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Circulating immune complexes as “tumor marker” in hepatoma-bearing rats (Yoshida AH 130)
lm.lumdt~gl I.cttcr~, 6 (1993) 59 61
EIse,,ier Biomedical Press
CIRCULATING I M M U N E COMPLEXES AS "TUMOR MARKER" IN HEPATOMA-BEARING RATS (YOSHIDA AH 130) Bruno NER1 Institute ~I (/in.a~ .~ledicme and Medical .S'cm('it~h,,~t. l 'niv('r.~itl ~1 Irh.'en(c. I-'lor(,nce 50139. hah"
(Received 23 August 1982) [Modified '.ersion reccixcd 17 Noxemb,,:r 19~2) (Accepted 23 Noxember 19~2)
1. S u m m a r y
ty. the extent and the clinical course of tumor clone
[i,2]. The presence of circulating immune complexes (CIC) in a large number of malignancies was investigated in relation to the activity, the extent and the clinical course of the tumor clone. The aim of this study was to explore the relation between CIC serum levels and tumor growth in rats bearing the 130 Yoshida ascites hepatoma. For the assay of C3biCIC levels all samples were tested by a new competitive immunoenzymatic test. Experiments carried out on normal rats and on hepatoma-bearing ones, showed that during tumor growth xalues of CIC serum levels expressed as percent coefficient of inhibition are significantly higher in hepatoma-bearing rats (mean = 60.2) than in the normal controls (mean = 29.5). Furthermore CIC level and tumor proliferation are strictly correlated r = 0.9559. In this view sequential measurements of CIC levels may be valuable in establishing diagnosis, estimating prognosis and monitoring the behavior of neoplasia.
2. Introduction The presence of circulating immune complexes (CIC) in a large number of malignancies was investigated, over a 5 year period, in relation to the activi-
[~el word.~, circulating immune complexes immuneenzymaticassa,, YoshidaAll 130
tumor marker
0165 2478 83 0(XX) 0~XX)S3.(X) © 1983 ElsevierScience Publishers
In fact, there is now increasing evidence that malignancy in both humans and experimental animals can bring about the formation of an host immune reaction, directed against the growing tumor [3,4]. Allied with these investigations, analysis of serum factors released during growth of transplanted animal tumor, such as carcinogen-induced hepatomas has demonstrated unequivocal])' that CIC-conraining tumor-specific antigens can be identified at particular stages of tumor growth [5,6]. More recently. the development of sensitive methods for the detection of immune complexes has allowed further investigation into the role of immune complexes in malignant diseases [7,8]. Many authors [9,10] have reported that the quantitation of CIC in sera from patients with malignant diseases may be valuable as a diagnostic a n d or prognostic marker and there exists a good degree of correlation between the extent of serum elevation of CIC and the growth or regression of neoplasia. In this view we decided to investigate whether rats with experimental hepatoma would react similar])'. If so, changing levels of CIC might provide an estimation of the rate of tumor growth both less subjective and less approximate than clinical observations. The aim of this stud)', therefore, was to explore systematically the relation between CIC levels and tumor growth in rats bearing the 130 Yoshida ascites hepatoma. Specifically, what we investigated, was the behavior of CIC levels during the course of the tumor development. 59
3. Materials and m e t h o d s
Male Albino rats of Wistar strain, weighing 180 -~_ 10 g u, ere used for this study. Unless otherwise indicated, they were kept on a stock diet and water available ad libitum until the time of experiment. I u m o r transplant w.as perfornted b \ Yoshida ascites hepatoma ( A l l 130), maintained by the intraperitoneal inoculation of 0.5 ml whole ascite lluid (ascitic plasma plus ascites cells) in an isologous rat, at the 7th day of the growth. With sterile procedures each animal received by intraperitoneal iniection 20 million cells (Yoshida ascites hepatoma) [11]. Blood samples (3 ml) were withdrawn bv heart puncture at different times of tumor growth: all the rats were anesthetized slightly with ether. All serum samples (controls and transplanted rats) w'ere taken from rats of the same age, and were kept at 30 ~'(" after centrifugation at 2000 × g for 15 min. After blood collection from each rat bearing tumor the sample was drained by a needle for ascitic fluid. l-he concentration and the number of tumor cells were measured during the whole evolution of the hepatoma in a Burker chamber. ]'he ascitic plasma was measured and separated from the tumor cells by centrifuging at low speed. 3.1. Circulating i m m u n e COmlfle.w's assay F o r the assay of C~biCIC all samples were tested in duplicate by an enzyme-linked immunosorbent assay, provided by Farmitalia Carlo Erba (Clinicals CIC kit) [12]. In this assay immobilized antigens or antibodies are used on a solid phase with enzyme-labelled antibody or antigen conjugates. A beta-galactosidase anti-beta-galactosidase probe immune complex (PIC) competes with circulating immune complexes for bovine conglutinin, adsorbed on polystyrene beads through C3bi complement fraction. The amount of beta-galactosidase enzymatic activity on the solid phase, measured spectrophotometrically at 420 nm (Saitron M O - C spectrophotometer), is inversely proportional to the ( ' I C concentration in the sample (Ae). ] h e results are expressed as an inhibition coefficient w'hich is a percentage related to a negative saline control (Acn): Inhibition coefficient CIC 60
I
-Acn
x 100
In a precedent study, a 4.1¢; coefticient of variation was obtained w.ith repeated assays (8 determinations} on the same sample, at different times [ 13].
4. Results
Experm~ents carried out on apparently normal rats (control group) and on hepatoma-bearmg ones, ha,,e yielded results concerning: the behavior of blood serum CIC in relation to cell proliferation oI the tUlylOr.
Fig. 1 clearl,, shov,.s that during the first I I davs ol observation, mean values of ( ' I C in serum are signilicantly higher in hepatoma-bearing rats mean (CIC = 60.2) than m the normal controls mean (CI(" = 29.5). We haxe discarded the results of the 3 days follo~ing transplant, since in this period the number of tumor cells, metabolically active, arc not correctly measurable. Previous studies, in fact, ha~e shown that it] this phase superimposed inllammatory processes might interfere leading to false results. Furthermore the increase in the number of hepatoma cells approaches zero on about the 12th day'; that is, around this time the number of cells formed equals the number of cells destroyed [111, and after this lime, around the 13th 16th day, we evidence the
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' 14
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Days after transplantation
Fig. I. Beha',ior ut ('1(" serum levels expressed :is percent cc~'lficient tfl" inhibition and tumor grtw.th expressed a~, rio cell,, . lip +
death of all rats. In this period we can observe a significant decrease of CIC levels (from 67.7 to 28.2). Moreover, data on possible mathematical correlation between the CIC blood levels and the growth of the hepatoma (expressed as number of t u m o r cells) show a good linear correlation (r 0.9559).
sured by this assay. In view of this. sequential measurements o1" CIC levels may be valuable in establishing diagnosis, estimating prognosis and monitoring the behavior of neoplasia.
Acknowledgements 5. Discussion l h e aim of this study is to veri~;, as reported by several authors, if there exists a close correlation between the circulating immune complexes concentration, the tumor size (number of hepatoma cells) and the stage of disease. In our stud,,' CIC levels in the control group of rats were found to be 29.5 .+_3.1 (inhibition coetficient percent), in rats bearing hepatoma, mean values of CIC vary from 39.0 to 68.9 ( 3 r d - l llb day) according to the degree of tumor growth. The first 3 davs after transplant have been discarded since, as previously mentioned, the xalues have a dubious interpretation [ l 1]. l-rom the 3rd day on, CIC levels undergo to a significant progressive increase. In this regard, the linear regression equation relating CIC serum levels to cell proliferation is valid only for the period of dynamic growth of the tumor (from 3rd to I lth day) and sho~s a ~ery high correlation (r =- 0.9559). A dramatic failure of CIC levels w'as observed on the 13th day, lower than normal values. This observation assumes a significant value if correlated with the extinction of the immunological response in the phase before the rats death, that usually comes on between 13th and 16th day from the tumor's transplant. These results are consistent with the clinical findings of many authors [9,10,13] that CIC levels are strictly related to tumoral growth: without that, as observed in a previous study [14], an increased level of C3 could be correlated with CIC levels, mea-
"lhe author expresses gratitude to Prof. Grazia Cini for her excellent technical assistance.
References [I] [2] [3]
[41 [5]
IN
[71 [8] [9] [I0]
Ill] [ 12 ] [13] [14]
llcicr. II. l!., ('arpentior. N.. langc, G.. l.ambert, P H. and Goal. "I. (1977) Int. J. ('anccr 20, 887 894. [.o'.~e. ,I.. Segal-Eiras. A.. lles. P B. and Baldy.in, R. \V. 11981) [ borax 36, 56 59. l:lald'0.in, R . W , P r i c e . M R. and Robbm. R A ( 1 9 7 2 ) Nalure Ne'0. Hiol. 238, 185 191}. I or, is. M. (;., I'hilhp, ] . M., Roy, den, (i. and Jerr.,.. I.. M. 11978) Progr.('anccr Res. lher. 5,245 251. (icrgcl.,., .I., ('hi Phi, N., Fhsl. (;., Medg)¢~,i, (i. A. and Puska~, 1. (lgg()) l a Ricerca ('hn. lab. 10, 27 34. Price, M. R. and Bald'.~in. R. W. (1977)l).,,namic Aspects ol ('ell Surface Organi,'atmn, (.'all Suclace re,.iev, s, (G. Postc and (i. I. Nicob, tm, [-2ds.), North-Holland. Amsterdam Robin.,,. R. A. and Baldwin, R. W (1978) ( a n c e r lmmunol. Immunolher. 4. I 3. lheofilopoulos. A N. and Dixon. l-.+l (1980) Ad~. Immunol. 28.89 22(I. [-Liras. A S.. Robins, R. A., Baldy.in. R. W and B~ers. V. S. (198()) Inst..I. ('anccr 25, 735 739. llerbcrman. R. B.. Borde~,, M..l.amN:rt. P. H..l.uthre. H. S.. Robins. R. A . Sizarct, P. and I h¢olilopoulo~,, A. (1982) Int. ,I. ('ancer 27, 569 576. I)e] Monte. t'. and Rossi. C. B (1963) Cancer Rcs. 2.t. .t63 367. ('clada, k.. [!llis. ,I.. Bodlund, K. and Rot man. B. ( 1971 ) J. Exp. Med. 134,751 757. Neri, B.. Comparini. I.. Bacalli. S.. De Scal,'i. M. and Brogioni, M. (1982) IRe'F, Med. Sch Biochem 10, 265 266. Neri. B.. 1 orcia. M., I)e Scal,,i, M. and Guidi, (i. (1982),1. Clin. Pilth. {in press).
61
EIse,,ier Biomedical Press
CIRCULATING I M M U N E COMPLEXES AS "TUMOR MARKER" IN HEPATOMA-BEARING RATS (YOSHIDA AH 130) Bruno NER1 Institute ~I (/in.a~ .~ledicme and Medical .S'cm('it~h,,~t. l 'niv('r.~itl ~1 Irh.'en(c. I-'lor(,nce 50139. hah"
(Received 23 August 1982) [Modified '.ersion reccixcd 17 Noxemb,,:r 19~2) (Accepted 23 Noxember 19~2)
1. S u m m a r y
ty. the extent and the clinical course of tumor clone
[i,2]. The presence of circulating immune complexes (CIC) in a large number of malignancies was investigated in relation to the activity, the extent and the clinical course of the tumor clone. The aim of this study was to explore the relation between CIC serum levels and tumor growth in rats bearing the 130 Yoshida ascites hepatoma. For the assay of C3biCIC levels all samples were tested by a new competitive immunoenzymatic test. Experiments carried out on normal rats and on hepatoma-bearing ones, showed that during tumor growth xalues of CIC serum levels expressed as percent coefficient of inhibition are significantly higher in hepatoma-bearing rats (mean = 60.2) than in the normal controls (mean = 29.5). Furthermore CIC level and tumor proliferation are strictly correlated r = 0.9559. In this view sequential measurements of CIC levels may be valuable in establishing diagnosis, estimating prognosis and monitoring the behavior of neoplasia.
2. Introduction The presence of circulating immune complexes (CIC) in a large number of malignancies was investigated, over a 5 year period, in relation to the activi-
[~el word.~, circulating immune complexes immuneenzymaticassa,, YoshidaAll 130
tumor marker
0165 2478 83 0(XX) 0~XX)S3.(X) © 1983 ElsevierScience Publishers
In fact, there is now increasing evidence that malignancy in both humans and experimental animals can bring about the formation of an host immune reaction, directed against the growing tumor [3,4]. Allied with these investigations, analysis of serum factors released during growth of transplanted animal tumor, such as carcinogen-induced hepatomas has demonstrated unequivocal])' that CIC-conraining tumor-specific antigens can be identified at particular stages of tumor growth [5,6]. More recently. the development of sensitive methods for the detection of immune complexes has allowed further investigation into the role of immune complexes in malignant diseases [7,8]. Many authors [9,10] have reported that the quantitation of CIC in sera from patients with malignant diseases may be valuable as a diagnostic a n d or prognostic marker and there exists a good degree of correlation between the extent of serum elevation of CIC and the growth or regression of neoplasia. In this view we decided to investigate whether rats with experimental hepatoma would react similar])'. If so, changing levels of CIC might provide an estimation of the rate of tumor growth both less subjective and less approximate than clinical observations. The aim of this stud)', therefore, was to explore systematically the relation between CIC levels and tumor growth in rats bearing the 130 Yoshida ascites hepatoma. Specifically, what we investigated, was the behavior of CIC levels during the course of the tumor development. 59
3. Materials and m e t h o d s
Male Albino rats of Wistar strain, weighing 180 -~_ 10 g u, ere used for this study. Unless otherwise indicated, they were kept on a stock diet and water available ad libitum until the time of experiment. I u m o r transplant w.as perfornted b \ Yoshida ascites hepatoma ( A l l 130), maintained by the intraperitoneal inoculation of 0.5 ml whole ascite lluid (ascitic plasma plus ascites cells) in an isologous rat, at the 7th day of the growth. With sterile procedures each animal received by intraperitoneal iniection 20 million cells (Yoshida ascites hepatoma) [11]. Blood samples (3 ml) were withdrawn bv heart puncture at different times of tumor growth: all the rats were anesthetized slightly with ether. All serum samples (controls and transplanted rats) w'ere taken from rats of the same age, and were kept at 30 ~'(" after centrifugation at 2000 × g for 15 min. After blood collection from each rat bearing tumor the sample was drained by a needle for ascitic fluid. l-he concentration and the number of tumor cells were measured during the whole evolution of the hepatoma in a Burker chamber. ]'he ascitic plasma was measured and separated from the tumor cells by centrifuging at low speed. 3.1. Circulating i m m u n e COmlfle.w's assay F o r the assay of C~biCIC all samples were tested in duplicate by an enzyme-linked immunosorbent assay, provided by Farmitalia Carlo Erba (Clinicals CIC kit) [12]. In this assay immobilized antigens or antibodies are used on a solid phase with enzyme-labelled antibody or antigen conjugates. A beta-galactosidase anti-beta-galactosidase probe immune complex (PIC) competes with circulating immune complexes for bovine conglutinin, adsorbed on polystyrene beads through C3bi complement fraction. The amount of beta-galactosidase enzymatic activity on the solid phase, measured spectrophotometrically at 420 nm (Saitron M O - C spectrophotometer), is inversely proportional to the ( ' I C concentration in the sample (Ae). ] h e results are expressed as an inhibition coefficient w'hich is a percentage related to a negative saline control (Acn): Inhibition coefficient CIC 60
I
-Acn
x 100
In a precedent study, a 4.1¢; coefticient of variation was obtained w.ith repeated assays (8 determinations} on the same sample, at different times [ 13].
4. Results
Experm~ents carried out on apparently normal rats (control group) and on hepatoma-bearmg ones, ha,,e yielded results concerning: the behavior of blood serum CIC in relation to cell proliferation oI the tUlylOr.
Fig. 1 clearl,, shov,.s that during the first I I davs ol observation, mean values of ( ' I C in serum are signilicantly higher in hepatoma-bearing rats mean (CIC = 60.2) than m the normal controls mean (CI(" = 29.5). We haxe discarded the results of the 3 days follo~ing transplant, since in this period the number of tumor cells, metabolically active, arc not correctly measurable. Previous studies, in fact, ha~e shown that it] this phase superimposed inllammatory processes might interfere leading to false results. Furthermore the increase in the number of hepatoma cells approaches zero on about the 12th day'; that is, around this time the number of cells formed equals the number of cells destroyed [111, and after this lime, around the 13th 16th day, we evidence the
40
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60
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20
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• E
21
20
0
10
/
J
I
I
i
i
I
~
I
0
2
4
6
8
10
12
' 14
0
Days after transplantation
Fig. I. Beha',ior ut ('1(" serum levels expressed :is percent cc~'lficient tfl" inhibition and tumor grtw.th expressed a~, rio cell,, . lip +
death of all rats. In this period we can observe a significant decrease of CIC levels (from 67.7 to 28.2). Moreover, data on possible mathematical correlation between the CIC blood levels and the growth of the hepatoma (expressed as number of t u m o r cells) show a good linear correlation (r 0.9559).
sured by this assay. In view of this. sequential measurements o1" CIC levels may be valuable in establishing diagnosis, estimating prognosis and monitoring the behavior of neoplasia.
Acknowledgements 5. Discussion l h e aim of this study is to veri~;, as reported by several authors, if there exists a close correlation between the circulating immune complexes concentration, the tumor size (number of hepatoma cells) and the stage of disease. In our stud,,' CIC levels in the control group of rats were found to be 29.5 .+_3.1 (inhibition coetficient percent), in rats bearing hepatoma, mean values of CIC vary from 39.0 to 68.9 ( 3 r d - l llb day) according to the degree of tumor growth. The first 3 davs after transplant have been discarded since, as previously mentioned, the xalues have a dubious interpretation [ l 1]. l-rom the 3rd day on, CIC levels undergo to a significant progressive increase. In this regard, the linear regression equation relating CIC serum levels to cell proliferation is valid only for the period of dynamic growth of the tumor (from 3rd to I lth day) and sho~s a ~ery high correlation (r =- 0.9559). A dramatic failure of CIC levels w'as observed on the 13th day, lower than normal values. This observation assumes a significant value if correlated with the extinction of the immunological response in the phase before the rats death, that usually comes on between 13th and 16th day from the tumor's transplant. These results are consistent with the clinical findings of many authors [9,10,13] that CIC levels are strictly related to tumoral growth: without that, as observed in a previous study [14], an increased level of C3 could be correlated with CIC levels, mea-
"lhe author expresses gratitude to Prof. Grazia Cini for her excellent technical assistance.
References [I] [2] [3]
[41 [5]
IN
[71 [8] [9] [I0]
Ill] [ 12 ] [13] [14]
llcicr. II. l!., ('arpentior. N.. langc, G.. l.ambert, P H. and Goal. "I. (1977) Int. J. ('anccr 20, 887 894. [.o'.~e. ,I.. Segal-Eiras. A.. lles. P B. and Baldy.in, R. \V. 11981) [ borax 36, 56 59. l:lald'0.in, R . W , P r i c e . M R. and Robbm. R A ( 1 9 7 2 ) Nalure Ne'0. Hiol. 238, 185 191}. I or, is. M. (;., I'hilhp, ] . M., Roy, den, (i. and Jerr.,.. I.. M. 11978) Progr.('anccr Res. lher. 5,245 251. (icrgcl.,., .I., ('hi Phi, N., Fhsl. (;., Medg)¢~,i, (i. A. and Puska~, 1. (lgg()) l a Ricerca ('hn. lab. 10, 27 34. Price, M. R. and Bald'.~in. R. W. (1977)l).,,namic Aspects ol ('ell Surface Organi,'atmn, (.'all Suclace re,.iev, s, (G. Postc and (i. I. Nicob, tm, [-2ds.), North-Holland. Amsterdam Robin.,,. R. A. and Baldwin, R. W (1978) ( a n c e r lmmunol. Immunolher. 4. I 3. lheofilopoulos. A N. and Dixon. l-.+l (1980) Ad~. Immunol. 28.89 22(I. [-Liras. A S.. Robins, R. A., Baldy.in. R. W and B~ers. V. S. (198()) Inst..I. ('anccr 25, 735 739. llerbcrman. R. B.. Borde~,, M..l.amN:rt. P. H..l.uthre. H. S.. Robins. R. A . Sizarct, P. and I h¢olilopoulo~,, A. (1982) Int. ,I. ('ancer 27, 569 576. I)e] Monte. t'. and Rossi. C. B (1963) Cancer Rcs. 2.t. .t63 367. ('clada, k.. [!llis. ,I.. Bodlund, K. and Rot man. B. ( 1971 ) J. Exp. Med. 134,751 757. Neri, B.. Comparini. I.. Bacalli. S.. De Scal,'i. M. and Brogioni, M. (1982) IRe'F, Med. Sch Biochem 10, 265 266. Neri. B.. 1 orcia. M., I)e Scal,,i, M. and Guidi, (i. (1982),1. Clin. Pilth. {in press).
61