Highly Efficient In Vivo Agonist-Induced Internalization of sst₂ Receptors in Somatostatin Target Tissues

Abstract

The successful peptide receptor imaging of tumors, as exemplified for somatostatin receptors, is based on the overexpression of peptide receptors in selected tumors and the high-affinity binding to these tumors of agonist radioligands that are subsequently internalized into the tumor cells in which they accumulate. Although in vitro studies have shown ample evidence that the ligand-receptor complex is internalized, in vivo evidence of agonist-induced internalization of peptide receptors, such as somatostatin receptors, is missing. Methods: Rats subcutaneously transplanted with the somatostatin receptor subtype 2 (sst 2 )-expressing AR42J tumor cells were treated with intravenous injections of various doses of the sst 2 agonist [Tyr 3 , Thr 8 ]-octreotide (TATE) or of the sst 2 antagonist 1,4,7,10-tetraazacyclododecane-N,N9,N$,N9$,-tetraacetic acid (DOTA)-Bass and were sacrificed at various times ranging from 2.5 min to 24 h after injection. The tumors and pancreas were then removed from each animal. All tissue samples were processed for sst 2 immunohistochemistry using sst 2 -specific antibodies. Results: Compared with the sst 2 receptors in untreated animals, which localized at the plasma membrane in pancreatic and AR42J tumor cells, the sst 2 receptors in treated animals are detected intracellularly after an intravenous injection of the agonist TATE. Internalization is fast, as the receptors are already internalizing 2.5 min after TATE injection. The process is extremely efficient, as most of the cell surface receptors internalize into the cell and are found in endosomelike structures after TATE injection. The internalization is most likely reversible, because 24 h after injection the receptors are again found at the cell surface. The process is also agonist-dependent, because internalization is seen with high-affinity sst 2 agonists but not with high-affinity sst 2 antagonists. The same internalization properties are seen in pancreatic and AR42J tumor cells. They can further be confirmed in vitro in human embryonic kidneysst 2 cells, with an immunofluorescence microscopy-based sst 2 internalization assay. Conclusion: These animal data strongly indicate that the process of in vivo sst 2 internalization after agonist stimulation is fast, extremely efficient, and fully functional under in vivo conditions in neoplastic and physiologic sst 2 target tissues. This molecular process is, therefore, likely to be responsible for the high and long-lasting uptake of sst 2 radioligands seen in vivo in sst 2 -expressing tumors.

Effect of amino acid infusion on potassium serum levels in neuroendocrine tumour patients treated with targeted radiopeptide therapy

“…This therapy relies on the use of radiolabelled peptides that preferentially bind to the somatostatin receptor 2 (SST 2 ). Following binding, the peptide-receptor complex is rapidly internalized, and the radioactive peptide is trapped in the cell and produces cytotoxic damage [2].…”

Strict rules are needed for validation of G-protein-coupled receptor immunohistochemical studies in human tissues

“…Internalization is even part of the physiological mechanism of action for many GPCR; a specific condition necessary for this phenomenon to occur is usually an acute receptor stimulation by agonist treatment. It should be understood that internalized receptors have a very particular intracellular distribution, as they are usually internalized in circumscript endosomes; thus, they are not diffusely distributed in the cytoplasm [9,10]. To precisely evaluate the cellular localization of receptors, an illustration at high magnification is, therefore, required.…”

Evaluation of 177Lu-DOTA-sst2 Antagonist Versus 177Lu-DOTA-sst2 Agonist Binding in Human Cancers In Vitro

“…Indeed, several excellent somatostatin agonists, among which are 90 Y-DOTATOC and 177 Lu-DOTATATE, have been developed in the last few years and are now routinely used in the clinic to treat patients with neuroendocrine tumors (7,8). An important feature of such agonist radioligands is that after binding to the receptor, they have to be internalized into the tumor cell by receptor-mediated endocytosis, leading to an accumulation of the radioactive agonist within the tumor cell (9,10). Interestingly, it has recently been shown for sst 2 and sst 3 in an animal tumor model that somatostatin antagonists with high affinity for the receptor can also have excellent properties for peptide receptor targeting (11).…”