Uptake of radiolabelled alpha-fetoprotein by experimental mammary adenocarcinoma and adenoma: in vivo and in vitro studies

The biodistribution of iodine-labelled alpha-fetoprotein ( I-AFP) in experimental mammary tumours was studied. C3H mice with subcutaneously transplanted mammary adenocarcinoma and Sprague-Dawley rats treated with -methyl- -nitrosourea for mammary adenoma induction were used as animal models. The accumulation of labelled I-AFP in mouse mammary adenocarcinoma was significantly higher than that in rat mammary adenoma. The tumour/muscle radioactivity ratios increased with time and, 48 h after intravenous injection, were estimated as 23.4 and 6.7, respectively. For experiments, extracts from both mammary tumours were prepared. The extracts were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes and incubated with I-AFP. A single major AFP-binding protein with a molecular weight of about 30 kDa was detected in both extracts. The amount of AFP-binding protein was clearly higher for adenocarcinoma than for adenoma. In the presence of cross-linking reagent, I-AFP formed a complex (about 100 kDa) with adenocarcinoma proteins.     

Detection of experimental atherosclerosis with indium-111 radiolabelled hematoporphyrin derivative

It has been observed that atherosclerotic lesions (AL) concentrate certain porphyrins. We evaluated the usefulness of 111In-labelled Photofrin II (PFII), a porphyrin derived from haematoporphyrin derivative, for detection of experimental AL in cholesterol fed rabbits. Three groups of rabbits were studied: non-atherosclerotic (n = 3), 6 and 12 week cholesterol fed (n = 4, 3). 111In-PFII was injected intravenously and gamma camera images were obtained at 24 and 48 h. At 48 h, explanted aortas were also imaged. Aortic arch (AA) to background (BKG) count ratios were calculated from images of the whole body and explanted aortas. AA/BKG ratios were significantly higher in the 12 week cholesterol fed rabbits (3.9 +/- 0.72 at 24 h) and (4.0 +/- 0.67 at 48 h) than in the non-atherosclerotic rabbits (2.2 +/- 0.07 at 24 h) and (2.3 +/- 0.18 at 48 h) (p less than 0.05). The AA/BKG ratio for the explanted aortas showed similar results. Additionally, in two of three 12 week cholesterol fed rabbits, focal count deposition was visible on the whole animal images at the site of aortic arch atherosclerosis. We conclude that 111In-PFII concentrates in AL as early as 24 h after injection and has the potential to be used as an imaging agent for experimental atherosclerosis.     

Biodistribution of radiolabelled human dendritic cells injected by various routes

Purpose The purpose of this study was to investigate the biodistribution of mature dendritic cells (DCs) injected by various routes, during a cell therapy protocol.Methods In the context of a vaccine therapy protocol for melanoma, DCs matured with Ribomunyl and interferon-gamma were labelled with ¹¹¹In-oxine and injected into eight patients along various routes: afferent lymphatic vessel (IL) (4 times), lymph node (IN) (5 times) and intradermally (ID) (6 times).Results Scintigraphic investigations showed that the IL route allowed localisation of 80% of injected radioactivity in eight to ten nodes. In three cases of IN injection, the entire radioactivity stagnated in the injected nodes, while in two cases, migration to adjacent nodes was observed. This migration was detected rapidly after injection, as with IL injections, suggesting that passive transport occurred along the physiological lymphatic pathways. In two of the six ID injections, 1–2% of injected radioactivity reached a proximal lymph node. Migration was detectable in the first hour, but increased considerably after 24 h, suggesting an active migration mechanism. In both of the aforementioned cases, DCs were strongly CCR7-positive, but this feature was not a sufficient condition for effective migration. In comparison with DCs matured with TNF-, IL-1, IL-6 and PGE2, our DCs showed a weaker in vitro migratory response to CCL21, despite comparable CCR7 expression, and higher allostimulatory and TH1 polarisation capacities.Conclusion The IL route allowed reproducible administration of specified numbers of DCs. The IN route sometimes yielded fairly similar results, but not reproducibly. Lastly, we showed that DCs matured without PGE2 that have in vitro TH1 polarisation capacities can migrate to lymph nodes after ID injection.     

Enhanced proton magnetic resonance imaging of experimental mammary tumors

The proton magnetic resonance imaging contrast of experimental mammary tumors in rats has been dramatically improved. The technique developed combines T2 weighting and chemical-shift imaging in order to suppress the thoracic wall, muscle, and subcutaneous fat contributions to the image. The technique is demonstrated using the R3230AC mammary adenocarcinoma in F344 rats at 4.7 T.     

Radiopharmaceutical development of radiolabelled peptides

Receptor targeting with radiolabelled peptides has become very important in nuclear medicine and oncology in the past few years. The overexpression of many peptide receptors in numerous cancers, compared to their relatively low density in physiological organs, represents the molecular basis for in vivo imaging and targeted radionuclide therapy with radiolabelled peptide-based probes. The prototypes are analogs of somatostatin which are routinely used in the clinic. More recent developments include somatostatin analogs with a broader receptor subtype profile or with antagonistic properties. Many other peptide families such as bombesin, cholecystokinin/gastrin, glucagon-like peptide-1 (GLP-1)/exendin, arginine-glycine-aspartic acid (RGD) etc. have been explored during the last few years and quite a number of potential radiolabelled probes have been derived from them. On the other hand, a variety of strategies and optimized protocols for efficient labelling of peptides with clinically relevant radionuclides such as (99m)Tc, M(3+) radiometals ((111)In, (86/90)Y, (177)Lu, (67/68)Ga), (64/67)Cu, (18)F or radioisotopes of iodine have been developed. The labelling approaches include direct labelling, the use of bifunctional chelators or prosthetic groups. The choice of the labelling approach is driven by the nature and the chemical properties of the radionuclide. Additionally, chemical strategies, including modification of the amino acid sequence and introduction of linkers/spacers with different characteristics, have been explored for the improvement of the overall performance of the radiopeptides, e.g. metabolic stability and pharmacokinetics. Herein, we discuss the development of peptides as radiopharmaceuticals starting from the choice of the labelling method and the conditions to the design and optimization of the peptide probe, as well as some recent developments, focusing on a selected list of peptide families, including somatostatin, bombesin, cholecystokinin/gastrin, GLP-1/exendin and RGD.     

Inhibition of the hepatocyte uptake of radiolabelled monoclonal antibodies by chelating agents

The imaging of small abdominal tumours with indium 111 labelled monoclonal antibodies (MAbs) is often obscured by the uptake of activity into the heptocytes of normal liver tissue. A model has therefore been developed to analyse reagents which may inhibit the hepatocyte uptake of 111In-MAb whilst preserving tumour uptake. Primary rat hepatocyte cultures and an epithelial membrane antigen (EMA) expressing tumour cell line (MCF7), recognised by the EMA-specific MAb ICR2, were obtained in tissue culture. Monolayers of both cells were incubated with the 111In-MAb with or without the additional reagents and the cell uptake then measured and expressed per milligram of cell protein using a Lowry protein assay. No preferential reduction in hepatocyte uptake was noted by incubating cells with either saturated or unsaturated transferrin. The chelating agent, diethylene triamine penta-acetic acid (DTPA), however, significantly reduced the uptake of activity in hepatocytes but not the tumour cell line (P less than 0.05). An optimum concentration and time period for incubating DTPA with labelled MAb was established. The mean hepatocyte uptake was reduced by 80% with a 1 h incubation with 1 mM DTPA. These results suggest that DTPA may have a role in reducing the liver uptake of radioactivity in patient studies using 111In-MAb.     

Elevation of radiolabelled thymidine uptake in RIF-1 fibrosarcoma and HT29 colon adenocarcinoma cells after treatment with thymidylate synthase inhibitors

Purpose We recently showed an increase in tumour uptake of 2-[¹¹C]thymidine in patients with gastrointestinal malignancies after thymidylate synthase (TS) inhibition. To understand the phenomenon in more detail, we investigated whether TS inhibition by different TS inhibitors leads to a dose- and time-dependent change in the uptake of radiolabelled thymidine, and whether radiotracer uptake is related to changes in cell viability resulting from treatment. Methods RIF-1 and HT29 cells were treated with the TS inhibitors 5-fluorouracil (5-FU) and AG337 (nolatrexed dihydrochloride), as well as cisplatin as control. The cell viability and net accumulation of [³H]thymidine after a 1-h pulse was determined at different times after drug treatment. Results In both cell lines, [³H]thymidine uptake increased after a 2-h treatment with 5-FU, in a dose- and time-dependent manner. [³H]thymidine uptake decreased at 24 and 48 h post treatment. AG337 also produced a similar effect. In contrast to the TS inhibitors, cisplatin decreased [³H]thymidine uptake in RIF-1 and HT29 cells at all time points. Cell viability was compromised only after 24 h. Conclusion Using two types of TS inhibitor, we have shown an increase in [³H]thymidine uptake, in a dose-dependent manner, a few hours after TS inhibition when the cell viability was not compromised. This effect was not seen with a non-TS inhibitor. These findings suggest that 2-[¹¹C]thymidine positron emission tomography can be used to study TS inhibition in vivo at early time points when cell viability is not compromised and may therefore be helpful in the development of new TS inhibitors and in differentiating between patients with tumours sensitive to TS inhibitors and those unlikely to respond.     

Inhibition of the hepatocyte uptake of radiolabelled monoclonal antibodies by chelating agents

The imaging of small abdominal tumours with indium 111 labelled monoclonal antibodies (MAbs) is often obscured by the uptake of activity into the hepto cytes of normal liver tissue. A model has therefore been developed to analyse reagents which may inhibit the he patocyte uptake of¹¹¹In-MAb whilst preserving tumour uptake. Primary rat hepatocyte cultures and an epithelial membrane antigen (EMA) expressing tumour cell line (MCF7), recognised by the EMA-specific MAb ICR2, were obtained in tissue culture. Monolayers of both cells were incubated with the¹¹¹In-MAb with or without the additional reagents and the cell uptake then measured and expressed per milligram of cell protein using a Lowry protein assay. No preferential reduction in hepatocyte uptake was noted by incubating cells with either saturated or unsaturated transferrin. The chelating agent, diethylene triamine penta-acetic acid (DTPA), however, significantly reduced the uptake of activity in hepatocytes but not the tumour cell line (TP<0.05). An optimum concentration and time period for incubating DTPA with labelled MAb was established. The mean hepatocyte uptake was reduced by 80% with a 1 h incubation with 1 mM DTPA. These results suggest that DTPA may have a role in reducing the liver uptake of radioactivity in patient studies using¹¹¹In-MAb.This work was presented at the Society of Nuclear Medicine annual meeting, St. Louis, USA, July 1989     

Molecular imaging of vascular cell adhesion molecule-1 expression in experimental atherosclerotic plaques with radiolabelled B2702-p

Purpose VCAM-1 plays a major role in the chronic inflammatory processes present in vulnerable atherosclerotic plaques. The residues 75–84 (B2702-p) and 84–75/75–84 (B2702-rp) of the major histocompatibility complex-1 (MHC-1) molecule B2702 were previously shown to bind specifically to VCAM-1. We hypothesised that radiolabelled B2702-p and B2702-rp might have potential for the molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) expression in atherosclerotic plaques. Methods Preliminary biodistribution studies indicated that ¹²⁵I-B2702-rp was unsuitable for in vivo imaging owing to extremely high lung uptake. ¹²³I- or ^99mTc-labelled B2702-p was injected intravenously to Watanabe heritable hyperlipidaemic rabbits (WHHL, n = 6) and control animals (n = 6). After 180 min, aortas were harvested for ex vivo autoradiographic imaging, gamma-well counting, VCAM-1 immunohistology and Sudan IV lipid staining. Results Robust VCAM-1 immunostaining was observed in Sudan IV-positive and to a lesser extent in Sudan IV-negative areas of WHHL animals, whereas no expression was detected in control animals. Significant 2.9-fold and 1.9-fold increases in ¹²³I-B2702-p and ^99mTc-B2702-p aortic-to-blood ratios, respectively, were observed between WHHL and control animals (p < 0.05). Tracer uptake on ex vivo images co-localised with atherosclerotic plaques. Image quantification indicated a graded increase in ¹²³I-B2702-p and ^99mTc-B2702-p activities from control to Sudan IV-negative and to Sudan IV-positive areas, consistent with the observed pattern of VCAM-1 expression. Sudan IV-positive to control area tracer activity ratios were 17.0 ± 9.0 and 5.9 ± 1.8 for ¹²³I-B2702-p and ^99mTc-B2702-p, respectively. Conclusion Radiolabelled B2702-p is a potentially useful radiotracer for the molecular imaging of VCAM-1 in atherosclerosis.     

A simple synthesis of radiolabelled bromoacetic acid

A facile procedure for the radiosynthesis of ³H- or ¹⁴C-labelled bromoacetate by reaction of sodium acetate with bromine in the presence of elemental sulfur is described. The effects of reaction time and temperature as well as bromine and sulfur concentration were investigated. Optimum conditions in which 1 mg sodium acetate (mass equivalent to 1 mCi (37 MBq) ¹⁴C) was allowed to react with 10 μL bromine and 0.1 mg sulfur at 105°C for 60 min afforded [¹⁴C]bromoacetate in greater than 90% radiochemical yield and [³H]bromoacetate in greater than 60% radiochemical yield based on initial ¹⁴C- or ³H-labelled sodium acetate concentration.     

Molecular imaging of hypoxia with radiolabelled agents

Tissue hypoxia results from an inadequate supply of oxygen (O₂) that compromises biological functions. Structural and functional abnormalities of the tumour vasculature together with altered diffusion conditions inside the tumour seem to be the main causes of tumour hypoxia. Evidence from experimental and clinical studies points to a role for tumour hypoxia in tumour propagation, resistance to therapy and malignant progression. This has led to the development of assays for the detection of hypoxia in patients in order to predict outcome and identify patients with a worse prognosis and/or patients that would benefit from appropriate treatments. A variety of invasive and non-invasive approaches have been developed to measure tumour oxygenation including oxygen-sensitive electrodes and hypoxia marker techniques using various labels that can be detected by different methods such as positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), autoradiography and immunohistochemistry. This review aims to give a detailed overview of non-invasive molecular imaging modalities with radiolabelled PET and SPECT tracers that are available to measure tumour hypoxia.     

Detection of inflammatory lesions with radiolabelled immunoglobulins

Previous reports on the use of radiolabelled immunoglobulins led us to undertake a pilot experiment in an animal model to investigate the potentials of Tc 99m-immunoglobulin scintigraphy in the detection of infectious foci. Mice infected in one leg with staphylococcus infection were injected with Tc 99m-immunoglobulin, Tc 99m-albumin or gallium citrate Ga 67. The results obtained by scintigraphy suggested a specific accumulation of radiolabelled immunoglobulin at the site of infection. Visualization of the infection and the image quality, especially the 6- and 24-h images, were clearly enhanced after the use of immunoglobulin preparations as compared with gallium.