Human SSTR2 Stable Cell Line - HCT 116

Dye design, which influences the ability of fluorescently labeled imaging agents to generate tumor contrast, has emerged as a key area of intense interest within the field of fluorescence-guided surgery (FGS). Here, researchers present a charge-balanced near-infrared fluorescent (NIRF) dye, FNIR-Tag, capable of significantly enhancing the imaging performance of fluorescently labeled somatostatin analogs. In vitro studies demonstrate that the optimized fluorescent conjugate, MMC(FNIR-Tag)-TOC, binds primarily via somatostatin receptor subtype 2 (SSTR2). In contrast, its negatively charged counterpart-labeled with IRDye 800CW-exhibits a higher rate of off-target binding. NIRF imaging results obtained from both cell-line-derived and patient-derived xenograft models reveal that MMC(FNIR-Tag)-TOC generates significantly higher tumor contrast-an advantage attributed to its superior tumor specificity. Ex vivo staining analyses of surgical biospecimens-including primary tumors, metastatic lesions, and involved lymph nodes-further validate the conjugate's ability to bind to human tumor tissues. Finally, utilizing an orthotopic tumor model, the researchers simulated a clinical workflow to highlight a unique capability of this study: the potential to use standard preoperative nuclear imaging techniques to identify patients likely to benefit from SSTR2-targeted FGS. These findings not only demonstrate the translational potential of MMC(FNIR-Tag)-TOC for intraoperative imaging applications but also underscore the broad prospects for the FNIR-Tag dye in the development of novel fluorescent probes.

To evaluate the binding characteristics of MMC(FNIR-Tag)-TOC, researchers conducted in vitro experiments, utilizing its IR800 counterpart as a control. Researchers selected HCT116-WT (no SSTR2), HCT116-SSTR2, BON–SSTR2, and NCI-H69 cells based on their varying SSTR2 expression (HCT116-SSTR2 ≫ BON–SSTR2 > NCI-H69) and established use for characterizing novel SSTR2-targeted agents. Flow cytometry results demonstrated that, in HCT116-SSTR2 cells, the uptake levels of both agents were comparable; however, when co-incubated with octreotide-or in the absence of SSTR2 expression (HCT116-WT cells)-drug uptake levels were significantly reduced (Figure 1A). Subsequently, leveraging the radiolabeling properties of MMC, the researchers compared the 67Ga-labeled FNIR-Tag and IR800 conjugates against the "gold standard" agent, 67Ga-DOTA-TOC. The results indicated that, in SSTR2-expressing HCT116 cells, the uptake levels of both radiolabeled conjugates were comparable to that of the positive control, 67Ga-DOTA-TOC (Figure 1B). Notably, across all cell lines tested, the binding characteristics of 67Ga-MMC(FNIR-Tag)-TOC more closely mirrored the performance of 67Ga-DOTA-TOC; its specific binding ratios were 9.2 (HCT116-SSTR2), 5.9 (BON–SSTR2), and 1.7 (NCI-H69), respectively. These values not only rivaled the performance of the clinical agent but also consistently exceeded the corresponding values observed for 67Ga-MMC(IR800)-TOC. To confirm that the conjugation of FNIR-Tag does not compromise the agonist properties of TOC (e.g., receptor-mediated endocytosis), researchers performed confocal microscopy on HCT116-SSTR2 cells and observed SSTR2-mediated endocytosis, exhibiting characteristics similar to those of MMC(IR800)-TOC (Figure 1C). Taken together, these in vitro data indicate that substituting IR800 with FNIR-Tag yields a biologically active conjugate that demonstrates high selectivity for SSTR2-expressing cells.

Figure 1. In vitro binding of dual-labeled conjugates. (Hernandez Vargas S, et al., 2022)