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Experimental Chemotherapy in Chemically Induced Mouse Tumors and Their Transplants I F. HOVIBURGER, , A. B. RUSSFIELDy J. R. BAKER,AND A. TREGIER Reprinted for private circulation from CANCER RESEARCH Vol. 22, No. 3, pp. 368-74, ApriP 1962 Copyxlght 1962 by Caum Rewrep, Ina nmxa w va.a.

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90 f/OD r1 T£PA-J WfEXSAFTER D.P. 1NJECTION I' (/DO f7YEPA- 6 WEEKS AFTER D:P. /NJECT/ON 2' -S(157) Af69) 18 «~ / r 79 { ' ~ r / r f ! . f r I X f t 10 II 12 13 14 15 16 liI6 19 2021 22 -WEEKS 10+11 12 13 14 15 16 17 18 19 20 21 I 22 (/00 j) TEPA- 7 WEEKSAFTER D.R, /NJEGT/ON 3' SUMMARY OF l. P, AND A •1213) 20 l~ 1 ~ r / R r r f r . ~ . i ' J . r r ,r r 10 IIf.12 13 14 15 16 17 IB 19 20 21 22 -WEEK9- 10 II 12 13 14 15 16 17 18 19 20 21 22 Cnanx L-The bar graphs show the weekly tumor inci- weeks (panel 4)', and 7 weeks (panel!S) after carcinogen injee- dence in untreated (black) and treated (open bars) mice. tiom. Pane14 summarizes these data: The line graphs show the Tteatment in these instances was thioTEPA injected intra- cumulative tumor incidence in treated (solid lines) and un- peritoneally on the dose schedule outlined in the text, begin- treated (broken lines) mice, ning 5weeksafter carcinogen injection. (Panel'I of chart), 6 TABLE 1 EFFECTS'OF THfOTEPA PRF.TREATME\'T ON DIBENZPIRENE-IiNDDCED AND TBAN'9Pt.ANTED TRMOBB' . AND THE@ SGD9EQHEL'T TItA.\SPIANTABD.ITY Top part of tabk: Five animals bearing 5,4,9,10-dibemspyrene-induced tumors received £ mg/kg thiofEPA in 0.1 ml. of Ringer e solution intraperitoneally for 7 days,, and their tumors were transplanted into fifteen animals of the same strain, allowed; to grow for 8 weeks, and the animals then killed. Another fifteen animals were given transplan ts of 8,4,9,10dibenapyrene-iuducedtumors treated with Ringer's solution. Notethe significant tumor inhibition when the original host received thioTEPA. Bodom: parB.of Wble: The experiment is the same as above, except that the tumor which was transplanted'after chemotherapy wasnot.the originally induced neoplasm, but instead a 54th-generation offspring of a 8,4,9,10-dibenzpyrene-iuduced tumor. These transplants grew more rapidlly, bnt at'the end of 2 weeks the tumors originating from treated animals had grown signifivaat- ly moreslowly tban those emanating from controls. Dbxntms Danos An '+'cnoa Smnnrraxca oe o. No. Pca cvvr rvxoa ro.va Av, ws~oar raaoe cz,aex ceeaaas Gaovr eice(..vmrar). I e,.-ivn~e nc..res anowra mu (a.J (~oa) (wc.) (ua.) I I (au.) P Treated (induced) 15 0: 14X17 4.SX 5.6 0.64 3 7.5 <0.01 Controls (induced). 15 0 1RXQ0 11.7X29.0 4.02 ~ (1.7-7.7) Treated (transplant) 15 0 ~ -- I 12XS2 I1.BXQ0.4 1.47 (0.6-4.0) 2 5.1 <0.01 Controls(transplant) 15 I 0 17XR8 14.OXQ4.4 ?..S I r&. (1.4-5.8) 19XS1 01197

I-Io.rnrxGEltet ad.-CA,e.notherapy of Induced Jlouse TuSnors' days. Tumor weights were then determined and compared with vehicle-treated controls. Tumor inhibitions of 50 per cent ormorer were obtained in 3,4,9,10-dibenepyrene-induced tumors of approximately 1 cm. in diameterby means of Miracil-D, mitomycin C and 5-fluorouracil (see Table 5)'. As judged by tumorweight,r amethop- terin yielded no significant effect; however, in terms of tumor diameter, the reduction of tumor size with amethopterin was less than 50 per cent,, which is in accordance with Sugiura's report (16) and with Clarke's' observations on Sarcoma 180. The ineffectiveness of ameLhopterin in induced tumors was also observed by Weiant (17) and by Clarke.' With 5-Huorouracil, Sugiura observed approximately 50 perr cent inhibition of Sarcoma 180:and even less with mitomycin C. DISCIISSIO\ linder present conditions of cancer chemothera- py screening, the extrapolotion of animal data to humans is hazard'ous (5) and, according to some, 373 may be expected to improve only uponn discovery of aclinically'effectiveagent (10). It is tempting: to hypothesize that biologic models resembling, more closely human cancer would, when used as screening systems, yield predictivce results applie cable. to humann chemotherapy. Spontaneous mouse mammary adenocarcinoma has indeed re- sisted most potential chemotherapeutic agents. (11, 12) as does human cancer, with the exception of certain, plant auxins which inhibited mouse breast cancer (1) but have as yet not been ade- quately tested in liumans. Unfortunately, the use of spontaneous.and methylcholanthrene-indhced mammary mouse cancers for screening iss imprac- tical (9, 10). Autochthonous tumors are the logical tool for chemothcrapy study (4), since its aim is the cure of autochthonous human cancer. Subcutaneous fitirosarcomas,, induced in mice by a single injec, tion of 3,4,9,10-dibenzpyrene, grow with remark- able uniformity.'1'his carcinogen is not excreted in ~ D.. Clarke, personal communication. TABLE 5 EFFECTS OF CHERIOTHEHAPEUTIC AGENTS'OV DIBEr-ZPPRENE-IN'DUCED TUSfons'. Groups of ten or twelve animals bearing$,4,9,10-dibenzpyrene-induced tumors of approximately 1 cm. in diameter received the chemotherapeutic agents.in the doses listed in Column lifor 7-8 days. Tumor weightB were thenAetermined and'compared with those ofvehicle-treated controls. Av. mvoe e.xc Ar my E 9iunmewxce oe GB9CP A\E l,fiqE \o. PeE ccxr Av..uam we,axa (m'c'1 wemex "e~on weecar cneucea s.\O1aL9 DEEtE-0 O}Ca.lV4F.1 C4a~1 (al\-OE) Start Autapey IOY.J S P f-~ !Ymethnpterin 12 8.3 -0..54(-4.0to}O~S) SX10. 6X9 '0~.64(0.1-1.3) 1.5 mg,'kg. Not sig nificsnt Controls ~ 1$ 0 }L1(O:.Oto-{-4.3). 8Xlu 14X12 1O19(0.4-1.9) Niracil D r- 67.5mgjkg 18 8.3 -4.05(-3.4to-0.7) 9X9 10X10 -0lR7(0.1-016) . 4.0 <0.01 Controls 12 0 }0.44(', 0./.to}1.4) 8X99 lOXll 0.85(O.Q-1.4) AIiracil D 5-Fmg/kg 10 10.0. -8.9(-4.Rto-r.9) SX10 9X11 0.5(0.1-1.0) j ' 1.9 <0.05 Controls 10 0 +0.8(0.Oto}1.7). 9X10 - 10X1B 0.8(0.8-1_5) Jlitomycin C. ~ 4mg/kg 12 . 8.3 -$.46(:-5.Sto-0.9) ! 9X10 7X8 0.44(01-0.5) 4.7 <0.01 Controls 12 0 }L1(0.Otot4.3) 8X10 1YX12 0.9(0.4-1_9) 5-Fluoronracil 35 mg/kg 14 0 -8.34(: 7.4 to -4.8) 8X9 4X5 0.Y(0.1-0.5) 5A Controls 19 0 }l.l(OLO[o}4.3): 8X10 14X'lE 0.0(0.4-1.9) i-Fluorouracil 44mg/kg, 10' 0 -1.Si(-5.Sto-0.4) 9X1.0 9X12 0.44(0.1-1.3) 3.1 <0.01 Controls 10 0 }0.8(O.Oto}U.7)'. 9X10 1OX14 0.8(0.3-1.5)

J HovisuxGEx et al.-Cheraotherapg of fnduced Jfou.4e Turrmors from the Jackson Memorial Laboratory in Bar Harbor, Maine. Unless stated otherwise, male. mice were used. All animals were kept in the same air-conditioned animal room in groups of ten per cage and received Purina chow and water ad libitum. San-I-CeI wass used as.bedding. 3,4'y9,10- d'ibenzpyrene caused subcutaneous.fibrosarcomass in all injected animals, which grew at comparable. rates. Serial histologic studies. beginning atthe time of injection of the carcinogen had shown that morphologically malignant cells were identifiable at the injection sites 4-5 weeks after administra. tion of the carcinogen (7) and that the tumors in- duced were fibrosarcomas of consistent morpho- logic and functional uniformity. Although it is known that 20-methylcholan- threne is rapidly excreted through the bile, no 8,4,9,10-dfbenzpyrenc was found in urine or feces of mice for many weeks after its injection, and the carcinogen~has been recovered by solvent extrac: tion from large tumors.which it.had produced. TRANSPr.ANTATION OF TUMORs' Transplants were made into male C57BL/6' mice from fibrosarcomas which had been induced by 3,4;9,I0-dibenzpyrene. Tumor fragments 1-$2 mm. in diameter were used and transferred into the recipients by the. usual trocar method. The take rate was 100 per cent,. and the growth rate of these. tumors was relatively uniform. There were no regressions, and the tumors killed the hosts in 2-4 weeks. One line of these.transplanted offspring of 3,4,9,10-dibenzpyrene-induced tumors was.kept by weekly transplants for 54 generations. To ob- tain slowly growing tumorss for more extended chemotherapy studies,.cytosieve transplants were used (14), with injection of 0.$ ml. of a 4 per cent tumor cell suspension in Ringer's solution. This method produced; tumors of 1-cm. diameter in R9 weeks and permitted sufficient survival time for S- to 14-day courses of therapy after tumors had reached as.much as 2 cm. im diameter. CIIE:rIOTHERAPYCompounds. and doses.-N-N'-N"-triethylene thiophosphoramide (thioTEPA) was used as the standard chemotherapeutic agent, since its effec- tiveness against transplantable mouse sarcomas had been well established (1.5).. Doses of 2 and 4mg/kgin Ringer's solution, given intraperitoneal- ly daily for 8-14 days, were used. Other chemo- therapeutic agents studied were: , 5-Fluorouracil 24 and 35 mg/kg .. Amethopterin 1.5 mg/kg Jlytomycin C 4 mg/kg Miracil D 54 and 67.5 mg/kg 369 RESULTS Effects of thioTEPA during tlre tumor induction period.-Four mg/kg of thioTEPA was given in- traperitoneally for 8 d'ays,, followed by a single injection 7'days'.later and by three more, beginning ]I days later. Thisempiricali dosage schedule thus stretched out over 47 days, beginning.5, 6, and 7 weeks after injection of the carcinogen. ThioTEPA was thus given during the induction period, at a time when previous histologic studies. had shown the presence of morphologically malignant cells (4r5 weeks after injection of carcinogen). This re- suited in asignificantlp delayed appearance of the tumors. These observations are illustrated in Chart 1. Effects of thioTEPA administered to animals bearing 3,4;9,10-dibenzpyrene-indueed tumors and F-54 transplantss of induced tumors upon their aub- sequent transpdantabitity.-Five donor mice bear- ing 3,4;9,10-dibenzpyrene-induced tumors were given 2 mg/kg of thioTEPA in 0.1 ml.ofRinger's solution intraperitoneally for 7 days, and their tumors were transplanted by trocar into fifteen animalsof the same strain. Another fifteen animals received transplants of 3,4,9,10-dibenzpyrene-in- duced tumors of comparable size, the hosts. of which had received a similar injection schedule of Ringer's solution only. After 3 weeks;e the animals were killed and the. tumors weighed. The growth rate of these transplants was' significantly re- duced,, and the average final Lumor weight was 0.6 gm., as eompared'with 4.0 gm.. in the controls, with a t value of 7.5 (P < 0.01). Anotherexperi- ment was done treating,, in similar fashion, mice bearing 54th-generation transplants derived from 3,4,9,10-dibenzpyrene-iuduced tumors after they had grown for 1 week. These tumors also showed a reduced transplantability, but to a lesser degree (Table 1). E,f'ecta of a single dose of thioTEPA ' onthe growth rate of induced tumors when measuring 1-1.5 cm. in diameter -A single dose of 4 mg/kg of thio- TEPA was given intraperitoneally to sixteen male and fifteen female mice with 3,4~,9,10-dibenzpy- rene-induced tumors measuring 1-1.5 em, in di- ameter. A similar group of animals received only Ringer's solution as a control. Weekly plas6o- grams (1) were made to obtain growth curves on treated and untreated tumors. Na effect was ob-served on either tumor growth rate or survival time. Effect of multiple doses of thioTEPA on.3,4,9,10- dibenzpyrene-induced tumorsmeasuring 1-1.5 cm. in diaraeter.-Forty seven animals with induced tumors measuring 1-1.5 em. in diameter were matched with 47 controls of similar tumor size and

Experimental Chemotherapy in Chemically Induced Mouse Tumors and Their Transplants* F. HOMBUAGER, A. B. RU55FIELD, J. R. BAKER,AND A. TREGIER wrrHTHE TECRNICALABBr9TANCE OY R, KENNEY. C. CEVULHiy H: RY9, AND S`VARNEH' (Bin-Rtsearcfi InstiEUk, Cambridge, SlaseacLveetta) SU41Af ARYUniform subcutaneous fibrosarcanas can be.induced by singlee injectionsof 3,4,9,10- dibenzpyrene into C57BL/6 mice at the rate of 400-300 tumors per week from a colony of approximately 3,000 animals (8) and are practical for chemotherapy screening, as shown by their response.to five different chemotherapeutic agents' previously tested in transplanted tumors..The animals are safe, since no carcinogen is exereted. Histology showed more marked responses (reduced: mitoses, fibrosis)) in induced than in trans- planted, tumors. Chemotherapy with thioTEPA administered during the induction period or latent phase.caused significant'.delays in the appearance of tumors, which isanalogous.to the tumor-inhibiting effect.claimed clinically forthioTEP_it when ad- ministered after radical surgeryf'or breast cancer. Transplanted mouse tumors, widelyused for experimental screeningof' potential chemothera- penticc agent.s,, represent a biologic model ofthef tumor-host system which is different in many re- spects from cancer in patients.`Ghereas human, cancers and spontaneouss animal tumors are autologous, transplanted tumors, especially during continuous transplantation, may losc their initial genetic and immunologic compatibility (10): Thee response of transplanted tumors to ehemothera- peutic agents represents the end-result of numer, ous interactions of host and tumor, complicated by estraneous.factors such as infection and chemi- cal and physical' effects on cell viability,, which enter into play at the time of tumor tYansplanta- tion. The results,of such experiments must there- fore be.interpreted with caution. Spontaneous mouse tumors such as breast adenocareinomas, which probably would constitute the most vigorous animal screening, test for a chemotlicrapcrd!ic agent (4, 10)4 present numerous .difficulties.. It is impractical too obtain at any one time sufficient numbers of such tumors of com- parable size for screening purposes (10, 11). Sufficient knowledge is now available on the virus-induced Rouschicken sarcoma. and Friend and Moloney mmtseleukemia (2, 3) to permit their use in experimental chemotherapy (Q), but this necessitat'es the introduction of tumor viruses into animal colonies. Tumors.induced by means of chemical carcino- gens arise from thee host's own cells and therefore resemble human clinical cancer much more closely than do transplanted ncoplasms. Complicating factors with chemically induced tumors are the possible effects of carcinogen upon the behavior of the tumor and thc hazards to other animals and to personnel which may arise from the excreted carcinogen or its metabolites in urine and feces. The carcinogens 3,4,9,10'-dibenzpyrene, when injected subcutaneously, induces uniform fibro- sarcomas and: is not excreted in urine or feces (8). Since these induced mouse.tumors are transplant- able, an opportunity exists to compare the re- sponse i.o chenmt'herapy of the originally induced tumor with the response of transplants derived, from the original' tttmor;, as well as with other transplantable tumor systcrns. MATERIALS AND ASETHODS IRDUCTIOY OF TUMURS The carcinogens used were 3,4,9,10-dibenzpy- reue and ?0-methylcholanthrene,.given subcuta- • Supportcd in part byUSPHS Grant CY-4B66. . neously in singlee doses of 500. µg. in peanut oil. Received fon publication October 90, 1961. The animals used were C57BL/6mice obtained 368 01197951

374 Cancer Research feces or urine and remains in t'he.induced tumors2 without apparently interfering with their response to chemotherapeutic agents. Whereas such tumors induced in a different strain were shown by others (17) to be susceptible to. caloric restriction, weight losses per se do not explain the tumor responses observed in this study. Judged by histologic study, induced neoplasms aremore responsive to thioTEPA than their trans- planted offspring, and the induced tumors grow more slowly; as do spontaneous mammary tumors, when compared with their transplants (13). Al- though the response of the fully developed 8,4y9,10-dibenzpyrene-induced tumors was analo- gous to responses observed with commonly used transplanted tumors, the inhibiting effects on tu- mor appearance rate obtained with thioTEPA given during the induction period suggest that this biologic system of chemotherapy during early cancerization behaves much as do patients given thioTEPA for cancer cells remaining after opera- tion (6). REFERENCES 1. ArFFEI, C. A.;.TREGren,.A.; and HomaURCER, F. Syn- thetic Auxins in the Chemotherapy of Tmnsplwried and Spontaneous Mouse Adenocarcinoma. J.. Nat'1. Cancer In.st., 26:1111-8o, 1960. 2. BarwN, W. R Problems in Viml Tumor Therapy. Nat'l. Cancer Inst. Monograph, 4:363-76, 1960. 3. Cmmcos;. M. A.; MOLONEY, J. B.;. HUUrImIES, S. R.; tiLAxrEE, N.; and Gol.DSN, A. Response ofVirus-indnced Murine Lymphoid Leukemia to Drug Therapy. Cancer Rese arc h,. 21: B03 -11,.1961. . 4. DAY,.E. D. Animal Cancer, the Primary Tool (Gue.u Edi- torial). CancerResearch, 2L•58L-fii, 1961.. 5: GEm.aoax;,A., and: HmsctmEaG, E: (ed.). Investigations ' Author a unpublished dataa and' personall communication from E. L'nseren and L. F. Fieser. Frc:.1.-Photomicmgraphof tmnsplanted (FI) tumor de- rived from 3,i,9,l0dibenspyrene-induced parent tumor (H. & E., X 1600) from control animal treated with Ringer's solution for 1@ d6,vs. Note numerous mitotic figures andlarge, vesicular nuclei. FIG. R.-Photomicrogmph of'. 3,4,9,10-dibenzpyrene-in- duavl tumor from mouse treated with tbioTEPA for 8 days Vol. 42, April 1964 of Diverse Systems for Cancer Chemotherapy Screening. Cancer Research Supplement No. 3, 1955. 6. Hor.L.xn,. J, F. Chemotherapy and Chemapmxis. Cancer Research, 31:1f188-99; 196L.. 7.. HoxEVneEn; F:, and TueolEa,A. Modifying Factors in Carcinogenesis. In: F', HoesaIIAGEa (ad.), Progress in Es- perimental Tumor Research,1:311-Z8. Base]/New York: S. Karger AG, 1960. 8.. Hoslavnoen; F., and TaeGIEn, A. Modifying Factons in 3.4,9,10-Dibenzpyrene Carcinogenicity. Proe Am, Assoc. Cancer Research, 3:140, 1960. 9.HvGGlus, C.; BmauREl.m, G.; and SornoN,. H., Jn-Rapid Induction of MammaryCarcinoma in the Rat and the In- fluence of Hormones.on the Tumom. J. Exp,.Jled., 109: 44-45, 1959. 10. Knacsossar, D. A. Experimental Cancer Chemotherapy. In: F.,Homsuacen (ed.), The PhysiopatYolbgy of Cancer. 4d ed.,, pp. 788-8,0. New York: Paul B.. Hoeber, Inc., 1953. 11.. SenoI.I.Ea,.J. Practical and Theoretical Considemtions in the Use of Induced and Spontaneous Mammaty Tumors in Cancer Chemotherapy. Ann. N.Y. Acad. Sci:, 76:855-00, 1958. 14.. Scnouwa, J., and BerruEn, J. J. Further Studies of Chemotherapeutic Agents in Spontaneous Mammary Adenocurcinomxs of Micc and imthe Transplants of Recent Origim Cancer Research, 18:464-69, 1956. 13. SclrolaEa, J.; Brrrvea, J. J.; and Pnrmra, F. S.. Chemo- therapeutic Studies with Transplants af'~ Spontaneous Mammary Tumors of Mice Grorving.ih Various Hosts. Cancer Research, 17:GOb-8..19n7: 14. SxEEU, C. D. A Cytosieve Permittfing$terile Preparation of Suspcnsions of Tumor Cells for Tmnsplantation. J. Nat'l. Cancer Inst., 13:1511-15; 19.i3. 15. SrocK, C. C.; CLenKa,.D. A.; PulErra, F. S.;andBaacner, R. K. Sarcoma ISO Screening Data.. Cancer Research, 20(8);.8-4, 1960. 16. Suomne, K. EAperimental Chemotherapy of Cancer-A Report of Progress. In: F...HWIBURGER (ed.)i Plogress in Experimental Tumor Research, 2:334-76. Basel/Nbw York: A. Kargcr AG, 1061. 17. WEIANT, E. A.; R'OUNaKV, IL;. E.EnsLEt; C. J.; and LElren, J. Studies on the Chemotherapy of Chemically Induced Tumors. Proc. Am.,.lssoc. Cancer Research, 3: 476, 1961. (total dose, 0.8.mg.)i (H. & E:,. X1600). Note dark spindle shaped nuclei and absence of mitotic figures: FIG. d.-Photomicrograph of 3,4,9,10-dibenapyrene-in- duaedlumor from a mouse treated %.ith thioTEPA (0.1 mg/ day for B days). Note numerous giant cells (H.. & E;, X800). FIG. 4:-Details of Fig. 3 at 160U.magnificatfon. Note ab- normal mitotic figure in giant cell. 0119'795r7

TABLE 3 EFFECIS OF TRIOTEFA ON DIBENZPYRENE-INDUCED TOMORS AND F-86 TBANSPLA!V18 MEASURING 1 Caf. AND 2 CM. INDiAMETER Eleven 3;4.9,10-dibenzpyrene-indOced tumors were selected when they measured 1 cm. in diameter.and were matched with fifteen transplanted tumors (36th transplant generation of.S,4,0,10iiibenspyrene-induced tumors) of comparable size. Both groups received 4 mg/kg tkdoTEFA intraperttoneally for 8 days. Tumor weights were determined at the end of treatment. Fifteen induced tumors were selected when they measured appruximately E cm. in diametcr and were matched with ten transplanted tttmors.(S6thgeneration of 3,4,9,10-dibenapyrene-induced tumors) of similar siee: These animals received the same treatment as the 1-cm. tumor group. Controls with comparable tumors were given Ringer's solution. (See text for interpretatfon of results.) SlanrCrCA.YCE Ur Gvoor No.. *pnyre Pv eWr x AvA aooT Wsraer A}f )~ ( ) Islrus ~em`ce ouYSTa6(Cn.) Av. ToYOe WalOnx(OY.). mr.oa Ws.cux csnaaee aas ee aLwx . aY. Ar. (aesos) (asnoe) I P Tteated(induced) 11 0'. -Q,77(-4.6to-1-S) 1 (0.8-1.R) 0.4(0.04-0.4) t-5.RT Controls(induced) 15 0 -0.27(:-4.8to+1.$) 1 (0.8-1.R) 8.5 <0.01 Treated(4ndumd)'. , 15 0 -4.44(:-5.8to-0.4) 4 l-1.0' Controls(iuduced) 15 0 to}1.R) 2 (1.9-9.4) 1_6(1. 0-2.5) 8.5 <0.01 Treated (transplant) 15 6.7 +0.49(-1.1 to }g,4) 1 (0.8-1.2) 0.7(0.4-1.1)) t-4.0* Controls(tmnsplant) 15 0 -1.3(-4.5to}0.8) 1 (0.8-1.2) 1.8(0.7-3.1) . ' 7.3 <0.01 Treated (transplant) 10 10.0 }0.59(-L.5 to }8.6) 2 (1.4-4.8) 1.4(0.6-R.1). f-0.9* Control'.(transplant): 10 0 }0A5(-eto}R.Y). 2 E.7(fl.4-3.4) 7.6 <0.01 Transplant/Induced (Treated). 1 0'..7/0.S 6.3 <0.01 Transplant/Induced(Treated) 2 (1.4~.5) 1.4/0.5 5.4 <0.01 Transplant/Indneed (Controls) 1 (0.8-1.2). 1,8/0.5 7.2 . <0.01 Transplant/Induoad(Controls) Y (1.4-4.5) 9.7/L6 8.1 <0.01 * These dvalues.are for the significance of diffcremeof body weight changes in controls versus treated,.animals- TABLE 4 EFFECTSOF THIOTEPA ON40.METHYLCHOLANTHRENE7NDUCED TOMORSINC57BL/6a'D' Fourteen animals with methylchoianthrene-induced tumors measuring approximately 1 cm. in diameter were given 4 mg/kg of thio'FEPA in Ringer's solution intraperitoneally daily for 9 days and matched with fourteen controls bearing comparable tumors.and receiving only Ringer's solution. GAOLr No. Pa. CENT AY. YOni wEI®X• Av, rovax ema (YY) Av. roaos We1WT Slmarsrc.uascs or TeY08 ALIOar CaLMGm *p1Y*Ly aedfae C}(BANOC) tOY.) Start Autopsy (ayNpa) (oY') S I P Tieated' 14 35.7 -1.6(-10.Oto}5.8) 10XIY IIX1fl 0.9 ~ (0.4-&.5) 1-1.2' 4.6 <0.01 Controls. 14 21.4 }0.1(-5.0to}Q-8) 9X14 18X15 5./. (0.8-4.4) * t is for the significance of bod¢ weight (change).difference between treated and untreated animals (not significant)'. 01197955

I-LoalBUROE,x el al. Clzemotlierapy of Induced Mouse Tumors. were given 4 mg/kg of thioTEPA intraperitoneal- ly, daily,, for 14 days. Significant inhibition of these induced tumors wasobserved (Tablc £). Effeets of multiple doses of tJaioTEPA on 9,.f,9,10- dibenzpyrene-induced tumors and F-.46 transplants measuring 1 cm. andt' cm. in diameter.-Eleven in- duced tumors (3,4;9,10-dlbenzpyrene) were select- ed when thev measured 1 cm. in diameter and were matched with fifteen transplanted tumors (36th transplant generation of 3,4,9,10-dibenzpyrene- induced tumors) of comparable size. Both groups received 40 mg/kg thioTEPA in Ringer's solution intraperitoneally for 8days.. Tumor weights' were determined at the end of treatment. All tumors weree sectioned, and their histology wass studied. Similar numbers of controls with tumors of similar size received only Ringer's solution. Untreated transplanted tumonsgrew more rapidly than un- 371 Fifteemindueed tumors were selected when they measured approximately Scm. in diameter and were matched with ten transplanted tumors (36th generation of 3,4,9,10tlibenzpyrene-induced tu- mors) of similar size. These animals received the same treatment as the1-em, tumor group. Con- trols with comparable tumors were given Ringer's solution. Induced, as well as transplanted, tumors meas- uring 2 cm. in diameter were affected by thio- TEPA (Table 3), but in this case, even judging by tumor weight,, the induced neoplasm wass some- what more inhibited than the transplanted tumor (Figs..1-4)~. In this experiment, differences of body weight changes in treated,, as compared with untreated, animals were not statistically significant in the case of animals bearing tumors measuring approxi- TABLE 2 EFFECIS ON THIOTEPA ON 3:4,9,10-DIBEYEPYRENH-I6DUCED TUMOBs IFC57BL/6dl cj' The3,4,9,10-dibenzpyrene-induced tumors in,a9 04 animals were of comparable size (1-1.5 cm~ in rGameter) when half of the mice received 4 mg/kg ofdhioTEPa daityin Ringer's solution intraperitoneallyfur 14 days. The controls received only Atinger s solution. The resulting.tumor inhibition m the treated animals wassignificant. Gsore N0, . r8N Ar. EOUi 4GION2 ' Av: muos nunsrna ~~') A...roaos w T S~om..c"cs or 'CCNOa W3IOFIT~ rnAnOflB y ~u nsersa A}(.ez) (cu.) . Start Autopey (a. oe) (ax) t P Treated 47 33. -3.66(-9.4to-0,S) 1.0-1.5 1.0-L5 0.7 I 3.8 <0.01 Controls 47 ?8' +0.81('-3.4to-F4.3) 1.0-1.5 1.5~.0 1.9 I (0.1-7.3) treated induced tumors (beginning, of measure- ments in both eases with tumors of 1 cm. in di- ameter)L However, the degree of inhibition result- ing from thioTEPA was the same in both cases as measured by tumor weight.. Histolbgical study showed that, in the induced tumors, thioTEPA appeared to decrease thenumberof mitotic.figures present as compared with those in the controls. Treatment also appeared toincrcase the number of'bizarre gjantcells present„ which are interpreted as being the result of incomplete cell division. (Occasional giant cells were a1so.folmd in the un- treated induced'tumors and might represent a re- action to the carcinogenpresent.).Individltal cells in t'hetYeated tumors were smaller and more uni- form than in the controls and: tended to resemble fibroblasts. In no case did the decrease in tumor size produced by thioTEPA appear to be caused by actual necrosis. In the.transplanted tumors, the histologic differences between the treated and un- treatedd tumors werc.less noticeable, and no giant cells were seen. mately 2 em. at.the.beginning of the experiment. The tumor reductions in thee instances with insig- nificant body weight change were highly signifi- cant. Effects of thioTEPA on methylcholanthrene-in- duced tumors.-Fourteen animals with methylcho- lanthrene-induced tumors measuring; approxi- mately I cm.. in diameter were given 4 mg/kgpf thioTEPA intraperitoneally daily for 9 days and matched' with fourteen controls bearing com- parablee tumors and receivingg only Ringer's solu- tion. TbioTEPA caused approximately 50 per cent tumor inhibition in 20-methylcholanthrene-in« dueed tumors, which is comparable to the depres- sion observed in3,4',9,10-dibenepyrenc-induced tumors (1'able 4). E,Yjects of other eTiemotherapeuticagents on in- duced tumors.-Groups of twelve animals bearing 3,4,9,10-dibenzpyrene-induced tumors of approxi- mately 1 em',, in diameter received the chemothera- pcut'ic agents in the doses listed above for 7-8