Studies on the67Ga uptake mechanism by Ehrlich ascites tumor cells

In order to obtain more information concerning the mechanism of Gallium (⁶⁷Ga) accumulation in malignant tissue, an investigation was carried out using Ehrlich ascites tumor cells. Chlorpromazine, a phospholipid stabilizer decreased the uptake of⁶⁷Ga and Calcium 45 (⁴⁵Ca) by the cells at low dose, but increased them at high dose. On the other hand, the uptake of both radionuclides by the cells was inhibited by ruthenium red, a Ca ATPase inhibitor in a dose dependent manner. The time course of⁶⁷Ga and⁴⁵Ca uptake were quite different from each other. Moreover, the subcellular distribution patterns of⁶⁷Ga and⁴⁵Ca were also different from each other;⁶⁷Ga accumulated in the lysosomal fraction and⁴⁵Ca mainly in the mitochondrial fraction. These results suggest that there may be a commonality for⁶⁷Ga and⁴⁵Ca uptake by the tumor cells, whereas the behaviour of these two radionuclides in the cells is dissimilar.     

In vitro binding of 67Ga to Ehrlich ascites tumor cells

The binding of ⁶⁷Ga to Ehrlich ascites tumor cells (ETC) was studied in vitro. Acid mucopolysaccharide (AMPS) present at the cell surface of ETC was identified as heparan sulfate (HS). The extent of ⁶⁷Ga binding to ETC reached a plateau (ca. 10% of the added dose) at 1–2 h after the start of incubation. The binding was higher under neutral or alkaline conditions than under acidic conditions. Heparin and heparitinase treatment both significantly decreased the extent of ⁶⁷Ga binding to ETC. Mild treatment with protease, including trypsin or papain, also decreased the binding. On the contrary, the treatment with trypsin under severe conditions markedly increased the extent of ⁶⁷Ga binding to ETC. These results support the hypothesis that HS plays an important role as a ⁶⁷Ga receptor in the mechanism of gallium binding to ETC.     

67Ga uptake and heparan sulfate content of Ehrlich solid tumor in mice

The relationship between ⁶⁷Ga uptake and heparan sulfate (HS) content in Ehrlich solid tumor (EST) of mice was investigated, and the effect of cyanomethylamine, papain, streptozotocin, or bleomycin pretreatment on ⁶⁷Ga uptake in EST was studied. ⁶⁷Ga uptakes in EST and kidney were much higher than other tissues, and these tissues also contained large amounts of HS. ⁶⁷Ga uptakes and HS synthesis in the EST were inhibited by pretreatment with cyanomethylamine or papain (inhibitors of fibrosis). Parallel reductions of ⁶⁷Ga uptake and HS synthesis in EST were observed in EST transplanted into streptozotocin-induced diabetic mice. The weight of EST in the bleomycin-injected group was decreased to less than half of the control, but no effect was observed on ⁶⁷Ga uptake per gram of EST. These results suggest that ⁶⁷Ga uptake in the tumor and inflammatory lesions are related to the quantity of HS in these tissues, and the correlation between the uptake of ⁶⁷Ga and the rate of cellular proliferation is secondary.     

67Ga uptake and heparan sulfate content of Ehrlich solid tumor in mice

The relationship between 67Ga uptake and heparan sulfate (HS) content in Ehrlich solid tumor (EST) of mice was investigated, and the effect of cyanomethylamine, papain, streptozotocin, or bleomycin pretreatment on 67Ga uptake in EST was studied. 67Ga uptakes in EST and kidney were much higher than other tissues, and these tissues also contained large amounts of HS. 67Ga uptakes and HS synthesis in the EST were inhibited by pretreatment with cyanomethylamine or papain (inhibitors of fibrosis). Parallel reductions of 67Ga uptake and HS synthesis in EST were observed in EST transplanted into streptozotocin-induced diabetic mice. The weight of EST in the bleomycin-injected group was decreased to less than half of the control, but no effect was observed on 67Ga uptake per gram of EST. These results suggest that 67Ga uptake in the tumor and inflammatory lesions are related to the quantity of HS in these tissues, and the correlation between the uptake of 67Ga and the rate of cellular proliferation is secondary.     

67Ga and 59Fe uptake by tumor cells treated with hyperthermia or hyperthermia plus lanthanum

The uptake of 67Ga-citrate and 59Fe-citrate by tumor cells treated with hyperthermia or with hyperthermia plus lanthanum has been studied. The comparison of these results with ion flux determinations (42K) appears to indicate that hyperthermia alone or combined with lanthanum modifies the ionic permeability of the plasma membrane. This phenomenon seems to be responsible for the increased accumulation of 67Ga and 59Fe.     

The mechanism of 67Ga uptake in animal and human tumours

The subcellular distribution of ⁶⁷Ga has been studied by differential centrifugation in 3 transplantable mouse tumours, 3 transplantable rat tumours, 1 dog tumour, 3 human tumour xenografts and 2 human tumours in situ at various times after injection of the citrate complex. From 24 h post injection the nuclide was located predominantly in lysosomal structures in all the tumours studied. Studies in two murine tumours showed marked differences in the rate of lysosomal accumulation of ⁶⁷Ga. In the ADJ/PC6 plasmacytoma lysosomal uptake of ⁶⁷Ga had reached a plateau within 15 min while in the S180 tumour lysosomal accumulation of the nuclide occurred over the first 24 h. Normal mouse liver showed a similar pattern to this latter tumour. It is postulated that these variations in the rate of lysosomal accumulation of ⁶⁷Ga reflect differences in the permeability of the lysosomal membrane. While large amounts of ⁶⁷Ga were found in the crude nuclear fraction of some tumours this was attributed to unbroken cells as studies with purified nuclei from 7 different tumours indicated that between 2 and 14% of the total tumour ⁶⁷Ga was associated with the nuclei.     

Relative tumour enhancement of 67Ga uptake by desferrioxamine

The administration of the iron- and gallium-chelating agent, desferrioxamine (DFO), to mice after the injection of 67Ga has been shown to improve tumour to normal tissue ratios. Time and dose parameters have been studied. The optimal time was about 7 h after 67Ga, when the tumour to blood ratio was increased 16 times compared with controls. The effect was due to the greater retention of 67Ga in tumour compared with other tissue. Tumour retention after DFO was 78% compared with controls at 7 h, giving a tumour to blood ratio of 25.9. Tumour retention of 67Ga correlated well with the proportion of cell-bound activity which was localised in the lysosomes. Prior administration of iron increased lysosomal accumulation of 67Ga in tumour cells, and hence the retention of the nuclide after the use of DFO.     

Relative tumour enhancement of67Ga uptake by desferrioxamine

The administration of the iron- and gallium-chelating agent, desferrioxamine (DFO), to mice after the injection of⁶⁷Ga has been shown to improve tumour to normal tissue ratios. Time and dose parameters have been studied. The optimal time was about 7 h after⁶⁷Ga, when the tumour to blood ratio was increased 16 times compared with controls. The effect was due to the greater retention of⁶⁷Ga in tumour compared with other tissue. Tumour retention after DFO was 78% compared with controls at 7 h, giving a tumour to blood ratio of 25.9. Tumour retention of⁶⁷Ga correlated well with the proportion of cell-bound activity which was localised in the lysosomes.Prior administration of iron increased lysosomal accumulation of⁶⁷Ga in tumour cells, and hence the retention of the nuclide after the use of DFO.