Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden.
Int J Oncol. 2010 Apr;36(4):757-63. doi: 10.3892/ijo_00000551.
Tumor cell internalization of targeting agents is of interest, since internalization influences the local retention time of a radionuclide and thereby imaging quality in PET and SPECT and effects of radionuclide therapy. In cases where nuclear methods are not applicable at the cellular level, quantitative fluorescent techniques are useful as described in this article. Two fluorescence-based methods to study cellular internalization were applied: the CypHer and the Alexa488-quenching methods, both utilized in fluorescence microscopy and flow cytometry. Two EGFR-binding Affibody molecules were analyzed in A431 cells: the monomer Z1907 and the dimer (Z1907)2. EGF, cetuximab and non-specific Affibody molecules were used as controls. For comparison, internalization of 111In-labeled Z1907 was studied with the acid wash internalization assay. The Cypher method is straightforward, but requires equal labeling of all compounds for accurate quantification. The Alexa488-quenching method is preferable since it is independent of the dye-to-protein ratio. According to this method, about 45% of EGF and 19-24% of the bound Affibody molecules and cetuximab were internalized within one hour. Similar results were seen with 111In-Z1907 in the acid wash method, while (Z1907)2 was not removed by acid and thus could not be studied this way. The fluorescence-based Alexa488-quenching method is well suited to quantitatively analyze internalization of targeting agents, also those that resist acid wash. The internalized fraction showed that both the monomeric and dimeric Affibody molecules are expected to give good uptake and thereby good retention of metallic radionuclides which will render good tumor to background values.
肿瘤细胞对靶向剂的内化作用很重要，因为内化作用会影响放射性核素的局部滞留时间，从而影响 PET 和 SPECT 的成像质量以及放射性核素治疗的效果。在细胞水平上不适用核方法的情况下，如本文所述，定量荧光技术很有用。两种基于荧光的方法被应用于研究细胞内化：CypHer 和 Alexa488 猝灭法，这两种方法都在荧光显微镜和流式细胞术中使用。两种 EGFR 结合的 Affibody 分子在 A431 细胞中进行了分析：单体 Z1907 和二聚体 (Z1907)2。使用 EGF、西妥昔单抗和非特异性 Affibody 分子作为对照。为了进行比较，还使用酸洗内化测定法研究了 111In 标记的 Z1907 的内化。Cypher 方法简单直接，但需要所有化合物的均匀标记才能进行准确的定量。Alexa488 猝灭法更可取，因为它独立于染料与蛋白质的比例。根据该方法，在一个小时内，约有 45%的 EGF 和 19-24%的结合 Affibody 分子和西妥昔单抗被内化。用酸洗法观察到类似的 111In-Z1907 结果，而 (Z1907)2 不能被酸去除，因此不能用这种方法研究。基于荧光的 Alexa488 猝灭法非常适合定量分析靶向剂的内化作用，也适合抵抗酸洗的靶向剂。内化部分表明，单体和二聚体 Affibody 分子都有望获得良好的摄取，从而使金属放射性核素保持良好的保留，这将使肿瘤与背景值产生良好的比值。
