The Action of Anti-Ehrlich Ascites Tumor Antibody*

MARTINH. FLAX

(Department of Pathology and the Argonne Cancer Research Hospital, The University of Chicago, Chicago, III.)

One of the basic problems in tumor chemotherapy is the development of agents that canact selectively. Because of the marked specificityof immunological reactions, it would seem thata differentiation on a serological basis might bea promising approach. Specific antitumor sera mayalso damage the neoplasm in a way analogous tothe action of specific antibacterial sera. Althougha great deal of work has been done along theselines (5,8,24), recent developments in immunological technics, leading to improved methods of preparation and purification of highly specific andpotent antisera and methods of isotopically labeling the antisera (20, 23), make a re-evaluation ofthis immunological approach worth while.

The Ehrlich ascites tumor offers several advantages for such a study because it is antigeni-cally "pure," i.e., connective tissue contamination

is negligible, and its intraperitoneal form of growthmakes it directly accessible to injected antiserum.It was therefore decided to investigate some ofthe serological characteristics of this tumor andto determine the nature of the effect of specificimmune sera on these cells.

MATERIALS AND METHODSNEOPLASM

The Ehrlich ascites tumor1 was maintained by weeklyintraperitoneal injections of 0.1 ml. of ascites tumor, approximately 7 days old, into young adult CF No. 1 mice. Themice were fed ad libitum on a diet of Purina Laboratory Chow-

IMMUNIZATIONPROCEDUREThe antitumor antisera were produced by repeated injection

of Ehrlich ascites (EA) cells into rabbits over a 3-week interval.A 5 per cent suspension of fresh ascites tumor cells was prepared once a week on the day of the first injection for thatweek. The suspension was first washed 4 times with 0.86 percent NaCl. For the first injection, 5 ml. of the suspension wasmixed with Freund's adjuvant and injected into multiple

intramuscular depots. On the next 2 days, the rabbits wereinjected with 1 ml. of the tumor suspension into each of fiveportals (intravenous, intramuscular, intraperitoneal, sub-

* This study was supported in part by Institutional GrantNo. 3 from the American Cancer Society.

1The tumor was provided by Dr. E. Simmons of the Ar-gonne Cancer Research Hospital, The University of Chicago.

Received for publication February 10, 1956.

cutaneous, and intradermal). The multiple-portal injectionswere repeated 3 times a week for the next 2 weeks, and therabbits were bled by cardiac puncture on the 4th, 5th, and6th weeks. Control sera from noninjected rabbits were collectedsimultaneously. The sera were lyophilized and stored at —¿�20°

C. over PjOs in a vacuum. Sera from the various immunizedanimals were pooled, and the gamma globulin fraction wasseparated by alcohol fractionation by means of the methodof Nichol and Deutsch (19) as it was needed.

ENZYMESTUDIES1. Neotetrazolium method.—Themethod depends upon the

capacity of the tctrazolium compounds to accept hydrogenions from various dehydrogenases, thus permitting a determination of the activities of these enzymes, if the corresponding substrates are furnished (2). The neotetrazolium wasprepared in a 1 per cent solution in M/10 phosphate-bufferedsolution at pH 7.4. In general, the procedure was as follows:after the EA cells were treated with the various immunologicalreagents, 0.25-ml. aliquots were removed and mixed with 0.75ml. of the neotetrazolium reagent and 0.25 ml. of l Msubstrate.The mixture was then incubated at 37°C. for 1 hour. The

amount of reduction was determined visually or by means ofa Coleman spectrophotometer. In the latter case, the reducedneotetrazolium was extracted by washing 6 times with 1-ml.aliquots of acetone, which removed all the reduced salt fromthe cells. The optical density of reduced neotetrazolium in theextract was determined in the spectrophotometer at a wavelength of 540 mix (the absorption curve of the reduced neotetrazolium had its peak at this wavelength).

2. Warburg method.—The Warburg flasks and manometers were first calibrated by the mercury method. Oxygenconsumption was determined by the direct method; a 10 percent solution of KOH was put in the well to absorb the COjproduced. The various serological reagents were placed in thesidearms and were added after a respiratory baseline hadbeen established. The amount of oxygen consumption wascorrected by means of the flask constants (see Umbreit forcalculations [22]).

CYTOCHEMICALSTUDIESThe tissues were fixed in either Carnoy's fluid or neutral

formalin, embedded, and sectioned at Sit. They were stainedwith either the methyl green-pyronin mixture or with theFeulgen-fast green method. The former method distinguishesribo- and deoxyribonucleic acids under the condition used,while the latter method stains deoxyribonucleic acid and protein differentially.

RESULTSEFFECT OF SPECIFIC 7-GLOBULiN ON EHRLICH

ASCITESTUMORCELLSin Vitro1. Direct observation of wet and dried smears

of treated tumor cells.—Thepurpose of this experi-

774

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FLAX—Anti-EhrlichAscüesTumorAntibody 775

ment was to determine if complement added toAEA2 had any greater effect on the tumor thanAEA alone, and, if so, what the optimal proportions for this reaction would be.

A double dilution series of both AEA and complement3 was prepared (beginning with 2 mg. ofAEA and 1 ml. of complement), and all possiblecombinations of the two components were made.Two-tenths ml. of fresh washed EA cells wasadded to each tube, and the mixtures were shakenfor 2 hours at 37°C. Aliquots were removed and

either examined directly under the microscope,or put on slides, dried, and stained with AzureB before examination. It was noted that withcertain combinations of 7-globulin and complementthe cells appeared swollen. After being dried andstained, the majority of these cells were fragmented, the cytoplasm appearing as irregularclumps streaming out from the intact nucleus.This was obviously an artifact introduced by drying, since cells fixed prior to drying retained theirshape; it did, however, apparently indicate anincreased fragility of those cells. These changeswere never observed in cells treated with eitherAEA or complement alone. The amounts of 7-globulin and complement found to cause the mostsevere alterations in cell structure were 1.5 mg.AEA and 1 ml. of complement. In subsequentexperiments mixtures of AEA and complementin these proportions are referred to as G-C.

2. Effect of specific ^-globulin on the viability of Ehrlich osciles tumor.—Ehrlich ascitescells were treated with various combinations ofspecific antibody and complement prior to transplantation of the tumor into adult CF No. I1mice.The survival time of the mice is given in Table1. Only one animal in the AEA-complement-treated group and one in the AEA-treated groupdied during the 60-day period of observation; allthe animals in the other groups died before thistime, most of them by the end of the 2d week.

3. Vital staining of treated tumor cells.—Furtherstudies were directed toward characterization ofthe AEA 7-globulin effect. According to Michaelis(16), living cells stain with certain acid or basicdyes only when the nucleoprotein complex is dissociated; this dissociation is not compatible withthe life of the cell. We decided to investigate theeffect of the specific 7-globulin-complement mixture on the vital stainability of the tumor cells.Figure 1 shows EA cells after treatment withAEA followed by staining with Azure B, a basic

1Anti-Ehrlich ascites -y-globulin.*The complement used in this work was a preparation of

lyophilized guinea pig serum obtained from Markham Laboratories.

dye. The cells remained essentially unstained. Theresults of treatment with AEA plus heat-inactivated complement were the same. The cellsin Figure 2 were treated for an equal time withG-C and were placed in the dye solution for thesame period of time as those in Figure 1. The nucleiand cytoplasm of these cells avidly took up thedye and stained intensely. These results furthersupport the role of complement in in vitro celldamage with the specific 7-globulin.

4. Enzyme studies: (a) Studies on the Utilizationof glucose and succinate by the neotetrazoliummethod.—The capacity of G-C-treated cells to

TABLE 1

EFFECTOFTREATMENTOFEA CELLSPRIORTOTRANSPLANTATIONONHOSTSURVIVAL*

Pretreatment (2 hours, 37°C.) Tube

AEA and complement aAEA and inactivated

complement bAEA cComplement dSaline e

Host animalsurvival

(60 days)13/14

0/1514/150/150/15

* Tube a contained 4 mg. of AEA in 1 ml. ofsaline and 2 ml. of restored guinea pig complement. Tube b contained 4 mg. of AEA in 1 ml. of0.85 per cent saline and 2 ml. of restored complement that had first been heated to 56°C. for30 minutes. Tube c contained 4 mg. of AEA in3 ml. of saline; tube d, 2 ml. of restored complement; and tube e, 3 ml. of saline. The tumor usedin this experiment was derived from a 7-daygrowth that was washed 3 times with cold salineand diluted to twice its initial volume. Aliquotsof 0.4 ml. were removed from each tube for injection. Fifteen mice were used for each of thefive groups, and each animal received 0.02 ml. ofthe initial tumor preparation.

utilize glucose or succinate as the sole exogenousenergy source was investigated by the neotetrazolium method described in "Materials and Methods." The degree of reduction was graded visually

from 0 to +5, and the results are summarized inTable 2. It is apparent that with succinate as thesubstrate, there is no appreciable difference in theamount of neotetrazolium reduced by tumor cellsin the four groups. However, with glucose as thesubstrate, there was no neotetrazolium reductionin the G-C-treated group. There was a slightincrease in reduction by the cells treated withnormal rabbit 7-globulin plus complement, ascompared with the other two control groups. Whenno exogenous energy source was added, the Ehrlichascites cells were capable of reducing neotetrazolium owing to the utilization of endogenoussubstrates. Utilization of this reserve was alsoinhibited by treatment with G-C, while this activity was not altered by either specific 7-globulin

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776 Cancer Research

TABLE iEFFECTOFT-GLOBULINANDCOMPLEMENT

ONNEOTETRAZOLIUMREDUCTIONBYEA (SUBSTRATEVARIED)*

AMODKTOFNEOTETRAZOUTHSEDUCTIONSubstrates

Glucose Succinite

0

PRETBEATMENT(«BODES,37°C.)

AEA and complement

AEANormal rabbit y-

globulin and complement

Saline* EA tumor cells were combined with each of

the serological reagents. (The following amountswere used—1.5mg -y-globulin and 1 ml. complement, and all volumes finally adjusted to 1 ml.)After 2 hours aliquot s were placed in the neotetrazolium reagent with either glucose or succhiateas energy source. After 1 hour, the amount ofreduced neotetrazolium was estimated visually,from Oto+ ++++.

or complement alone. In order to reduce the endogenous energy store to an insignificant level, thetumor cells were allowed to respire in the absenceof substrate before the experiments began.

6) Studies on the utilization of glucose and suc-cinate by the Warburg method.—To determinewhether the neotetrazolium was indicating actualimpairment of glucose utilization after treatmentwith G-C, direct measurements of oxygen consumption were made with a Warburg respirometer.The results are summarized in Charts 1 and 2.With glucose as the sole exogenous energy source,there was an almost immediate cessation of respiration following treatment with G-C. The difference in oxygen consumption of cells observedafter treatment with -y-globulin and heated complement or with complement alone, on the onehand, and those treated with 7-globulin or saline,

160

ISO

140

130

120

0 noi

100

¡90.o.

1 80z

o 70

0*60-

50

40

30

20-

IO

UTILIZATION OF GLUCOSE BY EA TUMOR CELLS

SIDEARM CONTENTS

•¿�Õ GLOBULIN 4- COMPLEMENT

» Õ GLOBULIN + HEATED COMP

•¿�1 GLOBULIN

•¿� COMPLEMENTo SALINE

SIDEARM TIPPED20 40 60 80 IOO 120

-T- RM TIPPED20 70"6*0 (TO ÎÕÕÔ120

TIME (MIN.)

CHART1.—¿�The effect of various serological reagents on theoxygen consumption of EA tumor cells with glucose as the soleexogenous energy source. (The data from two experiments areplotted together.) The sidearm contents were 1.5 mg. AEA

plus 1 ml. of complement; 1.5 mg. AEA plus heat-inactivatedcomplement; 1.5 mg. AEA; 1 ml. of complement; or 0.85 percent saline (all made up to 1.5 ml. volumes).

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FLAX—Anti-Ehrlich Ascites Tumor Antibody 111

on the other, was probably the result of the stabilizing effect of the guinea pig serum. (This mayalso account for the increased neotetrazolium reduction in these groups which was described in theprevious experiment.) With Buccinate as the substrate, there was no inhibition of respiration withG-C; the cells reacted in a manner similar tothose treated with complement alone. The difference in oxygen consumption in the presence orabsence of guinea pig serum was like that observedwith glucose.

c) Studies on the utilization of phosphorylatedglycolytic intermediates by the neotetrazolium method.—Attempts were made to determine the effect

of G-C on the utilization of various phosphorylated glycolytic intermediates, i.e., fructose-1,6-diphosphate, glucose-1-phosphate, and glucose-6-phosphate. It was not possible, however, to demonstrate that untreated EA tumor cells were ableto utilize this type of substrate; presumably, thesesubstances could not be transported across intactcell membranes.

EFFECTOFAEA 7-GLOBULiNONEHRLICHASCITESTUMOKCELLSin Vivo

Several experiments were performed in the attempt to damage tumor cells by means of repeatedinjections of G-C while the tumors were actively

225

200

175

_ 150_j

5

2 125 Ho.

S 100 •¿�

75 •¿�

50

25 .

UTILIZATION OF SUCCINATE BY EA TUMOR CELLS

SIDEARM CONTENTS

•¿�T GLOBULIN + COMPLEMENT

« T GLOBULIN

* COMPLEMENT

O SALINE

' / ^V ,* ^°—o—-° o—

SIDEARM TIPPED

20 40 60

TIME (MIN.)

8O 100 120

CHART2.—The effect of various serological reagents on the oxygen consumption of EA tumor cells with succhiate as thesole exogenous energy source. Sidearm contents as in Chart 1.

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778 Cancer Research

growing within the host mouse. On the 3d dayfollowing tumor transplantation, various combinations of the immunologie reagents were injected intraperitoneally, and these injections wererepeated the following 2 days. In one experiment,the cytologie effects were studied, while in theothers, the animals were allowed to survive, andboth the body weight and survival time of theanimals were determined.

1. Cytologie studies.—-Animals were sacrificed at12-hour intervals for 3 days following the initialtherapeutic injection, and 4 days later the animalsremaining in each group were sacrificed. Thetumors were removed and prepared for cytologiestudy (see "Materials and Methods"). The results

with the different dyes were found to be essentiallythe same, so that only the results with the methyl

TABLE 3

EFFECTOF TREATMENTWITHAEA ANDCOMPLEMENTONWEIGHTOFTUMOR-BEARINGANIMALS*

TREATMENTAEA and complement

(G-C)AEANormal rabbit -y-globulin

and complementSaline

Av. WT. INCREMENT OTEH INITIAL

ANIMAL WT.

Day St Day ä Day 8(gm.) (gm.) (gm.)

2.12.2

2.11.9

0.20.5

2.75.8

3.32.6

7.05.1

* Three days following the intraperitoneal injection of0.1 ml. of viable E A cells, the first intraperitoneal injectionswere given, consisting of either G-C (1.5 mg. AEA plus 1 ml.complement), 1.5 mg. AEA, 1.5 mg. normal rabbit -y-globulinplus 1 ml. complement, or 1 ml. of 0.85 per cent saline. Similarinjections were repeated on the next 2 days. Each groupinitially contained fifteen animals.

f Before treatment was begun.

green-pyronin combination will be described. Thechanges that were observed with G-C occurredgradually, and three stages of the alterations aredescribed below. It is important to emphasizethat the changes described below did not occurin all the tumor cells; there were always appreciable numbers of apparently undamaged cells remaining, although these were reduced to as low asabout 5 per cent of the total in several of theG-C treated series.

a) Saline-treated group (Fig. 3).—-As expected,

no change was observed following 0.85 per centsaline injections. The normal cytoplasmic materialand the prominent nucleoli were intensely pyro-nophilic owing to their high ribonucleic acid content. There were numerous mitotic figures.

6) G-C-treated group—12 hours (Fig. Jf).—There was, in general, a clumping of the pyronin-staining cytoplasmic particulates at the cell membrane (6), while other cells showed loss of cyto

plasmic ribonucleic acid staining, associated withswelling of the cells (a). The nucleolar stainingwas somewhat depressed because of the loss ofnucleolar ribonucleic acid (c).

c) G-C-treated group—36 hours (Fig. 5).—The

cellular changes progressed; the cytoplasmic ribonucleic acid staining was completely lost, andnucleoli were no longer apparent. Some cells hadmethyl green-staining material at the peripheryof the cell membrane (a), presumably consistingof nuclear material that had escaped from thenucleus.

d) G-C-treated group—72 hours (Fig. 6).—Thetumor cells no longer stained with methyl green-pyronin, indicating that the cells no longer contained either nucleic acid (or that, if it was present,it was in such a form that it was no longer stain-able). With the Feulgen-fast green stain, the cellsstain only with the fast green which reacts withproteins, indicating that some protein still remained. There were numerous polymorphonuclearleukocytes present among the nonstaining tumorcells; these were probably present in response tothe cellular debris.

e) AEA-treated cells.—No changes were ob

served until the 3d day, when two of the tumorsshowed early degenerative changes of the typesimilar to those described above in section (6).

2. Effect on tumor weight and survival of hostmice.—-The average increase in the weight of the

surviving animals above their initial weight priorto tumor transplantation is shown in Table 3.Weight increase was assumed to parallel theamount of tumor present (10, 11). There was amarked drop in the average weight of animalsgiven either G-C or AEA alone after 2 daysof treatment. The normal rabbit 7-globulin-treated group had a slight weight increase, whilethe saline-treated group underwent the greatestweight increase. By the 8th experimental day, thetumors in the G-C- and AEA-treated groups hadapparently begun to grow again, but the totaltumor present was still considerably less than inthe other two treated groups. Because of thesmall number of survivors and the fact that thosewith the greatest tumor weights, in general, diedearliest, the data on weight changes after the8th day tend to be misleading and will not bedescribed; the data for the saline-treated groupat 8 days give evidence of this trend.

Chart 3 and Table 4 show the duration of thesurvival of animals in the four groups. The G-Cgroup survived the longest, while the normal rabbit 7-globulin plus complement and the AEA-treated groups survived an appreciably shortertime. The G-C-treated group survived about twice

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FLAX—Anti-EhrlichAscites Tumor Antibody 779

as long as the saline-treated controls. One of theG-C-treated animals survived for 40 days; thetumor that persisted was located extraperitoneallyat the site of the original tumor implantation andwas thus not directly accessible to the G-Cmaterial.

SPECIFICITYOFTHEEFFECTOFG-C in Vitro1. Effect on the Krebs ascites tumor.—To deter

mine whether the effect of G-C was specific onthe Ehrlich ascites tumor, which was used as thesource of antigen for the initial immunization,the effect of G-C on a different ascites tumor, theKrebs ascites tumor, was investigated. An experiment exactly comparable to the one describedabove in Section 4a (page 775) was performed:the Krebs ascites cells were treated with eitherG-C, AEA, normal rabbit 7-globulin plus complement, or 0.85 per cent saline, and their capacityto utilize glucose or succinate was studied bymeans of the neotetrazolium method. The resultswere identical with those obtained with Ehrlichascites tumor cells; no neotetrazolium reduction

was observed after G-C treatment with glucoseas substrate, while normal reduction was observedwith succinate. Other alterations observed afterG-C treatment of EA cells were not investigatedwith the Krebs tumor, but the above data suggestthat the effect of G-C is not limited to the homologous tumor type.

2. Effect on normal mouse tissues.—Accordingto Wissler et al. (23), I1"-labeled AEA combinedwith various lyophilized normal antigens, such askidney and liver, although the per cent combining

TABLE 4

AVERAGESURVIVALTIME OFTUMOR-BEARINGMICEAFTERin Vim

TREATMENT*

Av. lurvival(days)16.510.3

11.28.2

* Experimental conditions as in Table 3.

TreatmentAEA and complement (G-C)AEANormal rabbit 7-globulin

and complementSaline

I

§

12

10

DAYS

SURVIVAL OF EA TUMOR BEARING MICE

AFTER IN VIVO TREATMENT

» ANTI EA - X GLOBULIN + COMPO ANTI EA - V GLOBULIN

•¿�NORMAL RABBIT.» GLOB + COMP

O SALINE

ÃŽÃŽÃŽINJECTIONS

I» 12 ¡6 fë TO ?2 ¡f4 25 Õ8—¿�70"

CHABT3.—The effect of in »troinjections of various sero- jections were on the 3d-5th days following tumor transplan -logical reagents on the survival of EA tumor-bearing mice. In- tation.

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780 Cancer Research

was appreciably less than with lyophilized EAtumor cells. It was decided to learn whether G-Cwould affect normal tissues in a manner comparable to the effect on the tumors. The resultantdata are given in Table 5. The utilization ofglucose by the liver and kidney homogenates wasfound to be inhibited by G-C, while utilizationby the spleen was decreased to a lesser degree.There was considerable variability, but the differences between the G-C-treated cells and thevarious controls seem to be significant.

3. Efeet of adsorption of AEA ivith lyophilizednormal tissue antigens.—The specificity of theactive constitutent of G-C was also studied by an

TABLE 5

EFFECTOFANTI-EHRLICHASCITES-»-GLOBULINANDCOMPLEMENTONNORMALTISSUES*

AMOUNTor NEOTETRAZOLIUMREDUCTION

PRETREATUENTAEA and comple

ment (G-C)AEAComplementSalineascites

tumor0.32

1.581.381.68

Liver

0.301.521.261.58

Kidney

0.300.911.101.22

Spleen

1.31.722.081.88

* CF No. 1 mouse kidney, liver, and spleen were homogenized with a loose-fitting plastic modified Potter-Elvehjemhomogenizer and diluted with buffered saline (pH 7.4) to approximately comparable cell concentrations. The homogenizedkidney, liver, spleen, or EA tumor cells were added to tubescontaining the various reagents. After 2 hours at 37°C.,aliquots were removed and placed in tubes with neotetrazoliumand glucose as the energy source for 2 hours. The reducedneotetrazolium was then extracted and the amount determinedspectrophotometrically.

indirect method : the AEA was mixed with variableamounts of lyophilized normal tissues Giver, kidney, and muscle), as well as with lyophilized E Acells, to determine the degree to which the activetumor-damaging constituent could be adsorbedby these antigens. The amount of the active constituent that was adsorbed was indicated by theloss of activity of the corresponding supernatantfluid as tested against living EA tumor cells. Theresults are summarized in Table 6. Under the conditions of the experiment, the amount of neotetrazolium reduced by untreated EA cells hadan extinction of almost 1.00. The cells treated withG-C that had not been mixed first with any antigen were, as usual, almost completely unable toreduce neotetrazolium (extinction was 0.09). Thelyophilized EA cells, as might be predicted, weremost active in adsorbing the active constituent:20 mg. of the antigen removed about 60 per centof the active factor. This was evident, since about60 per cent of the control amount of reducedneotetrazolium was formed by the treated living

EA cells under these conditions. Forty mg. oflyophilized EA cells removed all the active sero-logical constituent; the "test" EA cells reduced

the same amount of neotetrazolium as the untreated control cells. Kidney was the next mostactive antigen: about 80 per cent of the activefraction was removed by adsorption with 40 mg.of lyophilized kidney, and all the active constit-

TABLE 6

DECREASEOFACTIVEAEA ANTIBODYBYPRE-ABSORPTIONWITHVARIOUS

LYOPHILIZEDTISSUES*

Lyopbilized antigen Amt. of reduced neo-(mg.)

Ehrlich ascites 2040fiO80

Kidney

Liver

Muscle

804060802040608U

80406080

tetrazolium (ext.sio)0.651.041.051.15

0.190.831.051.250.230.730.761.150.240.460.750.79

0.090.95

NoneUntreated EA

cells* Lyophilized antigen (in 20-, 40-, 60-, or 80-

mg. amounts) was mixed with 1.5 mg. AEA and1 ml. of complement, and the mixture wasshaken at room temperature for 2 hours. Thetubes were then centrifuged, and the super-natants, containing the unadsorbed -y-globulin,were decanted. One-half ml. of complement wasadded to each of the solutions to assure thatlevels were still adequate, and their activity wastested by determining their effects on living EAcells: 0.2 ml. of EA cells was added to each ofthe solutions, and the reaction was allowed toproceed for 2 hours at 37°C. Aliquots were removed, and alterations in the dehydrogenaseactivities of the tumor cells were tested, usingthe neotetrazolium method with glucose as asubstrate. The reduced neotetrazolium wasmeasured spectrophotometrically (expressed asextinction or optical density).

uent was adsorbed with 60 mg. Liver was stillless active, requiring 80 mg. to adsorb all the active•¿�y-globulin.Muscle tissue was by far the leastactive. The explanation of the relatively low valuefor the untreated cells is not entirely clear butprobably represents a measure of the variabilityof the method. These findings are further supportfor the conclusion that the effective antibodyconstituent does not react only with the immunizing cell type, although it is apparently quantitatively most reactive with it. It is possible that the

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FLAX—Anti-EhrlichAscites TumorAntibody 781

complete adsorption of the active antibody bylarge quantities of unrelated antigens may, in part,be due to nonspecific adsorption.

DISCUSSIONMuch of the literature dealing with tumor im

munity has been concerned with cellular andhumoral factors produced by the host in responseto the growing tumor (5, 24). Some earlier workon the effect of such homologous antitumor seraseems to be pertinent to the present study.Lumsden (13) prepared an antitumor serum inrats by intraperitoneal injections of the Jensenrat sarcoma which, when added to a tissue cultureof the sarcoma, killed the cells. Kidd (9) describeda unique antibody found only in rabbits in whichBrown-Pearce tumors had been transplanted, butin which the tumor had failed to grow or regressed.When the tumor was treated with this antiserumin vitro prior to transplantation, the tumor wouldnot grow in the host rabbit. Cytologie changesfollowing such in vitro treatment, which have beendescribed recently (7), are strikingly similar tothe early in vivo changes described in this paper.It is of considerable interest that complement isrequired for the in vitro cytotoxic action of theanti-Brown-Pearce carcinoma serum; the activityof the serum was destroyed by heat and wasrestored by the addition of guinea pig complement.Alteration of the Ehrlich ascites tumor in vitroalso required complement as well as AEA, whilein the in vivoexperiments, the need for complementdoes not appear to be as marked. For example,AEA alone prevented tumor growth when the treated cells were injected into mice, while no alterationof the tumor cells with 7-globulin treatment alonewas ever observed in these in vitro experiments.Similarly, intraperitoneal injections of AEA inhibited the growth of actively proliferating tumor, asjudged by host weight and survival. This discrepancy may well be explained by postulating thepresence of endogenous complement (or a comparable material) in mouse serum or in the asciticfluid, thus eliminating the need for exogenouscomplement.

Experiments performed to determine the therapeutic possibilities of heterologous antitumor anti-sera have been described recently (6,18). In these,antisera, like AEA, were produced by immunization of animals other than the species from whichthe tumor was derived. Nettleship (18) injectedrabbits with protein and nucleoprotein fractionsof a rat lymphosarcoma. When lymphosarcoma-bearing rats were treated with these antisera,retardation or complete regression of the tumorswas observed. More recently, a number of Japa

nese workers have studied the effect of specificantitumor sera on the Yoshida ascites tumor ofthe rat. Both homologous sera from "immunized"

rats and heterologous rabbit antitumor sera werestudied. Neutralization experiments in which thetumor was treated with antisera before transplantation led to inhibition of growth (6). Following intraperitoneal injections of the antisera into48-hour tumor growth, "vacuolar degeneration"

was observed in the tumor, and several animalswere reported to be completely cured. The serum,however, was found to be extremely toxic, andsome of the animals usually died shortly afterits introduction. Absorbing the serum with ratred blood cells or various normal rat tissues succeeded in reducing the toxicity of the serum butalso decreased its antitumor activity (17). Toxicityof a similar nature was observed in therapeuticexperiments with G-C, when the number of injections was increased to more than one per day.As a result of this, a serious limitation was putupon the amount that could be injected safely.Thus, although all the tumor cells might theoretically be destroyed by injections of antibodies overa longer period of time, a more effective dosecould not be administered without lethal effects.It is possible that with smaller amounts of tumorand careful spacing of injections, complete curemight be achieved, but the recent observationthat one viable ascites tumor cell may be sufficientto give rise to a lethal carcinomatosis makes anytherapy particularly difficult.

One of the main purposes of the study was thecharacterization of the effect of an antitumor antibody on living tumor cells. The antibody used inthis study did not react specifically with the tumorcell of origin, but cross-reacted with other tumorcells and to a lesser extent with normal tissue cells.Cross-reactions between antitumor sera and normal tissue or unrelated neoplasms have beenobserved frequently. In general, the reaction hasbeen greatest with the specific tumor, whilecross-reaction was greater with unrelated tumorsthan with normal tissues. Only quantitative differences could be demonstrated serologically between intracellular particulates of mouse hepa-toma (14, 15) or leukemia (3) and their normalcounterparts. On the other hand, recent studieswith a rabbit antimouse lymphogenous leukemiaserum (21) have indicated that antibodies specificfor this leukemia could be demonstrated by complement fixation after the serum had been incubated with normal lymphocytic antigen. Thedemonstration of cross-reactions has been, mostoften, by relatively gross methods such as complement fixation or precipitin reactions. By utiliz-

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782 Cancer Research

ing a specific effect of the 7-globulin, e.g., the inhibition of utilization of gluscose described above,it was hoped that some qualitative differencesbetween normal and EA cells could be elucidated.The results indicate, however, that, again, onlyquantitative differences could be demonstrated.The nature of the common antigens is unknownat present, but the presence of qualitatively similarenzymatic proteins in normal and neoplastic tissue(4) suggest that these proteins may contributeto the observed antigenic similarities.

Studies of the mechanism of interaction of anti-tumor antibody and tumor cells are rare, and,though the effects of AEA may not be due to any"specific" interaction with the tumor cells, thecharacterization of this antibody-cell system wasundertaken. Unfortunately, the mechanism of action of AEA can still be only a subject of speculation at this time. Certain cytochemical and biochemical alterations were observed after suchtreatment. The cells became swollen, which suggested increased permeability of the cellular membrane. No lysis of the membrane was ever observed,though it was more fragile than normal. The stain-ability with Azure B is evidence that the nucleo-protein complex present in normal living cells hasbeen dissociated, thereby making the nucleic acidgroups available for association with the basic dye.The enzymatic studies are open to several interpretations. In describing the experiments in thepreceding sections, the difference between thebehavior of the G-C-treated cells in the presenceof glucose and succinate was interpreted to indicate that the treated cells could still utilize thesuccinate but not the glucose. This would suggestthat the inhibition may occur in the glycolyticportion of carbohydrate metabolism. However,these findings may also be interpreted to indicatethat succinate rather than glucose restores activityto the treated cells. Unfortunately, the effect ofG-C on the phosphorylated glycolytic intermedi

ates could not be accomplished. Finally, the cytochemical studies indicate that the damaged cellslose both desoxyribo- (DNA) and ribo-nucleic acid(RNA), the loss of RNA occurring first. Theobvious question arises as to whether thesechanges are primary changes or only reflect othermore basic ones. Kalfayan and Kidd (7), as described above, observed similar structural changesin Brown-Pearce tumor cells treated with specificantitumor serum. They attempted to reproducethese changes by a variety of other agents (i.e.,bacterial toxins, various enzymes, and hypotoniesolutions) with no success. Their data suggestthat perhaps the structural changes observed weresomewhat specific. In recent experiments on thechemical and enzymatic changes associated withmouse liver necrosis (1) it was observed that theRNA and DNA decreased steadily, the loss in theRNA occurring somewhat earlier and more rapidlythan that of the DNA. The two oxidative enzymesstudied, succinoxidase and cytochrome oxidase,underwent a rapid loss in activity, while the otherenzymes (alkaline and acid phosphatase, esterase,and L-leucylglycine peptidase) declined at a slowerrate. Such investigations of the changes that occurunder many other conditions of cell death arenecessary before the significance of changes following any specific reagent can be evaluated.

SUMMARYThe 7-globulin fraction of rabbit anti-Ehrlich

ascites tumor serum was prepared by alcoholicfractionation. Preliminary treatment of the livingascites tumor with the -y-globulin and guinea pigcomplement prior to transplantation preventedtumor growth. Heating the complement (56°C.,

30 min.) destroyed the inhibiting effect of thecombination.

Attempts were made to characterize this effecton the tumor cells. The use of several histo-chemical technics revealed differences between

Fio. 1.—Ehrlich ascites tumor celb after 5 minutes in 0.1per cent Azure B in pH 7.0 buffered saline following treatmentwith 1.5 mg. of AEA for 2 hours at 37°C.

Fia. 2.—Ehrlich ascites tumor cells after 5 minutes in theidentical solution of Azure B following treatment with 1.5 mg.of AEA plus 1 ml. of complement for 2 hours at 87°C.

Fios. 8-6.—Stages in the destruction of the Ehrlich ascitestumor by injections of G-C. The cells have been stained withmethyl green-pyronin and show progressive loss of nucleic acidstaining. The G-C mixture was injected intraperitoneally onthe 3d, 4th, and 5th days after the initial tumor transplantation. The last three figures depict changes occurring at 12, 36,and 72 hours following the first G-C injection.

Fio. 8.—Saline-treated group. Note the density of the nor

mal cytoplasmic staining and the prominent nucleoli (a).Fio. 4.—G-C-treated group after 12 hours. Note the clump

ing of the pyronin-staining cytoplasmic particulates at the cellmembrane ((>').In other cells there is a loss of the cytoplasmic

stainability (a). The nucleoli are still present (c).Fio. 5.—G-C-treated group after 36 hours. The cytoplasmic

ribonucleoprotein staining is completely lost and nucleoli areno longer apparent. Some cells show the appearance of methyl-green staining material at the cell membrane, presumably nuclear material that has escaped from the nucleus (a). Some ofthis material is outside the cells (6).

Fio. 6.—G-C-treated group after 72 hours. The tumor celbno longer stain with methyl green-pyronin. Note the poly-morphonuclear leukocytes between the tumor cell "ghosts" (a).

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FLAX—Anti-EhrlichAscites TumorAntibody 783

these -y-globulin-complement (G-C)-treated tumorcells and those treated with one or the other ofthe components. By the neotetrazolium methodand direct Warburg measurements, it was foundthat the G-C-treated cells were unable to utilizeeither their endogenous substrate or exogenousglucose; they were as capable of utilizing succinateas were the 7-globulin- or complement-treatedcells. This action of G-C also occured with theKrebs ascites tumor and, to a variable degree,with some normal mouse tissues.

When G-C was injected intraperitoneally intomice bearing actively growing ascites tumor, theaverage survival time relative to untreated controlswas approximately doubled, and intermediary effects were observed with the separate components.Progressive cellular degeneration with loss of cyto-plasmic, nucleolar, and finally chromatin baso-philia was observed in a histologie study of thein vivo G-C treated cells.

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7. KALFAYAN,B., and KIDD, J. G. Structural Changes Produced in Brown-Pearce Carcinoma Cells by Means of aSpecific Antibody and Complement. J. Exper. Med.,97:145-62, 1953.

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1956;16:774-783. Cancer Res   Martin H. Flax  The Action of Anti-Ehrlich Ascites Tumor Antibody

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