Endoplasmic Reticulum Mediates Prolonged Tumor Retention of FAPI Radioconjugates and Enhanced Radiotherapeutic Efficacy

Radiolabeled fibroblast activation protein inhibitors (FAPIs) face a critical challenge in radionuclide therapy due to their short tumor retention. While existing strategies to prolong retention are often limited in clinical translation, we hypothesized that targeting the endoplasmic reticulum (ER) could provide a solution. We therefore designed a novel agent for sequential targeting, first to fibroblast activation protein (FAP) and then to the ER, aiming to enhance both tumor retention and radiotherapy efficacy. The sequential targeting agent, FAPI-PEG₃-K-PTSA, was synthesized by conjugating a PTSA ER-targeting moiety to the FAPI-46 core via a PEG₃-K linker. The compound was radiolabeled with ¹⁷⁷Lu and synthesized with high radiochemical yield (>95%). Cellular assays demonstrated specific binding to FAP and successful ER localization. This sequential-targeting capability resulted in superior <i>in vivo</i> performance, demonstrating a 3-5-fold higher tumor uptake and a retention time exceeding 72 h compared to FAPI-46. Consequently, ¹⁷⁷Lu-FAPI-PEG₃-K-PTSA achieved remarkable tumor suppression. The developed compound pioneers a three-level active targeting mechanism. It utilizes FAP for primary tumor enrichment, followed by ER-mediated internalization and specific organelle localization. This strategy effectively circumvents rapid efflux, dramatically prolongs intratumoral retention, and significantly enhances radiotherapeutic outcomes. Our findings provide crucial insights into organelle-targeted radionuclide therapy (TRT) and highlight its strong translational potential.