Waser Bea, Tamma Maria-Luisa, Cescato Renzo, Maecke Helmut R, Reubi Jean Claude
Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Berne, Switzerland.
J Nucl Med. 2009 Jun;50(6):936-41. doi: 10.2967/jnumed.108.061457. Epub 2009 May 14.
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.
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,N',N'',N''',-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.
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 kidney-sst(2) cells, with an immunofluorescence microscopy-based sst(2) internalization assay.
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.
肿瘤的成功肽受体显像，如以生长抑素受体为例，是基于特定肿瘤中肽受体的过表达以及激动剂放射性配体与这些肿瘤的高亲和力结合，随后这些配体被内化到肿瘤细胞中并在其中积累。尽管体外研究已充分证明配体 - 受体复合物会被内化，但激动剂诱导的肽受体（如生长抑素受体）内化的体内证据却缺失。
将表达生长抑素受体亚型2（sst(2)）的AR42J肿瘤细胞皮下移植到大鼠体内，静脉注射不同剂量的sst(2)激动剂[酪氨酸(3)，苏氨酸(8)] - 奥曲肽（TATE）或sst(2)拮抗剂1,4,7,10 - 四氮杂环十二烷 - N,N',N'',N''' - 四乙酸（DOTA） - Bass，并在注射后2.5分钟至24小时的不同时间点处死大鼠。然后从每只动物身上取出肿瘤和胰腺。所有组织样本均使用sst(2)特异性抗体进行sst(2)免疫组织化学处理。
与未处理动物中位于胰腺和AR42J肿瘤细胞质膜上的sst(2)受体相比，静脉注射激动剂TATE后，处理动物中的sst(2)受体在细胞内被检测到。内化过程很快，因为在TATE注射后2.5分钟受体就已经开始内化。这个过程极其高效，因为大多数细胞表面受体在TATE注射后内化到细胞中，并在类似内体的结构中被发现。内化很可能是可逆的，因为注射后24小时受体又再次出现在细胞表面。这个过程也是激动剂依赖性的，因为高亲和力的sst(2)激动剂能观察到内化，而高亲和力的sst(2)拮抗剂则不能。在胰腺和AR42J肿瘤细胞中观察到相同的内化特性。它们可以在体外人胚肾 - sst(2)细胞中通过基于免疫荧光显微镜的sst(2)内化试验进一步得到证实。
这些动物实验数据有力地表明，在激动剂刺激后，体内sst(2)内化过程在肿瘤和生理性sst(2)靶组织的体内条件下快速、极其高效且功能完全正常。因此，这个分子过程可能是导致在表达sst(2)的肿瘤中体内观察到的sst(2)放射性配体高且持久摄取的原因。
