Abnormal Plasma Components in C3H Mice Bearing ......2 Manufactured by Stein, Hall & Co., Inc., New York. 1 rag. total protein per pound of body weight mixed with Freund's adjuvant,

Abnormal Plasma Components in C3H Mice Bearing Spontaneous Tumors*

ELIZABETH ESHELMAN MILLER AND PETER BERNFELD

(Bio-Researeh Institute, Cambridge 41, Mass.)

SUMMARY

The plasma proteins of C5tI mice bearing spontaneous mammary adenocarcinoma were fractionated by zone electrophoresis on cornstarch gel stiffened by additional amylose, and rabbits were immunized with purified protein fractions. Precipitin reactions by the agar gel diffusion technic clearly demonstrated the presence of an anomalous protein in the plasma of tumor-bearing mice which is absent in normal control animals. Differences in concentration of certain proteins, in particular of a-globulins, between the plasma of normal and of tumor-bearing animals were also evident.

In 34 out of 37 instances, tumor-bearing animals were correctly identified by the observation of an extra precipitin line, whereas 6.5 per cent of the observations were doubtful and only 1.5 per cent were incorrect. With another rabbit antiserum, producing a weaker extra line, 74 per cent correct, 14 per cent doubtful, and 1~ per cent incorrect observations were obtained. The significance of these data lies in the potentiality of this procedure as a diagnostic tool.

Plasma mucoproteins belonging to the group of a-globulins have been found in increased amounts in patients and experimental animals with neoplastic disease (1, 9, 1~-14). The first paper of this series (1) has demonstrated statistically significant increases of a-globulin in Sarcoma 180-bearing C57BL/6 mice. Preliminary studies with rabbit antisera to a-globulin fractions from Sarcoma 1- bearing A/Jax mice have indicated the presence of certain antigens in the plasma from tumor-bearing animals absent in normal animals of the same strain (10).

Since spontaneous tumors more closely re- semble the conditions found in human disease, experiments were initiated with the intention of searching for anomalous proteins in the plasma of CJH mice bearing spontaneous mammary gland adenocarcinoma. Electrophoretic studies of whole plasma in this host-tumor system had revealed no increases in the a-globulin components (6). 1 Con-

* Supported in part by research grant C-885~ from the Na- tional Cancer Institute, National Institutes of Health, U.S. Public Health Service, and by a gift from Maurice Gordon, Boston, Massachusetts.

1 p. Bernfeld, unpublished data.

Received for publication January 15, 1960.

sequently, immunological technics were selected as a tool for the present study, because their sen- sitivity is considered to be far superior to tha t of the electrophoretic methods. In the expectation of increasing the antigenicity of small or minute amounts of anomalous plasma proteins, purified plasma fractions have been used for the preparation of antisera instead of whole plasma, and alteration of biological activity during the purification was reduced to a minimum by the use of mild fractionation procedures, such as zone electrophoresis.

The present paper describes changes occurring in the plasma of CSH mice bearing spontaneous tumors and reports on the reliability with which individual plasma from tumor-bearing mice could be distinguished from normal plasma by the detection of an antigenic component absent in normal controls.

MATERIALS AND M E T H O D S

Plasma.--The plasma was obtained from inbred CSH mice purchased from the R. B. Jackson Memorial Laboratories, Bar Harbor, Maine. Blood was drawn from retired female breeders bearing spontaneous mammary gland carcinomas

1149

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1150 Cancer Research Vol. 90, September 1960

ranging in size from 1 to 8 gan. and, for the controls, from normal males which had been mated to the females, as well as from young C3H males and females, 5-6 weeks old. Blood was collected by severing the brachial plexus and drawing the blood into a pipette wetted with saturated sodium citrate.

Fractionation of plasma proteins.--The method for fractionation of plasma by zone clectro- phoresis on a cornstarch gel stiffened by additional amylose has previously been described (2). In the present experiments a commercial preparation of dried amylose called "Superlose ''2 was used, and a concentration of 3 per cent Superlose was found to give a gel of suitable consistency for fractionation.

Superlose was suspended in 1.5 times its weight of ethyl alcohol and put into solution in distilled water by autoclaving the mixture for 1 hour at 17 lbs. per sq. in. pressure, whereby most of the ethanol evaporated. The hot solution was diluted with enough warm water and veronal citrate buffer of pH 8.6, 0.1 ionic strength, to give a final concentration of 0.05 ionic strength. Filter cel and dry cornstarch were immediately added to prepare the gel. After electrophoresis of the plasma in the gel for approximately 60 hours in a field of about 1 volt/era, the gel was cut into 16 segracnts, �89 or 1 era. wide.

The protein was recovered from each segment by squeezing the liquid out of the gel through Whatman No. 1 filter paper on centrifugal filters with Buchner funnels No. 0, fitted into aluminum adapters and bottles for ~50-ml. trunnion cups of a refrigerated International centrifuge. Thirty minutes of centrifugation at 3,000 r.p.m, yielded a clear filtrate and an almost dry starch cake on the filter.

Characterization of the protein fractions.--The eluate from each segment was analyzed by paper electrophoresis for uniformity and mobility of protein at pH 8.6 and for total protein according to the method of Ix)wry (8). Eluates which thus were found to contain components of the same mobilities were combined, This resulted in the col- lection of eight fractions, representing the major electrophoretically distinguishable plasma proteins, or mixtures thereof, i.e., (a) albumin, (b) albumin with al-globulin, (c) al- and a2-globulins, (d) a2-globulin, (e) a3 -and ~-globulins, (f) /~- globulin, (g) fl- and 7-globulins, and (h) ~,- globulin.

Production of rabbit antisera.--Rabbits weigh- ing 4-6 pounds were given a single injection of

2 Manufactured by Stein, Hall & Co., Inc., New York.

1 rag. total protein per pound of body weight mixed with Freund's adjuvant, as described by Cohn (3). Depending on their availability, each protein fraction was injected into one to four rabbits. Blood was collected from the marginal ear vein of the rabbit 6 weeks after injection.

Since the isolated fractions contained soluble starch and buffer salts from the gel media, the maximal amount of these substances expected to be found in any one fraction was injected into control animals in the absence of protein, and neither toxicity nor antigenicity was found.

Precipitin tests in agar.--The Ouchterlony agar plate diffusion method (11) as modified by Korn- gold (7) was used for the precipitin tests. Rabbit antiserum to the isolated protein fractions was placed in the center cup of the agar plate and various antigen solutions in four surrounding cups. Eight different antigen concentrations covering a 1:512 range of dilution were used for each antigen. On each agar plate a set of two different antigens was opposed to one another at two corresponding dilutions. This permitted the direct comparison between the two antigens of a set, as well as the detection of extra lines in either one or the other. I)recipitin lines were observed after incubation at room temperature for ~ weeks. To eliminate sub- jective errors, this procedure was carried out by two investigators, neither of whom had knowledge of the identities or distribution of the individual antigens on the plate.

The antiserum from each of the twenty inl- munized rabbits was thus tested against sets of antigens consisting of pooled plasma from tumor- bearing animals on one side of the agar plate and of pooled plasma from normal old littermates on the other side. With the use of those two rabbit antisera showing the most marked difference between tumor-bearing females and male littermates, the suitability of using plasma from normal old males as controls was ascertained in a series of experiments in which individual plasma from fifteen normal old male C3H mice was compared with individual plasma from each of ~ young male and female C3H mice of 5-6 weeks of age. To test the frequency of the occurrence of extra antigens in plasma from tumor-bearing animals, as well as the absence of these extra antigens in normal animals, plasma from individual tumor-bearing mice was opposed to plasma from individual nor- real animals on each of 66 agar plates. The antigen dilutions were 1:8 and 1:32, which had been found in preliminary experiments to be within the range of optimum concentration, and the antisera were again those that produced extra precipitin lines with plasma from tumor-bearing animals,



~[ILI~ER AND BEt~:xFELD--..lbnornlal Plasma ("Oml~ouenls in Mice 1151

i.e., sera from rabbits immunized with an a._,- globulin fraction and a fraction containing a~- anti ao.-globulins, respectively. Eleven plates of the same experiment contained mixtures of plasma from a normal mouse with tha t of a tumor-bearing mouse on both sides. Tile existence of these plates was unknown to the investigators reading the plates anti, thus, considerably increases the significance of their observations.

In some instances, isolated protein fractions have been used as antigens.

Experiments in which plasma from individual nornlal young C'HI males and females was tested against plasma of the old males, used as control animals, showed tha t there were no significant antigenic differences between the plasmas of these different types of normal CSH mice.

Dist inct differences were observed, however, when plasma from tumor-bearing and from nornml mice were compared with each other. These differences include marker variations in tile distant.es of the precipitin lines from the center cup for

T A B L E 1

I)iFFI.:RENCFS IN ANTIGEN C()N( 'ENTRATI(JN FROM THE DISTAN( 'ES

BFTVCEFN I )RE( ' I I ' IT IN LINES AND (_'.ENTER CUP

]{ABllI T I M M V -

NIZ~:D W I T H :

Albumin

Albumin+ a l-t.;lobulin

c,.,-Globulin

~'- (; lobulin

3,-Globulin

.AtN TI ( ; EN

]) l ] ,U TION

(IN A ( ; A I ~

1 : It56 1:51~2

1 :64

1 :4

n o

1:3~

L I N E

IL

14

b C

R

b I t

b

a

b C

I t

b C

d

] ) I ~ T A N C E IN M M . I-'I~OM

( : E N T E R (-:UI' TO LINI ' : [4*

Tumor Normal plasma plasma.

1`2,4 11. (; 14.1 14.1

20 .8 1 7 8 1~.5 11 .5 11 .0 10 .7

17 .7 18 .7 15 .0 15 .0 15.1~ 16 .~ 1~.5 1"2.5

2"2.5 ~`2.5 20 .0 "20.0 14 .3 14 .3

~1 .5 ~1 .0 19 .0 19.0 16 .8 17."2 11.9 11.9

I ) I . F I - ' E HEN (lG

I ~ E T W E E N I)l,~l -

T A N ( ' E IN " / l i e

o sLmrsl"

- - 0 . 8

- - ~ 5 - 1 o - -0 3

-4-1.0

+1 .o

- 0 . 5

+0.4

* M e a s u r e d on 1.7-fold p h o t o g r a p h i c en la rgemen t s .

t Pos i t i ve sign deno tes increased concen t r a t i on of the c o m p o n e n t in the tu- m o r - b e a r i n g an imal .

RESULTS

Eighteen out of the twenty rabbits immunized with protein fractions gave good ant ibody reactions. Precipitating antibodies were obtained to all eight fractions, but the antigenic complexity, as observed by the Ouchterlony technic, varied greatly among the different fractions. Albumin was the simplest fraction antigenically, giving only two lines of precipitation, while the 7-globulin fraction was very complex, exhibiting at least six distinct lines. When albumin or ~-globulin fractions were applied on the agar as antigen against antisera from rabbits immunized with ao_- globulin, lines of precipit:ation were obtainr with ~-globulin, bu t not with albumin.

normal and tumor antigens, as seen from the data in Table 1, indicating considerable differences in the concentrat ions of the components in normal and pathological sera. The most significant difference appears to be the fact tha t an t i seraf rom rabbits imnmnizcd with certain a-globulin fractions from tumor-bearing mice produced an adtti- tional precipitation line with plasma from tumor- bearing mice which was absent when normal mouse plasma was used as the antigen.

In every instance in which this extra line was observed its position on the agar plates was found to be characteristic for each of the two protein fractions used for immunization. A strong extra line in addition to a single normal line was pro-



115~ Cancer Research Vol. ~0, Sep t embe r 1960

duced by the antiserum of the rabbit immunized with a~- and a2-globulins, as seen in :Figure 1. A much fainter extra line, in addition to two or three strong normal lines, was observed with antiserum from the rabbit immunized with a2-globulin.

By the presence of these lines, plasma from tumor-bearing animals could be distinguished from that of normal animals with a high degree of certainty. Table ~ shows that the high percentage of correct readings, in which the observers identified most of the tumor-bearing animals, is opposed by only 1.5 and 1~ per cent wrong negatives, and by no wrong positives. The statistical significance of these results is considerably increased by the presence in this series of experiments of a certain number of plates, unknown to the observers, on which the antigen was the same on both sides.

which is absent in the plasma of normal mice of the same strain. The present data have shown that such an anomalous plasma component, a protein with the eleetrophoretie mobility of an a-globulin, is present in animals bearing a spontaneous tumor, while it had earlier been found also in mice with transplantable tumors (10). The possibility that normal tissue antigens from the host, or unspecific antibacterial antigens introduced by tumor graft- ing, may be involved in this phenomenon is greatly reduced, however, in inbred animals bearing spontaneous tumors. The use of old male littermates as normal control animals appears justi- fiable, since no difference in the antigenic response was found between old males, young males, and young females. If young CgH mice had been used as controls, the possibility of age differences would

TABLE

OCCURRENCE OF EXTRA PRECIPITIN LINES WITH PLASMA FROM TUMOR-BEARING ~IICE

FRACTION USED

FOR IMMUNIZA-

TION OF RAnBIT

al-&a2-globulin al-&a~-globulin ~2-globulin a.~-globulin

DISTRIBUTION" OF

ANTIGENS ON

AGAR PL.K TE $

T v s . ~ "

T + N vs. T + N T v s . N T + N vs. T + N

No. PLATES

O n c o r -

r e c t s i d e

37 34, 34 o 0, 0

~9 ~0, ~3 9 0, 0

EXTRA LINES OBSERVED t

Dou]~tful on ec rreet None

si, le

~, 3 1, 0 0 ,0 ~,~ 4, 4 5, 0, 0 9, 9

(In

si, te

0 ,0 0, 0 0, 0 0, 0

Av. RATING OF BOTH OBSERVERS

(PER CENT)

Correct Doubtful readings readings

9~2 6.5 100

74 14 lOO

Wrong readings

1.5

lC2

* T = Plasma from tumor-bearing animals. N = Normal plasma. T + N = Mixed tumor and normal plasma. The two figures represent individual readings by two independent observers.

These plates were correctly identified (lines and 4). I t can also be seen from this table that the blind readings made by two independent investigators agreed very closely.

DISCUSSION

The demonstration of an extra preeipitin line on agar diffusion plates when certain rabbit antisera are brought into contact with plasma from tumor- bearing mice appears to indicate tha t the plasma of the tumor-bearing animals contains an antigen

not have been excluded. Whether the anomalous plasma component is a unique feature in all tumor-bearing mice independent of mouse strain and tumor type, or may occur in certain other conditions totally unrelated to neoplastic growth, is not known at the present time.

Eleetrophoretie analyses of the plasma proteins from C3H mice bearing a spontaneous adenocarcinoma did not reveal any increase in a-globulin (6). 1 The detection of an anomalous plasma component in this system by immunological methods

FIG. 1.--Preeipitin pat tern on an agar diffusion plate. Cen- ter cup: rabbit antiserum to a protein fraction from the plasma of tumor-bearing mice, containing al- and a2-globulins. Cups on upper right and lower right: plasma from an individual C3H mouse bearing a sp ontaneous adenoeareinoma, at the dilutions of 1:8 and 1:8~, respectively. Cups on upper left and lower left: plasma from an individual normal CSH mouse (male litter-mate), a t the same dilutions.



~[ILLER AND BERNFELD . . . . Abnormal Plasma Components in Mice 115'~

indicates, therefore, that the anomaly nmst be present at concentrations below the limit of per- ceptibility of the electrophoretic technic. I t may be concluded that an anomalous plasma component, detectable by immunologic procedures, may be present in a host-tumor system, although electrophoretic plasma protein analyses have been found to be normal.

I t also appears evident from the present data (Table 1) that a certain component in the a2- globulin fraction is increased during the growth of a spontaneous mouse tumor, a fact which had previously been shown in mice with transplantable tumors (1, 9). Increased Inucoproteins belonging to the group of a-globulins have repeatedly been reported in patients with malignancies (1~-14). The increase of a-globulin in tumor-bearing A/Jax mice, which was observed by moving boundary electrophoresis (10), was due no doubt to both an increase of a normal plasma component and to the appcarancc of small amounts of an anomalous protein.

In addition, the present results clearly show that a certain constituent of 3,-globulin is increased, while another 7-globulin, at least two al-globulin components, and albumin arc de- creased in the tumor-bearing animals. The de- crease in albumin in tumor-bearing mice has been described (1) and is commonly observed in patients with canccr.

Increases in antigenic components in whole plasma of tumor-bcaring animals were observed by Darcy (4, 5). However, no at tempt was made by this worker to fractionate the plasma and to locate specifically the proteins responsible for the antigenic reactions.

Sincc the use of whole plasma from tumor-bearing hosts as antigen was found to produce highly complex antigenic patterns, 3 we believc that the results described in the present paper were due in large part to thc fact that purified plasma fractions served as antigens instead of whole plasma. Thus, the anomalous protein was libcrated from the bulk of competing antigens and became a major component of the material used for immunization. I t appcars possible that its antigenicity was thereby increased. Although the technic of purification uscd in this work may not yicld pure protein fractions, this mild method nevertheless appears logical and suitable for the purpose of concentrating anomalous plasma proteins from tumor-bearing mice into partially purified fractions suitable for immunological studies.

P. Bernfeld and E. E. Miller, unpublished results.

The fact that the anomalous component could be detected with a very high degree of accuracy in individual tumor-bearing animals and not at all in normal controls makes this immunological procedure a model experiment of diagnostic tests. Its application to humans, i.e., the combination of the immunological analysis with electrophoretic, or possibly chemical purification of plasma proteins, may become a significant approach to diagnostic procedures in cancer patients.

A C K N O W L E D G M E N T S

We arc indebted to Dr. Max Goldfrank of Stein, IIall & Co. for the generous supply of "Superlose" for our investigation.

R E F E R E N C E S

1. BEm'~F~LD, P., and HOMBrJRGER, ~'. The Influence of Tu- inor Growth on the Plasma Proteins in Mice. Cancer Re- search, 15:359-64, 1955.

~2. BEltNFELD, P., and NlSSr;LBAb'M, J. S. A Modified Method for Protein Separation by Zone Electrophoresis on a Starch Gel. J. Biol. Chem., 220:851-60, 1956.

3. Con~', M. In: A. C. COUCORAN (cd.), Methods in Medical Research, 5:o~71-83. The Year Book Publishers, Inc., Chicago, 195~.

-~. I)AUCY, D. A. Immunological Discrimination between the Blood of Normal and Tumor-bearing Rats. Nature, 176: 643-44, 1955.

5. Immunological I)emonstrat ion of a Substance in Rat" Blood Associated with Tissue Growth. Brit. J. Cancer, 11:137-47. 1957.

6. ,JOItNSO.~, R. M.; ALBERT, S.; and PI_~'KUS, II. Serum Pro- teins in Mice Bearing Induced and Spontaneous Mam- mary Gland Carcinomas. Cancer Research, 14:830-36, 1954.

7. KOI~N(:;OLD, L. The I)istribution and Immunochcmical Properties of Human Tissue and Tumor Antigens. Ann. New York Acad. Sc., 69:681-97, 1957-58.

8. LowRy, 0. H.; ROSEBUO~'aH, N. J.; FARR, A. L.; ~nd RANDALI~, n . J. Protein Measurement with the Foliu Phenol Reagent. J. Biol. Chem., 193:265-75, 1951.

9. NISSELBAUM, ,I. S., and BERNFELD, P. The Properties of Two (;lycoproteins Isolated from the Plasma of Normal and Tumor-bearing Mice. J. Am. Chem., Soc. 78:687-89, 1956.

10. . Immunological Studies on Anomalous Plasma Proteins from Tumor-bearing Mice. Proc. Am. Assoc. Cancer Research, 2 : ~235, 1957.

11. O~-CItTV:aLO~CY, O. Antigen-antibody Reactions in Gels. IV. Types of Reactions in Coordinated Systems of Diffusion. Acta Pathol. & Microbiol. Scandinav., 32:~231-40, 1958.

1~. PETEItMANN, ~[. L., and HOGNESS, K. R. Elcctrophoretic Studies on the Plasma Proteins of Patients with Neoplastic Disease. II. An Acid Protein Present in the Plasma. Cancer, 1:104-8, I948.

1,~{. SEIIIERT, F. B.; SEIBEH.T, M. V.; ATNO, A. J.; and ChirP- nELL, H. J. Variation in Protein and Polysaccharide Con- tent of Scra in the Chronic Discuses, Tuberculosis, Sar- coidosis, and Carcinoma. J. Clin. Investigation, 26:90-10-2, 1947.

14. WINZLP;H, R. J.; DOyEn, A. W.; MEIIL, J. W.; and SMYTIL i. M. Studies on the Mucoprotcins of I Iuman Plasma. I. l )etermination and Isolation. J. Clin. Investigation, 27: 609-16, 1948.



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1960;20:1149. Cancer Res Elizabeth Eshelman Miller and Peter Bernfeld Spontaneous TumorsAbnormal Plasma Components in C3H Mice Bearing

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Abnormal Plasma Components in C3H Mice Bearing ......2 Manufactured by Stein, Hall & Co., Inc., New York. 1 rag. total protein per pound of body weight mixed with Freund's adjuvant,