Dimeric CCK2R radiotheranostic tracers synergize with mTOR inhibition for enhanced tumor therapy

<b>Purpose:</b> The cholecystokinin-2 receptor (CCK2R) is highly expressed in several neuroendocrine cancers, particularly in medullary thyroid carcinoma (MTC) and small cell lung cancer (SCLC) and represents a promising target for radiotheranostic applications. Several minigastrin-derived analogs, such as DOTA-MGS5 and DOTA-CCK-66, have demonstrated favorable tumor targeting and imaging performance. Building on these advances, we developed and evaluated a novel dimeric CCK₂R-targeted radiotracer, and further investigated its radiosensitization potential in combination with mTOR inhibition. <b>Experimental Design:</b> We designed a dimeric CCK₂R-targeted agent, DOTA-CCK₂R-dimer, labeled with ⁶⁸Ga for PET imaging and ¹⁷⁷Lu for radionuclide therapy. Furthermore, we combined [¹⁷⁷Lu]Lu-DOTA-CCK₂R-dimer with the mTOR inhibitor RAD001 and used single-cell RNA sequencing (scRNA-seq) to investigate the mechanisms of radiosensitization. <b>Results:</b> Compared with its monomeric counterpart [⁶⁸Ga]Ga-DOTA-CCK-66, [⁶⁸Ga]Ga-DOTA-CCK₂R-dimer demonstrated superior tumor targeting <i>in vivo</i>. Tumor uptake reached 26.13 ± 6.21 %ID/g at 2 h post-injection, which was significantly greater than that of the monomeric tracer (19.63 ± 3.35 %ID/g, <i>p</i> < 0.05). Additionally, [¹⁷⁷Lu]Lu-DOTA-CCK₂R-dimer selectively eliminated highly proliferative and poorly differentiated tumor cell subpopulations. Combination treatment with RAD001 improved therapeutic efficacy by suppressing glutathione-mediated detoxification and increasing oxidative stress. Furthermore, glutathione S-transferase kappa 1 (GSTK1) was identified as a key regulator that modulates radiosensitivity. Conclusions: DOTA-CCK₂R-dimer exhibits favorable <i>in vivo</i> stability, notable tumor retention, and excellent imaging performance. Combining this agent with mTOR inhibition offers a synergistic strategy to sensitize tumors to radiotherapy, providing a promising approach for treating refractory CCK₂R-positive malignancies.