Stroet Marcus C M, Dijkstra Bianca M, Dulfer Sebastiaan E, Kruijff Schelto, den Dunnen Wilfred F A, Kruyt Frank A E, Groen Rob J M, Seimbille Yann, Panth Kranthi M, Mezzanotte Laura, Lowik Clemens W G M, de Jong Marion
Department of Radiology and Nuclear Medicine/Molecular Genetics, Erasmus Medical Centre, 's-Gravendijkwal 230, 3015 CE, Rotterdam, The Netherlands.
Department of Molecular Genetics, Erasmus MC, Rotterdam, The Netherlands.
EJNMMI Res. 2021 May 10;11(1):47. doi: 10.1186/s13550-021-00789-4.
There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys(IRDye800CW)-Tyr-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery.
Binding of 800CW-TATE could be blocked with DOTA-Tyr-octreotate (DOTA-TATE) on cultured SSTR-positive U2OS cells and was absent in SSTR negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR negative cells. No SSTR-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE.
This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents.
越来越多的核造影剂被重新用于荧光引导手术。新的造影剂是通过用荧光花菁取代放射性标记或在抗体或肽的分子结构中添加荧光花菁而获得的。这使得术中能够对癌组织进行荧光检测，从而实现更彻底的肿瘤切除。然而，这些荧光花菁对药代动力学和肿瘤摄取可能有显著影响，尤其是当标记到较小的靶向载体如肽上时。在此，我们展示了花菁介导的Ac-Lys(IRDye800CW)-Tyr-奥曲肽（800CW-TATE）与死细胞结合的作用，以及如何将其用作荧光引导手术的优势。
在培养的SSTR阳性U2OS细胞上，800CW-TATE的结合可被DOTA-酪氨酸-奥曲肽（DOTA-TATE）阻断，而在SSTR阴性U2OS细胞中则不存在结合。然而，观察到与死细胞有强烈结合，这不能被DOTA-TATE阻断，并且在死的SSTR阴性细胞中也存在。在冷冻肿瘤切片中未观察到SSTR介导的结合，这可能是由于在将组织切片并暴露于造影剂之前的过程中细胞被破坏。DOTA-TATE阻断导致小鼠NCI-H69肿瘤的荧光摄取不完全降低61.5±5.8%。对切除肿瘤的石蜡切片进行近红外成像和死细胞染色显示，在用DOTA-TATE阻断后，坏死区域仍存在荧光摄取。
本研究表明，用花菁标记肽可导致与死细胞结合。这并不妨碍荧光引导手术的最终目的，因为坏死组织在大多数实体瘤中都会出现。因此，与坏死组织的结合可增加肿瘤的总体摄取。此外，坏死组织应尽可能清除：它无法挽救，会引起炎症，并且具有致瘤性。然而，在用核探针常规进行的对膜完整性受损细胞的结合实验中，这种与死细胞的结合可能类似于非特异性结合。本研究将有助于荧光造影剂的开发。
