Two Transplantable Mouse Hepatomas Associated withan Increase of Metal-combining ß-Globulin

(Transferrin) in Serum*

J. CLAUSEN,R. RASK-NIELSEN,H. E. CHRISTENSEN,ANDT. MUNKNER

(University Institute of Bioc/iemistry; Laboratory for K/ieiimatic Research, University Institute of Pathological

Anatomy; and Department of Radiology, Isotope Laboratory, University Clinic, Copenhagen, Denmark)

SUMMARY

The growth of two transplantable lines of mouse hepatoma was found to be associated with an increase of the beta and often of the alpha2 globulin as seen in paperelectrophoresis. In immunoelectrophoresis the predominant feature was a stronglyincreased concentration of beta-2-I globulin. Other beta fractions were constantly foundchanged, and some alpha-2 fractions were found changed more occasionally. Hypo-gammaglobulinemia was invariably seen. Au to radiography showed the beta-2-Ifraction of normal and hepatomatous serum to be iron-binding, thus demonstratingthe correspondence of this fraction to the metal-combining beta-I globulin (transferrin,siderophilin) of human serum.

The microscopic appearances of the hepatomas and of the organs of the hosts aredescribed. In one of the hepatoma lines numerous mast cells were constantly observedin the tumor tissue and in the connective tissue of the hosts.

The results are discussed in relation to earlier findings.

Hepatomas in rats and mice are rather commonly observed. Their development may be eitherspontaneous or induced by different chemical compounds. From a biochemical point of view themetabolic functions of the malignant liver cells areof particular interest, since they may possiblyelucidate some of the numerous properties andfunctions of the normal liver cells. Several investigations with this purpose have been carried out,especially studies on differences in various enzymatic activities of normal and malignant livercells.

As part of a series of experiments (9) we haveexamined two lines of transplantable hepatomas inmice which appeared to be associated with the occurrence of an abnormally high level of metal-combining beta globulin in serum. Since observations of this kind have not been described previously, we feel justified in reporting our findingsin the present paper.

* These investigations have been supported by grants fromKing Christian X Foundation, The Leukemia Society, Inc.,and from the Danish League against Rheumatism.

Received for publication June 22, 1959.

METHODS AND RESULTSANIMALMATERIAL

The two lines of hepatoma in question, lines 41and 44, arose spontaneously in male (CBA XDBA/2) Fi hybrid mice, age 30 and 21 months.Both lines were continued by subcutaneous inoculation of hepatomatous tissue into mice of thesame genetic constitution. Line 41 was followed forfive transfer generations, line 44 through three passages so far, comprising 25 and fourteen mice, respectively.

The percentage of takes was 100 except in thefirst passage, where the graft failed to grow in onemouse of each line.

Survival time was about 100-150 days for line41, 150-250 days for line 44.

In line 41 the number of white blood cells wasoften observed to be slightly increased (10,000-

20,000/cc). In line 44 normal values were found.Differential counts were normal until the hepatomas reached a large size; then a relative graniilo-

cytosis developed. During tumor development thenumber of basophilic erythrocytes increased, as isoften seen in mice bearing large, slow-growing tu-

178

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CLAUSEXet al.—MouseIlepatomas with Increase in Tranxfemn 179

mors. No nucleated red cells were seen in theperipheral blood.

At autopsy encapsulated, rather soft, andcoarsely lobuled tumors measuring 20-50 mm. indiameter were found at the site of inoculation.The cut surface was greyish and often revealedcystic spaces filled with blood or a clear, yellowishfluid. By gross examination of the organs nométastases and no other changes of relevance wereobserved, although hypertrophy of the heart wasseen in some cases.

SERUM PROTEIN CHANGESThe technic used for these investigations has

been described in a previous paper (10).Total amount of serum protein.—In both lines of

hepatoma total serum protein increased from normal values (6.1-6.7 gm. per cent protein [10]) up to8-9 gm. per cent in the later stages of tumor devel

opment. In the very last days of survival a slight decrease of total protein could be observed, mainlydue to a decrease of the albumin fraction.

Serum protein changes as seen in paper electropho-resis.—During the development of the implanted

tumors a rather marked increase of the beta globulin gradually developed. This was particularly truefor line 44. Furthermore, a more or less pronouncedincrease of the alpha2 fraction could also be found,most frequently in line 44. An example of thesechanges is given in Figure 1.

Serum protein chatiges as seen in immunoelectrophoresis.—The immunoelectrophoretic technic

and the pattern of our normal (CBA X DBA/2)FI mice has previoxisly been described in detail (6).It is, therefore, necessary to mention only that thismethod, which was introduced by Grabar and Williams (4) and which combines electrophoretic separation of the serum proteins in an agar gel with asucceeding antigen-antibody precipitation, hasbeen used to demonstrate the existence of 21 different proteins in normal mouse serum (6). Severalof these, developed with our antiserum 4HV, areshown in Chart 1.

Immunoelectrophoresis of serum from numerous mice of our lines of hepatoma have revealedchanges in several of these proteins. In principle,these changes were alike in the two transplantation lines, and a common description will thereforebe given, divided into (a) the main changes localized in the beta region, corresponding to those constantly seen in paper electrophoresis, (6) changesin the gamma globulin, and (c) changes concerningthe alpha proteins. These findings are shown onChart 1. On the upper part a conventional agar-electrophoresis is seen. The central part shows thesame hepatomatous serum as seen in Figure 1 com

pared with normal serum. The lower part demonstrates immunoelectrophoresis of the same sera,diluted 1:4. By this dilution abnormally increasedproteins will appear much more distinctly than thecorresponding normal ones.

1. Changes in the ß-proteins: The predominantfeature is that the beta-2-I globulin precipitationbow was closer to the antibody reservoir and extended much farther into the alpha area and intothe intermediate part of the gamma area than thecorresponding normal bow. These findings indicate that the concentration of this protein wasmuch higher than occurs normally.

Furthermore, the beta-2-III showed a larger extension and a slightly higher mobility than didnormal serum, the anodical part of this bow reaching far into the alphai area.

The beta-3-I was also changed. In immunoelectrophoresis the bow of this protein normallyhas the form of a double curvature which is partlysuperimposed by the anodical part of the gammabow. In all hepatomatous sera an accentuation ofboth the anodical and the cathodical curves of thisbow, especially of the latter, was seen. This cathodical curve was found closer to the antibodyreservoir than occurs normally, and the anodicalend of the anodical curve was localized moreanodically than is true for normal serum. Bothfindings indicate an increased concentration of thisprotein.

The immunoelectrophoreses of the diluted sera(lower part of Chart 1) distinctly show that beta-2-I, beta-2-III, and beta-3-I were increased considerably in the pathological serum.

2. Changes in the gamma area: In all hepatomatous sera a hypogammaglobulinemia could beobserved, characterized by a weak 7 bow mainlylocalized to the intermediate part of the gammaarea, as seen in Chart 1.

3. Changes in the alpha proteins: The changesin the alpha area occurred rather irregularly, incontrast to those of the other zones, which wereconstantly present In some sera the most remarkable finding was an extremely high concentrationof both alpha-2-I and alpha-2-II globulins, manifesting themselves as two vastly extended bows.The cathodical extremities of these bows, which innormal serum end just before the central fillinghole, reached far to the cathodical side of this hole(Chart 1, central part). In some cases both bowswere much stronger than normal and nearer to theantibody reservoir. In other sera these bows werefound normal or very weak; the alpha-2-II couldeven be completely lacking.

Sometimes the alpha-2-III and alpha-2-V werefound of higher concentrations than normal, as

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CHART1.—Upper part: Conventional agar electrophoresisof the same hepatomatous sera as seen in Figure 1.

Central part: Iminunoelectrophoresis of—above: the samehepatomatous serum as seen in Figure 1; below: normal serum.

Lower part: Iminunoelectrophoresis of the same sera asseen in Figure 1, diluted 1:4; above: hepatomatous serum;

below: normal serum.The electrophoreses were run on 2 /«I.of serum for 3J-4

hours at 4 v/cm, a Veronal buffer, pH 8.6, ionic strength 0.05being used. The immunoelectrophoreses were developed withantiserum for 24 hours, washed, dried, and stained withAmidoschwartz 10 B.

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CLAUSEN et al.—Mouse Hepatomas with Increase in Transferrin 181

shown by the fact that these bows showed a widerextension and were closer to the antibody reservoir.

The general impression was that the changes inthe alpha proteins tended more toward an increase in line 44 and more toward a decrease inline 41.

Autoradiography of immunoeledrophoretic pat-ferns.—In mouse serum the main protein of the

beta region, as judged from dilution experiments(6), is the beta-2-I. In human serum the main protein of this region is the beta-I-metal-combiningprotein (transferrin, siderophilin). By using a combined diffusion method (the Ouchterlony techniccombined with immunoelectrophoresis) it has beenshown (1) that certain groups were antigeneticallyidentical for these two proteins. It was, therefore,tempting to try to identify these proteins witheach other. Furthermore, it was desirable to demonstrate properties common to the beta-2-I of normal and hepatomatous sera, thus confirming theidentity 01 this protein in both sera For identification we chose to investigate the iron-binding capacity, which we did by performing an auto-radiography of immunoelectrophoretic tracings ofnormal and hepatomatous sera that had been incubated in vitro with Fe59.

For the incorporation of iron, 50 /il. of serumwas mixed with 10 /al. of Fe69Cl3 (Philips-Roxane)with a specific activity of 1 mc/mg Fe+++, (pH =1-2); thus, 10 /il. contained 80 X 10~3 /ig. Fe

(or 0.08 /ic.). Five /tl of incubated serum were usedin immunoelectrophoresis. The immunoelectrophoretic slide was developed as usual (6), coloredwith sudan black, and finally decolorized in a mixture of water and ethanol. The slide was nowplaced directly on a film (Kodak safety 3 film).Three days later it was easy to localize the iron,and after 8 days the tracings were very pronounced. It was found that the beta-2-I globulin ofnormal and hepatomatous serum was the main carrier of the iron. Further information obtained fromthese results was that the beta-2-I globulin of mouseserum corresponded to the beta-I-metal-combiningprotein = transferrin = siderophilin of human serum. In the upper part of Figure 2 is shown thepicture of such an autoradiograph of the immunoelectrophoretic tracings of hepatomatous serum;the lower part of Figure 2 shows the same tracingsphotographed in the conventional way.

Serum protein changes as seen in starch gel elec-trophoresis.—The main changes observed in paper

and in immunoelectrophoresis were in the alphaand beta regions and in the zone between them.Since the starch gel electrophoresis of Smithies(11) has a high resolutive power in these regions,

we thought it appropriate to investigate our hepatomatous serum by means of this method.

Sera from the same case of hepatoma as seen inFigure 1 and in Chart 1 and from a normal mouse,undiluted and diluted 1:2 and 1:4, were run instarch gel electrophoresis. Figure 4 shows thetracings of the dilutions 1:2, in which the pathological changes were most distinctly observable.Several changes were seen. An abnormal zone, extremely strong and broad, was localized in and justbehind the beta area. Probably this zone represents transferrin. At any rate, the human transferrin has been found only in this location (8). Infront of this zone was a distinct band with someincrease in mobility when correlated with normalserum. We suppose that this band, which we havealso observed in sera from mice carrying othertransplantable neoplasias (10), corresponds to thebeta-2-III in immunoelectrophoresis. Furthermore, there was an increase in the F-alpha-2 zone,and to a smaller extent in the S-alpha-2 zone.These findings probably correspond to the changesin the alpha area observed in immunoelectrophoresis.

HlSTOLOGICAL EXAMINATIONS

Specimens from tumor, spleen, liver, kidney,and lungs were routinely taken for histological examination. Occasionally lymph nodes, intestine,ovaries, adrenals, tongue, and heart were also examined. Staining with hematoxylin-eosin, methylgreen-pyronine (method of Unna-Pappenheim),periodic acid-Schiff (method of Hotchkiss), and byvan Gieson's method was regularly done. In addi

tion, several specimens were stained with carbol-fuchsin, toluidine blue, and alcian blue (12). Intwo mice the Gomori lead phosphate and calciumcobalt methods for demonstration of acid andalkaline phosphatases were used. The method ofMarshall for demonstration of metalophil reticu-lum cells (7) was employed in some cases.

Histologically, the grafted hepatomas of ourtransplantation lines 41 and 44 had the same appearance, although small differences could be seenfrom animal to animal. The tumors (Fig. 4a) werecomposed of lobules of different sizes, each with anarrow rim of connective tissue. Some areas of thetumors were solid, the tumor cells being large andpolygonal without intervening vascular structuresresembling sinusoids. Other areas contained moreor less cystic spaces or "sinusoids" and had smaller

tumor cells with hyperchromatic nuclei closelypacked in villi or narrow cords. Areas with atrabecular appearance were also found.

The tumor cells in general had a rather highcontent of pyroninophilic substance (interpreted

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182 Cancer Research Vol. 20, February 1960

as ribonucleic acid), most often found as small particles in the periphery of the cells (Fig. 46). However, this content was smaller than in the livercells of the tumor-bearing mice and of normalmice. Adjacent to the nucleus was a "perinuclearhof." The nuclei were round or oval, with well

marked nuclear membranes and a fine chromatinnetwork. Two or three round nucleoli, moderatelypyroninophilic, were seen. The number of mitoseswas moderate. Foci of extramedullary hemato-poiesis (Fig. 4e) and also megakaryocytes wereoften found in the tumor tissue.

The organs of the hepatoma-bearing miceshowed only small deviations from normal appearance. In the liver the Kupffer cells seemed to havehypertrophiée!nuclei and sometimes a pyroninophilic and/or PAS-positive cytoplasm. Extra-medullary hematopoiesis was occasionally seen.The spleen was the seat oíextramedullary hematopoiesis and of an immtmoreaction in the form ofcollections of pyroninophilic reticulum cells in thered pulp and around the artery of the follicularcenters and of perifolliciilar proliferations of lesspyroninophilic reticulum cells. In line 41 the extra-medullary hematopoiesis was heavy, the immu no-reaction weak. In line 44 the opposite was the case.The glomeruli of the kidneys occasionally showeda few pyroninophilic cells, and in addition in a fewmice some glomerular tufts showed PAS-positivehyalinizations. Other organs examined did notshow relevant changes. Métastaseswere not found,in spite of the rather malignant histological appearance of the tumors. However, in some casesthe pulmonary arteries contained emboli of from afew to about twenty tumor cells, which, however,were never seen to have become attached to thevessel walls.

No alkaline phosphatase activity could be demonstrated in the hepatoma tissue. Acid phosphatase activity was found to be greater than that ofliver tissue from the tumor-bearing mouse, whoseactivity was slightly higher than that of liver tissue from a normal mouse. These findings are in accordance with those of others (5).

In only one respect did the two transplantationlines differ significantly from each other. In line 44,

but not in line 41, numerous hypertrophied mastcells were seen in all connectiva tissue. Mast cellswere also seen in the tumor tissue, in some mice inlarge numbers, located as lining cells in the villousor sinusoidal areas (Fig. 4¿)and in the connectivetissue between the tumor cells. In the impregnation of metalophil reticulum celts according to themethod of Marshall, these mast cells were impregnated only to a slight degree or not at all. Nobasophilic granulocytes were found in the blood.Investigations concerning the function of thesemast cells are being currently carried out bv one ofus (H. E. C.).

DISCUSSIONThe serological changes observed during the

growth of malignant hepatic tissue were similar inour two transplantation lines. The most outstanding feature was an increase of the beta fraction asseen in paper electrophoresis. Immunoelectro-phoresis showed this increase to be due first of allto a strong increase of the beta-2 I fraction, whichwas identified by autoradiography as the iron-binding protein, corresponding to transferrin(siderophilin) in human serum. This finding wasfurthermore confirmed in starch gel electrophoresis.

The rise of the transferrin level was found in allhepatomatous mice examined. We have also observed an increase of this beta fraction in two linesof plasma-cell leukemia (2, 10), but here this protein fraction was only moderately or slightly enlarged, and the increase did not occur constantlyas it did in the hepatomatous sera. In several othertransplantable leukemias and tumors of variousother types we have found a normal or decreasedlevel of transferrin (unpublished investigations).

Furthermore, other proteins of the hepatomatous serum were also found changed, as for instance the beta-3-I, the alpha-2-I, and the alpha-2-II. It should be mentioned that changes in thealpha proteins were seen as the main changes in atransplantable hepatoma in rats.1 On the otherhand, the alpha-2 proteins are known to be

1Personal communication from Dr. A. Deckers, The CancerInstitute, University of Louvain, Belgium.

FIG. 1.—Scanningdiagram and paper-strip electroplioresisof serum from a characteristic case of hepatoma and from anormal mouse.

The electrophoreses were run on 10 ¡j\.of serum for 17-18hours at 100 v. An acetate-Veronal buffer, pH 8.6, ionicstrength 0.05, was used. Bromphenolblue was used for staining.

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Papere/e ctrophoreses:1Hep ato m a

Normal mouse serum.

!Normal ser\um-.

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FIG. 2.—Upper part: Photo of autoradiogram of immuno-cleetroplioretic tracings of hepatoinatous serum.

Lower part: Conventional photo of the same immunoclec-troplioretic tracings as seen in the autoradiogram. Concerningthe technic, see the text.

FIG. 3.—Photo of starch gel electrophoresis.Above: the same hepatomatous serum as seen in Fig. 1 and

on Chart 1, diluted 1:2.Below: normal serum; diluted 1:2.The electroplioreses were run on 25 //I. of diluted serum for

5 hours at 6 v/cm in a starch gel soaked with 0.01,5M IIsBOsand O.OOGMXaOII, pH 8.5.

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Immimoelectrophoresis of hepatomatous mouse serum

I. Seen as autoradiogram with Fe"9

II. The same tracing seen in conventional photo

FIG. •¿�

Starch gel elect rophoresis

Hepatoma

S-a ß

Xonnal mouse serum

F-a All)

FIG. 3

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FIG. 4.—a. Area of grafted hepatoma with cells arrangedin cords separated by more or less cystic vascular spaces. VanGioson, X70.

/). (¡rafted hepatoma. The pyroninophilic substance isprincipally situated in the periphery of the tumor cells, leavinga clear space adjacent to the nucleus which shows a distinctnuclear membrane, a fine diramatili reticuliiin, and two orthree slightly pyronine-positive nucleoli. In the center of thefigure a vena centralis-like structure above a megakaryocyteis seen. Methyl green-pynmine, method of Unna-Pappenheim,X1075.

c. An extramcdullary hematopoietic focus between hepatoma cells. Methyl green-pyronine, method of I nna-1'appeii-

hcim, X1075.d. Mast cells between hepatoma cells. Methyl green-pyro

nine, method of Unna-l'appenheim, X^o'i. The metachromasiaof the granules was verified with toluidine blue staining afterfixation in 4 per cent alkaline lead acetate.

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CLAUSEN et al.—-Mouse Hepatomas with Increase in Transferrin 183

changed in other diseases—e.g., in experimentalamyloidosis (3). So far it is impossible to decidewhether the changes in the alpha globulins shouldbe considered primary to the hepatomatousgrowth or secondary, due to secondary changes inthe host.

The changes in the beta-2-III have been observed in mice with all types of spontaneous andtransplanted tumors. Most probably this has to beconsidered an immunoglobulin, the answer of thehost to the conveyance of foreign (tumor) protein(3, 10).

Our two transplantable hepatomas were slow-growing tumors of identical structural andcellular morphology. The cytoplasmic content ofpyroninophilic material, presumably ribomicleicacid, was rather high, although lower than in normal liver cells. This could be compatible with thecapacity of the tumor cells, present in large numbers in the huge grafts, to synthesize the excess oftransferrin, and need not be indicative only of tumor growth, since these tumors grow slowly. Furthermore, it should be considered that the tissuesand organs of the hepatoma-bearing mice showedno changes incompatible with the assumption thatthe tumor cells may produce the excess of transferrin.

The dominating extramedullary hematopoiesisin line 41 and the immunoreaction and the masto-cytosis of line 44 could not be correlated with theobserved serum protein changes.

REFERENCES1. CLAUSEN,J., and HEREMANS,J. An Immunological and

Chemical Study of the Similarities between Mouse andHuman Serum Proteins. J. Immunol. (in press).

2. CLAUSEN*,J.; HEHEMAXS,J.; HEREMANS,M. T., and

RASK-NIELSEN, R. Immunoelectrophoretic Studies ofSerums from Mice Carrying Two Transplantable Plasma-Cell Leukemias. J. Nat. Cancer Inst., 22:57-81, 1959.

3. CLAUSEN,J.; RASK-NIELSEN%R.; and CHBISTENSEN,H. E.Biochemical Changes in Experimental Amyloidosis. III.Immunochemical Investigations on Sera and Urines fromMice with Experimental Amyloidosis. In: H. PEETEHS(ed.), Proceedings of the VII Colloquium on the Protidesof the Biological Fluids, Bruges, 1959. Amsterdam:Elsevier Press, Inc., 1960.

4. GRABAR,P., and WILLL\MS,C. A., JR.: Méthodepermettant l'étudeconjugéedes propriétésélectrophorétiquesetimmunochimiques d'un mélangede protéines;applicationau sérumsanguin. Biochim. et Biophys. acta, 10:193-94,1953.

5. GREENSTEIN,J. P. Biochemistry of Cancer. New York:Academic Press, 1954.

6. HEHEMANS,J.; CLAUSEX,J.; HEHEMANS,M. T.; andRASK-NIELSEN,R. Immunoelectrophoretic Characteristicsof Normal Mouse Serums as a Basis for Studying Pathological Changes in Serums from Mice Carrying Transplantable Malignant Growths. J. Nat. Cancer Inst., 22:45-55, 1959.

7. MARSHALL,A. H. E. An Outline of the Cytology andPathology of the Reticular Tissue, p. 258. Edinburgh andLondon: Oliver & Boyd, 1956.

8. MORETTI,J.; BOUSSIER,G.; HUGOU,M. P.; and HART-MAXN,L. Recherches des correspondances entre les résultats des électrophorèsessur papier, à travers un geld'amidon et de l'immunoélectrophorèseen gélosedes protéinesde sérum.Bull. Soc. Chim. biol., 41:79-87, 1959.

9. RASK-NIELSEN,R. Role of Implanted Inbred-Strain Thy-mic Tissue in the Development of Leukemia in FI HybridMice. J. Nat. Cancer Inst., 21:1083-98, 1958.

10. RASK-NIELSEN,R.; GORMSEN,H.; and CLAUSEN,J. ATransplantable Plasma-Cell Leukemia in Mice Associatedwith the Production of beta-Paraprotein, J. Nat. CancerInst., 22:509-41, 1959.

11. SMITHIES,O. Zone Electrophoresis in Starch Gels. GroupVariations in the Serum Proteins of Normal HumanAdults. Biochem. J., 61:629-41, 1955.

12. WAGNER,B. M., and SHAPIRO,S. H. Application of AlcianBlue as a Histochemical Method. Lab. Investigation, 6:472-77, 1957.

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