Development and Evaluation of [⁶⁸Ga]Ga-Labeled Riboflavin Derivative for RFVT3-Targeted PET Imaging of Melanoma in Mice

The limited progress in treatment options and the alarming survival rates in advanced melanoma emphasize the significant research importance of early melanoma diagnosis. RFVT3, a crucial protein at the core of energy metabolism reprogramming in melanoma, might play a pivotal role in early detection. In this study, [⁶⁸Ga]Ga-NOTA-RF, based on riboflavin (RF), was rationally developed and validated, serving as an innovative tool for positron emission tomography (PET) imaging of RFVT3 expression in melanoma. The <i>in vitro</i> assays of RFVT3 specificity of [⁶⁸Ga]Ga-NOTA-RF were performed on B16F10 melanoma cells. Then, PET imaging of melanoma was investigated in B16F10 allograft mouse models with varying volumes. Biodistribution studies are used to clarify the behavior of [⁶⁸Ga]Ga-NOTA-RF <i>in vivo</i>. [⁶⁸Ga]Ga-NOTA-RF was obtained with high radiochemical purity (>95%). A significant uptake (37.79 ± 6.86%, <i>n</i> = 4) of [⁶⁸Ga]Ga-NOTA-RF was observed over time in B16F10 melanoma cells, which was significantly inhibited by RFVT3 inhibitors RF or methylene blue (MB), demonstrating the specific binding of [⁶⁸Ga]Ga-NOTA-RF. At 60 min postinjection, the tumor-to-muscle (T/M) ratio of [⁶⁸Ga]Ga-NOTA-RF was 4.03 ± 0.34, higher than that of the RF-blocked group (2.63 ± 0.19) and MB-blocked group (2.14 ± 0.20). The T/M ratios for three distinct tumor volumes-small (5 mm), medium (10 mm), and large (15 mm) were observed to be 5.25 ± 0.28, 4.03 ± 0.34, and 3.19 ± 0.55, respectively. The expression of RFVT3 was validated by immunohistochemical staining in various tumor models, with small B16F10 tumors exhibiting the highest expression. [⁶⁸Ga]Ga-NOTA-RF demonstrates promising properties for the early diagnosis of melanoma and the examination of minute metastatic lesions, indicating its potential to assist in guiding clinical treatment decisions.