that were specifically immunized before UY treat· ment.-J Natl Cancer Inst 57: 211-215, 1976.
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KRIPKE AND FISHER
the failure of a primary host to reject its own tumor is a local phenomenon, restricted to the site of tumor development, or a phenomenon that is expressed systemically. Primary tumors were surgically excised and immediately returned sc to the ventral (nonirradiated) side of the autochthonous host. At the same time, tumor fragments were implanted sc in groups of normal and immunosuppressed (ATX) syngeneic recipients. Since the primary hosts were often more than 12 months old when tested, normal controls of the same age as the primary tumor donors were usually used as recipients. Table 1 shows that all 13 of the tumors tested grew progressively upon retransplantation to their autochthonous hosts. Only 2 of these tumors grew in normal recipients (#91, 1 of 5; #81, 3 of 5); 12 of the 13 tumors grew in I or more ATX recipients. From this, we conclude that the primary host is systemically altered in a way that permits tumor growth.
We then asked whether the susceptibility of the primary host to tumor growth was restricted to autochthonous tumors or extended to other, non-cross-reacting tumors induced with UV. In the first experiment, primary tumors were excised and immediately transplanted to groups of normal and A TX mice, to the autochthonous host, and to a different primary host. Thus each primary host received an autograft and an isograft. In all 7 isografted mice (syngeneic primary hosts) the tumors grew progressi"ely, which indicated that a primary host is susceptible to the growth of its autochthonous tumor and to other UV-induced tumors (table 1). To determine whether a primary host is more suscepTABLE I.-Transplantation of primary UV-induced tumors into various syngeneic recipients Number of mice with progressively growing tumor/No. challenged Tumor No.
41 91 53 45 126 913 115
Histologic type
Spindle cell tumor "
Squamous cell carcinoma Myxosarcoma Spindle cell tumor
230 231 81 " 82 71 102 Total number of progressively growing implants/No. ofrecipients Total number of progressively growing tumors/No. of tumors tested a
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NT=not tested.
NATL CANCER INST
Autoch- Normal thonous . mIce host
ATX mice
Syngeneic primary host a
1/1 1/1 1/1 1/1 1/1 1/1 1/1
0/5 1/5 0/5 0/5 0/5 0/5 0/5
2/4 3/5 1/4 0/5 3/5 1/5 1/5
NT NT NT NT NT NT NT
1/1 1/1 1/1 1/1 1/1 1/1 13/13
0/5 0/5 3/5 0/5 0/5 0/3 4/63
2/5 3/5 5/5 5/5 1/5 1/3 28/61
1/1 1/1 1/1 1/1 2/2 1/1 7/7
13/13
2/13
12/13
6/6
Tumor No.
Number of weeks between implantation and development of a palpable tumor Autograft
82 102 230 231 81 71 a
2 2 4
2 2 2
Isograft 4
5 8 5 2 2,2a
This tumor was tested in two syngeneic primary hosts.
tible to the growth of its own tumor than to a different primary one, we compared the growth of the 6 tumors as autografts versus isografts to a different primary host. Table 2 shows that, after transplantation, 4 of the 6 tumors were detectable earlier in the autochthonous hosts than in the syngeneic recipients. In these 4 cases, the tumors grew more rapidly in their autochthonous hosts than in the syngeneic hosts, but in the remaining 2, no differences were detectable in the time of appearance or in the growth rates of the tumors transplanted into autologous or syngeneic hosts. No tumor grew better as an isograft than an autograft. Although only a few comparisons were made, the data suggest that, in general, primary hosts may be more susceptible to the growth of their own than to syngeneic tumors. Similar experiments were performed with transplanted, rather than primary, UV-induced tumors. Autochthonous tumors were excised from primary hosts, and these animals were challenged with another (UV-induced) tumor that had been passaged in vivo in ATX mice. Normal mice the same age as the primary hosts were used as controls. The results, depicted in table 3 (expts 1,2), again indicate that primary hosts are more susceptible to the growth of UV-induced tumors than are normal mice. Growth of Syngeneic UV-Induced Tumors in UV-Irradiated Mice
We next asked whether the state of susceptibility to tumor transplantation in the primary host was brought about by the presence of the primary tumor or by the chronic exposures to UV light. Groups of C3Hf mice were UV-irradiated for various periods before challenge with a syngeneic UV-induced tumor. After tumor challenge, the UV treatment was continued until the experiment ended. Groups of age-matched normal mice and A TX mice were included at each testing. The results are summarized in table 3. In experiments 3-16, all UV-treated recipients were free of macroscopic primary tumors. 7 Nevertheless, in all mice treated with UV light for 17 or more weeks, the transplanted tumors continued to grow. Most animals treated for less than 17 weeks also developed progressively growing implants, and even in mice treated for only 2 weeks before chal7 The mean time of tumor appearance for this regimen of UV treatment is approximately 45 weeks, with a range of 27-60 weeks (SpikesJD. Kripke ML. Eichwald EJ: Unpublished data).
VOL. 57, NO. I, JULY 1976
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Growth of Syngeneic UV-Induced Tumors in Primary Hosts
TABLE 2.-Time of appearance of transplanted tumors in autochthonous or syngeneic primary hosts
213
IMMUNOLOGIC PARAMETERS OF ULTRAVIOLET CARCINOGENESIS
Expt No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Totals
TABLE 3.--Growth of transplanted UV-induced tumors in UV-irradiated, normal, and ATX recipients Weeks ofUV Recipients b Transplant gen- treatment a before Challenge tumor eration challenge UV-treated Normal" 231 82 45 231 231 231 231 154 231 231 861 231 231 861 231 231
50 d 45 d 51 50 30 26 23 21 17 15 15 8 8 8 4 2
2 2 1 2 5 4 2 1 5 4 2 4 4 4 3 3
3/3 4/4 4/4 2/2 5/5 5/5 4/4 5/5 10/10 6/9 9/10 3/5 9/10 7/10 5/5 3/5 84/96(88%)
0/5 2/5 0/5 0/5 0/5 0/5 0/5 1/4 1/10 0/10 1/10 0/5 0/10 0/10 0/5 0/5 5/104(5%)
ATX 2/5 5/5 0/5 2/5 5/5 4/5 2/5 5/5 5/5 3/5 5/5 6/10 10/10 9/12 3/7 2/10 68/104(65%)
b
TABLE 4.-Duration of susceptibility to tumor challenge after 3 months of UV treatment Time of tumor challenge, mob Group
UV treatment"
I II III
3 mo Continuous None
6
7
8
5/5" 5/5 0/5
5/5 5/5 0/5
5/5 5/5 2/5
" 60 sec three times/week. b Mice tested at 6 and 7 mo received implants of tumor #231 from the 4th passage in ATX mice; those tested at 8 mo received implants of tumor #231 from the 5th passage. C Number of mice with progressively growing tumor/No. challenged.
lenge (expt 16), 3 of 5 recipients were susceptible to tumor challenge. Since chronic treatment with UV light rendered mice susceptible to challenge with UV-induced tumors, we tested the duration of this effect after cessation of UV ir~adi.ation. In the following preliminary experiment, mICe m groups I and II were irradiated for 60 seconds three times/week. After 3 months, group I received no f~rther U,:, treatment. UV treatment of group II contmued untIl the end of the experiment. Group III received no UV irradiation (age-matched controls). At 3, 4, and 5 months after the end of UV treatment in group I (months 6, 7, and 8, respectively), mice from each group were tested for their ability to reject a transplanted UV-induced tumor. As shown in table 4 all mice treated continuously with UV developed tu~ors when challenged (group II). Mice treated with UV for 3 months and given implants at 6,7, or 8 months were also susceptible to tumor challenge (group I). Animals receiving no UV treatment were resistant to tumor challenge at the 6- and 7-month testing, but 2 animals developed tumors after the 8-month testing (group III). However, these 2 tumors in group III appeared at 6 weeks after challenge, whereas those in groups I and II were apparent at 2 weeks after the 8-month challenge. VOL. 57, NO.1, JULY 1976
TABLE 5.--Effect of UV irradiation on skin allograft rejection Months ofUV treatment" before grafting 2 4 6 8 10
Sham-treated
UV-treated
Numberb
Median (range)"
Number
Median (range)
16 15 16 16 12
11(9-18) 11(10-14) 13(8-21) 11(9-18) 14(9-20)
15 16 13 11 9
10(8-20) 12(8-17) 13(11-19) 12(9-18) 13(9-16)
" 20 sec three times/week. b Number of mice given grafts. C Median and range of graft survival times, in days. Graft donors were female A/J (H-2 incompatible) mice. Effect of UV Irradiation on In Vivo Cell-Mediated Immunity
Since UV irradiation rendered mice susceptible to with highly antigenic syngeneic tumors, we next mvestigated the possibility that these animals had a general immunologic impairment. First we tested the in vivo rejection response against skin allografts. In this experiment, C3H/He] males and females were exposed to UV irradiation for 20 seconds three times/ weekS for 2,4,6,8, or 10 months before ~rafting. Agematched controls were shaved once a week and sham treated to subject them to the same amount of stress and handling as the irradiated mice. Strain A/] females served as H-2-incompatible graft donors. The results are .given in table 5. Analysis of the data by the MannWhItney U test detected no significant differences in graft survival times between UV-treated and control mIce. chall~nge
8 This UV treatment produced tumors in 100% of the mice with an average latent period of 60 weeks and a range in time of appearance from 48 to 74 weeks (Spikes jD, Kripke ML, Eichwald EJ: Unpublished data).
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NATL CANCER INST
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60 sec three times/week. Number of mice with progressively growing implants/No. challenged. : Norma~ ~ecipi~nts were the same age as the ~V-treated mice in each experiment. All recIpIents In the UV-treated group had prImary tumors that were excised just before challenge. a
214
KRIPKE AND FISHER
TABLE 6.-Effect of UV irradiation on tumor allograft rejection Recipients
UV treatment, wk a
C3Hf UV-treated C3Hfnormal C3Hf ATX A/J normal C3Hf UV-treated C3Hfnormal C3Hf ATX A/J normal
UV-induced challenge tumorb C3Hf
A/J
8
3/5 0/5 7/10
0/5 0/5
17
10/10 1/10 5/5
5/5 0/10 0/10 5/5
60 sec three times/week. Number of mice with progressively growing tumors/No. challenged. a b
TABLE 7.-Effect of UV irradiation on preimmunized mice Challenge tumor a Recipients Normal ATX UV-treated b 231-immunized + UV -treated c
#231
#861
0/10 3/5 6/9 0/10
1/10 5/5 8/10 6/10
a Number of mice with progressively growing implants/No. challenged. b 60 sec of UV three times/week x 15 weeks. C Mice immunized against tumor #231, then given 60 sec of UV three times/week x 15 weeks.
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NATL CANCER INST
tion response against the syngeneic UV -induced tumors. DISCUSSION
These experiments are directed to the question of why highly antigenic UV-induced tumors are not immunologically rejected by the autochthonous host. Although the answer to this question is still incomplete, our studies have established several points: 1) The primary host is systemically altered in such a way that it is unable to immunologically eliminate transplanted UV -induced tumors. It is possible that this systemic alteration is responsible for, or at least contributes to, the inability of the primary host to deal with its autochthonous tumor in situ. 2) The failure of immunologic rejection in a primary host is not limited to the autochthonous tumor but extends to others induced with UV as well. Since these tumors do not exhibit any detectable cross-reactivity in vivo (1,2), it is unlikely that the progressive growth of an isograft in a primary host results from immunologic tolerance to tumor-specific transplantation antigens or from the presence of specific serum blocking substances. These factors may account for our finding that UV-induced tumors tend to grow better as autografts than as isografts in a different primary host (table 2); however, the failure of the primary host to reject all transplanted UV-induced tumors suggests a lack of immunologic specificity in the major mechanism responsible for this phenomenon. 3) A systemic alteration that results in an inability to reject transplanted syngeneic UV-induced tumors can be detected in UV -irradiated mice free of detectable primary tumors (table 3). This demonstrates that the carcinogen itself can alter host reactivity against a tumor in addition to initiating the neoplastic transformation. It seems reasonable to assume that this UV-induced alteration is responsible for the progressive growth of autochthonous tumors as well as transplanted UV tumors but, as yet, we have no direct proof of this. 4) UV irradiation does not alter the host immune response against strong histocompatibility antigens, nor does it abrogate the secondary response against syngeneic UV-induced tumors in preimmunized mice (tables 5-7). This demonstrates that UV -irradiated mice are capable of mounting an in vivo rejection response to certain antigens, even though they do not reject syngeneic UV-induced tumors upon first challenge. Thus their failure to reject the latter cannot be attributed to total immunologic suppression. Several explanations are possible for our findings. First, the systemic alteration induced by UV irradiation may not be an immunologic one. Conceivably, UV irradiation alters the host environment (i.e., hormonally or by the endogenous production of growth-promoting substances) in a way that permits UV-induced tumors to outgrow the immune response. This possibility was raised by Potter et al. (6, 7) who, in their studies with mineral oil-induced plasmacytomas, found that primary plasmacytomas grow poorly in normal syngeneic recipiVoL. 57, NO. I,JULY 1976
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We then tested the ability of UV -treated mice to reject a tumor allograft, which was induced in an A/] mouse by chronic UV treatment and was selected because of its relatively low antigenicity. This tumor exhibited progressive growth when transplanted into normal A/] recipients. Groups of C3Hf/Sm mice were treated with 60 seconds of UV three times/week for 8 or 17 weeks before challenge with the allogeneic or a syngeneic UV-induced tumor. Even though the UV-treated mice were susceptible to the growth of a syngeneic UV-induced tumor, they uniformly rejected a tumor allograft (table 6). We also examined the effect of UV treatment on established immunity to a syngeneic UV -induced tumor. We immunized the mice by implanting tumor fragments and then allowing these to regress. Two weeks after implantation, these animals and a group of unimmunized mice were started on a course of UV irradiation for 60 seconds three times/week. After 15 weeks of treatment, these mice and groups of normal and ATX mice were challenged with the immunizing tumor (#231) or with a different UV-induced tumor (#861). As indicated in table 7, UV irradiation did not abrogate tumor rejection in specifically immunized mice, but most of the unimmunized animals and those given tumor #231 and challenged with tumor #861 were susceptible to challenge. The preceding experiments indicated that the first-set rejection of allografts and the second-set rejection of syngeneic UV tumors were unaffected by UV treatment. The only alteration that we detected in the UVirradiated mice (aside from eventual carcinogenesis) was their inability to mount an effective first-set rejec-
IMMUNOLOGIC PARAMETERS OF ULTRAVIOLET CARCINOGENESIS
VOL. 57, NO. I, JULY 1976
nous and UV-irradiated hosts are highly susceptible to challenge with syngeneic UV-induced tumors rejected by normal animals. In the UV and MeA tumor systems, the primary host clearly is not equivalent to a normal animal that has received a tumor transplant. In fact, a normal animal will cure itself of a transplanted syngeneic UV-induced tumor by immunologic means, whereas the autochthonous host will succumb to progressive tumor growth. These studies also show that a relatively short course of UV irradiation makes mice susceptible to challenge with UV-induced tumors which, in addition to its carcinogenic action, induces a systemic alteration that changes the outcome of this particular host-tumor interaction. Further, it raises the possibility that this systemic alteration might also affect the balance between host and tumor during viral or chemical carcinogenesis as well as during UV carcinogenesis. Since UV light is an important environmental carcinogen, this possibility warrants further investigation. REFERENCES (1) KRIPKE ML: Antigenicity of murine skin tumors induced by
ultraviolet light. J Nat! Cancer Inst 53:1333-1336, 1974 (2) PASTERNAK G, GRAFFI A, HORN K-H: Der Nachweis individualspezifischer Antigenitiit bei UV-induzierten Sarkomen der Maus. Acta BioI Med Ger 13:276-279, 1964 (3) ST]ERNSWARD J: Immune status of the primary host toward its own methylcholanthrene-induced sarcomas. J Natl Cancer Inst 40: 13-22, 1968 (4) BASOMBRIO MA, PREHN RT: Immune status of autochthonous and adoptively protected mice toward spontaneous and chemically induced tumors. Cancer Res 32:2545-2550, 1972 (5) BILLINGHAM RE, MEDAWAR PG: The technique of free skin grafting in mammals. J Exp BioI 28:385-402, 1951 (6) POTTER M, PUMPHREY JG, WALTERSJL: Growth of primary plasmacytomas in the mineral oil-conditioned peritoneal environment. J Nat! Cancer Inst 49:305-308, 1972 (7) POTTER M, WALTERS JL: Effect of i"ntraperitoneal pristane on established immunity to the Adj-PC-5 plasmacytoma. J Nat! Cancer Inst 51:875-881,1973 (8) HEPPNER GH: Blocking antibodies and enhancement. Ser Haematol 4:41-66, 1972
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ents but can be successfully transplanted in carcinogen (mineral oil)-pretreated mice. Potter et al. suggest that these findings might be due to an environmental change in the carcinogen-treated animals, rather than an immunologic change. A second possibility is that UV irradiation causes only a moderate reduction in the number of immunologically competent cells. This reduction might be insufficient to alter the rejection of skin and tumor tissue bearing strong histocompatibility antigens, or to eliminate all the reactive cells generated by immunization against a syngeneic UV-induced tumor before UV irradiation. However, it could still be sufficient to prevent the first-set rejection of relatively less antigenic syngeneic tumors. Third, specific serum blocking factors [tumor antigens, antitumor antibodies, or tumor antigen-antibody complexes; see (8) for review] or immunologic tolerance to tumor antigens could account for our findings. However, this seems unlikely, since the UV -irradiated animals would have to be made tolerant to or have blocking factors against all possible tumor antigens that could be expressed on UV-transformed cells. The fourth hypothesis is that UV irradiation selectively suppresses a subpopulation of cells that plays a decisive role in the primary response to syngeneic tumors, but not in the secondary response to syngeneic tumors or in first-set allograft rejection. Although our findings in the UV-induced tumor system are quite striking, they do not differ qualitatively from the results of others who studied the response of the autochthonous host after chemical carcinogenesis. Stjernsward (3) and Bosombrfo and Prehn (4) also reported that the autochthonous host was more susceptible to the growth of its own MeA-induced tumor than were normal mice. In addition, Stjernsward showed that primary hosts were more susceptible to autochthonous tumors than to isografts of primary MeA tumors and, further, that MeA-treated mice showed increased susceptibility to tumor challenge compared with untreated controls. Our experiments indicated that autochtho-
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