CuET Radiosensitizes FAP-Targeted Radioligand Therapy in Lung Cancer by inhibiting DNA Repair

CuET Radiosensitizes FAP-Targeted Radioligand Therapy in Lung Cancer by inhibiting DNA Repair | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CuET Radiosensitizes FAP-Targeted Radioligand Therapy in Lung Cancer by inhibiting DNA Repair Jiahao Xie, Yijin Zou, Yuzhao Zheng, Dazhi Shi, Wenlan Zhou, Ying Tian, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9032718/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background The efficacy of fibroblast activation protein (FAP)-targeted radionuclide ligand therapy (RLT) is often limited by the activation of DNA damage repair pathways in tumor cells. This study explores whether bis-(diethyldithiocarbamate)-copper (CuET, a NPL4 inhibitor and also a DNA repair inhibitor) enhances FAP-targeted RLT efficacy. Results While both [ 177 Lu]Lu-FAP-2286 and CuET monotherapies significantly inhibited tumor growth and prolonged survival compared to vehicle (P < 0.05), their efficacy was limited. Notably, [ 177 Lu]Lu-FAP-2286 monotherapy markedly upregulated NPL4 expression in tumors. The full-dose combination therapy resulted in profound tumor regression (-65.1% volume change), significantly outperforming monotherapy at day 13 (P < 0.05). This translated into a striking survival benefit, with a 77.8% survival rate at 50 days post-treatment versus 0% for monotherapy (P < 0.001). Mechanistically, the combination led to a significant increase in DNA damage, elevated apoptosis and autophagy, and suppressed proliferation (P < 0.05). Critically, the half-dose combination achieved short-term tumor control superior to full-dose monotherapy and yielded a comparable median survival (41.3 vs. 38.7 days, P = 0.41), demonstrating a significant radiosensitizing effect. Conclusion CuET acts as a potent radiosensitizer for [ 177 Lu]Lu-FAP-2286 by exacerbating DNA damage and exploiting therapy-induced NPL4 upregulation. This dual-mechanism combination presents a promising strategy to enhance the efficacy of FAP-targeted RLT, with the potential for dose reduction to mitigate toxicity. Targeted radionuclide therapy radiosensitizer FAP Lung cancer CuET Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Radionuclide ligand therapy (RLT), an innovative internal radiotherapy strategy, offers a promising alternative for treating widespread metastatic disease. RLT employs ligands conjugated to therapeutic radionuclides such as 177 Lu or 225 Ac via macrocyclic chelating agents, enabling selective delivery of radiation to cancer cells following intravenous administration. These radionuclides exert sustained therapeutic effects through localized emission of α- and β-particles while sparing normal tissues. RLT has emerged as an effective treatment for disseminated metastatic tumors ( 1 – 3 ). For example, 177 Lu-PSMA-617 targeting prostate-specific membrane antigen (PSMA) and 177 Lu-DOTATATE targeting somatostatin receptors have demonstrated that these RLTs significantly improve overall survival in patients with advanced prostate cancer and metastatic neuroendocrine tumors ( 4 , 5 ). Fibroblast activation protein (FAP) is a cell surface marker of cancer-associated fibroblasts (CAFs), a critical component of the tumor stroma. FAP is highly expressed in CAFs across various tumor types, including lung cancer( 6 , 7 ). In lung cancer, its high expression in both primary and metastatic lesions, coupled with minimal expression in healthy tissues, makes FAP an ideal candidate for RLT in lung cancer ( 8 – 12 ). Radiolabeled FAP inhibitors (FAPIs), such as [ 177 Lu]Lu-FAP-2286, [ 177 Lu]Lu-DOTAGA-(SA-FAPI) 2 , and [ 225 Ac]Ac-FAPI, have shown the ability to deliver targeted radiotherapy to FAP-expressing tumors ( 13 – 15 ). Although [ 177 Lu]Lu-FAP-2286 has shown initial clinical promise, therapeutic responses can be heterogeneous, and durable control remains a challenge, likely due to inherent or acquired resistance mechanisms( 10 , 16 – 19 ). Therefore, further exploration of novel therapeutic strategies is necessary to improve the therapeutic efficacy of this treatment. The limited antitumor efficacy of [ 177 Lu]Lu-FAP-2286 may be largely attributed to efficient repair of the predominantly single-strand DNA breaks induced by its continuous low-dose-rate β-irradiation. ( 2 ). Diethyldithiocarbamate (DDC) is an anti-cancer metabolite derived from disulfiram (DSF), an old and cheap FDA-approved drug primarily used to treat alcoholism. It can chelate with copper ions to form the ditiocarb-copper complex Cu(DDC)2, also known as CuET. This complex binds to NPL4 with extremely high affinity (Kd = 4.647×10–8M), effectively immobilizing NPL4 and disrupting the critical p97–NPL4–UFD1 pathway, which inhibits the very important proteasome/poly-Ub protein degradation pathway ( 20 ). Concurrently, CuET has been shown to impair the ATRIP-ATR-CHK1 signaling axis, a master regulator of the DNA replication stress response, leading to replication fork stalling, accumulation of single-stranded DNA, and defective DNA repair ( 21 , 22 ). This dual activity positions CuET as a potent radio- and chemosensitizer ( 23 – 25 ). This study aims to evaluate whether CuET can overcome resistance to [ 177 Lu]Lu-FAP-2286 RLT. We specifically investigate a two-pronged hypothesis: ( 1 ) CuET-mediated inhibition DNA repair, converting repairable single-strand DNA breaks (SSBs) into lethal double-strand breaks (DSBs); and ( 2 ) RLT-induced upregulation of NPL4, will render tumor cells more vulnerable to CuET's proteotoxic effects, creating a therapeutic feedback loop. By combining [ 177 Lu]Lu-FAP-2286 with CuET, we sought to demonstrate a synergistic antitumor effect and elucidate its underlying mechanisms. Methods Detailed information on materials and methods including radiolabeling of [ 177 Lu]Lu-FAP-2286 and [ 68 Ga]Ga-FAP-2286, their radiochemical purity, in vitro and in vivo stability, binding affinity and cellular uptake studies are described in the Supplementary data according to the methods previously reported ( 26 , 27 ). 1. Tumor Xenograft Models of Lung Adenocarcinoma Male BALB/c nude mice (aged 4 to 6 weeks; GemPharmatech Co., Ltd.) were subcutaneously injected with A549-FAP cells on the right flank, with each injection containing 5×10 6 cells in 100 µL of phosphate-buffered saline (PBS). For biodistribution studies and micro-PET/CT imaging, mice were used when the tumor diameter reached 7 − 9 mm. For therapeutic studies, mice were selected when the tumor diameter was 5 − 6 mm, corresponding to a tumor volume of approximately 100 mm 3 . All animal experiments were performed in compliance with the legislation on the use of laboratory animals and approved by Ethics Committee. 2. Micro-PET Imaging of [Ga]Ga-FAP-2286 Micro-PET/CT imaging of tumor-bearing mice was conducted using a micro-PET/CT scanner (Siemens, Erlangen, Germany). Mice were intravenously injected with 5.55–7.40 MBq of [ 68 Ga]Ga-FAP-2286 and subsequently imaged with PET/CT. To elucidate the in vivo pharmacokinetics and tumor retention of [ 68 Ga]Ga-FAP-2286 over an extended period, a series of static micro-PET/CT scans were performed at 1, 2, and 4 hours post-injection (n = 3). The metabolic tumor volume (MTV) was measured using the threshold method (45.0% of SUV max ) on [ 68 Ga]Ga-FAP-2286 PET/CT images ( 9 ). [ 68 Ga]Ga-FAP-2286 image was reconstructed using the three-dimensional ordered-subset expectation maximum algorithm (Siemens, Erlangen, Germany), and the uptake intensity in tumors and normal organs was quantified as %ID/g. 3. In Vivo Radioactivity Biodistribution Study of [Lu]Lu-FAP-2286 The in vivo radioactivity biodistribution of [ 177 Lu]Lu-FAP-2286 was evaluated in A549-FAP tumor-bearing mice. Each mouse received an intravenous injection of approximately 1.85 MBq (50 µCi) of [ 177 Lu]Lu-FAP-2286. At 1, 4, 24, 72, and 168 hours post-injection, the mice were euthanized, and tumors along with various organs of interest were collected. These organs included blood, lung, heart, brain, bone, muscle, gallbladder, liver, kidney, spleen, intestine, stomach, and tumor tissue. A γ-counter was employed to measure the radioactivity in the collected samples. The uptake intensity of [ 177 Lu]Lu-FAP-2286 was quantified as %ID/g. 4. Treatment Protocols A549-FAP tumor-bearing mice were randomly assigned into five groups (n = 12/group) and treated with the following regimens: PBS (vehicle control), CuET alone, full-dose alone [ 177 Lu]Lu-FAP-2286 (37 MBq), half-dose [ 177 Lu]Lu- FAP-2286 (18.5 MBq) plus CuET(half-dose combined treatment), and full-dose [ 177 Lu]Lu-FAP-2286 (37 MBq) plus CuET༈full-dose combined treatment). For CuET treatment, mice received intraperitoneal injections of 2 mg/kg CuET, as referenced in the literatures ( 20 , 28 ). [ 177 Lu]Lu-FAP-2286 was administered via intravenous injection at doses of 37 MBq (full-dose) or 18.5 MBq (half-dose) for RLT. The injection frequency and time intervals are illustrated in Supplementary Fig. S1 . 5. Efficacy Evaluation To assess treatment efficacy, tumor size and body weight were measured every three days starting from the first day after treatment. The tumor volume was calculated (V=width 2 × Length × 0.5; mm 3 ). The changes in tumor volume and body weight were calculated as V X /V 0 and W X /W 0 , respectively. Meanwhile, at 13 days post-treatment, [ 68 Ga]Ga-FAP-2286 PET/CT imaging was conducted to assess the therapeutic efficacy for 5 groups (n = 3/group). Then, the mice underwent the immunohistochemistry (IHC) essay. The tumor-bearing mice (n = 9/group) were used to continuously evaluate their long-term therapeutic efficacy. The evaluation was terminated if the tumor volume exceeded 1500 mm 3 , if active ulceration occurred in the tumor, or if body weight loss exceeded 15% of the initial weight. Survival rate was calculated as the proportion of mice alive before reaching the evaluation endpoint relative to the total number of mice. 6. Hematoxylin-Eosin (HE) Staining and IHC At 13 days post-treatment, three tumor-bearing mice per group were euthanized after PET/CT scans, and their heart, liver, spleen, lung, kidney, and tumor were collected. For HE staining, the hearts, livers, spleens, lungs, and kidneys of mice were collected at 13 days post-treatment. For IHC analysis, after processing A549-FAP tumor samples, IHC staining was performed targeting FAP, NPL4, γ-H2AX (a marker for DNA double-strand breaks), cleaved caspase-3 (an apoptosis marker), LC3B (an autophagy marker), survivin(an anti-apoptotic protein), Bcl2 (an anti-apoptotic protein), and Ki-67 (a protein related to cell proliferation), following the standard protocol. The primary antibodies used for IHC staining were sourced from the following suppliers: FAP (Cell Signaling Technology), NPL4 (Proteintech), cleaved caspase-3 (BioWorld), LC3B (Affinity), γ-H2AX (Abcam), Ki-67 (Abcam), Bcl2 (BioWorld), and survivin (BioWorld). The IHC results were further analyzed using ImageJ software. The H-score, a reliable metric for evaluating staining intensity, was calculated as follows: (1 × percentage of weak staining) + (2 × percentage of moderate staining) + (3 × percentage of strong staining) within the target region, with a range of 0 to 300 ( 29 ). 7. Statistical Analysis Data were reported as mean ± standard deviation (SD) and analyzed accordingly. All calculations were performed using SPSS Statistics version 26 (IBM). T-tests and Wilcoxon Signed-Rank Test were used to compare the different groups. Survival analysis was performed with the Kaplan–Meier method and Log-rank (Mantel-Cox) test. Statistical significance was set as P < 0.05. Results 1. Successful Synthesis and High Stability of [Lu]Lu-FAP-2286 [ 177 Lu]Lu-FAP-2286 was successfully synthesized, achieving a molar activity of up to 27.2 MBq/nmol and a radiochemical purity exceeding 95%. It demonstrated excellent in vitro stability, maintaining over 95% radiochemical purity after 24 hours of incubation at 37°C in both PBS and FBS. Additionally, [ 177 Lu]Lu-FAP-2286 exhibited remarkable in vivo stability in mouse blood, with radiochemical purity exceeding 90% at 24 hours post-injection (Supplementary Fig. S2). [ 68 Ga]Ga-FAP-2286, the theranostic pair of [ 177 Lu]Lu-FAP-2286, was also successfully radio-synthesized. The non-decay corrected radiochemical yield was 69.2% ± 4.5% (n = 3). After purification, [ 68 Ga]Ga-FAP-2286 exhibited high molar radioactivity (24.7–31.0 GBq/µmol) and radiochemical purity (> 95%). 2. High Affinity and High Uptake of [Lu]Lu-FAP-2286 in A549-FAP Cells and Tumors In vitro cell experiments were conducted to evaluate the cellular uptake capacity and binding affinity of [ 177 Lu]Lu-FAP-2286 to A549-FAP cells. As shown in Supplementary Fig. S3, [ 177 Lu]Lu-FAP-2286 exhibited high-level cellular uptake (40.9 ± 5.1% ID/1 mio cells at 48 hours post-incubation), which increased gradually with prolonged incubation time. The competitive binding assay revealed that DOTA-FAP-2286 had a strong binding affinity to A549-FAP cells (IC 50 = 6.9 ± 0.6 nM). Over-expression of FAP in A549-FAP tumor xenografts was confirmed using IHC (Fig. 1 A). Consistent with the high FAP expression, micro-PET/CT imaging showed strong uptake (7.5 ± 1.0% ID/g) of [ 68 Ga]Ga-FAP-2286 in A549-FAP xenograft tumors at 4 hour after intravenously administration (Fig. 1 B, 1 C). The uptake of [ 177 Lu]Lu-FAP-2286 was evaluated using an in vitro radioassay following a 1 hour intravenous administration to A549-FAP tumor xenografts. Consistent with the findings for [ 68 Ga]Ga-FAP-2286, [ 177 Lu]Lu-FAP-2286 demonstrated significant accumulation within the tumors, reaching an uptake level of 19.0 ± 1.8% ID/g. This pronounced uptake was maintained over an extended period, lasting up to 72 hours. Meanwhile, low uptake of [ 177 Lu]Lu-FAP-2286 was found in normal organs (Fig. 1 D and Supplementary Table S1 ). 3. Anticancer Efficacy of [ 177 Lu]Lu-FAP-2286 and CuET Treatment and Upregulation of Tumor NPL4 Expression Induced by [ 177 Lu]Lu-FAP-2286 Treatment As a single agent, [ 177 Lu]Lu-FAP-2286 effectively stalled tumor growth at day 13 (Table 1 and Fig. 2 ). In the vehicle group, tumor volume rapidly increased from 97.4 ± 14.3 mm 3 to 530.2 ± 148.8 mm 3 after 13 days of treatment, representing a 468.1% ± 184.9% volume increase. In contrast, in the [ 177 Lu]Lu-FAP-2286 monotherapy group, tumor volume remained nearly unchanged, from 102.0 ± 11.1 mm 3 to 103.2 ± 40.1 mm 3 after 13 days of treatment, with a minimal volume increase (1.5% ± 43.2%), which was significantly lower than that of the vehicle group (P < 0.05). Moreover, the survival time of [ 177 Lu]Lu-FAP-2286 monotherapy was significantly longer than that of the vehicle group (38.7 ± 6.0 vs. 21.7 ± 2.5 days, P < 0.001) (Supplementary Table S2). Table 1 The tumor volume and their changes of A549-FAP xenografts mice measured at 1 day and 13 days after the treatments. Treatment Tumor volume, Day 1 (mm 3 ) Tumor volume Day 13 (mm 3 ) Change of tumor volume (%) P -value PBS 97.4 ± 14.3 530.2 ± 148.8 +, 468.1 ± 184.9 < 0.001 CuET 94.3 ± 11.5 176.5 ± 58.4 +, 86.3 ± 54.5 0.003 177 Lu-FAP-2286 (37 MBq) 102.0 ± 11.1 103.2 ± 40.1 +, 1.5 ± 43.2 0.933 CuET+ 177 Lu-FAP-2286 (18.5 MBq) 93.0 ± 9.7 63.2 ± 35.5 -, 33.9 ± 34.0 0.038 CuET+ 177 Lu-FAP-2286 (37 MBq) 95.1 ± 10.0 34.6 ± 33.6 -, 65.1 ± 31.5 0.001 +: increase, -: reduce. CuET treatment effectively delayed tumor growth and prolonged the survival time of A549-FAP lung adenocarcinoma xenografts. As shown in Table 1 , in the CuET treatment group, the increase in tumor volume in 13 days post-treatment was significantly smaller than that in the vehicle group (86.3 ± 54.5% vs. 468.1 ± 184.9%, P < 0.001). As shown in Supplementary Table S2, regarding long-term efficacy, the survival time of the CuET treatment group was also significantly longer than that of the vehicle group (33.1 ± 3.9 days vs. 21.7 ± 2.5 days, P < 0.001). However, its efficacy was significantly lower than that of [ 177 Lu]Lu-FAP-2286 (P < 0.05). High NPL4 expression was identified in A549-FAP tumors before treatment. On the 13th day post-treatment, IHC showed that [ 177 Lu]Lu-FAP-2286 treatment significantly upregulated NPL4 expression compared with the vehicle group (P < 0.05), with the NPL4 H-score increasing by 119.1% (Fig. 1 E). 4. Enhanced Anticancer Efficacy of [Lu]Lu-FAP-2286 Combined with CuET for A549-FAP Tumor Xenografts. In the short-term efficacy evaluation, tumor volumes in the full-dose combined treatment group decreased from 95.1 ± 10.0 mm 3 to 34.6 ± 33.6 mm 3 after 13 days treatment, representing a 65.1% ± 31.5% reduction. In contrast, the tumor volume in the [ 177 Lu]Lu-FAP-2286 treatment group had a slight increase from 102.0 ± 11.1 mm 3 to 103.2 ± 40.1 mm 3 during this period (Table 1 ). On the 13th day post-treatment, the tumor volume of the full-dose combined treatment group was significantly lower than that of the [ 177 Lu]Lu-FAP-2286 treatment group (t = 3.937, P = 0.001). [ 68 Ga]Ga-FAP-2286 PET/CT imaging was performed to measure the MTVs of the treatment groups on day 13 post-treatment, showing similar changes in MTV, as depicted in Fig. 2 B, I. This early profound response translated into a remarkable long-term survival benefit. In the long-term efficacy evaluation, a distinct difference in therapeutic efficacy was observed between the full-dose combined treatment groups and the [ 177 Lu]Lu-FAP-2286 monotherapy group (Fig. 3 ). After 50 days post-treatment, all mice had died in the [ 177 Lu]Lu-FAP-2286 monotherapy group, whereas 77.8% of mice were still alive in the full-dose combined treatment groups (Table 2 ). As shown in Supplementary Table S2, the survival time of the full-dose combined treatment groups was 55.3 ± 9.7 days, which was significantly longer than that of the [ 177 Lu]Lu-FAP-2286 groups (38.7 ± 6.0 days) (P < 0.001). Meanwhile, tumor complete resolution was observed 44.4% of the mice in full-dose combined treatment groups (Table 2 ). Table 2 The survival rates of A549-FAP xenografts mice at 20, 30, 40, 50 and 60 days after treatment and tumor complete resolution rate. Treatment (n = 9) Survival rate, Day 20 Survival rate, Day 30 Survival rate, Day 40 Survival rate, Day 50 Survival rate, Day 60 Tumor complete resolution (%) PBS 77.8% 0% 0% 0% 0% 0/9 (0) CuET 100% 88.9% 11.1% 0% 0% 0/9 (0) 177 Lu-FAP-2286 (37 MBq) 100% 100% 33.3% 0% 0% 0/9 (0) CuET+ 177 Lu-FAP-2286 (18.5 MBq) 100% 100% 66.7% 0% 0% 1/9 (11.1%) CuET+ 177 Lu-FAP-2286 (37 MBq) 100% 100% 88.9% 77.8% 22.2% 4/9 (44.4%) 5. CuET Acts as a Potent Radiosensitizer and Enables Radiation Dose Reduction Compared with full-dose combined treatment, half-dose combined treatment demonstrated inferior efficacy in both short-term and long-term evaluations (P < 0.05) (Table 2 ). Strikingly, the half-dose combination achieved short-term tumor control superior to full-dose [ 177 Lu]Lu-FAP-2286 monotherapy (tumor volume change: -33.9% vs. +1.5%, t = 2.238, P = 0.04) and yielded a comparable median survival (41.3 days vs. 38.7 days, P = 0.41, Fig. 3 ). This demonstrates a potent radiosensitizing effect of CuET, capable of enhancing the efficacy of a suboptimal radiation dose to a level equivalent to a full-dose treatment. 6. Safety No adverse reactions were observed in A549-FAP tumor xenografts during the above treatments. As depicted in Supplementary Fig. S4, mild body weight loss (< 15%) was observed only in the CuET group during the early treatment phase and in the full-dose combined treatment group during the late phase. As shown in Supplementary Fig. S5, HE staining of heart, liver, spleen, lung, and kidney tissues from mice in the full-dose combined treatment group revealed no histological abnormalities at 13 days post-treatment. 7. Potential Mechanisms Underlying the Synergistic Antitumor Effect of CuET Combined with [Lu]Lu-FAP-2286 Treatment To elucidate the potential mechanisms of the synergistic antitumor effect, IHC analysis was conducted to evaluate the expression levels of proteins associated with DNA damage response, apoptosis, autophagy, and cell proliferation (Fig. 4 ). The full-dose combination led to a marked increase in DNA damage, as evidenced by a 213.5% higher γ-H2AX H-score compared to RLT alone (P < 0.001). This was accompanied by a significant increase in apoptotic (cleaved caspase-3, P < 0.001) and autophagic (LC3B, P = 0.022) markers. Conversely, the full-dose combination treatment profoundly suppressed tumor cell proliferation (Ki-67, P < 0.001) and downregulated the anti-apoptotic proteins survivin and Bcl-2 (P = 0.036, and P = 0.028, respectively). These data suggest that CuET not only exacerbates RLT-induced DNA damage but also dismantles key survival pathways, shifting the cellular balance decisively towards cell death. Discussion This study demonstrates that both [ 177 Lu]Lu-FAP-2286 and CuET treatments can significantly retard tumor growth and extend the survival time of A549-FAP tumor xenografts compared with the vehicle group (P < 0.05). However, their anticancer efficacy is far from optimal. Given that previous studies have identified CuET as a potent radiosensitizer that enhances the efficacy of antitumor therapy ( 24 , 25 ), the present study investigated whether CuET can inhibit the DNA damage repair triggered by [ 177 Lu]Lu-FAP-2286 treatment, thereby enhancing the anticancer efficacy. Our results demonstrated that the combination therapy induced profound tumor regression and significantly prolonged survival. CuET not only enhanced cancer cells' susceptibility to [ 177 Lu]Lu-FAP-2286 treatment but also achieved a dose-reduction effect, highlighting its translational relevance. The radiosensitizing effect of CuET for [ 177 Lu]Lu-FAP-2286 is highly encouraging. In the long-term therapeutic efficacy evaluation, after 50 days post-treatment, the survival rates were 0.0% for [ 177 Lu]Lu-FAP-2286 treatment and 77.8% for full-dose combined treatment (P < 0.001). The combination treatment significantly prolonged survival time compared to [ 177 Lu]Lu-FAP-2286 alone (55.3 ± 9.7 days vs. 38.7 ± 6.0 days, P < 0.001). Similar to our research, Dirk Zboralski et al. demonstrated in preclinical studies that the therapeutic efficacy of [ 177 Lu]Lu-FAP-2286 as a monotherapy was limited ( 13 ). They further combined [ 177 Lu]Lu-FAP-2287 with anti-PD-1 therapy and found this combination enhanced the sensitivity of tumor cells to radionuclide therapy by modulating immune cells within the tumor microenvironment and significantly prolonged survival( 30 ). Therefore, combined treatment may be a good strategy to improve the efficacy of [ 177 Lu]Lu-FAP-2286 for the treatment of FAP-positive tumors. Additionally, the ability of the half-dose combination to match the efficacy of a full-dose RLT monotherapy (41.3 ± 3.8 days vs. 38.7 ± 6.0 days, P = 0.41) suggests that CuET could allow for dose reduction of the radioligand in a clinical setting, potentially mitigating off-target toxicity and broadening the therapeutic window. This radiosensitizing effect, coupled with the excellent safety profile of CuET observed here, positions it as an ideal candidate for combination strategies. The anticancer efficacy of [ 177 Lu]Lu-FAP-2286 is derived from the of β-rays emitted from [ 177 Lu], which primarily induces single-strand breaks rather than double-strand breaks in DNA. This DNA damage, in turn, activates the DNA repair mechanisms of tumor cells, thereby diminishing the therapeutic efficacy of [ 177 Lu]Lu-FAP-2286 ( 31 , 32 ). CuET, a multimodal inhibitor of both the ATR-CHK1 DNA repair pathway and the NPL4-p97 proteostatic pathway, is a powerful radiosensitizer ( 20 – 22 ) In this study, we investigated the mechanisms by which CuET enhances the therapeutic effect of [ 177 Lu]Lu-FAP-2286. First, CuET likely impairs the ATR-CHK1-mediated DNA damage response, which is critical for coping with replication stress induced by low-dose-rate ionizing radiation from [ 177 Lu]Lu. The marked increase in γ-H2AX in the combination group suggests that CuET effectively traps RLT-induced DNA lesions, preventing their repair and promoting their conversion into lethal DSBs. This DNA damage sequentially induces apoptosis and autophagy in cancer cells, and yield a better therapeutic effect for tumors. This is analogous to the successful strategy of combining PARP inhibitors with RLT, as recently demonstrated with olaparib and [ 177 Lu]Lu-DOTAGA.(SA.FAPi) 2 ( 33 ). Second, [ 177 Lu]Lu-FAP-2286 RLT itself significantly upregulated NPL4 expression in tumors. NPL4 upregulation may represent a cellular stress response to ionizing radiation or proteotoxic stress. This is a critical finding, as it renders the surviving tumor cells exquisitely sensitive to CuET, whose primary mechanism of action is high-affinity NPL4 binding and subsequent inhibition of the p97-NPL4-UFD1 pathway ( 20 ). Thus, the RLT primes the tumors for CuET attack by both inflicting DNA damage and increasing the concentration of its molecular target. This mutual collaboration is a powerful concept for overcoming resistance and achieving deep responses. Although the radiosensitizing effect of CuET in combination with [ 177 Lu]Lu-FAP-2286 is highly encouraging, the ideal treatment effect has not yet been fully achieved in this study (only 44.4% of the mice with tumor complete resolution). Further efforts are needed to identify strategies that can achieve complete tumor elimination. In our study, only a single [ 177 Lu]Lu-FAP-2286 treatment was used; multiple cycles of [ 177 Lu]Lu-FAP-2286 treatment may yield even greater therapeutic benefits. Furthermore, although our immunohistochemical findings strongly support the proposed mechanism, further in‑depth mechanistic studies are still required to fully elucidate the underlying molecular events. Conclusion This preclinical study establishes that combining CuET with [ 177 Lu]Lu-FAP-2286 yields a powerful synergistic antitumor effect in lung cancer. The combination enhances DNA damage, promotes apoptosis, and suppresses proliferation, leading to superior tumor control and prolonged survival compared to monotherapy. The radiosensitizing effect of CuET, together with the RLT- induced upregulation of NPL4 observed in our study, represents a promising novel strategy for clinical translation to improve the efficacy of FAP‑targeted radionuclide therapy. Declarations Consent for publication Not applicable. Conflict of interest The authors declare that they have no conflict of interest. Ethics approval and consent to participate All animal experiments were performed in compliance with the relevant regulations governing the use of laboratory animals and were approved by the Animal Experiment Ethics Committee of Nanfang Hospital, Southern Medical University (Approval Number: IACUC-LAC-20240119-001). Funding This study was supported by funding received from the Hubing Wu (the National Natural Science Foundation of China, grant number: 82371995, 12326616), Lijuan Wang (the National Natural Science Foundation of China, grant number: 82402326) and Wenlan Zhou (the President Foundation of Nanfang Hospital, Southern Medical University, grant number: 2023A028). Author Contributions Conceptualization and study design: HBW. Data acquisition: JHX and YJZ. Data analysis and interpretation: JHX, YJZ, YZZ, and DZS. Data curation: JHX, WLZ, and YT. Drafting of the manuscript: JHX. Manuscript revision: LJW and HBW. All authors have read and approved the final manuscript. Acknowledgements We gratefully acknowledge the support provided for this research by all colleagues from the Department of Nuclear Medicine, Nanfang Hospital, Southern Medical University and the Key Laboratory Project of Guangdong Provincial Department of Education for Ordinary Universities and GDMPA Key Laboratory for Quality Control and Evaluation of Radiopharmaceuticals. Data Availability The presented data is available from the corresponding author upon reasonable request. References Zhu T, Hsu JC, Guo J, Chen W, Cai W, Wang K. Radionuclide-based theranostics - a promising strategy for lung cancer. Eur J Nucl Med Mol Imaging. 2023;50(8):2353–74. Sgouros G, Bodei L, McDevitt MR, Nedrow JR. Radiopharmaceutical therapy in cancer: clinical advances and challenges. Nat Rev Drug Discovery. 2020;19(9):589–608. Jadvar H. Targeted Radionuclide Therapy: An Evolution Toward Precision Cancer Treatment. AJR Am J Roentgenol. 2017;209(2):277–88. 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Cancer Discov. 2022;12(11):2606–25. Zhao Y, Jia Y, Wang J, Chen X, Han J, Zhen S, et al. circNOX4 activates an inflammatory fibroblast niche to promote tumor growth and metastasis in NSCLC via FAP/IL-6 axis. Mol Cancer. 2024;23(1):47. Zboralski D, Hoehne A, Bredenbeck A, Schumann A, Nguyen M, Schneider E, et al. Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging. 2022;49(11):3651–67. Bao G, Wang Z, Liu L, Zhang B, Song S, Wang D, et al. Targeting CXCR4/CXCL12 axis via [(177)Lu]Lu-DOTAGA.(SA.FAPi)(2) with CXCR4 antagonist in triple-negative breast cancer. Eur J Nucl Med Mol Imaging. 2024;51(9):2744–57. Taddio MF, Doshi S, Masri M, Jeanjean P, Hikmat F, Gerlach A, et al. Evaluating [(225)Ac]Ac-FAPI-46 for the treatment of soft-tissue sarcoma in mice. Eur J Nucl Med Mol Imaging. 2024;51(13):4026–37. Rao Z, Zhang Y, Liu L, Wang M, Zhang C. [(177)Lu]Lu-FAP-2286 therapy in a case of right lung squamous cell carcinoma with systemic metastases. Eur J Nucl Med Mol Imaging. 2023;50(4):1266–7. Baum RP, Schuchardt C, Singh A, Chantadisai M, Robiller FC, Zhang J, et al. Feasibility, Biodistribution, and Preliminary Dosimetry in Peptide-Targeted Radionuclide Therapy of Diverse Adenocarcinomas Using (177)Lu-FAP-2286: First-in-Humans Results. Journal of nuclear medicine: official publication. Soc Nuclear Med. 2022;63(3):415–23. Banihashemian SS, Akbari ME, Norouzi G, Nikkholgh B, Amini H, Divband G, et al. The complete metabolic/molecular response to chemotherapy combined with [(177)Lu]Lu-FAP-2286 in metastatic breast cancer. Eur J Nucl Med Mol Imaging. 2024;51(13):4185–7. Banihashemian SS, Akbari ME, Pirayesh E, Divband G, Abolhosseini Shahrnoy A, Nami R, et al. Feasibility and therapeutic potential of [(177)Lu]Lu-FAPI-2286 in patients with advanced metastatic sarcoma. Eur J Nucl Med Mol Imaging. 2024;52(1):237–46. Skrott Z, Mistrik M, Andersen KK, Friis S, Majera D, Gursky J, et al. Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4. Nature. 2017;552(7684):194–9. Majera D, Skrott Z, Chroma K, Merchut-Maya JM, Mistrik M, Bartek J. Targeting the NPL4 Adaptor of p97/VCP Segregase by Disulfiram as an Emerging Cancer Vulnerability Evokes Replication Stress and DNA Damage while Silencing the ATR Pathway. Cells. 2020;9(2). Krastev DB, Li S, Sun Y, Wicks AJ, Hoslett G, Weekes D, et al. The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin. Nat Cell Biol. 2022;24(1):62–73. Lee YE, Choi SA, Kwack PA, Kim HJ, Kim IH, Wang KC, et al. Repositioning disulfiram as a radiosensitizer against atypical teratoid/rhabdoid tumor. Neurooncology. 2017;19(8):1079–87. Wang K, Michelakos T, Wang B, Shang Z, DeLeo AB, Duan Z, et al. Targeting cancer stem cells by disulfiram and copper sensitizes radioresistant chondrosarcoma to radiation. Cancer Lett. 2021;505:37–48. Yao W, Qian X, Ochsenreither S, Soldano F, DeLeo AB, Sudhoff H et al. Disulfiram Acts as a Potent Radio-Chemo Sensitizer in Head and Neck Squamous Cell Carcinoma Cell Lines and Transplanted Xenografts. Cells. 2021;10(3). Huang J, Zhang X, Liu Q, Gong F, Huang Y, Huang S, et al. (68)Ga/(177)Lu-Labeled Theranostic Pair for Targeting Fibroblast Activation Protein with Improved Tumor Uptake and Retention. J Med Chem. 2024;67(19):17785–95. Li H, Ye S, Li L, Zhong J, Yan Q, Zhong Y, et al. (18)F- or (177)Lu-labeled bivalent ligand of fibroblast activation protein with high tumor uptake and retention. Eur J Nucl Med Mol Imaging. 2022;49(8):2705–15. He Y, Yang M, Yang L, Hao M, Wang F, Li X, et al. Preparation and anticancer actions of CuET-nanoparticles dispersed by bovine serum albumin. Colloids Surf B Biointerfaces. 2023;226:113329. Wen Z, Luo D, Wang S, Rong R, Evers BM, Jia L, et al. Deep Learning-Based H-Score Quantification of Immunohistochemistry-Stained Images. Mod pathology: official J United States Can Acad Pathol Inc. 2024;37(2):100398. Zboralski D, Osterkamp F, Christensen E, Bredenbeck A, Schumann A, Hoehne A, et al. Fibroblast activation protein targeted radiotherapy induces an immunogenic tumor microenvironment and enhances the efficacy of PD-1 immune checkpoint inhibition. Eur J Nucl Med Mol Imaging. 2023;50(9):2621–35. Dadgar H, Pashazadeh A, Norouzbeigi N, Assadi M, Al-Balooshi B, Baum RP, et al. Targeted radioligand therapy: physics and biology, internal dosimetry and other practical aspects during (177)Lu/(225)Ac treatment in neuroendocrine tumors and metastatic prostate cancer. Theranostics. 2025;15(10):4368–97. Gudkov SV, Shilyagina NY, Vodeneev VA, Zvyagin AV. Targeted Radionuclide Therapy of Human Tumors. Int J Mol Sci. 2015;17(1). Bao G, Zhou H, Zou S, Chen L, Zhang B, Wang Z, et al. Inhibition of Poly(ADP-ribose) Polymerase Sensitizes [(177)Lu]Lu-DOTAGA.(SA.FAPi)(2)-Mediated Radiotherapy in Triple-Negative Breast Cancer. Mol Pharm. 2023;20(5):2443–51. Supplementary Files SupplementaryData.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 09 Mar, 2026 Reviewers invited by journal 09 Mar, 2026 Editor invited by journal 06 Mar, 2026 Editor assigned by journal 05 Mar, 2026 First submitted to journal 04 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9032718","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":602974266,"identity":"d2f7d791-3d6c-41e5-9653-7ecc775803a2","order_by":0,"name":"Jiahao Xie","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiahao","middleName":"","lastName":"Xie","suffix":""},{"id":602974267,"identity":"c636b775-439c-43d7-8658-f4b1e8e4ac88","order_by":1,"name":"Yijin Zou","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yijin","middleName":"","lastName":"Zou","suffix":""},{"id":602974268,"identity":"7128ab66-5b60-4680-810f-3bcbeac6b608","order_by":2,"name":"Yuzhao Zheng","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuzhao","middleName":"","lastName":"Zheng","suffix":""},{"id":602974269,"identity":"486cd0d1-8219-4f5a-a9db-43e1ed8254a5","order_by":3,"name":"Dazhi Shi","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dazhi","middleName":"","lastName":"Shi","suffix":""},{"id":602974270,"identity":"45c82b1f-5963-4b35-8aaa-10ed06615a7f","order_by":4,"name":"Wenlan Zhou","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenlan","middleName":"","lastName":"Zhou","suffix":""},{"id":602974271,"identity":"53d2284c-4892-4dc3-a17a-1ab52cf2a878","order_by":5,"name":"Ying Tian","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Tian","suffix":""},{"id":602974272,"identity":"94fc0cde-263c-4c13-a86c-3632dbd8634a","order_by":6,"name":"Lijuan Wang","email":"","orcid":"","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lijuan","middleName":"","lastName":"Wang","suffix":""},{"id":602974273,"identity":"e4b4c65b-2945-4bef-ba1a-0478f611e7c2","order_by":7,"name":"Hubing Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoklEQVRIiWNgGAWjYFACHjYgYcPDz99AmpY0GckZB0jTctjGoCGBSA0GN3KPPebNOc9jwHCA8cPHHKK05KUb8267zWPO3MAsOXMbEVrMbuSYSYO0WDYcYGPmJUHLOR6DAwmkaTlAghb7M2/MDeduS+aRnHGwmTi/SLbnmD14u83Onp+/+eCHj8RoAQEmHjDF2ECkepDaH8SrHQWjYBSMgpEIALhnNKPFBJQRAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7546-5430","institution":"Southern Medical University Nanfang Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hubing","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-03-04 17:10:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9032718/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9032718/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104794561,"identity":"dddc183f-c5ea-4c2e-a867-975e73072d93","added_by":"auto","created_at":"2026-03-17 09:21:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7178981,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative images of FAP and NPL4 expression in mouse A549-FAP xenograft tumors by immunohistochemical staining (left, FAP; right, NPL4; 50 µm). (B) The representative MIP images of [\u003csup\u003e68\u003c/sup\u003eGa]GA-FAP-2286 at 1, 2, and 4 h after intravenous injection in A549-FAP tumor-bearing mice. Tumors were indicated inside the red circles.\u0026nbsp;(C) The uptake intensity (%ID/g) of [\u003csup\u003e68\u003c/sup\u003eGa]GA-FAP-2286 in A549-FAP tumor-bearing mice at different time points (1h, 2h, and 4h). (D) Radioactivity biodistribution of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 in tumors and selected organs.\u0026nbsp;(E) Immunohistochemical staining of NPL4 expression in the tumors tissue from the tumor xenografts\u003cstrong\u003e \u003c/strong\u003eof vehicle group and [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment group after 13 days treatment. Scale bar: 50 µm. %ID/g, percent injected dose per gram; Data are mean ± SD, n = 3. MIP: maximum intensity projection.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9032718/v1/da523a5143e82e362353f750.png"},{"id":104794565,"identity":"9578ff30-4884-4c78-884f-420f85c64343","added_by":"auto","created_at":"2026-03-17 09:21:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9432149,"visible":true,"origin":"","legend":"\u003cp\u003e(A,B) Representative [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 PET/CT of the A549-FAP models of different treatment groups on days 1 and 13 after treatment (n=3). (C,D) Representative pictures of A549-FAP models of different treatment groups at days 1 and 13 after treatment. Tumors were indicated inside the red circles. (E) Representative pictures of excised A549-FAP tumors of different treatment groups at day 13 after treatment. (F) Tumor volume of 5 treatment groups measured on days 1 and 13 (n = 9) after treatment. (G) Changes of tumor volume of 5 treatment groups after 13 days treatment (n = 9). (H) Tumor uptake of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 of 5 treatment groups measured on PET/CT on days 1 and 13 after treatment (n = 3). (I) MTV of 5 treatment groups measured on PET/CT on days 1 and 13 after treatment (n = 3). MTV: Metabolic tumor volume. Group 1 = PBS; Group 2 = CuET; Group 3 = [\u003csup\u003e177\u003c/sup\u003eLu]Lu\u003csup\u003e \u003c/sup\u003e-FAP-2286 (37MBq); Group 4 = CuET + [\u003csup\u003e177\u003c/sup\u003eLu]Lu\u003csup\u003e \u003c/sup\u003e-FAP-2286 (18.5MBq); Group 5 = CuET + [\u003csup\u003e177\u003c/sup\u003eLu]Lu\u003csup\u003e \u003c/sup\u003e-FAP-2286 (37MBq).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9032718/v1/e85ed7229b977f54ea47e486.png"},{"id":104794564,"identity":"5964fd37-9831-4cc3-a487-f49bf92069a4","added_by":"auto","created_at":"2026-03-17 09:21:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1168098,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic effect evaluation. (A−E) The changes in tumor volume (V\u003csub\u003ex\u003c/sub\u003e/V\u003csub\u003e0\u003c/sub\u003e) of every individual of different treatment groups after treatments; each line represents one mouse. (F) The changes of tumor volume of mice of different treatment groups after treatments. (G) The Kaplan−Meier survival curves for different treatment groups. Data are mean ± SD, n = 9 for each treated group. (H) The survival times of the PBS, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 group and the full-dose combination group.\u0026nbsp;\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9032718/v1/eff74a0b5bcb47d9d7ab489e.png"},{"id":104835267,"identity":"cab22c1c-db00-45a2-89a9-5ad6b325d6ef","added_by":"auto","created_at":"2026-03-17 17:42:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5187393,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Immunohistochemistry of γ-H2AX, Cleaved caspase-3, LC3B, Survivin, Bcl2, and Ki-67. Scale bar for immunohistochemistry, 50 µm. (B) The H-score for γ-H2AX, Cleaved caspase-3, LC3B, Survivin, Bcl2, and Ki-67 were quantified and compared (n = 3). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt;0 .01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9032718/v1/e7419967deaa228c21b01a7b.png"},{"id":104836023,"identity":"60f1b5d1-ca92-48ee-865d-061b1d3e4f76","added_by":"auto","created_at":"2026-03-17 17:51:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21947937,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9032718/v1/837e9f7d-2a57-4509-a1d3-da310dd46e52.pdf"},{"id":104808772,"identity":"3e6a2dd7-8a62-4974-bfe1-c51d2206c6ab","added_by":"auto","created_at":"2026-03-17 12:39:56","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1603252,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.docx","url":"https://assets-eu.researchsquare.com/files/rs-9032718/v1/6fef753470755cfc19f3f4f0.docx"}],"financialInterests":"","formattedTitle":"CuET Radiosensitizes FAP-Targeted Radioligand Therapy in Lung Cancer by inhibiting DNA Repair","fulltext":[{"header":"Background","content":"\u003cp\u003eRadionuclide ligand therapy (RLT), an innovative internal radiotherapy strategy, offers a promising alternative for treating widespread metastatic disease. RLT employs ligands conjugated to therapeutic radionuclides such as \u003csup\u003e177\u003c/sup\u003eLu or \u003csup\u003e225\u003c/sup\u003eAc via macrocyclic chelating agents, enabling selective delivery of radiation to cancer cells following intravenous administration. These radionuclides exert sustained therapeutic effects through localized emission of α- and β-particles while sparing normal tissues. RLT has emerged as an effective treatment for disseminated metastatic tumors (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). For example, \u003csup\u003e177\u003c/sup\u003eLu-PSMA-617 targeting prostate-specific membrane antigen (PSMA) and \u003csup\u003e177\u003c/sup\u003eLu-DOTATATE targeting somatostatin receptors have demonstrated that these RLTs significantly improve overall survival in patients with advanced prostate cancer and metastatic neuroendocrine tumors (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFibroblast activation protein (FAP) is a cell surface marker of cancer-associated fibroblasts (CAFs), a critical component of the tumor stroma. FAP is highly expressed in CAFs across various tumor types, including lung cancer(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In lung cancer, its high expression in both primary and metastatic lesions, coupled with minimal expression in healthy tissues, makes FAP an ideal candidate for RLT in lung cancer (\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Radiolabeled FAP inhibitors (FAPIs), such as [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTAGA-(SA-FAPI)\u003csub\u003e2\u003c/sub\u003e, and [\u003csup\u003e225\u003c/sup\u003eAc]Ac-FAPI, have shown the ability to deliver targeted radiotherapy to FAP-expressing tumors (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Although [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 has shown initial clinical promise, therapeutic responses can be heterogeneous, and durable control remains a challenge, likely due to inherent or acquired resistance mechanisms(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Therefore, further exploration of novel therapeutic strategies is necessary to improve the therapeutic efficacy of this treatment.\u003c/p\u003e \u003cp\u003eThe limited antitumor efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 may be largely attributed to efficient repair of the predominantly single-strand DNA breaks induced by its continuous low-dose-rate β-irradiation. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Diethyldithiocarbamate (DDC) is an anti-cancer metabolite derived from disulfiram (DSF), an old and cheap FDA-approved drug primarily used to treat alcoholism. It can chelate with copper ions to form the ditiocarb-copper complex Cu(DDC)2, also known as CuET. This complex binds to NPL4 with extremely high affinity (Kd\u0026thinsp;=\u0026thinsp;4.647\u0026times;10\u0026ndash;8M), effectively immobilizing NPL4 and disrupting the critical p97\u0026ndash;NPL4\u0026ndash;UFD1 pathway, which inhibits the very important proteasome/poly-Ub protein degradation pathway (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Concurrently, CuET has been shown to impair the ATRIP-ATR-CHK1 signaling axis, a master regulator of the DNA replication stress response, leading to replication fork stalling, accumulation of single-stranded DNA, and defective DNA repair (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). This dual activity positions CuET as a potent radio- and chemosensitizer (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aims to evaluate whether CuET can overcome resistance to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 RLT. We specifically investigate a two-pronged hypothesis: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) CuET-mediated inhibition DNA repair, converting repairable single-strand DNA breaks (SSBs) into lethal double-strand breaks (DSBs); and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) RLT-induced upregulation of NPL4, will render tumor cells more vulnerable to CuET's proteotoxic effects, creating a therapeutic feedback loop. By combining [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 with CuET, we sought to demonstrate a synergistic antitumor effect and elucidate its underlying mechanisms.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eDetailed information on materials and methods including radiolabeling of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 and \u003cb\u003e[\u003c/b\u003e\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286, their radiochemical purity, in vitro and in vivo stability, binding affinity and cellular uptake studies are described in the Supplementary data according to the methods previously reported (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e1. Tumor Xenograft Models of Lung Adenocarcinoma\u003c/h3\u003e\n\u003cp\u003eMale BALB/c nude mice (aged 4 to 6 weeks; GemPharmatech Co., Ltd.) were subcutaneously injected with A549-FAP cells on the right flank, with each injection containing 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells in 100 \u0026micro;L of phosphate-buffered saline (PBS). For biodistribution studies and micro-PET/CT imaging, mice were used when the tumor diameter reached 7\u0026thinsp;\u0026minus;\u0026thinsp;9 mm. For therapeutic studies, mice were selected when the tumor diameter was 5\u0026thinsp;\u0026minus;\u0026thinsp;6 mm, corresponding to a tumor volume of approximately 100 mm\u003csup\u003e3\u003c/sup\u003e. All animal experiments were performed in compliance with the legislation on the use of laboratory animals and approved by Ethics Committee.\u003c/p\u003e\n\u003ch3\u003e2. Micro-PET Imaging of [Ga]Ga-FAP-2286\u003c/h3\u003e\n\u003cp\u003eMicro-PET/CT imaging of tumor-bearing mice was conducted using a micro-PET/CT scanner (Siemens, Erlangen, Germany). Mice were intravenously injected with 5.55\u0026ndash;7.40 MBq of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 and subsequently imaged with PET/CT. To elucidate the in vivo pharmacokinetics and tumor retention of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 over an extended period, a series of static micro-PET/CT scans were performed at 1, 2, and 4 hours post-injection (n\u0026thinsp;=\u0026thinsp;3). The metabolic tumor volume (MTV) was measured using the threshold method (45.0% of SUV\u003csub\u003emax\u003c/sub\u003e) on [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 PET/CT images (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 image was reconstructed using the three-dimensional ordered-subset expectation maximum algorithm (Siemens, Erlangen, Germany), and the uptake intensity in tumors and normal organs was quantified as %ID/g.\u003c/p\u003e\n\u003ch3\u003e3. In Vivo Radioactivity Biodistribution Study of [Lu]Lu-FAP-2286\u003c/h3\u003e\n\u003cp\u003eThe in vivo radioactivity biodistribution of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 was evaluated in A549-FAP tumor-bearing mice. Each mouse received an intravenous injection of approximately 1.85 MBq (50 \u0026micro;Ci) of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286. At 1, 4, 24, 72, and 168 hours post-injection, the mice were euthanized, and tumors along with various organs of interest were collected. These organs included blood, lung, heart, brain, bone, muscle, gallbladder, liver, kidney, spleen, intestine, stomach, and tumor tissue. A γ-counter was employed to measure the radioactivity in the collected samples. The uptake intensity of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 was quantified as %ID/g.\u003c/p\u003e\n\u003ch3\u003e4. Treatment Protocols\u003c/h3\u003e\n\u003cp\u003eA549-FAP tumor-bearing mice were randomly assigned into five groups (n\u0026thinsp;=\u0026thinsp;12/group) and treated with the following regimens: PBS (vehicle control), CuET alone, full-dose alone [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 (37 MBq), half-dose [\u003csup\u003e177\u003c/sup\u003eLu]Lu- FAP-2286 (18.5 MBq) plus CuET(half-dose combined treatment), and full-dose [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 (37 MBq) plus CuET༈full-dose combined treatment). For CuET treatment, mice received intraperitoneal injections of 2 mg/kg CuET, as referenced in the literatures (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 was administered via intravenous injection at doses of 37 MBq (full-dose) or 18.5 MBq (half-dose) for RLT. The injection frequency and time intervals are illustrated in Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003e5. Efficacy Evaluation\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo assess treatment efficacy, tumor size and body weight were measured every three days starting from the first day after treatment. The tumor volume was calculated (V=width\u003csup\u003e2\u003c/sup\u003e \u0026times; Length \u0026times; 0.5; mm\u003csup\u003e3\u003c/sup\u003e). The changes in tumor volume and body weight were calculated as V\u003csub\u003eX\u003c/sub\u003e/V\u003csub\u003e0\u003c/sub\u003e and W\u003csub\u003eX\u003c/sub\u003e/W\u003csub\u003e0\u003c/sub\u003e, respectively. Meanwhile, at 13 days post-treatment, [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 PET/CT imaging was conducted to assess the therapeutic efficacy for 5 groups (n\u0026thinsp;=\u0026thinsp;3/group). Then, the mice underwent the immunohistochemistry (IHC) essay. The tumor-bearing mice (n\u0026thinsp;=\u0026thinsp;9/group) were used to continuously evaluate their long-term therapeutic efficacy. The evaluation was terminated if the tumor volume exceeded 1500 mm\u003csup\u003e3\u003c/sup\u003e, if active ulceration occurred in the tumor, or if body weight loss exceeded 15% of the initial weight. Survival rate was calculated as the proportion of mice alive before reaching the evaluation endpoint relative to the total number of mice.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e6. Hematoxylin-Eosin (HE) Staining and IHC\u003c/h3\u003e\n\u003cp\u003eAt 13 days post-treatment, three tumor-bearing mice per group were euthanized after PET/CT scans, and their heart, liver, spleen, lung, kidney, and tumor were collected. For HE staining, the hearts, livers, spleens, lungs, and kidneys of mice were collected at 13 days post-treatment. For IHC analysis, after processing A549-FAP tumor samples, IHC staining was performed targeting FAP, NPL4, γ-H2AX (a marker for DNA double-strand breaks), cleaved caspase-3 (an apoptosis marker), LC3B (an autophagy marker), survivin(an anti-apoptotic protein), Bcl2 (an anti-apoptotic protein), and Ki-67 (a protein related to cell proliferation), following the standard protocol. The primary antibodies used for IHC staining were sourced from the following suppliers: FAP (Cell Signaling Technology), NPL4 (Proteintech), cleaved caspase-3 (BioWorld), LC3B (Affinity), γ-H2AX (Abcam), Ki-67 (Abcam), Bcl2 (BioWorld), and survivin (BioWorld). The IHC results were further analyzed using ImageJ software. The H-score, a reliable metric for evaluating staining intensity, was calculated as follows: (1 \u0026times; percentage of weak staining) + (2 \u0026times; percentage of moderate staining) + (3 \u0026times; percentage of strong staining) within the target region, with a range of 0 to 300 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e7. Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eData were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analyzed accordingly. All calculations were performed using SPSS Statistics version 26 (IBM). T-tests and Wilcoxon Signed-Rank Test were used to compare the different groups. Survival analysis was performed with the Kaplan\u0026ndash;Meier method and Log-rank (Mantel-Cox) test. Statistical significance was set as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\n\u003ch3\u003e1. Successful Synthesis and High Stability of [Lu]Lu-FAP-2286\u003c/h3\u003e\n\u003cp\u003e[\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 was successfully synthesized, achieving a molar activity of up to 27.2 MBq/nmol and a radiochemical purity exceeding 95%. It demonstrated excellent in vitro stability, maintaining over 95% radiochemical purity after 24 hours of incubation at 37\u0026deg;C in both PBS and FBS. Additionally, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 exhibited remarkable in vivo stability in mouse blood, with radiochemical purity exceeding 90% at 24 hours post-injection (Supplementary Fig. S2).\u003c/p\u003e \u003cp\u003e[\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286, the theranostic pair of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286, was also successfully radio-synthesized. The non-decay corrected radiochemical yield was 69.2% \u0026plusmn; 4.5% (n\u0026thinsp;=\u0026thinsp;3). After purification, [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 exhibited high molar radioactivity (24.7\u0026ndash;31.0 GBq/\u0026micro;mol) and radiochemical purity (\u0026gt;\u0026thinsp;95%).\u003c/p\u003e\n\u003ch3\u003e2. High Affinity and High Uptake of [Lu]Lu-FAP-2286 in A549-FAP Cells and Tumors\u003c/h3\u003e\n\u003cp\u003eIn vitro cell experiments were conducted to evaluate the cellular uptake capacity and binding affinity of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 to A549-FAP cells. As shown in Supplementary Fig. S3, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 exhibited high-level cellular uptake (40.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1% ID/1 mio cells at 48 hours post-incubation), which increased gradually with prolonged incubation time. The competitive binding assay revealed that DOTA-FAP-2286 had a strong binding affinity to A549-FAP cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 nM).\u003c/p\u003e \u003cp\u003eOver-expression of FAP in A549-FAP tumor xenografts was confirmed using IHC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consistent with the high FAP expression, micro-PET/CT imaging showed strong uptake (7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0% ID/g) of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 in A549-FAP xenograft tumors at 4 hour after intravenously administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe uptake of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 was evaluated using an in vitro radioassay following a 1 hour intravenous administration to A549-FAP tumor xenografts. Consistent with the findings for [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 demonstrated significant accumulation within the tumors, reaching an uptake level of 19.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8% ID/g. This pronounced uptake was maintained over an extended period, lasting up to 72 hours. Meanwhile, low uptake of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 was found in normal organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. Anticancer Efficacy of [\u003c/b\u003e \u003csup\u003e \u003cb\u003e177\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eLu]Lu-FAP-2286 and CuET Treatment and Upregulation of Tumor NPL4 Expression Induced by [\u003c/b\u003e \u003csup\u003e \u003cb\u003e177\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eLu]Lu-FAP-2286 Treatment\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs a single agent, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 effectively stalled tumor growth at day 13 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In the vehicle group, tumor volume rapidly increased from 97.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3 mm\u003csup\u003e3\u003c/sup\u003e to 530.2\u0026thinsp;\u0026plusmn;\u0026thinsp;148.8 mm\u003csup\u003e3\u003c/sup\u003e after 13 days of treatment, representing a 468.1% \u0026plusmn; 184.9% volume increase. In contrast, in the [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 monotherapy group, tumor volume remained nearly unchanged, from 102.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1 mm\u003csup\u003e3\u003c/sup\u003e to 103.2\u0026thinsp;\u0026plusmn;\u0026thinsp;40.1 mm\u003csup\u003e3\u003c/sup\u003e after 13 days of treatment, with a minimal volume increase (1.5% \u0026plusmn; 43.2%), which was significantly lower than that of the vehicle group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, the survival time of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 monotherapy was significantly longer than that of the vehicle group (38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 vs. 21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 days, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Supplementary Table S2).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe tumor volume and their changes of A549-FAP xenografts mice measured at 1 day and 13 days after the treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTumor volume, Day 1 (mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTumor volume Day 13 (mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChange of tumor volume (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e97.4\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e530.2\u0026thinsp;\u0026plusmn;\u0026thinsp;148.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e+, 468.1\u0026thinsp;\u0026plusmn;\u0026thinsp;184.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCuET\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e94.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e176.5\u0026thinsp;\u0026plusmn;\u0026thinsp;58.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e+, 86.3\u0026thinsp;\u0026plusmn;\u0026thinsp;54.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu-FAP-2286 (37 MBq)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e102.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e103.2\u0026thinsp;\u0026plusmn;\u0026thinsp;40.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e+, 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;43.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.933\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCuET+\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu-FAP-2286 (18.5 MBq)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e93.0\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e63.2\u0026thinsp;\u0026plusmn;\u0026thinsp;35.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-, 33.9\u0026thinsp;\u0026plusmn;\u0026thinsp;34.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.038\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCuET+\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu-FAP-2286 (37 MBq)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e95.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;33.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-, 65.1\u0026thinsp;\u0026plusmn;\u0026thinsp;31.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e+: increase, -: reduce.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCuET treatment effectively delayed tumor growth and prolonged the survival time of A549-FAP lung adenocarcinoma xenografts. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, in the CuET treatment group, the increase in tumor volume in 13 days post-treatment was significantly smaller than that in the vehicle group (86.3\u0026thinsp;\u0026plusmn;\u0026thinsp;54.5% vs. 468.1\u0026thinsp;\u0026plusmn;\u0026thinsp;184.9%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). As shown in Supplementary Table S2, regarding long-term efficacy, the survival time of the CuET treatment group was also significantly longer than that of the vehicle group (33.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 days vs. 21.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 days, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, its efficacy was significantly lower than that of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eHigh NPL4 expression was identified in A549-FAP tumors before treatment. On the 13th day post-treatment, IHC showed that [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment significantly upregulated NPL4 expression compared with the vehicle group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the NPL4 H-score increasing by 119.1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\n\u003ch3\u003e4. Enhanced Anticancer Efficacy of [Lu]Lu-FAP-2286 Combined with CuET for A549-FAP Tumor Xenografts.\u003c/h3\u003e\n\u003cp\u003eIn the short-term efficacy evaluation, tumor volumes in the full-dose combined treatment group decreased from 95.1\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0 mm\u003csup\u003e3\u003c/sup\u003eto 34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;33.6 mm\u003csup\u003e3\u003c/sup\u003e after 13 days treatment, representing a 65.1% \u0026plusmn; 31.5% reduction. In contrast, the tumor volume in the [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment group had a slight increase from 102.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1 mm\u003csup\u003e3\u003c/sup\u003e to 103.2\u0026thinsp;\u0026plusmn;\u0026thinsp;40.1 mm\u003csup\u003e3\u003c/sup\u003e during this period (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). On the 13th day post-treatment, the tumor volume of the full-dose combined treatment group was significantly lower than that of the [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment group (t\u0026thinsp;=\u0026thinsp;3.937, P\u0026thinsp;=\u0026thinsp;0.001). [\u003csup\u003e68\u003c/sup\u003eGa]Ga-FAP-2286 PET/CT imaging was performed to measure the MTVs of the treatment groups on day 13 post-treatment, showing similar changes in MTV, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, I. This early profound response translated into a remarkable long-term survival benefit. In the long-term efficacy evaluation, a distinct difference in therapeutic efficacy was observed between the full-dose combined treatment groups and the [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 monotherapy group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After 50 days post-treatment, all mice had died in the [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 monotherapy group, whereas 77.8% of mice were still alive in the full-dose combined treatment groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As shown in Supplementary Table S2, the survival time of the full-dose combined treatment groups was 55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7 days, which was significantly longer than that of the [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 groups (38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 days) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Meanwhile, tumor complete resolution was observed 44.4% of the mice in full-dose combined treatment groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe survival rates of A549-FAP xenografts mice at 20, 30, 40, 50 and 60 days after treatment and tumor complete resolution rate.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvival rate, Day 20\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSurvival rate, Day 30\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSurvival rate, Day 40\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSurvival rate, Day 50\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSurvival rate, Day 60\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTumor complete resolution (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/9 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCuET\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/9 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu-FAP-2286 (37 MBq)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/9 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCuET+\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu-FAP-2286 (18.5 MBq)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1/9 (11.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCuET+\u003c/b\u003e\u003csup\u003e\u003cb\u003e177\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eLu-FAP-2286 (37 MBq)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4/9 (44.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e5. CuET Acts as a Potent Radiosensitizer and Enables Radiation Dose Reduction\u003c/h3\u003e\n\u003cp\u003eCompared with full-dose combined treatment, half-dose combined treatment demonstrated inferior efficacy in both short-term and long-term evaluations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Strikingly, the half-dose combination achieved short-term tumor control superior to full-dose [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 monotherapy (tumor volume change: -33.9% vs. +1.5%, t\u0026thinsp;=\u0026thinsp;2.238, P\u0026thinsp;=\u0026thinsp;0.04) and yielded a comparable median survival (41.3 days vs. 38.7 days, P\u0026thinsp;=\u0026thinsp;0.41, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This demonstrates a potent radiosensitizing effect of CuET, capable of enhancing the efficacy of a suboptimal radiation dose to a level equivalent to a full-dose treatment.\u003c/p\u003e\n\u003ch3\u003e6. Safety\u003c/h3\u003e\n\u003cp\u003eNo adverse reactions were observed in A549-FAP tumor xenografts during the above treatments. As depicted in Supplementary Fig. S4, mild body weight loss (\u0026lt;\u0026thinsp;15%) was observed only in the CuET group during the early treatment phase and in the full-dose combined treatment group during the late phase. As shown in Supplementary Fig. S5, HE staining of heart, liver, spleen, lung, and kidney tissues from mice in the full-dose combined treatment group revealed no histological abnormalities at 13 days post-treatment.\u003c/p\u003e\n\u003ch3\u003e7. Potential Mechanisms Underlying the Synergistic Antitumor Effect of CuET Combined with [Lu]Lu-FAP-2286 Treatment\u003c/h3\u003e\n\u003cp\u003eTo elucidate the potential mechanisms of the synergistic antitumor effect, IHC analysis was conducted to evaluate the expression levels of proteins associated with DNA damage response, apoptosis, autophagy, and cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The full-dose combination led to a marked increase in DNA damage, as evidenced by a 213.5% higher γ-H2AX H-score compared to RLT alone (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This was accompanied by a significant increase in apoptotic (cleaved caspase-3, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and autophagic (LC3B, P\u0026thinsp;=\u0026thinsp;0.022) markers. Conversely, the full-dose combination treatment profoundly suppressed tumor cell proliferation (Ki-67, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and downregulated the anti-apoptotic proteins survivin and Bcl-2 (P\u0026thinsp;=\u0026thinsp;0.036, and P\u0026thinsp;=\u0026thinsp;0.028, respectively). These data suggest that CuET not only exacerbates RLT-induced DNA damage but also dismantles key survival pathways, shifting the cellular balance decisively towards cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that both [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 and CuET treatments can significantly retard tumor growth and extend the survival time of A549-FAP tumor xenografts compared with the vehicle group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, their anticancer efficacy is far from optimal. Given that previous studies have identified CuET as a potent radiosensitizer that enhances the efficacy of antitumor therapy (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), the present study investigated whether CuET can inhibit the DNA damage repair triggered by [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment, thereby enhancing the anticancer efficacy. Our results demonstrated that the combination therapy induced profound tumor regression and significantly prolonged survival. CuET not only enhanced cancer cells' susceptibility to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment but also achieved a dose-reduction effect, highlighting its translational relevance.\u003c/p\u003e \u003cp\u003eThe radiosensitizing effect of CuET for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 is highly encouraging. In the long-term therapeutic efficacy evaluation, after 50 days post-treatment, the survival rates were 0.0% for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment and 77.8% for full-dose combined treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The combination treatment significantly prolonged survival time compared to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 alone (55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7 days vs. 38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 days, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similar to our research, Dirk Zboralski et al. demonstrated in preclinical studies that the therapeutic efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 as a monotherapy was limited (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). They further combined [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2287 with anti-PD-1 therapy and found this combination enhanced the sensitivity of tumor cells to radionuclide therapy by modulating immune cells within the tumor microenvironment and significantly prolonged survival(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Therefore, combined treatment may be a good strategy to improve the efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 for the treatment of FAP-positive tumors. Additionally, the ability of the half-dose combination to match the efficacy of a full-dose RLT monotherapy (41.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 days vs. 38.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 days, P\u0026thinsp;=\u0026thinsp;0.41) suggests that CuET could allow for dose reduction of the radioligand in a clinical setting, potentially mitigating off-target toxicity and broadening the therapeutic window. This radiosensitizing effect, coupled with the excellent safety profile of CuET observed here, positions it as an ideal candidate for combination strategies.\u003c/p\u003e \u003cp\u003eThe anticancer efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 is derived from the of β-rays emitted from [\u003csup\u003e177\u003c/sup\u003eLu], which primarily induces single-strand breaks rather than double-strand breaks in DNA. This DNA damage, in turn, activates the DNA repair mechanisms of tumor cells, thereby diminishing the therapeutic efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). CuET, a multimodal inhibitor of both the ATR-CHK1 DNA repair pathway and the NPL4-p97 proteostatic pathway, is a powerful radiosensitizer (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) In this study, we investigated the mechanisms by which CuET enhances the therapeutic effect of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286. First, CuET likely impairs the ATR-CHK1-mediated DNA damage response, which is critical for coping with replication stress induced by low-dose-rate ionizing radiation from [\u003csup\u003e177\u003c/sup\u003eLu]Lu. The marked increase in γ-H2AX in the combination group suggests that CuET effectively traps RLT-induced DNA lesions, preventing their repair and promoting their conversion into lethal DSBs. This DNA damage sequentially induces apoptosis and autophagy in cancer cells, and yield a better therapeutic effect for tumors. This is analogous to the successful strategy of combining PARP inhibitors with RLT, as recently demonstrated with olaparib and [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTAGA.(SA.FAPi)\u003csub\u003e2\u003c/sub\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSecond, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 RLT itself significantly upregulated NPL4 expression in tumors. NPL4 upregulation may represent a cellular stress response to ionizing radiation or proteotoxic stress. This is a critical finding, as it renders the surviving tumor cells exquisitely sensitive to CuET, whose primary mechanism of action is high-affinity NPL4 binding and subsequent inhibition of the p97-NPL4-UFD1 pathway (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Thus, the RLT primes the tumors for CuET attack by both inflicting DNA damage and increasing the concentration of its molecular target. This mutual collaboration is a powerful concept for overcoming resistance and achieving deep responses.\u003c/p\u003e \u003cp\u003eAlthough the radiosensitizing effect of CuET in combination with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 is highly encouraging, the ideal treatment effect has not yet been fully achieved in this study (only 44.4% of the mice with tumor complete resolution). Further efforts are needed to identify strategies that can achieve complete tumor elimination. In our study, only a single [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment was used; multiple cycles of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 treatment may yield even greater therapeutic benefits. Furthermore, although our immunohistochemical findings strongly support the proposed mechanism, further in‑depth mechanistic studies are still required to fully elucidate the underlying molecular events.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis preclinical study establishes that combining CuET with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 yields a powerful synergistic antitumor effect in lung cancer. The combination enhances DNA damage, promotes apoptosis, and suppresses proliferation, leading to superior tumor control and prolonged survival compared to monotherapy. The radiosensitizing effect of CuET, together with the RLT- induced upregulation of NPL4 observed in our study, represents a promising novel strategy for clinical translation to improve the efficacy of FAP‑targeted radionuclide therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e All animal experiments were performed in compliance with the relevant regulations governing the use of laboratory animals and were approved by the Animal Experiment Ethics Committee of Nanfang Hospital, Southern Medical University (Approval Number: IACUC-LAC-20240119-001).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by funding received from the Hubing Wu (the National Natural Science Foundation of China, grant number: 82371995, 12326616), Lijuan Wang (the National Natural Science Foundation of China, grant number: 82402326) and Wenlan Zhou (the President Foundation of Nanfang Hospital, Southern Medical University, grant number: 2023A028).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eConceptualization and study design: HBW. Data acquisition: JHX and YJZ. Data analysis and interpretation: JHX, YJZ, YZZ, and DZS. Data curation: JHX, WLZ, and YT. Drafting of the manuscript: JHX. Manuscript revision: LJW and HBW. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe gratefully acknowledge the support provided for this research by all colleagues from the Department of Nuclear Medicine, Nanfang Hospital, Southern Medical University and the Key Laboratory Project of Guangdong Provincial Department of Education for Ordinary Universities and GDMPA Key Laboratory for Quality Control and Evaluation of Radiopharmaceuticals.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe presented data is available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhu T, Hsu JC, Guo J, Chen W, Cai W, Wang K. 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Clin Nucl Med. 2024;49(9):830\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrout JA, Sirven P, Leader AM, Maskey S, Hector E, Puisieux I, et al. Spatial Positioning and Matrix Programs of Cancer-Associated Fibroblasts Promote T-cell Exclusion in Human Lung Tumors. Cancer Discov. 2022;12(11):2606\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Jia Y, Wang J, Chen X, Han J, Zhen S, et al. circNOX4 activates an inflammatory fibroblast niche to promote tumor growth and metastasis in NSCLC via FAP/IL-6 axis. Mol Cancer. 2024;23(1):47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZboralski D, Hoehne A, Bredenbeck A, Schumann A, Nguyen M, Schneider E, et al. Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging. 2022;49(11):3651\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao G, Wang Z, Liu L, Zhang B, Song S, Wang D, et al. Targeting CXCR4/CXCL12 axis via [(177)Lu]Lu-DOTAGA.(SA.FAPi)(2) with CXCR4 antagonist in triple-negative breast cancer. Eur J Nucl Med Mol Imaging. 2024;51(9):2744\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaddio MF, Doshi S, Masri M, Jeanjean P, Hikmat F, Gerlach A, et al. Evaluating [(225)Ac]Ac-FAPI-46 for the treatment of soft-tissue sarcoma in mice. Eur J Nucl Med Mol Imaging. 2024;51(13):4026\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao Z, Zhang Y, Liu L, Wang M, Zhang C. [(177)Lu]Lu-FAP-2286 therapy in a case of right lung squamous cell carcinoma with systemic metastases. Eur J Nucl Med Mol Imaging. 2023;50(4):1266\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaum RP, Schuchardt C, Singh A, Chantadisai M, Robiller FC, Zhang J, et al. Feasibility, Biodistribution, and Preliminary Dosimetry in Peptide-Targeted Radionuclide Therapy of Diverse Adenocarcinomas Using (177)Lu-FAP-2286: First-in-Humans Results. Journal of nuclear medicine: official publication. Soc Nuclear Med. 2022;63(3):415\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanihashemian SS, Akbari ME, Norouzi G, Nikkholgh B, Amini H, Divband G, et al. The complete metabolic/molecular response to chemotherapy combined with [(177)Lu]Lu-FAP-2286 in metastatic breast cancer. Eur J Nucl Med Mol Imaging. 2024;51(13):4185\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanihashemian SS, Akbari ME, Pirayesh E, Divband G, Abolhosseini Shahrnoy A, Nami R, et al. Feasibility and therapeutic potential of [(177)Lu]Lu-FAPI-2286 in patients with advanced metastatic sarcoma. Eur J Nucl Med Mol Imaging. 2024;52(1):237\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkrott Z, Mistrik M, Andersen KK, Friis S, Majera D, Gursky J, et al. Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4. Nature. 2017;552(7684):194\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMajera D, Skrott Z, Chroma K, Merchut-Maya JM, Mistrik M, Bartek J. Targeting the NPL4 Adaptor of p97/VCP Segregase by Disulfiram as an Emerging Cancer Vulnerability Evokes Replication Stress and DNA Damage while Silencing the ATR Pathway. Cells. 2020;9(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrastev DB, Li S, Sun Y, Wicks AJ, Hoslett G, Weekes D, et al. The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin. Nat Cell Biol. 2022;24(1):62\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee YE, Choi SA, Kwack PA, Kim HJ, Kim IH, Wang KC, et al. Repositioning disulfiram as a radiosensitizer against atypical teratoid/rhabdoid tumor. Neurooncology. 2017;19(8):1079\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Michelakos T, Wang B, Shang Z, DeLeo AB, Duan Z, et al. Targeting cancer stem cells by disulfiram and copper sensitizes radioresistant chondrosarcoma to radiation. Cancer Lett. 2021;505:37\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao W, Qian X, Ochsenreither S, Soldano F, DeLeo AB, Sudhoff H et al. Disulfiram Acts as a Potent Radio-Chemo Sensitizer in Head and Neck Squamous Cell Carcinoma Cell Lines and Transplanted Xenografts. Cells. 2021;10(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang J, Zhang X, Liu Q, Gong F, Huang Y, Huang S, et al. (68)Ga/(177)Lu-Labeled Theranostic Pair for Targeting Fibroblast Activation Protein with Improved Tumor Uptake and Retention. J Med Chem. 2024;67(19):17785\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Ye S, Li L, Zhong J, Yan Q, Zhong Y, et al. (18)F- or (177)Lu-labeled bivalent ligand of fibroblast activation protein with high tumor uptake and retention. Eur J Nucl Med Mol Imaging. 2022;49(8):2705\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Y, Yang M, Yang L, Hao M, Wang F, Li X, et al. Preparation and anticancer actions of CuET-nanoparticles dispersed by bovine serum albumin. Colloids Surf B Biointerfaces. 2023;226:113329.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen Z, Luo D, Wang S, Rong R, Evers BM, Jia L, et al. Deep Learning-Based H-Score Quantification of Immunohistochemistry-Stained Images. Mod pathology: official J United States Can Acad Pathol Inc. 2024;37(2):100398.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZboralski D, Osterkamp F, Christensen E, Bredenbeck A, Schumann A, Hoehne A, et al. Fibroblast activation protein targeted radiotherapy induces an immunogenic tumor microenvironment and enhances the efficacy of PD-1 immune checkpoint inhibition. Eur J Nucl Med Mol Imaging. 2023;50(9):2621\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDadgar H, Pashazadeh A, Norouzbeigi N, Assadi M, Al-Balooshi B, Baum RP, et al. Targeted radioligand therapy: physics and biology, internal dosimetry and other practical aspects during (177)Lu/(225)Ac treatment in neuroendocrine tumors and metastatic prostate cancer. Theranostics. 2025;15(10):4368\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGudkov SV, Shilyagina NY, Vodeneev VA, Zvyagin AV. Targeted Radionuclide Therapy of Human Tumors. Int J Mol Sci. 2015;17(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBao G, Zhou H, Zou S, Chen L, Zhang B, Wang Z, et al. Inhibition of Poly(ADP-ribose) Polymerase Sensitizes [(177)Lu]Lu-DOTAGA.(SA.FAPi)(2)-Mediated Radiotherapy in Triple-Negative Breast Cancer. Mol Pharm. 2023;20(5):2443\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Targeted radionuclide therapy, radiosensitizer, FAP, Lung cancer, CuET","lastPublishedDoi":"10.21203/rs.3.rs-9032718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9032718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe efficacy of fibroblast activation protein (FAP)-targeted radionuclide ligand therapy (RLT) is often limited by the activation of DNA damage repair pathways in tumor cells. This study explores whether bis-(diethyldithiocarbamate)-copper (CuET, a NPL4 inhibitor and also a DNA repair inhibitor) enhances FAP-targeted RLT efficacy.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWhile both [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 and CuET monotherapies significantly inhibited tumor growth and prolonged survival compared to vehicle (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), their efficacy was limited. Notably, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 monotherapy markedly upregulated NPL4 expression in tumors. The full-dose combination therapy resulted in profound tumor regression (-65.1% volume change), significantly outperforming monotherapy at day 13 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This translated into a striking survival benefit, with a 77.8% survival rate at 50 days post-treatment versus 0% for monotherapy (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Mechanistically, the combination led to a significant increase in DNA damage, elevated apoptosis and autophagy, and suppressed proliferation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Critically, the half-dose combination achieved short-term tumor control superior to full-dose monotherapy and yielded a comparable median survival (41.3 vs. 38.7 days, P\u0026thinsp;=\u0026thinsp;0.41), demonstrating a significant radiosensitizing effect.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCuET acts as a potent radiosensitizer for [\u003csup\u003e177\u003c/sup\u003eLu]Lu-FAP-2286 by exacerbating DNA damage and exploiting therapy-induced NPL4 upregulation. This dual-mechanism combination presents a promising strategy to enhance the efficacy of FAP-targeted RLT, with the potential for dose reduction to mitigate toxicity.\u003c/p\u003e","manuscriptTitle":"CuET Radiosensitizes FAP-Targeted Radioligand Therapy in Lung Cancer by inhibiting DNA Repair","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-17 09:21:36","doi":"10.21203/rs.3.rs-9032718/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-03-09T12:07:13+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-09T09:06:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"EJNMMI Research","date":"2026-03-06T06:41:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-05T23:32:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Research","date":"2026-03-04T23:28:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejre","sideBox":"Learn more about [EJNMMI Research](http://ejnmmires.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ejre/default.aspx","title":"EJNMMI Research","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1e01ee2-219a-4d6a-8136-e334bfc68078","owner":[],"postedDate":"March 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-17T09:21:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-17 09:21:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9032718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9032718","identity":"rs-9032718","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}