A transferrin-mediated uptake of gallium-67 by EMT-6 sarcoma. I. Studies in tissue culture

We have studied the in vitro uptake of gallium-67 by exponentially growing EMT-6 sarcoma cells in long-term tissue culture. In this system, the addition of transferrin to the medium was required before an appreciable cellular uptake of Ga-67 occurred. The transferrin effect was complex, with an initial stimulation to a peak cell-to-medium ratio of 8--10:1 at low concentrations of transferrin (0.2 mg/ml), followed by a gradual decline in uptake as transferrin in the medium was increased further. EMT-6 tumor-cell uptake of Ga-67 was probably mediated by a specific cellular receptor for transferrin. Scatchard analysis of the EMT-6 cellular binding of human transferrin labeled with iodine-125 indicated a cellular receptor with affinity for transferrin of 5 X 10(6) l/mole and abundance of 500,000 receptors per cell. Over the experimental range of transferrin concentration in the medium, the observed uptake of Ga-67 was closely correlated with the degree of formation of Ga-67-labeled transferrin and the fraction of transferrin bound to the cellular receptor (N = 69, r = 0.86, p less than 0.0001).     

Preparation, biodistribution, and small animal PET of 45Ti-transferrin.

Investigation of 45Ti-transferrin was pursued to provide insight into the mechanism of action of titanocene dichloride, a chemotherapeutic agent currently in clinical trials. METHODS: Plasma protein-binding studies of processed 45Ti were performed by solubilizing the 45Ti residue in 0.05N HCl, of which 1.22 MBq (33 microCi) in 10 microL were added to 250 microL of dog plasma. 45Ti-Transferrin was prepared by redissolving the processed 45Ti in 25 micromol/L apotransferrin or by in vivo incorporation through preparation and introduction of 45Ti-citrate. Biodistribution studies were performed on normal Sprague-Dawley rats and EMT-6 tumor-bearing BALB/c mice with 45Ti-transferrin coinjected with 67Ga-citrate for direct comparison. microPET was performed on mice bearing EMT-6 tumors and the images were analyzed for tumor-to-muscle uptake ratios. RESULTS: Direct labeling of apotransferrin in situ with 45Ti was achieved as well as in vivo incorporation by 2 h after injection with 45Ti-citrate. The biodistribution of 45Ti-transferrin and 67Ga-citrate showed similar trends. In Sprague-Dawley rats, initial blood uptake was higher for the 45Ti-transferrin, whereas bone uptake increased more for the 67Ga-citrate. EMT-6 tumor uptake in both cases was relatively high (14.6 +/- 1.83 %ID/g for 45Ti and 8.72 +/- 0.98 %ID/g for 67Ga [%ID/g = percentage injected dose per gram]) and remained elevated even out to 24 h after injection. The tumor-to-muscle ratio of the 67Ga-citrate reached 6.7 at 24 h, whereas the ratio of the 45Ti-transferrin increased to 4.3 at this time point. Uptake of 45Ti-transferrin was visualized in the EMT-6 murine mammary carcinoma tumor with microPET. In all cases, the tumor was clearly delineated from the surrounding tissue with tumor-to-muscle ratios on the order of 1.6. CONCLUSION: 45Ti forms a complex with apotransferrin that remains intact in vivo. Results of the biodistribution in mice showed that the tumor had increased uptake compared with nontarget organs (e.g., muscle). The microPET images of tumor-bearing mice clearly delineate the tumors from the surrounding tissue. Comparison of the data suggests that tissue uptake is similar whether injecting 45Ti-transferrin directly or as 45Ti-citrate, which transchelates to transferrin before the time of imaging.     

Ga-67 and Fe-59 distributions in mice

Tissue distributions of i.v.-injected Ga-67 citrate and [59Fe] ferric citrate were measured in normal mice and in lymphoid-tumor hosts. The study arose out of previously reported tissue-culture work showing marked transferrin stimulation of Ga-67 and Fe-59 uptakes by cultured cells from mouse lymphoid tumors. In vivo, however, no obvious correlation was found between Ga-67 and Fe-59 tissue distributions; indeed, Ga-67 showed high affinity for tumor tissue and low affinity for hemopoietic tissues, while for Fe-59 the reverse applied. Taken together, these comparisons of kinetics and distributions for Ga-67 and Fe-59 suggest that a tissue's avidity for Ga-67 is strongly influenced by other factors besides the cell population's capacity for transferrin interactions.     

Investigation into 64Cu-labeled Bis(selenosemicarbazone) and Bis(thiosemicarbazone) complexes as hypoxia imaging agents

BACKGROUND: Cu-diacetyl-bis(N4-methylthiosemicarbazone) [Cu-ATSM], although excellent for oncology applications, may not be suitable for delineating cardiovascular or neurological hypoxia. For this reason, new Cu hypoxia positron emission tomography (PET) imaging agents are being examined to search for a higher selectivity for hypoxic or ischemic tissue at higher oxygen concentrations found in these tissues. Two approaches are to increase alkylation or to replace the sulfur atoms with selenium, resulting in the formation of selenosemicarbazones. METHODS: Three 64Cu-labeled selenosemicarbazone complexes were synthesized and one was screened for hypoxia selectivity in vitro using EMT-6 mouse mammary carcinoma cells. Rodent biodistribution and small animal PET images were obtained from BALB/c mice implanted with EMT-6 tumors. One alkylated thiosemicarbazone was synthesized and examined. RESULTS: Of the three bis(selenosemicarbazone) ligands synthesized and examined, only 64Cu-diacetyl-bis(selenosemicarbazone) [64Cu-ASSM] was isolated in high-enough radiochemical purity to undertake cell uptake experiments where uptake was shown to be independent of oxygen concentration. The bis(thiosemicarbazone) complex synthesized, 64Cu-diacetyl-bis(N4-ethylthiosemicarbazone) [64Cu-ATSE], showed hypoxia selectivity similar to 64Cu-ATSM although at a higher oxygen concentration. Biodistribution studies for 64Cu-ASSM and 64Cu-ATSE showed high tumor uptake at 20 min (64Cu-ASSM, 10.33+/-0.78% ID/g; 64Cu-ATSE, 7.71+/-0.46% ID/g). PET images of EMT-6 tumor-bearing mice visualized the tumor with 64Cu-ATSE and revealed hypoxia selectivity consistent with the in vitro data. CONCLUSION: Of the compounds synthesized, only 64Cu-ASSM and 64Cu-ATSE could be examined in vitro and in vivo. Although the stability of bis(selenosemicarbazone) complexes increased upon addition of methyl groups to the diimine backbone, the fully alkylated species, 64Cu-ASSM, demonstrated no hypoxia selectivity. However, the additional alkylation present in Cu-ATSE modifies the hypoxia selectivity and in vivo properties when compared with Cu-ATSM.     

Administered dose and tumor dose of bleomycin labeled with cobalt-57 in mice and men.

Tumor concentrations of the chemotherapeutic drug, bleomycin, labeled with cobalt-57 (Co-bleo) were compared in mouse tumor models and in human lung tumors using quantitative single-photon emission computed tomography. Drug concentrations in histologically similar human tumors showed marked variability for the same injected dose (ID). Small cell carcinomas showed concentrations between 1.09 and 8.85 %ID/cc x 10(-3) while non-small cell lung tumors showed a concentration variation between 0.36 and 6.75 %ID/cc x 10(-3). In contrast to the situation in human tumors, uptake in mouse tumors showed only slight variability in animals with the same tumor model. EMT-6 tumors in mice showed at 6 hr significantly higher uptake of Co-bleo (p less than 0.001) and significantly higher tumor-to-lung ratio (p less than 0.001) when compared to murine fibrosarcomas. The EMT-6 tumors in contrast to the fibrosarcomas responded to bleomycin treatment in a dose dependent manner. The results indicate that while in mice the tumor dose closely follows the administered dose, in humans, the tumor dose and the tumor-to-lung ratio in the individual patient cannot be predicted from the administered dose.     

Intense Ga-67 uptake in adenosquamous carcinoma of the pancreas

Gallium-67 citrate (Ga-67) scintigraphy was performed in a patient with adenosquamous carcinoma of the pancreas. Intense and homogeneous uptake was observed in the tumor. Few reports have dealt with Ga-67 findings in pancreatic cancers. Ga-67 uptake in the tumor was assumed to be due to accumulation in the component of squamous cell carcinoma. This case suggested that Ga-67 citrate scintigraphy may be useful in detecting adenosquamous carcinoma of the pancreas. To our knowledge, no report has described findings of Ga-67 citrate scintigraphy of adenosquamous carcinoma of the pancreas. Radiologists should remember adenosquamous carcinoma of the pancreas when encountering such scintigraphic findings.     

Radioiodinated 1-(5-iodo-5-deoxy-beta-D-arabinofuranosyl)-2-nitroimidazole (iodoazomycin arabinoside: IAZA): a novel marker of tissue hypoxia

1-(5-Iodo-5-deoxy-beta-D-arabinofuranosyl)-2-nitroimidazole (IAZA) has been synthesised and labeled with 125I. Radioiodinated IAZA was shown to undergo hypoxia-dependent binding in EMT-6 cells in vitro and to have an initial binding rate of 284 pmole/10(6) cells/hr at a substrate concentration of 30 microM. This binding rate is more than three times that of the reference compound, misonidazole (89 pmole/10(6) cells/hr). The elevated binding rate was accompanied by in vitro cytotoxicity 30-40 times greater than that observed for misonidazole. Whole-body elimination and biodistribution studies in BALB/c mice bearing implanted, subcutaneous EMT-6 tumors showed a rapid excretion (greater than 98% in 24 hr) with moderate tissue levels which, in general, declined as a function of blood clearance. Tumor-to-blood ratios of 4.6 (4 hr) and 8.7 (8 hr), with respective tumor uptake values of 2.08% and 1.22% ID/g of tissue, form a rational basis for evaluation of this and related 2-nitroimidazole analogs as radiopharmaceuticals suitable for scintigraphic evaluation of tissue (tumor) hypoxia.     

The effect of amphotericin B on 67Ga biodistribution

The effect of Amphotericin B (AMB) on radiogallium biodistribution in mice was studied at various time intervals. Two doses of AMB (1 mg/kg and 10 mg/kg) were administered intraperitoneally into mice bearing EMT-6 mammary sarcomas. The tumor and tissue uptakes of 67Ga were calculated when the radioactivity was administered either as 67Ga-citrate or 67Ga-transferrin (Ga-Tf). The role of transferrin was also investigated by using doubly-labeled 67Ga-125I-Tf. Our results indicate that both the doses of AMB and the chemical form of the radiogallium administered affect the pattern of distribution in some distinct fashion. It was also found that although the administration of AMB results in an increase in tumor-associated transferrin, this does not necessarily result in a concomitant increase in radiogallium uptake. In general, the effects of AMB on various tissues uptake of 67Ga appear to be related to both acute and subacute toxicity.     

Gallium-67 uptake in Bellini duct carcinoma of the kidney

Bellini duct carcinoma is a rare variant of renal cell carcinoma and usually has a poor prognosis. In this article, we report the Gallium-67 citrate (Ga-67) uptake in Bellini duct carcinoma. To our knowledge, this is the second reported case of Bellini duct carcinoma in which Ga-67 uptake was positive. We suggest that Ga-67 scintigraphy has potential utility in detecting Bellini duct carcinoma of the kidney. And if a hypovascular tumor of the kidney shows Ga-67 uptake, Bellini duct carcinoma should be included in the differential diagnosis.     

Positron imaging feasibility studies: selective tumor concentration of 3H-thymidine, 3H-uridine, and 14C-2-deoxyglucose

The potential usefulness of substrates for glycolysis and nucleic acid synthesis as tumor imaging agents was compared to that of 67Ga-citrate. In separate experiments, 3H-thymidine, 3H-uridine, 14C-2-deoxyglucose, and 67Ga-citrate were injected intraveneously into BALB/c mice with solid subcutaneous EMT-6 sarcomas. For the 3H- and 14C-labeled substrates, absolute uptakes in tumor and tumor-to-blood ratios were as high 1 hour after injection as the comparable maximum values achieved for 67Ga-citrate after 48 hours. These studies suggest that positron-labeled analogues of thymidine, uridine, and 2-deoxyglucose should be useful radiopharmaceuticals for tumor imaging by positron-emission tomography.     

Ga-67 tumor scan in malignant diffuse mesothelioma —Comparison with CT and pathological findings—

Malignant diffuse mesothelioma is characterized by more difficult diagnosis and worse prognosis than other pleural tumors. In the Department of Thoracic Surgery, Hyogo Medical Center for Adults, 11 patients underwent panpleuropneumonectomy for this disease between January, 1988 and March, 1993. In 7 of these cases, Ga-67 scans were obtained before the operation. To clarify the factors affecting Ga-67 uptake in the pleural tumor, we compared Ga-67 uptake on the involved side of the thorax with CT and the pathological findings of the tumor. Regarding the use of Ga-67 scan imaging for the diagnosis of this disease, a number of related findings must be considered, such as an encircled wide Ga-67 uptake in the thickened pleural involvement and a diffuse slight Ga-67 uptake on the affected side with very slight involvement of the pleura. When the involved pleural thickness was over 6 mm, a definite correlation was found between the degree of Ga-67 uptake and the macroscopic thickness of mesothelioma in resected specimens. Thickness of the pleura on CT images demonstrates the real tumor thickness in the case of thickened involvement but in the case of thin involvement the real thickness of active mesothelioma could not be identified. No definite correlation was found between the degree of Ga-67 uptake and the histological type, or among microscopic findings, such as the extent of tumor parenchyma, interstitial volume and tumor vascularity. Our results suggest that the Ga-67 scan is very useful for revealing the extent of pleural involvement, especially when this involvement is more than 6 mm thick.     

Resolution of Ga-67 citrate uptake in the left neck mass of Hodgkin's disease and reversion of double scoliosis of cervical-thoracic and lower lumbar vertebrae

A 6-yr-old boy underwent a total body Ga-67 citrate imaging study because of a large mass of Hodgkin's lymphoma in the left neck and the left anterior chest wall region. The images showed intense uptake in the left neck extending anteroinferiorly to the left upper chest wall corresponding to the left neck and chest region. In addition, there was mild cervical-upper thoracic scoliosis with convexity to the right and mild scoliosis of the lower lumbar scoliosis with concavity to the left. After three cycles of chemotherapy, in the follow-up Ga-67 citrate total body images seven months after his first Ga-67 citrate imaging, the intense uptake in the left neck and the left upper chest wall had been resolved and the scoliosis of the cervical-thoracic and lower lumbar spine had also been reversed to normal. This case shows that a Ga-67 citrate imaging study is useful for first diagnosis and subsequent monitoring of the therapeutic effects in a follow-up imaging. Also Ga-67 citrate imaging provided evidence that the scoliosis had been reversed.     

Scintigraphic visualization of extraosseous xanthomatosis

A histologically proven case of extraosseous xanthomatosis is presented. Both Ga-67 citrate and Tc-99m-MDP imagings demonstrated tumor uptake of radiotracers. A second Ga-67 imaging taken after excisional biopsy of the left knee lesion revealed disappearance of the lesional uptake of the tracer seen on the preoperative Ga-67 scan. Radiotracer accumulation in extraosseous xanthomatosis has not been reported previously.     

Neuroblastoma: imaging evaluation by sequential Tc-99m MDP, I-131 MIBG, and Ga-67 citrate studies

Fourteen children with histopathologically confirmed neuroblastoma underwent sequential correlative imaging studies using I-131 MIBG, Tc-99m MDP, and Ga-67 citrate during various stages of the disease. Of the patients 86% showed I-131 MIBG accumulation in the primary tumoral site, whereas 71% showed Tc-99m MDP and 79% Ga-67 citrate uptake. In 86% at least one of the two latter radiopharmaceuticals concentrated in the primary tumor. The use of all three radiopharmaceuticals raised the detection rate to 93%. Of the osseous or extraosseous metastases 100% were detected by Tc-99m MDP studies. The I-131 MIBG studies were positive in 71% of the osseous metastases and in 70% of the extraosseous metastases. No Ga-67 citrate uptake was demonstrated in osseous metastases, although one extraosseous lung metastasis concentrated this radiopharmaceutical. Tc-99m MDP bone imaging was the best method for diagnosing metastatic spread of the disease and for monitoring the results of treatment. Primary tumor uptake was best indicated by I-131 MIBG. Both Ga-67 citrate and I-131 MIBG were superior to Tc-99m MDP with regard to accurately demonstrating the extent of primary tumors. Only Tc-99m MDP indicated the relationship of these tumors to the kidneys and neighboring osseous structures, providing early screening of kidney compression. Ga-67 citrate study was mainly indicated in tumors with catecholamine depletion, which failed to concentrate the other two radiopharmaceuticals. I-131 MIBG proved especially useful in detecting neuroblastoma with negative Tc-99m MDP and Ga-67 citrate studies and also proved to be helpful with those cases in which I-131 MIBG was planned for therapy. The following strategy is suggested for evaluating neuroblastoma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ga-67 citrate in diagnosing tracheoesophageal fistula in a patient with AIDS. Case report.

Increased radiopharmaceutical uptake in the mediastinum and/or hilum on Ga-67 citrate scan in patients with AIDS has been attributed, to date, mostly to infectious etiologies. Because other complications are being reported in these patients, awareness of their presentation in diagnostic imaging is important. Tracheoesophageal fistula can also be accompanied by a focal area of increased uptake in the mediastinum. In this patient, a Ga-67 citrate scan was the first modality of diagnostic imaging to raise suspicion of such a diagnosis.     

Ga-67 citrate scintigraphy for the early detection of radiation pneumonitis.

To evaluate the usefulness of Ga-67 citrate scintigraphy in the early detection of radiation pneumonitis, scintigrams from 103 patients who were treated with radiation therapy for lung cancer were retrospectively analyzed with regard to abnormal Ga-67 uptake after radiation therapy in sites other than the original or recurrent tumors. Sixteen (20%) of the 80 asymptomatic cases showed positive Ga-67 scintigrams. All of the other 23 symptomatic cases showed positive Ga-67 scintigrams. Thirteen (57%) of these 23 cases had negative chest radiographs although their Ga-67 scintigrams were positive. Detection of radiation pneumonitis by Ga-67 scintigraphy was significantly earlier than that possible by chest radiography (P less than 0.01). These data suggest that Ga-67 scintigraphy is more useful and sensitive than chest radiography for the early detection of radiation pneumonitis.     

Osteoblastic osteosarcoma of the mandible: findings on Tc-99m HMDP bone and Ga-67 citrate scintigraphy

A 25-year-old woman presented with a disturbance in the opening of her mouth 5 months before admission. On admission, painful swelling of the right preauricular region was revealed. Computed tomography (CT) demonstrated a soft tissue density mass around the right condylar process of the mandible. Tc-99m hydroxymethylene diphosphonate (HMDP) bone scintigraphy and Ga-67 citrate scintigraphy showed avid uptake in the mass. The tumor was histologically identified as an osteoblastic osteosarcoma of the right mandible. There are few reports of Ga-67 citrate scintigraphy findings of osteoblastic osteosarcoma of the mandible. The accumulation patterns on Tc-99m HMDP bone scintigraphy and Ga-67 citrate scintigraphy are possibly characteristic of osteoblastic osteosarcoma of the mandible.     

Ga-67 scan to detect intra-abdominal infection in patients after colorectal surgery

PURPOSE: Intra-abdominal infection is still an important issue in colorectal surgery. The Ga-67 scan is relatively inexpensive, but the delay in diagnosis and the intense physiologic bowel activity are considerable drawbacks to its use in abdominal investigations. In this study, the authors performed 24-hour Ga-67 scans and read the images with the surgeon to detect abdominal infection in patients who underwent colorectal surgery. METHODS: Sixty-one patients who underwent colorectal surgery were enrolled in this study. Ga-67 scintigraphy was performed 24 hours, and later as needed, after the injection of 111 MBq (3 mCi) Ga-67 citrate. All scans were interpreted together by a nuclear medicine specialist and the surgeon. The intensity of Ga-67 uptake was recorded as grade 0: no uptake; grade 1: less than liver uptake; grade 2: equal to liver uptake; or grade 3: greater than liver uptake. When the intensity of Ga-67 uptake was equal to or greater than grade 2, the pattern of Ga-67 uptake was classified as changing or unchanging. A positive Ga-67 scan was defined as having an intensity of grade 2 or 3 with an unchanging pattern. RESULTS: Nineteen abdominal infections were diagnosed, all by Ga-67 scans, for a diagnostic sensitivity rate of 100%. Of the 42 patients with no intra-abdominal infection, only 2 had positive Ga-67 scans. This resulted in a specificity rate of 95.2%. The overall accuracy of the Ga-67 scan in the detection of intra-abdominal infection after colorectal surgery was 96.7%. CONCLUSIONS: The Ga-67 scan is useful for detecting intra-abdominal infection after colorectal surgery. Reading the images with the surgeon and using a lateral view can decrease the false-positive rate. A negative Ga-67 scan can rule out intra-abdominal infection, whereas a positive Ga-67 scan indicates the need for further patient evaluation after colorectal surgery.     

Chelate conjugates of monoclonal antibodies for imaging lymphoid structures in the mouse

Radiolabeling of a mouse monoclonal antibody (MoAb) specific for the mouse histocompatibility alloantigen IAk expressed by the B lymphocytes of BALB/k and C3H mice but not BALB/c mice was performed by mixing the chelate-labeled anti (alpha) IAk MoAb with purified, no-carrier-added 111In citrate. Labeling efficiency was 85-95%, and the labeled alpha IAk MoAb retained its antigen binding properties in vitro and in vivo. The organ, spleen, and lymph node distribution of intravenously and subcutaneously administered 111In alpha IAk MoAb was compared in mice, two IAk positive and one IAk negative strains, and to 125I alpha IAk MoAb in one IAk positive strain. The 111In alpha IAk MoAb was more stable in vivo compared to 125I alpha IAk MoAb, as shown by a much slower excretion and a higher absolute uptake in lymph nodes and spleen. Lymph node to blood ratio was increased twofold by intravenous anti-EDTA MoAb. Subcutaneous injection permitted clear images of the tiny lymph nodes in the mouse. Potential clinical applications of 111In alpha lymphocyte MoAb include localization of normal lymph nodes and T & B cell leukemias and lymphomas, as well as detecting lymphatic metastases of other cancers. Therapy may also be possible using MoAbs labeled with beta-emitting metal ions such as yttrium-90.     

Altered biodistribution of Ga-67 by intramuscular gold salts

The authors observed a deviation from the normal scintigraphic pattern of Ga-67 citrate biodistribution. An 8-year-old black girl with juvenile rheumatoid arthritis, who had been treated with intramuscular injections of gold salts, had a Ga-67 study as part of her workup. The study demonstrated no hepatic uptake, but showed elevated skeletal and renal activity. This characteristic biodistribution of Ga-67 may be due to inhibition of lysosomal enzymes by gold and/or to accumulation of gold in lysosomes. To study these possibilities, the authors reviewed the mechanisms of Ga-67 localization and gold metabolism. Alteration of the Ga-67 citrate scintigraphic pattern due to earlier treatment with gold salts has not been reported previously.