Document ZJQxz4wXnmwM62rKkL2BNqNkZ

'^i^pntholagy and Applied Neurobiology 1981, 7, 68-75 ^ A SURVEY OF ETHYLNITROSpUREA-INDUCED RAT ^tfOMAS FOR THE PRESENCE OF TUMOUR REJECTION AHTIGENS EXPRESSED IN VIVO ALEX M. SPENCE AND GREGORY PRIESTLEY pepartments of Medicine (Neurology) and Pathology (Neuropathology), Univergilyof Washington School of Medicine, Seattle, Washington, USA Accepted for publication 23 June 1980 V' *- Spence A.M. & Priestley G. (1981) Neuropathology and Applied Neurobior logy 7,63-75. I' A survey of ethylnitrosourea-induced ret gliomas for the presence of tumour rejection antigens expressed in vivo t Transplanted lines of seven F-344 (Fischer) rat malignant gliomas induced ' transplacentally with ethylnitrosourea (ENU) were surveyed by in vivo im1 munoprotection assays for the presence of tumour rejection antigens. These gliomas were representative of commonplace histological types of human primary brain tumours and were analyzed in early transplantation passages. The classical tumour ligation method of immunizing animals was attempted with five glioma lines, but was found unusable in four of these because of a high incidence of local tumour recurrences and distant metastases. In most experiments the animals were immunized by repeated inoculations ofheavilyirradiated tumour cells. Two gliomas, a glioblastoma multiforme and a mixed astrocytoma-ependymoma, demonstrated weak but statistically significant tumour rejection responses. Immunization with three other tumours, a mixed oligodendroglioma-astrocytoma and two glioblastomas multiforme, led to enhanced outgrowth of the challenge cell inocula. Neither a rejection nor an enhancement response was observed in assays of the remaining two neo plasms, a glioblastoma multiforme and a mixed astrocytoma-oligodendrog lioma. Immunization With a 3-methylcholanthrene-induced urinary bladder Introduction carcinoma line, used as a control in assays of six gliomas, had no effect on the outgrowth of transplanted glioma cells. These results suggest that ENU- Whether maligninadnuctegdlimomaliagsnaenxt prraetssgliotummasoudro-snpoet cuifniciforamnltyigeelnicsit tshtraotngactutimvoautertumoricidal anti-regjleioctmionariemspmonusensitiyn vinivov.ivo, i.e. are immunogenic, is a question of Address for correspondence: Dr A. M. Spence, RG-20 Neurology, University of Washington, Seattle, WA. 98195, USA. 06-1846/81/0100-0063$02.00 1981 Blackwell Scientific Publications 63 64 A. M. Spence and G. Priestley fundamental importance to understanding the role of immunosurveillance controlling the development of these neoplasms, the role of the immune pr lege of the central nervous system (CNS) in limiting anti-glioma immunity, i the potential role of immunotherapeutic management of these tumou Although there is now a large body of evidence on the immunobiology ofhi and experimental brain tumours (Brooks & Roszman, 1978; Cravioto, 197] 1978b; Wikstrand & Bigner, 1980), it remains uncertain whether gliomas oft common histological types, namely, glioblastoma multiforme, malignant ast cytoraa and mixed glioma, uniformly express tumour rejection antigens in i and, if they do so, whether these antigens are weak or strong. Several chemically-induced rodent gliomas have been reported to eli] strong tumour rejection responses in vivo. These tumours include at least th 3-methylcholanthrene (MCA)-induced C57/B1 mouse ependymal and aatrocy gliomas (Long, O'Conner & Jelsma, 1962; Scheinberg et al,, 1962; Wilkins] Ketcham, 1963) and three methylnitrosourea (MNU)-induced F-344 rat astro tic gliomas (Blume, Wilson & Vasquez, 1974; Cravioto, 1972a; Denlinger et < 1975; Morantz et al., 1979; Tracey et al,, 1978). However, the immunogenicity j most of these tumours was examined after they had been transplanted for ms generations so that antigenic changes in the tumour lines or genetic drift in < animal strains of tumour origin may have supervened (Fellis & Kahan, 1976).] is necessary to be cautious, therefore, in concluding from these observatioi that most gliomas are immunogenic. We have recently studied seven malignant gliomas induced in F-344 raj with the potent neurotropic carcinogen, ethylnitrosourea (ENU) (Koestna Swenberg & Wechsler, 1971) using conventional transplantation assays (He berman, 1977; Sjogren, 1965) to test for tumour rejection antigens. All seve tumours represent histological types of gliomas commonly encountered human neuro-oncology, and all were investigated early in their transplantatid history in order to minimize potential immunological changes due to a lonf term serial transplantation maintenance. I'repc Tabk Materials and methods Animals F-344 (Fischer) rate (Simonson Laboratories, Gilroy, California) were used. Tumours were i duced with ENU in 'foundation line' animals produced by brother-sister matings which had I documented. Limited skin transplantation tests in our laboratory have shown that Simons F-344 rats accept grafts from one another. Tumors Pregnant rats were injected intravenously with 50 mg/kg of ENU (gift of Dr T. Lloyd Fletcherjj between the 17th and 20th days of gestation in order to induce neurogenic neoplasms in th* c. OLI 7057 -i-lmmunogenicity of ENU-induced rat gliomas 65 lillance jn nun('-'';V|. 1unity, and tumours, y of human oto, 1978a: imas of the nant astroens in viUQ d to elicit least three astrocytic Wilkins & at astrocy. nger et ai ?enicity 0f d for nu ny drift in: he n, 1976,. It servati- ns F-344 r..t$ Koesti: ?r, says (h r- ^^lterec n dama* n to a lc }- offapdag (Koe8tner etal., 1971). When these offspring manifested signs of tumour growth between 196 and 405 days of life, they were killed by exsanguination under ether anaesthesia. Intrinsic brain and spinal cord tumours were isolated under sterile conditions and then divided into portions for routine histology processing and for transplantation or explantation in culture. Transplantation was performed in sex-matched syngeneic recipients at the subcu taneous site. Cell cultures were maintained in Waymouth's medium containing 15 or 30% fetal jjlfserum plus additives as previously described (Spence & Coates, 1978). Seven glioma lines were *lected for further study. Four of these were maintained exclusively by subcutaneous transplan tation in rats, and three others were initially explanted in vitro followed by transplantation in as described by Benda et al. (1971). Table 1 provides the histological diagnoses of the icven original gliomas from which the transplanted lines were established. It also shows the transplant stage reached by the individual tumour lines at the time they were studied in immunoprotection assays: C, and As refer to culture or animal passage number. For example, C3A3 Indicates that a tumour, used for either immunization or transplantation challenge, was removed from its parent autochthonous glioma by 3 culture passages followed by three animal passages. |Kf" A syngeneic MCA-induced urinary bladder carcinoma line, BC158, donated by Dr Warren Chapman, was used at Aj-A# passages in several experiments to immunize control animals against $ non-gliogenous neoplasm. This neoplasm expresses tumour-specific transplantation antigens `(personal communication). I "hnmunoprotection assays assay for tumour rejection antigens immunization ofsyngeneic animals was usually performed by ligation or excision of growing tumour grafts, or by inoculation of either radiation-killed tumour cells or live tumour cells at sublethal doses (Heberman, 1977, Sjogren, 1965). Thereafter groups of immunized or unimmunized control animals were challenged with viable tumour cells and tumour outgrowth in experimental and control groups compared. Preparation ofcell suspensions For both immunization and challenge, cell suspensions were prepared from non-necrotic subcu taneous tumour implants, never directly from material in tissue culture, to avoid any potential Table 1. The identifying numbers and histological diagnoses of the seven autochthonous gliomas examined, and the passage numbers of their transplanted derivatives used in immunoprotection assays its were- -> -h had l n t Simor. n d Fletch. ri sms in tie Tumour number Histological diagnosis Passage numbers T131B T161B T175A T180A T181A T181B T185A Spinal mixed oligodendroglioma-astrocytoma Cerebral glioblastoma Cerebral glioblastoma Cerebral mixed astrocytoma-oligodendroglioma Cerebral glioblastoma Cerebral glioblastoma Cerebral mixed astrocytoma-ependymoma CftAi - - CftAi C3A2 * - CjA* Aj - - As A] - * A* Aj--A( CjA| * - C0A3 As-- Aj C culture, A - animal. immunologic effects of cu If scrum components in tissue culture medium. All cell suspensions wen' prepared hy mechanical dispersion methods- to avoid trvpsinization. Briefly, the tumours were finely minced with iris srissots in chilled si rum-free Waymouth's medium. The resulting frag ments were penth pressed through .'Mini nil g.-mpe stainless sled screens This yielded a suspen sion ol single cells mixed with filial I clomps foe immuni/.at ion proccduri s In a li clumps and 'inglt cells were retained and counted together to determine a total cell concentration. For challeng* " procedures, however, further steps were taken. Clumped cells were removed by allowing the cell suspensions to settle in test tubes for 5 to 10 minutes after which tile upper lay ers wen-1 etamsfl The yield of viable cells was determined hy the trypan blue dye exclusion method and found tobt. generally in the 25 to 40 % range. on f.* follow beyni it site- i (h) do mm ( pa!; voluir the C asse~ Tumour immunization We first tried to immunize animals by ligation of subcutaneous tumour grafts when they were 5-15 mm in diameter. This was attempted with T131B, T161B, T180A, T181A, and T181B. However, except with T181B this method failed because of local recurrences and the frequent development of lymph node and lung metastases. With T181B. only sixteen of thirty-six animals did not develop recurrences following tumor ligation. Other investigators have reported similar difficulties in working with nitrosourea-induced gliomas (Cravioto, 1978a; Morantz et at., 1979). > Consequently, with all tumors except T181B immunization with radint ion-ktiled tumour colls became the principal method we adopted. Tumour cell suspensions were immediately irradiated with "Vo giving a total dose of 15 000 rad. and were then inoculated in a volume of 1-2 ml at one site per animal. The immunizing dose per animal in each immunization ranged from 2 0 * 10' to 9 0 x 107 cells. The immunizing inocula were given in the dorsal subcutaneous tissue between the shoulder blades or i.p. (Price et al., 1978). Despite treatment with 15 000 rad, these immunizing , inocula produced palpable nodules which often attained 5 10 mm in diameter before they disap peared after 7--14 days. From 2 to 6 glioma cell immunizations were performed in all assay*. ; Animals immunized with the BC158 control tumour were similarly treated with irradiated coll* subcutaneously. In initial experiments with T180A.T181 A. and T185A, some animals received live cells but did not develop tumours. These animals were incorporated as immunized hosts into new experiment! after thev had received additional radiation-killed cell inocula. Rest The two men deto 1 and of n ar.ir wuImrr car.1 reje Tabl Tumour challenge Our atm was to test each glioma at one or two challenge doses close to, but greater than, the cell dose producing tumours in 50% of control animals (referred to as MTD) (Gross. 1943; Pellisi . Kahan, 1976). The proper tumour cell dosages were estimated from multiple pilot experiments in which graded doses of tumour cells were injected into untreated control animals. The MTD value for seveial glioma lines varied widely in early transplant passages (Mennell & Groneck, 1977) This frequently resulted in over- or under-estimations of optimal challenge doses and necessitated repetition of several assays. Challenge of immunized and control animals with viable cells from the respective glioma lines was performed 7 18 days after the last immunization. All cell challenges were injected s.c. to either the left or right flank. In the majority n( 1 experiments each animal was challenged at two sites (Hellstrom & Hellstrom, 1978; Vaage. 1972!. a lower dose being presented at one site and a higher dose contralaterally. All challenges were administered in a 0-2 ml volume. i Data gathering and statistical ecnluatwn Tumour challenge sites were monitored weekly to determine the incidence, volume, and latency of tumour outgrowths. All calculations and statistical assessments reported below were performed Cro,, Co;.: tur Tib'. tnv BC1 irr turn da`foot Immunogenicity of ENU-induced rat gliomas 67 ! suspensions were ` i he tumours were he resulting frags yielded a suspen clumps and single 'on-For challenge * - allowing the cell , . ers were retained. >>d and found to be on the measurements taken on the day the latest challenge outgrowths appeared, i.e. 6 to 9 weeks following the challenge date. However, the animals were kept under observation 2 to 3 weeks beyond this time to be certain that challenge sites recorded as negative remained so. Incidence was calculated as the ratio of number of sites showing tumour growth to the total sites challenged with live cells. Growing tumours were measured in their longest (a) and shortest (b) dimensions, and tumour volumes were calculated by means of the formula, 0-4ab2 in units of mm1 (Attia & Weiss, 1966). Latency was the number of days elapsed before the first detection of palpable tumour growth, i.e. 4 mm. Challenge sues with no tumour growth were assigned a volume of0 and were omitted from the calculation of latency. Statistical analysis was by means of the Chi square test with Yates' correction. Mean tumour volumes and latency periods were assessed for significance by means of Student's t tests. ten they were 5-15 riSIB. However, ient deve' pmem ' did not develop ir difficrhies in o>. 'ed tumour cells ( iridiated 2 nil at one `rum 2 0 * 10' to ^suebet. eenthe se immunizing . tore tlv'v disap- j -d in a ` assays. \ rradiated cells ' 'ive cells but did tew experiments Results The results on the seven glioma lines are presented below in the following order: two with tumour rejection responses (T181B and T185A), three with enhance ment following immunization (T175A, T131B, and Tl6lB), and two with no detectable responses (T180A and T181A). T181B (Table 2): The incidence of tumour outgrowth in Tl8lB-immunized and control groups was not significantly different. However, the mean volume of outgrowths at both the 1000 and 3000 cell challenge sites in immunized animals was significantly reduced, and the latency period at the 3,000 cell sites was prolonged. No tumour at positive outgrowth sites was observed to regress. Immunization with the bladder carcinoma control tumour produced no signifi cant effects. Thus T181B showed a slight., but statistically significant, tumour rejection response. Tatale 2. Tumour rejection response in rats immunized against T181B >:er than, the cell s, 1943; Pelhs & > experiments in L. The MTD value Groneck, 1977) *nd necessitated , , j , * i- the mn'ontv nf 478; Vaage. 1972), challenges were Challenge dataf Dosage: 10* lO'cellsIsite Dosage. 3 0* lQ3cells/site Groups* Tumour Tumour Tumour Tumour incidence volume Latency incidence volume Latency Control 14/24(58%) 1290 35 22/24(92%) 5025 30 (unimmunized) Tlrtill 6/16(38%) 159$ 31 15/16(94%) 555$ 37 immunized BC158 8/12(67%) 3063 33 12/12(100%) 4229 31 :mm inized me. and latency w were performed * TlHB-immumzed animals were prepared by ligation of lion-irradiated palpable subcutaneous t.imour nodules 7 days prior to the challenge rime tTwo challenge sites |ier rat were used The lata shown are from measurements taken on the 55th day following challenge. XI' 0 05 (All footm te P values represent comparisons with unimniunizcd controUi. IjP ~ 0-005. OLI 7060 68 A. M Spence and 0. I'ricstli-y T185A (Table 3): Of all the seven gliomas studied, T185A showed the highest MTD and the longest latency. Two groups of Tl85A-immunized animals were prepared, one immunized twice and the other four times. Statistically signifv cant results were recorded in both groups, but only at challenge sites that received the higher dose of 24 0(H) cells. The twice immunized and the four-times immunized groups showed a lower incidence of tumour outgrowth than did the unimmunized controls. Tumour volumes in immunized animals were slightlj smaller, but not significantly so. Although we again did not observe regression of any tumours at positive outgrowth sites, the tumour incidence data do denote that immunization with T185A induced a rejection response. T175A (Table 4): Two extra groups of rats were included in the analysis of this tumour: one that was immunized against T175A and additionally treated with 450 rad total-body irradiation (TBI) one day prior to challenge, and another that was unimmunized, but similarly irradiated prior to challenge (Hellstromfi Hellstrom, 1978). A comparison of the Tl75A-immunized group to the unimmunized control reveals that immunization led to enhanced tumour growth since the mean volume of tumours at the 3000 cell challenge sites was significantly increased (2025 mm3 vs. 688 mm3). The incidence and latency periods demonstrated a trend consistent with this. There was also definite enhancement recorded in the group that was both immunized and subsequently irradiated (third in the table) as compared to the group that received irradiation alone (fourth in the table). That is, at the 12 000 cell sites the volume and latency period values of these two Table 4. Enl.a Groups* Control (unimmuniz T175A immunized T175A immunized -TBI Unimmunized -TBI BC158 immunized All T175A-i before the cf irradiation or shown are fro P values are < Table 3. Tumour rejection response in rats immunized against T185A Challenge datai Dosage: 8 0 * 103 cells/site Dosage: 24 0 * 101 cells/site Groups* Tumour Tumour Tumour Tumour incidence volume Latency incidence volume Latency Control 6/24(25%) (unimmunized) T185A 5/23(22%) immunized 'l times T185A 7/19(37%) immunized 4 times 951 275 400 50 21/24(88%) 4970 51 10/23(43%)* 1500 44 46 44 10/19(53%) 3569 39 Animals were immunized two or four times with live and irradiated cells between 96 and 11 days before the challenge date. +Two challenge sites per rat were used. The data shown are froo measurements taken on the 58th day after challenge. No effects from immunization with BC15? were recorded in a previous experiment which included this tumour. *P<001. P<005. groups wet latency: If growth at . These d with total hancement capacity t non-glioma T131B (' at both the slightly urn doseoflOOC and a great ment. A sir cell dose s T161B: estimates reliably p: ments wer Immunogenicity of ENU-induced rat gliomas 69 ed the highest Table 4. Enhanced outgrowth of tumours in rats immunized against Tl 75A animals were tically signifinge sites that Challenge dataf thefour-timesh than did the Dosage: 3 0 x UP cells!site dosage: 120 *. UP cellsjsite were slightly ' >ve regression Groups* Tumour Tumour incidence volume Latency Tumour incidence Tumour volume Latency lata do denote Control 6/23(26%) 688 24 14/23(61%) mi 24 (unimmunized) he analysis of ' T175A 12/25(48%) onally treated immunized 2025$ 21 19/25(75%) 4263 22 e, and another *(Hellstrdm& T175A immunized +TB1 20/23(87%) 38I3 24 23/23(100%)** 9660** 19++ Unimmunized mized control \ + TB1 tee the mean BC1SS 13/21(62%)$ 2367$ 8/21(38%) 2037 23 24 21/21(100%)** 14/21(67%) 5817 5562 22 21 itly Hcreased 1 immunized f. ri. * a trend d in the group *AU Tl75A-immunized animals twice received radiation-killed cell inocula at 27 and 14 days the table) as be table). That ! of these two before the challenge date. TBI indicates that rats in these groups received 450r total-body irradiation one day before transplant challenge. (Two challenge sites per rat were used. The data shown are from measurements taken on the 42nd day following challenge. $P < 0 05 (All footnote Pvalues are comparisons with the unimmunized controls). P<0 001. **P<0 005. t+P< 0 02. ey letweon 96 and \1 n are from 'ation with BC158 P< 0 05. ' groups were significantly different (volume: 9660 mmJ vs. 5817 mm3, P<0 05; latency: 19 vs. 22, P< 0 05). Immunization with BC158 did not influence out growth at sites challenged with either dose of T175A. These data indicate that immunization alone with T175A or in combination with total body irradiation led to significant tumour enhancement. That en hancement did not follow immunization with BC158 further indicates that the capacity to enhance T175A challenge outgrowth was not shared with this non-gliomatous neoplasm. T131B (Table 5): The incidence of tumour outgrowth in the control animals at both the 1000 and 4000 cell challenge sites was below 50%, indicating that we slightly underestimated the MTD of this tumour. Nevertheless, at the challenge dose of 1000 cells per site the immunized animals showed both a higher incidence and a greater mean tumour volume than did controls, denoting tumour enhance ment. A similar but not statistically significant trend was recorded at the higher cell dose sites. TlfilB: This glioma line proved difficult to work with because repeated estimates of the MTD value were low, between 100 and 150 cells, and did not reliahly predict the outgrowth in the assays. Consequently, repeated experi ments were necessary. In one experiment the challenge outgrowths in T161B- Table 5. Enhanced outgrowth of tumours in rats immunized against T131B Challenge datai Dosage: 10* 1111 cells!site Dosage: 4 O'* 10' cellsjsite Groups* Tumour Tumour Tumour Tumour incidence volume Latency incidence volume Latency Control (unimmunized) T131B immunized 2/18(11%) 9/20(45%)* 170 246 It 31 7/19(37%) 2388 28 12/20(60%) 3506 28 31 *Tl31B-immunized rats twice received radiation-killed cells at 31 and 18 days before the challengt date. This experiment did not include a bladder carcinoma control group. tEither one or two chellange sites per rat were used. The data shown are from measurements taken on the 48th da; after challenge, tP < 0 05. immunized animals showed a significantly greater mean volume compared to controls, but with no effects on incidence or latency. In a repeat experiment neither rejection nor enhancement effects on tumour challenges resulted from immunization. T180A: The MTD value was low, between 150 and 300 cells, and also difficult to pinpoint in this glioma line. Animals that failed to develop tumors after live cell challenges in the first and second of three experiments were carried over into a third experiment. This resulted in two groups of animals that were immunized four or six times with both live and radiation-killed cells. These multiple immunizations produced no detectable enhancement or rejection on the challenge outgrowth in any of the three experiments. T181A: This tumour showed a MTD value of about fifty to 100 cells. Similar to T180A, it was assessed in two experiments which included animals that were immunized two or three times with irradiated and live cells. The challenge outgrowth data revealed neither enhancement nor rejection. Discussion The purpose of this investigation was to survey malignant gliomas induced with ENU in F-344 rats for the expression of tumour rejection antigens in vivo. Two glioma lines, namely T181B derived from a glioblastoma multiforme and T185A derived from a mixed malignant astrocytoma-ependymoma, showed significant but weak tumour rejection responses. By contrast, immunization with the bladder carcinoma control tumour produced no effect on outgrowth of chal lenges with T181B or T185A cells. This indicates that the capacity to induce rejection ever, Tltil zation-ch; ;jendent.l> be immui sponses o uncontestumours CNS tissi In tlnprocedur bladder * such enh. irradiatit together feeder ef growth. 1 1978) doi Enhu investig; tumour < challeng tion-kilb In these and the enhance cells. Si purpose: both sol The pret contribi mined w cell-men bodies (i factors ( irradiate From maligna tested b this cap a lack process 1977), c pressor Immunogenicity of ENU-induced rat gliomas 71 tlB 1 rejection of these gliomas was not shared by a non-gliomatous neoplasm. How ler, T181B and T185A were not tested against each other in criss-cross immuni zation-challenge procedures because these two glioma lines were assessed inde IlaIsite pendently and simultaneously before we had any indication that either would be immunogenic. Our results nevertheless do suggest that the rejection re- ; sponses observed in these two tumours were tumour-specific. To establish this Latency ancontestably, though, will require further assays which compare these two ' tumours to each other and include control groups immunized against normal 28 CMS tissue. In the assays of three tumours (T175A, T131B, and T161B) the immunization 31 procedures used led to enhancement of tumour outgrowth. Immunization with bladder carcinoma cells in the T175A and T161B experiments did not produce such enhancement, and the enhancement effect was not reduced by whole-body ,WSltEththechalleng* irradiation of immunized animals in the T175A experiment. These findings - taken on'thelsth 'W together tend to suggest that immunological processes rather than non-specific e 1 dar 1 feeder efFects (Revesz, 1958) were involved in the acceleration of tumour out- . growth. However, a radiosensitive suppressor cell population (Hellstrom et al., I 1978) does not appear to have been responsible for the enhancement, iui. compared to Enhanced outgrowth of tumour challenges has been observed by some repeat experiment investigators who immunized animals with certain doses of cell free extracts of ages resulted from tumour cells (Pellis & Kahan, 1975; 1976; Tracey et al., 1978) and by others who ; challenged tumour-immunized animals with live tumour grafts to which radia- s. and also difficult tion-killed tumour cells were purposefully added (Helstrom & Hellstrom, 1978). p tumors after live ! In these studies soluble antigens in the cell free extracts used for immunization were carried over , and the non-viable cells included in the challenge inocula may have produced inimals that were enhancement by functioning as blocking factors or by activating suppressor billed cells. These : cells. Since we prepared tumour cells for both immunization and challenge nt or rejection on purposes by mechanical methods, our cell suspensions undoubtedly contained both soluble antigens and non-viable, mechanically-traumatized tumour cells. > 100 cells. Similar i The presence of these constituents in the cell suspensions, therefore, may have animals that were i contributed to the enhancement we observed. However, it remains to be deter- ls. The challenge ! mined whether the enhancement results in our experiments were due to specific j cell-mediated immunostimulation (Prehn, 1972; 1977), tumour-enhancing anti- j bodies (Shearer, Philpott & Parker, 1973; Shin et al., 1978), specific blocking ' factors (Hellstrom, Hellstrom & Nepom, 1977), suppressor cells resistant to f irradiation, or some other mechanism. from the evidence we have generated, it appears that some ENU-induced mas induced with ` malignant rat gliomas are capable of inducing weak rejection responses as gens in vivo. Two tested by conventional assays. However, only two of our seven tumours showed forme and T185A this capacity. We conclude that the majority of ENU-induced rat gliomas either "towed significant a lack immunogenic determinants, b induce nonspecific tumour-stimulatory >7.r t with the processes, c carry weak determinants that elicit immunostimulation (Prehn, itgivwth of chal- ` 1977), or d express antigens that induce immunosuppression by means of sup- ipacity to induce pressor cells or blocking factors. I 1 OLI 7064 72 A. M. Spence and G. Priestley There is only a limited amount of recorded data on ENU-indueed glioma.with which our results can he compared. Morantz et al. (1979) examined a F-3H rat glioma, labelled F. by means of iminunoprotection assays in which surgicai removal of viable immunizing inocula was attempted. Similar to our experience. 92% of surgically removed tumours recurred at the operative site. There was ik protective effect from immunization. Cornain et al. (1975) reported that a BDIX rat glioma (GV1A1) demonstrated low but antigenically specific immunogem city in vivo. However, they did not publish any in vivo assay data. A further claim that ENU-induced rat gliomas are immunogenic in in vivo assays ha* appeared, but without data to substantiate it (Morantz, Shain & Cravioto, 1978) The other type of neurogenic tumour that commonly arises in rats treated transplacentally with ENU is the malignant Schwannoma of cranial nerve V. the cauda equina, or other spinal nerve roots (Koestner el al., 1971). Rainbiri and Ridley (1977) assessed the antigenicity of six of these tumours in vivo and found that only one of the six elicited a tumour-rejection response. These findings, therefore, are consistent with ours on gliomas induced with ENU. The ENU rat neurogenic tumour model is of great interest for studies of immunosurveillance because there is a long latency period between transpla cental tumour induction and the development of symptomatic neoplasms Several procedures applied during the latency period and aimed at stimulatinf or counteracting the immune apparatus include a treatment with immune stimulating agents, Bacille-Calmette-Guerin (BCG), bovine albumin, and com plete Freund's adjuvant (CFA); b active specific immunization with glioma or Schwannoma inocula in CFA; c administration of immunosuppressive drugs, amethopterin, cyclophosphamide, or hydrocortisone; and d treatment with anti-rat lymphocyte serum (ALS) alone or in combination with neonatal thy mectomy (Cravioto et al., 1975; Habs & Schmahl, 1976; Morantz et al., 1978: Schmahl, Mundt & Schmidt, 1974; Spence et al,, 1979). None of these procedures has been shown to alter the yield or latency period of neurogenic tumours induced with ENU. It can be argued that these procedures were all without effect because the antigens of the target neoplasms were shielded from the immune apparatus by virtue of being located within the immunologically privi leged confines of the central nervous system (Morantz et al., 1978). However, drawing upon our tumour rejection assay results and those of Rainbird & Ridley (1977), one can equally plausibly argue that these procedures were ineffective because too few of the target neoplasms expressed tumour rejection antigens, or the supposed antigens were too weak to induce any immunological defense mechanisms. In contrast to ENU-induced rat gliomas, a small number of rat gliomas induced with the closely related carcinogen, methylnitrosourea (MNU), have been documented to be highly immunogenic in vivo. They include glioma 528 reported by Morantz et al. (1979). glioma T9 (Benda et al., 1971) investigated by Denlinger et al. (1975) and others (Blume et al., 1974; Morantz et al., 1979; Tracey et al., 1978) and an additional glioma produced and reported by Cravioto (1978a) All three ENU-indi cannot bt develop o tically de The r. thoseind follows: ; similar tr Cooper, 1 administi (Benda ei doses in Koestner treated a carcinog chemical cinogen ( well be Alternat prenatal whereas difFerem from the formatic degree tl ENU-inc whereas tion ant stand th Acknow This resi Cancer Hellstrc advice a Refere, Ama M. Cane I Immunogenicity of ENU-induced rat gliomas 73 /-induced gliomas' All three of these originated in Fischer rats. Our findings, indicating that ) examined a F-344 , ENU-induced F-344 rat gliomas are weakly or non-immunogenic, therefore, in which surgical cannot be attributed to an inherent incapacity of glial cells in this rat strain to to our experience, develop or express strong tumour-rejection antigens, or to strain-related, gene site. There was no * tically determined immunological unresponsiveness of F-344 rats. orted that a BDIX j The reason that MNU-induced rat gliomas are highly immunogenic while cific immunogeni- those induced with ENU are not is unknown. A conjectural explanation is as ty data. A further * follows: although MNU and ENU are both resorptive carcinogens and are n vivo assays has similar in chemical structure and action (Goth & Rajewsky, 1974; Kleihues & & Cravioto, 1978). t Cooper, 1976), they are administered at different doses and schedules. MNU is -es in rats treated administered at a dose of 5 mg/kg given weekly for 30 or more weeks postnatally *' cranial nerve V, /Benda et al., 1971), whereas ENU is administered only once late in gestation at /., 1971). Rainbird doses in the range of 20 to 50 mg/kg (Cornain et al., 1975; Cravioto, 1978a; sours in uioo and Koestner et al., 1971). A conservative rough estimation indicates that MNU- response. These ireated animals are exposed to approximately twenty to fifty times greater total <..*d with ENU. carcinogen dosage than are ENU-treated animals. Since the immunogenicity of est for studies of chemically-induced neoplasms is greater with increasing doses of a given car "t n transpla- 1 cinogen (Baldwin, 1973; Prehn, 1975), the substantially larger MNU dosage may neoplasms, i well be the reason MNU-induced gliomas are more strongly immunogenic. d at stimulating Alternative speculations are based on the fact that ENU is administered during nt with immune j prenatal development when the target glial cells are not fully differentiated, 'humin, and com ' whereas MNU is administered during a period of the rat's life when glial .n with glioma or < differentiation is completed. Conceivably, immature glia, deviated prenatally tppressive drugs, j from the normal pathway of differentiation by ENU-induced neoplastic trans i treatment with ; formation, may not develop the capacity to form strong rejection antigens to the ith neonatal thy- '. degree that more mature MNU-transformed glial cells do; or the neoantigens in antz et a1978; ! ENU-induced neoplasms are inherently weak but altered embryonic antigens, these procedures whereas those generated by MNU are inherently stronger altered differentia t-ogenic tumours ! tion antigens. Only through further experimentation will we be able to under were all without l stand these interesting possibilities more fully. .ielded from the 1 tologically privi- j 1978). However, | Acknowledgements ainbird & Ridley ^ were ineffective 1 tion antigens, or * 'logical defense This research was supported by Grant No. CA 18385 awarded by the National Cancer Institute. DHEW. We thank Drs Ellsworth C. 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