Eur J Nucl Med (1985) 10:262-268 European M I i r , l,",,,-Jr Journal of I ~llglt,~lE;;iEl./

Medicine © Springer-Verlag 1985

Tumor and liver uptake models of 67Ga-citrate Atsushi Ando 1, Itsuko Ando l, Shigeru Sanada 1, Tatsunosuke Hiraki 1, and Kinichi Hisada 2 The School of Allied Medical Professions 1, School of Medicine 2 Kanazawa University, Kanazawa, Japan

Abstract. After administration 67Ga concentrates with time in lysosomes from the cytoplasm of liver cells. The lysoso- real role in the accumulation of 67Ga in the liver cell is weakened upon transformation of the liver cell into a malig- nant tumor cell. In malignant tumors (except for hepatoma) the lysosome does not play a major role in the tumor con- centration of 67Ga.

67Ga is bound to acid mucopolysaccharides (keratan polysulfate, etc.) in both tumor and liver. In liver cells, large amounts of 67Ga are transported into lysosomes with these acid mucopolysaccharides, and in hepatoma cells, quite large amounts of 67Ga are transported into lysosomes with these acid mucopolysaccharides. In malignant tumor cells (except for hepatoma) the effect is much smaller, the acid mucopolysaccharides transporting very little 67Ga into lysosome.

The 67Ga is concentrated in viable tumor tissue within malignant tissue but hardly at all in necrotic tumor tissue, and concentrates avidly in inflammatory infiltration around tumor cells. Plenty of 67Ga is found in liver but very little in connective tissue associated with the liver.

Key words: Tumor - Liver - Uptake model - Ga-67 - Acid mucopolysaccharide

that lysosome did not play a major role in the tumor con- centration of 67Ga, although it played an important role in the liver concentration of 67Ga. In the case of hepatoma AH109A, it was presumed that lysosome played quite an important role in the tumor concentration of 67Ga, hepa- toma AH109A possessing some residual features of the liver.

On the other hand, we considered that 67Ga binding substances among body constituents were the strongest ca- tion-exchange resin-like substances, because 67Ga can be a trivalent cation in solution, and this cation binds strongly to cation-exchange resin (Ando et al. 1974) and heparin (Ando et al. 1983). From this working hypothesis, Ando (1979) originally determined that 67Ga, lalIn, and 169yb were bound to the acid mucopolysaccharides in two species of tumor tissues (Ehrlich tumor and Yoshida sarcoma). It was also reported by Ando and co-workers (1980, 1982a and 1983) that a 67Ga-binding acid mucopolysaccharide had been separated by cellulose acetate electrophoresis from tumor tissue and liver lysosome, and 67Ga-binding acid mucopolysaccharide in tumor and liver were very similar.

Based on this earlier work and the present results, we (Ando and Ando 1983b) outlined models for tumor and liver uptake of 67Ga-citrate. The present paper describes the details of these models.

Hayes and co-workers (1970) observed that 67Ga concen- trates mainly in viable rather than in necrotic tumor cells and is most likely bound to an intercellular cytoplasmic component. The distribution of 67Ga in the tumor tissues determined by macroautoradiogram indicated that the con- centration of 67Ga was predominant in connective tissue (especially inflammatory tissue) rather than in viable tumor tissue, regardless of the time after administration, and we presumed that 67Ga was bound to acid mucopolysaccha- rides (Ando et al. 1977). Swartzendruber et al. (1971), origi- nally determined that the intracellular localization of 6VGa occurred in lysosomes or lysosome-like granules of cells. Aulbert et al. (1976), confirmed lysosomal localization of 67Ga in the liver. Ito et al. (1971) and Deckner et al. (1971) showed significant quantities of 67Ga in the soluble fraction in tumor cells. Later, it was concluded (Ando et al. 1982c)

Offprint requests to: Atsushi Ando PhD, The School of Allied Medical Professions, Kanazawa University, 5-11-80, Ko- datsuno, Kanazawa City, 920 Japan

Materials

The following animals and transplanted tumors were used: Male normal Wistar rats (body weight 158-220 g). Male Donryu rats (body weight 153-207 g) underwent SC im- plantation of Yoshida sarcoma (2 × 108 cells/0.5 ml) into the right thigh. Donryu rats underwent SC implantation of hepatoma AH109A(2 × 108 cells/0.5 ml) into the right thigh. An appropriate amount of radioactive nuclide was administered 6-7 days later, at which time the tumor had grown to 1.5-2.0 cm in diameter. Male ddY mice (body weight 32-36 g) received subcutaneous transplantation of Ehrlich tumor (5 x l07 cells/0.1 ml) into the right thigh. These mice were used in experiment 7-10 days later, at which time the tumor had grown to about 1 cm in diameter.

67Ga-citrate solution (10-20 gCi/ml) was prepared from carrier-free 67Ca-citrate solution (Daiichi Radio-Isotope Laboratories Ltd., Japan) and 0.08 M sodium citrate solu- tion. Carrier-free sodium sulfate-35S solution, pH 6.0-8.0 (800gCi/ml) was prepared from sulfuric acid-35S in 0.05 N HC1 solution (Japan Atomic Energy Research [nsti-

tute, Japan) and 0A N NaOH solution, and was adjusted with NaC1 solution to the correct osmotic pressure for intra- venous injection into the animals.

Methods

Tumor and liver uptake of 67 Ga

67Ga-citrate solution (0.4 ml) was injected IV into the rats and IP into the mice. These animals were killed at various time intervals from 10 rain to 10 days (Fig. 1) after the ad- ministration of 67Ga_citrate ' Liver was excised from normal rats and tumor was excised from tumor-bearing animals. Thes e tissues were weighed and counted on a well-type scin- tillation counter (Aloka JDC-701). The radioactivity in these tissues was expressed as a percentage of the injected dose per g tissue (% dose/g). This value was normalized to a body weight (BW) of 100 g by multiplying by BW/100.

Subcellular distribution o f 67 Ga in tumor and liver

67Ga-citrate solution (0.4 ml) was injected IV into the rats and IP into the mice. These animals were killed at various time intervals from 10 rain to 10 days (Fig. 2) after the ad- ministration of 67Ga-citrate. Liver was excised from normal animals and tumors were excised from tumor-bearing ani- mals. As described previously (Ando et al. 1982c), these tissues were homogenized in cold (5 ° C) 0.25 M sucrose containing 0.01 M Tris-HC1 buffer, pH 7.6 (10% W/V) in a Potter-Elvehjem type homogenizer. According to the modified method (Hogeboom 1955) of Hogeboom and Schneider, subcellular fractionation was carried out at 4 ° C. Fractions from the centrifugation were assayed for 67Ga using the scintillation counter.

Distribution of 67Ga in tumor tissues

6VGa-citrate solution (0.4 ml) was injected IP into the mice and IV into the rats implanted with Yoshida sarcoma. The tumor tissues were excised at 3 h, 24 h, and 48 h after ad- ministration of 67Ga-citrate. The tissues were frozen imme- diately after excision in n-hexane ( - 70 ° C) cooled with dry ice + acetone. The frozen tumor tissues were then cut into serial thin sections (10 gm) in a cryostat ( - 2 0 ° C). One of these sections was placed on X-ray film and this film was developed after exposure of several days. Every second section was fixed in ethanol, and was stained with hematox- ylin + eosin.

6 7 Ga binding substances in tumor and liver

Preparation of subcellular fractionation, digestion, and gel filtration. As mentioned above, 67Ga-citrate solution (0.4 ml) and sodium sulfate-35S (0.4 ml) were injected into the above animals separately. Twenty-four hours later, tu- mor was excised from tumor-bearing animals, while liver was excised from normal animals 3 h after administration. The tissues were rinsed in 0.9% NaC1 solution.

Tumor tissues were homogenized with 10 volumes of 0.15 M KC1 containing 0.01 M Tris-HC1 buffer (pH 7.6) in a Potter-Elvehjem type homogenizer. The homogenates were centrifuged for 15 rain at 400 g and the sediments (cell debris and nuclear fraction) were discarded. The homoge- nates (7 ml) from which the nuclear fractions were removed,

263

were adjusted to pH 7.8 8.2 with 0.1 M NaOH and were incubated with 60 mg proteinase (pronase E, Kaken Chemi- cal Co., Japan) at 37 ° C. After 24 h, the reaction mixture was adjusted again to pH 7.8 8.2 with 0.1 M NaOH, and 30 mg pronase E was added to each. They were further incubated for 24 h at 37 ° C. The liver was homogenized and subcellular fractionation was carried out according to the modified method of Hogeboom and Schneider as indi- cated above. Mitochondrial fractions (lysosomes are con- tained in these fractions), microsomal fraction, and super- natant fraction were separately incubated with pronase E by the same method as that performed on tumor tissue. After digestion, the reaction mixtures were centrifuged at 3000 rpm (1500 g) for 20 min, and the sediments were dis- carded. The supernatants (5-ml aliquots) were applied to a column of Sephadex G-100 particle size 40-120g (1.2 x 75 cm) that had been equilibrated with 0.15 M NaC1 containing 0.1 M acetic acid-sodium acetate buffer, pH 5.0. The radioactivity was eluted with the same buffer (120 ml). The flow rate was 0.3 ml/min. Fractions (3 ml) were collect- ed to measure radioactivity, uronic acids, protein, and sialic acids.

Measurements of substances in eluates. Radioactivity of 67Ga in the eluates was measured by the scintillation counter, and that of 35S was measured by a liquid scintilla- tion counter (Aloka, LSC-673). Protein was determined by the method of Lowry et al. (1951). Uronic acids and acid mucopolysaccharides were measured by the orcinol reaction (Brown 1946), while sialic acids were assayed by the thio- barbituric acid method (Aminoff 1961).

Results

Tumor and liver uptake o f 6 7 Ga

Figure 1 shows the retention values (% dose/g) for 67Ga in Ehrlich tumor, Yoshida sarcoma, hepatoma AH109A, and normal rat liver at various time intervals from 10 min to 10 days. In the cases of three tumors, the retention values were at their the highest 24 h after administration of 67Ga- citrate and then proceeded to decrease rapidly.

That in normal liver increased until 48 h after injection, and then decreased slowly. The main cause for the excretion of 67Ga from tumor tissues would be due to the expansion of necrotic tumor tissue.

Subcelhdar distribution of 6 7 Ga in tumor and liver

When the 67Ga activity in the nuclear fraction, mitochon- drial fraction (containing lysosome), microsomal fraction, and supernatant fraction, was expressed as A(cpm), B (cpm), C (cpm), and D(cpm), respectively, the 67Ga (per- centage) of the nuclear fraction can be calculated by the following formula:

A A + B + C + D x 100 (%)

the 67Ga of mitochondrial fraction, microsomal fraction, and supernatant fraction was calculated by substitution of A with B, C, and D in the numerator.

67Ga for both mitochondrial and supernatant fractions of the normal rat liver sample are shown in Fig. 2. In liver, 67Ga in the mitochondrial fraction rapidly increased with time after the administration of 67Ga-citrate. After 24 h,

264

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i0.5 O a.

.L-

,o.h,da..rco°a ,,, ;T2om':°2.0..

i 3hrs T i m e a f te r in ject ion 9days10

Fig. l . Uptake rates for 6VGa in normal liver, Yoshida sarcoma, hepatoma AH109A, and Ehrlich tumor. Uptake rates for 6VGa in tissues are expressed as % of administered dose per gram tissue weight. Tissue values are normalized to a body weight (BW) of 100 g by multiplying by BW/100. Each value is a mean value of data from five animals

Ehrlich tumor Yoshida sarcoma

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411 4(

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Hepatoma AHI09A °/o

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Normal liver

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Fig. 2. Distribution of 6VGa (%) between mitochondrial (containing lysosome) and supernatant fractions. It is shown that 67Ga was transported from supernatant fraction to mitochondrial fraction. Transposition of 67Ga from supernatant to mitochondrial fraction readily occurred in the liver but only slightly in Ehrlich tumor and Yoshida sarcoma

about 60% of the total 6VGa was accumulated in this frac- tion, and this value was hardly altered for 10 days. Cont rary to da ta for the mi tochondr ia l fraction, 67Ga of the superna- tant fraction rapidly decreased with time until 24 h after administrat ion, and this value was hardly altered over the succeeding 10 days. But in the cases of Yoshida sarcoma and Ehrlich tumor, most of the 67Ga was localized in the supernatant fraction, with only a small amount o f 67Ga

localized in the mi tochondr ia l fraction. Concerning the ac- cumulat ion of 67Ga in the mi tochondr ia l fraction and the decrease from the supernatant fraction, hepa toma AH109A indicated intermediate values between liver and the two tu- mors studied•

It is concluded that the lysosome does not play a major role in the tumor concentrat ion of 67Ga, al though it plays an impor tan t role in the liver concentrat ion of 67Ga. In

A : Macroautoradiogram

B: Hematoxy l in -Eos in staining

C: Ske tch i l lus t ra t ion [ ~ ]

Viable tumor tissue

Tissue containing viable and necrotic tumor tissue

Necrotic tumor tissue

Connective tissue(contain inflammatory tissue)

265

Ehrlich tumor

24hrs after injection of 67Ga-citrate

Yoshida s a r c o m a

3hrs after injection of 67Ga-citrate

A B C A B C

Fig. 3. Morphological specimens. Concentration of 67Ga was predominant in connective tissue rather than in viable tumor tissue

the case of hepatoma AH109A, it is presumed that the lysosome plays quite an important role in the tumor concen- tration of 67Ga, hepatoma AH109A possessing some resid- ual features of liver. From these points of view, it is pre- sumed that the lysosomal role in the accumulation of 67Ga in liver cells diminishes with the transformation of liver cells into malignant tumor cells.

Distribution o f 67 Ga in tumor tissues

Hematoxylin + eosin stained sections were classified into the following four categories: Viable tumor tissue, tissue con- taining viable and necrotic tumor tissue, necrotic tumor tissue, and connective tissue which contains inflammatory tissue. The distribution of those four groups is indicated in the sketch illustration. A typical autoradiogram concern- ing two tumors is indicated in Fig. 3. In the case of Ehrlich tumor injected with 67Ga-citrate, the concentration of 67Ga was predominant in connective tissue (especially inflamma- tory tissues) rather than in viable tumor tissue, regardless of time after administration. 67Ga was not seen in necrotic tumor tissue. In Yoshida sarcoma treated with 67Ga-citrate, the accumulation of 67Ga in the tissue was similar to that seen in Ehrlich tumor. But there were some cases where the concentration of 67Ga in inflammatory tissue was less than in viable tumor tissue.

67 Ga binding substances in tumor and liver

For Yoshida sarcoma, radioactivity from 59%-67% 67Ga remained in the supernatant on centrifugation, after diges- tion with pronase E. Figure 4 indicates gel filtration of the supernatant that was produced from Yoshida sarcoma by pronase E treatment. The eluates containing 6 VGa produced three peaks. Considering the relation between the molecular weight and the fraction number in the Sephadex G-100 column, the first peak of 67Ga was eluted in the void vol- ume, the third peak was eluted with low molecular weight substances, and the second peak was eluted with intermedi- ate molecular weight substances. In Yoshida sarcoma, 19.2%, 19.5%, and 50.8% of 67Ga applied to the Sephadex G-100 column were eluted in the first, second, and third peak, respectively. Uronic acids, protein, and sialic acids

in the eluates are also indicated in this figure. As described in the cited references (Ando et al. 1982b and 1983), the first peak contained a species whose molecular weight ex- ceeded 40000, the second peak consisted of substances with a molecular weight of about 10000, and radioactivity in the third peak was liberated 67Ga. The results of Sephadex G-100 gel filtration on Yoshida sarcoma treated with sodi- um sulfate-35S are also indicated in this figure. Three peaks for 67Ga were identical with peaks for 35S, respectively. In the case of Ehrlich tumor and hepatoma AHI09A, simi- lar results were obtained.

In the cases of the three liver fractions mentioned below, 64% 87% of 67Ga remained in the supernatant on centrifu- gation after digestion with pronase E. Figure 4 shows the gel filtration of the supernatants which were produced from the mitochondrial fraction, microsomal fraction, and super- natant fraction through pronase E treatment. In the mito- chondrial fraction, 10.3%, 25.5%, and 45.9% of 67Ga ap- plied to a Sephadex G-100 column were eluted in the first, second, and third peaks, respectively. Those values for the microsomal fraction were 9.4%, 22.8 %, and 44.1%, respec- tively, and for the supernatant fraction 3.7%, 18.1%, and 59.3%, respectively. Uronic acid, protein, and sialic acids in the eluates are also indicated in this figure. The results of Sephadex G-100 gel filtration on normal rat liver treated with sodium sulfate-35S are indicated in this figure. As shown, the three peaks of 67Ga were identical with the peaks of 35S, respectively. It is presumed that the sulfur-35S content of the first (fraction number 10-14) and second (fraction number 18-20) peaks was comprised of sulfated acid mucopolysaccharides. The third 3sS peak (fraction number 23-25), would be a component of the sulfated car- bohydrate chain of sulfated glycoprotein, as this peak is identical to that of sialic acids. The fourth 35S peak was free sulfate. As previously reported (Ando and Ando 1983a), it is reasonable that 67Ga in the second peak (mol.wt. about 10000) was bound to acid mucopolysaccha- rides (keratan polysulfate, etc.) which contained no uronic acid, as the second peak 67Ga was not identical to the peak produced by the orcinol method.

In the case of Yoshida sarcoma and liver, it was deduced that 67Ga-binding acid mucopolysaccharides in the second peak are keratan polysulfate for the reason described above. In the case of Ehrlich tumor and hepatoma AH109A, simi- lar results were obtained.

266

Yoshida sarcoma i ~ a act iv i ty

~S act iv i ty

Mat ochondr ia l , f ract ion

= ; 355 act iv i ty

" - - - - " Orcinol method

0 o Lowr y's method

O u ¢ m J:

, Q <

110 t5 20 25 30 35 t0 15 20 25 30 35 Fract ion number Fract ion number

i Microsomal fraction / ~ : : - 17Ga activi ty

-- : SSS act iv i ty / \ Superna tan t f rac t i on

. . . . Orc no m.th~ I k ; " O . . c . v t y O O Lowry's method .L ~. = : 3$s activity M

" [ o D Thiobarbituric a C i d m e t h o d f . . . . Orcinol method / ~

D o Thiobarbituric acid / method .J. .~L

"tk,! i /"%o "°"o, I t l k ~ . . . . f ; ; :" 1 .o "5 . \ ~ ' <~ , , ,

10 15 20 2s 30 35 to 15 ze 25 3e 3S Fract ion number Fract ion number

Fig. 4. Sephadex G-100 chromatography profiles for Yoshida sarcoma, mitochondrial fraction, microsomal fraction, and supernatant fraction of liver digested with pronase E at 37 ° C for 48 h

267

Tumor cell Liver cell

s o

6~Ga Acid mucopolysaccharide Protein

Liver tissue Tumor t i ssue

• i ! if" :

IIs

b Inflammatory/ INecrotic tumor infiltration tissue

Fig. 5a, b. Schematic models for tumor and liver cells, and the tissues of tumor and liver on 67Ga accumulation, a In liver cells, 67Ga is transported into lysosome with acid mucopolysaccharide. The lysosomal role in the accumulation of 67Ga in liver cells diminishes with the transformation of liver cell into malignant tumor cell. b 67Ga (black dots) is avidly concentrated into inflammatory infiltration around tumor cells and is hardly accumulated in necrotic tumor tissue. But in liver, a large amount of 6VGa is concentrated in liver cells

Schematic models of tumor and liver cells

From our results and those in the cited references, the fol- lowing conclusions are obtained concerning cell uptake of 67Ga in tumor and liver as shown in Fig. 5. (1) 67Ga is concentrated into the lysosome, from cytoplasm of liver cells progressively after administration. (2)The lysosomal role in the accumulation of 67Ga in liver cells diminishes with the transformation of liver cells into malignant tumor cells. (3) In tumors (except for hepatoma) the lysosome does not play a major role in the tumor concentration of 67Ga. (4) 67Ga is bound to acid mucopolysaccharides (keratan polysulfate, etc.) in tumor and liver, and is transported into lysosome with these acid mucopolysaccharides. These con- clusions are supported by the report of Aronsen and David- son (1968) that rats treated by injection of the radioactive acid mucopolysaccharides (35S-chondroitin sulfate A, C, 3H-chondroitin sulfate B) have been shown to be capable of concentrating about 1% of these materials within their liver lysosomes, and is also supported by the 'idealized ver- sion of the assemblage of glycolipids and glycoproteins in surface membranes ' of Hakomori (1975).

Considering that some of the affinity of 6VGa to lyso- some in hepatoma AH109A is due to the residual liver characteristics possessed by hepatoma AHI09A, the extent of the variation of hepatoma from liver may be determined by using this phenomenon as an indicator.

Schematic models for tumor and liver tissues

The models shown in Fig. 5 for the tissue uptake of 67Ga in tumor and liver, are obtained for the following reasons. It was observed by Hayes et al. (1970) and by us (Ando et al. 1977) that 67Ga is concentrated in viable tumor tissue, but is almost absent from necrotic tumor tissue. It has been shown by us that 67Ga is avidly concentrated into inflam- matory infiltration around tumor cells, as is described above. Considering the abundant concentration of 67Ga in liver tysosome, it is obvious that plenty of 6VGa exists in liver cells but very little is found in connective tissue around the liver cells.

Discussion

Since Edwards and Hayes reported that 67Ga-citrate local- ized in tumor, the mechanism for 67Ga localization in malignant tumor has been extensively investigated. Trans- ferrin, ferritin, 45000 molecular weight glycoprotein, and lactoferrin were reported as 6VGa-binding substances in tis- sues. Most of the currently popular proposed mechanisms for 67Ga localization assume that 67Ga acts in part as an iron analog. But we proposed that the binding of 67Ga to substances in the body should not be considered physio- logically but chemically, because iron is an essential element for animals, while Ga is not essential. Ga can be a trivalent cation in solution, and naturally, this cation can bind strongly to cation-exchange resins (Ando et al. 1974) and heparin (Ando et al. 1983) in solution, although Ga is pres- ent as negatively charged complexes under certain circum- stances. Among body constituents, the strongest cation-ex- change resin-like substances are sulfated acid mucopolysac- charides which have sulfonic groups in their structures. Under these circumstances, it was reported by Ando et al. (1980, 1982a, 1983) that a 67Ga-binding acid mucopolysac- charide had been separated by cellulose acetate electrophor- esis from tumor and liver, and that 67Ga-binding acid mu- copolysaccharides in tumor and liver were very similar. Later, our results were supported by in vitro experiments of Kojima et al. (1982).

Since localization of 67Ga in inflammatory lesions was shown by Lavender et al. (1971), many investigators re- ported that 67Ga was concentrated in inflammatory lesions. The mechanism for localization of 67Ga in inflammatory tissue was considered to be controlled by increased vascu- larity, protein accumulation (67Ga bound transferrin), la- beled leukocytes, and microorganisms. But we think that 67Ga is bound to the sulfated acid mucopolysaccharides in inflammatory tissue, because fibroblast in inflammatory infiltration produces a large amount of sulfated acid muco- polysaccharides as the intercellular substances in inflamma- tory tissue (Ando et al. 1977).

Models have been proposed concerning 67Ga accumula- tion in tumor, liver, and inflammatory lesion by Larson (1978) 'A proposed mechanism for 67Ga uptake into tumor

268

cells', Hayes et al. (1981) 'Proposed scheme of initial entry of 67Ga into tumor and normal tissues', and by Hayes et al. (1982) 'Schematic model of two basic pathways that may be involved in uptake of 67Ga by soft-tissue tumors, normal soft-tissues, and inflammatory processes'. Contrary to their models, we persist in the rationality of our own, based on the data presented here and cited in the references.

In clinical tumor scanning with 67Ga-citrate, at tention should be paid to the extent of necrotic tumor tissue and to the degree of inflammatory infiltration around the tu- mor.

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Received April 30, 1984 / July 20, 1984