Short Communication IMMUNIZATION AGAINST ETHYLNITROSOUREA-INDUCED AUTOCHTHONOUS NEUROGENIC RAT TUMOURS

THE POTENT oncogenic compound ethyl-nitrosourea (ENU) selectively induces a high incidence of neurogenic neoplasms in the offspring of pregnant rats after i.v. The ENU-treated offspring survive a latency period of 100 to several hundred days and then succumb either to malignant gliomas of the brain or spinal cord, or to malignant Schwannomas located predominantly in Cranial Nerve V or spinal nerve roots. Whether these gliomas and Schwan-nomas are immunogenic, and whether the gliomas, located in the relativelv immuno-logically privileged brain (Scheinberg et al., 1964, 1965) are protected from immune surveillance comprise two questions pertaining to the role of immunity in the patho-genesis of brain tumours. Among several experimental approaches to these questions , one consists of immunizing against carcinogenesis (Prehn, 1961) i.e., inocu-lating the offspring of ENU-treated rats with glioma or Schwannoma antigens to determine whether this type of immunization increases the lifespan, or reduces the incidence of tumours, in susceptible animals. The feasibility of attempting to immunize against chemical carcinogenesis in animal tumour systems was established by the successful work of Taranger et al. (1 972) who demonstrated that immunization of Fischer rats with syngeneic bladder-tumour cells significantly decreased the incidence of papillomas induced by subsequent implantation of methylcholan-threne in the bladder.Encouraged by these observations, we investigated whether immunization with tumour cells during the latent period affects the process of ENU neuro-oncogenesis in rats, and present our findings in this report. Twenty pregnant F-344 rats (Tyler's Laboratories, Bellevue, Washington) received single i.v. injections of ENU (gift of Dr T. Washington) at 50 mg/kg body wt between the 16th and 19th days of gestation (Koest-ner et al., 1971). The 181 offspring (90 males and 91 females) at 5 weeks of age were divided into 6 groups and immunized as indicated in Table I. The glioma-cell immunizing inocula administered to Group I rats were prepared from 4 separate ENU-induced syn-geneic glioma lines, previously generated in our laboratory by the alternate culture and transplantation method (Benda et al., 1971). These glioma lines originated from a cerebral glioblastoma multiforme, a cerebral mixed oligodendroglioma-astro-cytoma, and 2 astrocytomas, one cerebral and the other from spinal cord. Early

THE POTENT oncogenic compound ethylnitrosourea (ENU) selectively induces a high incidence of neurogenic neoplasms in the offspring of pregnant rats after i.v. administration late in gestation (Ivankovic & Druckrey, 1968;Koestner et al., 1971). The ENU-treated offspring survive a latency period of 100 to several hundred days and then succumb either to malignant gliomas of the brain or spinal cord, or to malignant Schwannomas located predominantly in Cranial Nerve V or spinal nerve roots. Whether these gliomas and Schwannomas are immunogenic, and whether the gliomas, located in the relativelv immunologically privileged brain (Scheinberg et al., 1964(Scheinberg et al., , 1965 are protected from immune surveillance comprise two questions pertaining to the role of immunity in the pathogenesis of brain tumours. Among several experimental approaches to these questions, one consists of immunizing against carcinogenesis (Prehn, 1961) i.e., inoculating the offspring of ENU-treated rats with glioma or Schwannoma antigens to determine whether this type of immunization increases the lifespan, or reduces the incidence of tumours, in susceptible animals. The feasibility of attempting to immunize against chemical carcinogenesis in animal tumour systems was established by the successful work of Taranger et al.
(1 972) who demonstrated that immunization of Fischer rats with syngeneic bladder-tumour cells significantly decreased the incidence of papillomas induced by subsequent implantation of methylcholanthrene in the bladder.Encouraged by these observations, we investigated whether immunization with tumour cells during the latent period affects the process of ENU neuro-oncogenesis in rats, and present our findings in this report.
Twenty pregnant F-344 rats (Tyler's Laboratories, Bellevue, Washington) received single i.v. injections of ENU (gift of Dr T. Lloyd Fletcher, University of Washington) at 50 mg/kg body wt between the 16th and 19th days of gestation (Koestner et al., 1971). The 181 offspring (90 males and 91 females) at 5 weeks of age were divided into 6 groups and immunized as indicated in Table I.
The glioma-cell immunizing inocula administered to Group I rats were prepared from 4 separate ENU-induced syngeneic glioma lines, previously generated in our laboratory by the alternate culture and transplantation method (Benda et al., 1971). These glioma lines originated from a cerebral glioblastoma multiforme, a cerebral mixed oligodendroglioma-astrocytoma, and 2 astrocytomas, one cerebral and the other from spinal cord. Earlygeneration stocks of the 4 glioma lines maintained in vitro in Waymouth's medium (Hellstrom & Hellstrom, 1971) were washed wvith phosphate-buffered  saline (PBS) dislodged witil rulbber policemen, suspended in PBS, and counted by means of the trypan-blue dye-exclusion method. Concentrations were adjuLsted and equtal portions of each glioma line were combined to yield a final suspension of 106 total dye-excluding cells per ml (i.e., each ml of final suspension contained 2*5 x 105 cells of each of the 4 glioma lines). Aliquots of 1 0 ml were stored frozen in liquid N2 until needed; they were then thawed, immediately suspended with 0-5 ml of complete Freund's adjuvant (CFA) (Grand Island Biologicals, Grand Island, New York), and injected s.c. into the right flank of Group I animals. Each animal of this group thereby received a single immunization with 106 glioma cells in CFA.
The immunizing inocula for Group II animals were similarly prepared from early cultures of 3 separate ENU-induced malignant Schwannomas, 2 from the trigeminal nerve and 1 from the cauda equina. The 1ml aliquots administered to each rat of this group contained 3-33 x 105 cells of each Schwannoma cell line.
Normal glial cell (Group III) and fibroblast (Group IV) suspensions at concentrations of 106 cells in I ml aliquots were prepared from newborn rat spinal cord or lung cultures, established and maintained by conventional methods.
Group V animals received only a single dose of 0 5 ml of CFA at 5 weeks of age. Group VI served as an unimmunized control population.
Following immunization all animals were monitored daily. After euthanasia of moribund animals or spontaneous death, all viscera, particularly the brain, spinal cord, and cranial and spinal nierves, were carefully examined in situ before removal and fixation in I 0o buffered formalin. Age at death was recorded in days. The fixed viscera were again carefully inspected and sectioned. One to 2mm thick coronal sections of the brain, and transverse sections of the spinal cord and roots, were examined with a hand lens. This method permitted detection of tumours 2 mm or greater in diameter. Tumours in the brain or spinal cord were recorded as gliomas, and tumours in the cranial nerves, spinal roots, or other peripheral nerves were recorded as Schwannomas. In selected instances microscopic sections were prepared in order to ascertain the correct location and histological diagnosis. However, only gross neoplasms are reported (Denlinger et al., 1973). Within the individual groups the meani lifespan, the mean number of gliomas or Schwannomas per rat, and the mean of the total number of tumours per rat were calculated. Among all the immunization groups the means of these 4 variables w-ere assessed for statistically significant differences by an unweighted analysis of variance, retaining sex as a second factor.
The results are presented in Table 11. XVith respect to lifespan, number of gliomas or number of Schwannomas per rat, or total number of tumours per rat, there were no differences among Groups I-VI, no differences attributable to sex, and no significant interaction between sex and groups; that is, the various immunization procedures were all without significant effect on the outcome of ENU neurooncogenesis under the conditions of this experiment.   The mean number of Schwannomas per rat was greater in the 90 males (0.69) than in the 91 females (0.43) (P<0 01 by analysis of variance). Although the mean lifespan (226 days) and mean number of gliomas per rat (1.30) in the entire group of males were less than in females (246 days and 1 58 gliomas respectively), these differences were not statistically significant.
That no immunological effect on ENU neuro-oncogenesis was detected in the present study is probably explained by the following considerations. First, with few exceptions chemically induced immunogenic neoplasms express individually unique rather than cross-reacting transplantation antigens (Herberman, 1977). Although some investigators have demonstrated cross-reacting antigens shared among certain chemically induced experi-mental tumours (Reiner & Southam, 1967;Steele & Sjogren, 1974;Hellstrom et al., 1978) such antigens probably do not play a dominant role in eliciting tumoricidal immunity in vivo (Hellstrom & Brown, 1979). It is unlikely, therefore, that our immunizing preparations, despite being derived from several tumours, contained major immunogenic constituents in common with the ENU-induced neoplasms that arose in our immunized rats. Second, regarding the gliomas, their location within the relatively immunologically privileged CNS parenchyma (Scheinberg et al., 1964(Scheinberg et al., , 1965 probably protected them from exposure to cellular and humoral immune elements. Third, the carcinogenic action of ENU takes place late in foetal development, a time at which immunological tolerance to potential tumour-associated transplantation antigens could evolve. Fourth, our methods may not have presented sufficient antigenic material to induce effective immunity in the tumourdeveloping rats. Fifth, since many chemical carcinogens including methylnitrosourea, which is closely related to ENU (Parmiani et al., 1971) display immuno-suppressive activity, there is a distinct possibility that ENU vitiated the capacity of host animals to respond immunologically to tumour development. Sixth, the immunogenicity of ENU-induced tumours may be so weak as to be undetectable.
To what degree these factors individually affected our experimental results is open to speculation. All probably contributed but, in our view, the immunogenicity of ENU-induced rat tumours is the most important consideration. Rainbird & Ridley (1977) evaluated the immunogenic strength of 6 ENU-induced rat Schwannomas in in vivo tumourrejection assays (Sjogren, 1965) and determined that only 1 of these 6 Schwannomas was immunogenic. On the other hand, Cornain et al. (1975) claimed to have demonstrated with similar methods that 2 ENU-induced rat tumours, one glioma and one Schwannoma, both manifested low immunogenicity. However, no supporting data from in vivo tests accompanied this claim. In our laboratory, ongoing investigations of this type have revealed that only 1 of 5 ENU-induced gliomas elicits detectable transplantation immunity in vivo (unpublished observations). In concert with the findings on Schwannomas (Rainbird & Ridley, 1977) these observations suggest that the incidence of tumour-rejection antigens in transplantable ENU-induced neurogenic tumours, gliomas as well as Schwannomas, is low. It is likely that the incidence of such antigens is similarly low in autochthonous ENU-induced gliomas and Schwannomas, indeed, low enough to explain adequately why the immunological measures reported in this communication influenced neither the latency nor the incidence of tumours in ENU neuro-oncogenesis in the rat.