Fibroblast activation protein-targeted radionuclide therapy (FAP-TRT) has emerged as a novel strategy for modulating the tumour microenvironment (TME) by selectively eradicating FAP-expressing cancer-associated fibroblasts (CAFs). Although preclinical studies have demonstrated promising results across various tumour models using diverse radiolabelled FAP inhibitors, clinical translation remains limited by modest efficacy, short tumour retention, and highly heterogeneous responses. This review aims to provide an overview of recent advances in radiopharmaceutical design to enhance tumour targeting and prolong retention. Furthermore, we summarise early clinical findings and ongoing trials, while emphasising the potential translational challenges of FAP-TRT. Special emphasis is placed on the radiobiological underpinnings of FAP-TRT, including the impact of CAFs heterogeneity, potential pro-tumorigenic effects of sublethal irradiation, and the uncertain contribution of bystander and abscopal effects. We further highlight the need for the development of translationally relevant tumour models, optimised dosimetry, predictive biomarkers, and refined patient selection criteria. Finally, we propose future directions such as combination therapy with immune checkpoint inhibitors (ICIs). Together, these insights aim to bridge the gap between promising preclinical efficacy and limited clinical outcomes in FAP-TRT.

In recent years, numerous studies have investigated the use of poly (ADP-ribose) polymerase (PARP) inhibitors, labelled with various radionuclides, as agents for targeted radionuclide therapy. This review discusses current advances in studies with radiolabelled PARP inhibitors for targeted radionuclide therapy, focusing on radionuclide selection and (radio)biological properties. We highlight differences between radionuclides and their efficacy in killing cancer cells, while safeguarding healthy tissue. Furthermore, important biomarkers and dosimetry are explored, providing insights and future directions for advancing radiolabelled PARP inhibitors.

The histone methyltransferase SETD2 and its associated histone mark H3 lysine 36 trimethylation (H3K36me3) are frequently lost in cancer, identifying SETD2 tumour suppressor loss as an important therapeutic target. Here we show that SETD2-deficient cancer cells are profoundly sensitive to RITA (2,5-bis[5-hydroxymethyl-2-thienyl] furan; NSC652287). Exposure of SETD2-deficient cancer cells to RITA results in significant p53 induction and apoptosis. However, TP53-deficient cells also exhibit RITA sensitivity suggesting p53 induction is an effect rather than a cause of RITA sensitivity. We find that RITA sensitivity is dependent on the phenol sulfotransferase SULT1A1, which is highly upregulated in SETD2-deficient cells. Accordingly, structural modifications of RITA, predicted to compromise its sulfation, ablated its activity. Further, SETD2-deficient cells can be targeted with YC-1, another SULT1A1-dependent anti-cancer agent. RITA sensitivity was associated with defects in DNA replication, leading to delays in S-phase progression, increased recruitment of replication stress markers, and reduced replication fork progression. Consistent with this, global target deconvolution using thermal profiling (2D-TPP) identified a broad range of RITA target proteins, including many involved in DNA replication stress. Together, these findings support the exploitation of SULT1A1 expression as a novel therapeutic strategy to target SETD2-deficient cancers.

Fibroblast activation protein-targeted radionuclide therapy (FAP-TRT) shows promise across multiple cancers, but clinical responses remain variable. Here, we aim to investigate how the presence, abundance, and spatial organisation of FAP-expressing cancer-associated fibroblasts (CAFs) relative to tumour cells influence the efficacy of FAP-TRT, to further optimisation. Spatial heterogeneity of FAP expression and radioligand uptake was assessed in human pancreatic ductal adenocarcinoma (PDAC) tissues using immunofluorescence and autoradiography with [1⁶1 Tb]Tb-FAPI-46. Mechanistic studies were conducted in vitro using PSN-1 tumour cells and FAP-expressing CAFs in 2D cultures and 3D spheroids, including mono- and co-culture systems with defined tumour-to-CAF ratios and spatial configurations. Cellular uptake and nuclear absorbed dose were quantified, and radiobiological effects were evaluated using DNA damage (γH2AX), clonogenic survival, spheroid growth, and cytokine profiling. PDAC tissues exhibited pronounced spatial heterogeneity in FAP expression and radioligand uptake. In vitro, uptake alone did not predict absorbed dose or biological response. In 2D models, [1⁶1 Tb]Tb-FAPI-46 did not significantly reduce clonogenic survival or increase DNA damage response. In 3D spheroids, FAP-TRT induced dose-dependent DNA damage and growth inhibition. Mixed tumour–CAF spheroids showed more uniform DNA damage and stronger growth suppression than layered models. CAF-containing models attenuated tumour-cell DNA damage and growth inhibition compared to models without CAFs (P < 0.001), associated with increased IL-6 and TGF-β secretion. FAP-expressing CAFs have a dual, context-dependent role in FAP-TRT, enhancing tumour irradiation through crossfire while also limiting tumour control potentially through radioligand partitioning towards the stromal compartment and CAF-derived paracrine signalling. These findings identify spatial organisation and CAF radiobiology as key determinants of FAP-TRT efficacy.

Intra-abdominal desmoid tumours (IADT) can closely mimic gastrointestinal stromal tumours (GISTs), particularly in recurrence and metastases. However, differentiating these two diseases using radiological methods remains challenging. This study aimed to investigate the value of [18F]FAPI-42 PET/CT in differentiating IADT and GISTs compared to [18F]FDG PET/CT. This study retrospectively included a total of 24 patients (12 patients with IADT and 12 patients with recurrent/metastatic GISTs) who underwent two separate PET/CT scans using [18F]FAPI-42 and [18F]FDG, respectively. The differences in tumour SUVmax on [18F]FAPI-42 PET/CT (SUVmax-FAPI) and [18F]FDG PET/CT (SUVmax-FDG) and the ratio of tumour SUVmax on FAPI PET/CT to SUVmax on FDG PET/CT [SUVmax ratio (FAPI/FDG)] were compared between IADT and GISTs. Receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic performance of these parameters in differentiating IADTs from GISTs. Immunohistochemistry was used to verify FAP expression in all IADT lesions and GISTs lesions. The SUVmax of IADTs on [18F]FAPI-42 PET/CT was significantly higher than that of GISTs [8.8 (7.2, 10.6) vs. 3.9 (2.8, 7.6), P = 0.045], whereas the SUVmax of IADTs on [18F]FDG PET/CT was significantly lower than that of GISTs [2.5 (1.9, 3.5) vs. 6.1 (3.6, 8.3), P = 0.007]. The SUVmax ratio (FAPI/FDG) of IADTs was significantly higher than that of GISTs [3.3 (2.2, 4.1) vs. 0.8 (0.3, 1.4), P < 0.001]. SUVmax-FAPI, SUVmax-FDG, and the SUVmax ratio (FAPI/FDG) achieved AUCs of 0.743, 0.819, and 0.896, with corresponding accuracies of 75.0

Abstract We investigated whether the antitumor effects of [123I]CC1, a PARP-binding radiopharmaceutical emitting very short-range ionising Auger electrons with potential use in Targeted Radionuclide Therapy (TRT), are driven by PARP trapping. Trapping will prolong its residence time on DNA and triggers more DNA damage. We tested this in PSN1 and U87 human cancer cell lines exposed to [123I]CC1, by assessing PARP levels in the chromatin fraction via western blot and quantifying 123I counts in nuclear and cytoplasmic fractions using a gamma counter. Downstream effects were evaluated using γH2AX immunofluorescence as a marker of DNA damage and DNA fiber assays to measure replication speed. Furthermore, we modulated PARP association with DNA by using methyl methanesulfonate (MMS) to increase and the PARG inhibitor JA2131 to decrease trapped PARP, using western blot and gamma counter to analyze how [123I]CC1 tracks PARP movements from chromatin to nuclear soluble fraction. Finally, γH2AX immunofluorescence was used to assess whether combinatory treatments with MMS or PARGi affect the ability of [123I]CC1 in producing DNA damage. [123I]CC1 elevated trapped PARP levels on DNA in PSN1 and U87 cells, followed by a rapid increase in γH2AX foci. Interestingly, DNA fiber assays showed that replication was markedly hindered in PSN1 at 30 min and 24 h, indicating substantial DNA damage. Pretreatment with 0.01% MMS before [123I]CC1 addition further elevated PARP levels compared to [123I]CC1 alone, resulting in greater chromatin 123I accumulation. In contrast, PARG inhibitor treatment reduced PARP residence time on DNA, and in combination with [123I]CC1 decreased DNA 123I delivery. Finally, assessment of γH2AX at 30 min and 24 hours showed that MMS pretreatment enhanced DNA damage induced by [123I]CC1 greater damage than MMS alone, demonstrating an additive effect, whereas PARGI pretreatment had no significant effect.The results indicate that the cytotoxic effect of [123I]CC1 is mediated by PARP trapping, which underlies its potent antitumoral activity previously observed by our group. Furthermore, modulation of the PARylation cycle alters delivery of radioactivity to DNA and the extent of DNA damage, suggesting a strategy to enhance the therapeutic benefit of [123I]CC1 and its potential for clinical translation. Citation Format: Luis Hernandez Cano, Nerea Delgado Mayenco, Hilleen Kramer, Elmar Diekstra, Francesca Amoroso, George Alachouzos, Wiktor Szymanski, Frank A. Kruyt, Bart Cornelissen. [123I]CC1, a radiopharmaceutical for Targeted radionuclide therapy (TRT), exploits PARP binding and trapping to amplify DNA damage and cytotoxicity in human cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5276.

Radiopharmaceuticals targeting poly(ADP-ribose) polymerase (PARP), a nuclear protein that plays a role in DNA damage repair, are promising candidates for targeted radionuclide therapy to treat PARP-expressing cancers. Here, we evaluated the potential of a PARP inhibitor with an olaparib-like structure, labelled with the radiometals 161Tb or 68Ga through a DOTA chelator, for targeted radionuclide therapy and PET imaging using in vitro models of cancer. [161Tb]TbCl3 and [68Ga]GaCl3 were used for radiolabelling a DOTA-conjugated derivative of the PARP inhibitor olaparib, yielding [161Tb]Tb-DOTA-olaparib and [68Ga]Ga-DOTA-olaparib, respectively. Both compounds were evaluated in vitro for cell uptake, internalisation, selectivity, and effect on clonogenic survival across a panel of PARP-expressing human and murine cancer cell lines. [161Tb]Tb-DOTA-olaparib and [68Ga]Ga-DOTA-olaparib were successfully produced with > 95

177Lu-rhPSMA-10.1 is a novel radiohybrid prostate-specific membrane antigen (PSMA)-targeted radiopharmaceutical therapy for prostate cancer. We conducted preclinical analyses on non-tumor-bearing BALB/c mice and on prostate cancer human xenograft mouse models (LNCAP and 22Rv1 xenografts) to evaluate its biodistribution and therapeutic efficacy. Methods: Longitudinal biodistribution of 177Lu-rhPSMA-10.1 was evaluated in BALB/c mice and 22Rv1 xenografts. Tissues of interest were harvested, and radioactivity was measured 1-168 h after injection of 1 MBq of 177Lu-rhPSMA-10.1 (4 per time point). Longitudinal biodistribution was compared with 177Lu-PSMA-I&T (1 MBq) in BALB/c mice and at a single time point (15 h) in 22Rv1 xenografts. The therapeutic efficacy of a single administration of 15, 30, or 45 MBq of 177Lu-rhPSMA-10.1 in LNCaP xenografts and 30 MBq of 177Lu-rhPSMA-10.1, 177Lu-PSMA-617, or 177Lu-PSMA-I&T in 22Rv1 xenografts (8 per group) was evaluated. Efficacy versus vehicle was evaluated on the basis of relative tumor volume and survival up to 49 d after administration. Statistical significance was evaluated with t testing (biodistribution data), 2-way repeated-measures ANOVA (tumor volume [analyzed until 3 per group remained]), or Kaplan-Meier log-rank analyses (survival). Results: Biodistribution of 177Lu-rhPSMA-10.1 in the BALB/c and 22Rv1 xenografts showed rapid clearance from blood and other normal tissues within 48 h, with the kidney showing the highest normal-organ uptake. Kidney uptake and retention were lower for 177Lu-rhPSMA-10.1 than for 177Lu-PSMA-I&T (6.5-fold lower at 12 h in BALB/c mice and 6.4-fold lower at 15 h in 22Rv1 xenografts; P < 0.01). High and sustained 177Lu-rhPSMA-10.1 tumor uptake was observed in 22Rv1 xenografts. This uptake was 2.3-fold higher than that of 177Lu-PSMA-I&T (15 h; P < 0.05). When efficacy was evaluated, 177Lu-rhPSMA-10.1 significantly suppressed tumor growth versus vehicle from day 11 (P < 0.05) in LNCaP xenografts in a dose-dependent manner and from day 18 (P < 0.05) in 22Rv1 xenografts and significantly prolonged median survival versus vehicle in both models. In 22Rv1 xenografts, 177Lu-rhPSMA-10.1 suppressed tumor growth versus vehicle to a greater extent than did 177Lu-PSMA-I&T (significant growth inhibition from day 25 [P < 0.05]) and similarly in extent to 177Lu-PSMA-617 (from day 18 [P < 0.05]). Overall, compared with 177Lu-PSMA-I&T, 177Lu-rhPSMA-10.1 suppressed tumor growth for longer than 177Lu-PSMA-617 (inhibition from day 39 onward [P < 0.05] versus on day 49 only [P < 0.05]). Conclusion: In preclinical models, 177Lu-rhPSMA-10.1 shows a favorable tumor-to-kidney uptake ratio, and significant antitumor effects, indicating it to be a promising next-generation radiopharmaceutical therapy.

PURPOSE:The prostate-specific membrane antigen (PSMA)-targeted radiohybrid ligand [177Lu]Lu-rhPSMA-10.1 is a promising next-generation radiopharmaceutical therapy in prostate cancer. This preclinical evaluation comprised an in vitro screen of potential novel synergistic drug combinations with [177Lu]Lu-rhPSMA-10.1, and an in vivo efficacy analysis of the lead drug combination in PSMA-expressing prostate cancer xenografts. METHODS:In total, 177 anticancer drugs were screened in a clonogenic survival assay of 22Rv1 cells which used 5-fold serial dilutions of the test drug (≤ 20 μM) to determine the half-maximal inhibitory concentration (IC50), compared to incubations of the test drug plus [177Lu]Lu-rhPSMA-10.1 (15 MBq) after 10 days. A subsequent focused screen assessed the impact of [177Lu]Lu-rhPSMA-10.1 (0-25 MBq/mL) on drug IC50. Synergy scores were determined using the zero interaction potency (ZIP) reference model (ZIP scores >5 % indicate high synergistic potency) and the multidimensional synergy of combinations (MuSyC) platform (log α >0 indicates synergistic potency). Therapeutic efficacy of the lead drug combination was evaluated in vivo: intravenous [177Lu]Lu-rhPSMA-10.1 (30 MBq, single dose) and oral cobimetinib (0.25 mg/day for 21 days) (alone/in combination) were administered to 22Rv1 tumor-bearing NMRI nude mice (eight mice/group plus untreated controls). Tumor volume was measured twice weekly for 69 days (two-way ANOVA and Tukey's multiple comparisons test: data analyzed until three mice/group remained). KaplanMeier Log-rank survival analyses were performed. RESULTS:In vitro screening identified cobimetinib (a mitogen-activated extracellular signal-regulated kinase inhibitor) as a lead candidate for synergistic combination with [177Lu]Lu-rhPSMA-10.1 across a wide concentration range (ZIP score=13 %). MuSyC analysis suggested synergistic efficacy from enhanced potency of both drugs in the combination (both log α>3). Combination treatment significantly suppressed tumor growth in vivo versus untreated controls (from Day 13-30; p<0.01) and [177Lu]Lu-rhPSMA-10.1 (from Day 17-30; p<0.001). Median survival was significantly longer with combination treatment (49 days) versus untreated controls (23 days; p=0.001) and [177Lu]Lu-rhPSMA-10.1 monotherapy (36 days; p=0.002). No major compound-related toxicity for cobimetinib ± [177Lu]Lu-rhPSMA-10.1 was observed. CONCLUSIONS:The combination of cobimetinib and [177Lu]Lu-rhPSMA-10.1 demonstrated enhanced preclinical therapeutic efficacy versus single agents, supporting clinical investigation of this novel drug combination in prostate cancer.

The growing interest and investments in targeted radionuclide therapy (TRT) have expanded research efforts across preclinical and clinical domains. Researchers from diverse fields, including nuclear medicine, radiochemistry, radiopharmacy, radiotherapy, biology, and physics, are increasingly focusing on TRT. Despite this, a lack of standardization in preclinical radiobiological studies hinders the evaluation and comparison of therapeutic radiopharmaceuticals across laboratories. Recognizing this, participants at the second International Workshop on Radiobiology of Molecular Radiotherapy, held in London, UK, in March 2023, emphasized the need for a consensus on nomenclature and standardized reporting guidelines in this field. The recommendations outlined here aim to address this gap by promoting consistent reporting. By adopting these standards, we hope to enhance the reproducibility, inter-laboratory comparability and visibility of preclinical TRT research, ultimately accelerating progress and amplifying its impact on the field and clinic.

Abstract Purpose: Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) has been shown to extend survival in men with advanced prostate cancer (PCa). Novel radiohybrid (rh) PSMA-targeted 177Lu-rhPSMA-10.1 has shown promising preclinical efficacy and advantageous radiation dosimetry in humans. We conducted an in vitro screen to identify known anticancer drugs with potential for synergistic interaction with 177Lu-rhPSMA-10.1. Here we present key screening data and a subsequent in vivo efficacy analysis of the lead novel drug combination in PSMA-expressing 22Rv1 PCa xenografts. Methods: Over 150 FDA-approved anticancer drugs were screened in a clonogenic survival assay of 22Rv1 cells using the test drug alone, at a range of concentrations <20 µM to determine the IC50, and results compared to incubations of the drug + 15 MBq/mL 177Lu-rhPSMA-10.1 after 10 d. A focused screen of 5 lead candidates was then conducted to determine the impact of 177Lu-rhPSMA-10.1 (0-25 MBq/mL) on the drug IC50. A synergy score was determined using the zero interaction potency (ZIP) reference model and the multi-dimensional synergy of combinations (MuSyc) platform. Subsequently, to evaluate the efficacy of the lead combination, 177Lu-rhPSMA-10.1 (single 30 MBq iv dose) and Cobimetinib (0.25 mg orally per day for 21 d) alone and in combination were administered to 22Rv1 tumor-bearing NMRI nude mice (n = 8 per group plus untreated controls). Tumor volume was measured 2x week for 69 d. Two-way ANOVA and Tukey’s multiple comparisons test (data analyzed until n = 3 remained per group) and Kaplan-Meier Log-rank survival analyses were performed. Results: The in vitro screen identified MEK inhibitor Cobimetinib as a lead candidate for synergistic combination with 177Lu-rhPSMA-10.1 across a wide concentration range, with a ZIP synergy score of 13.25% (95% CI ± 2.17) and promising results on MuSyc analysis. The 177Lu-rhPSMA-10.1 + Cobimetinib combination significantly suppressed tumor growth in vivo vs untreated controls (from day 13-30; p<0.01) and 177Lu-rhPSMA-10.1 alone (from day 17-30; p<0.001). The median survival in the combination group (49 d) was significantly longer vs the untreated group (23 d; p=0.001) and the group treated with 177Lu-rhPSMA-10.1 alone (36 d; p=0.002). Conclusions: Through an extensive in vitro screen, we identified Cobimetinib to have potential for a synergistic anti-tumor effect in combination with 177Lu-rhPSMA-10.1. This may be due to inhibition of the MEK-MAPK pathway by Cobimetinib during DNA damage response, resulting in radiosensitization of cancer cells to 177Lu-labeled RLT agents such as 177Lu-rhPSMA-10.1. This novel combination showed an enhanced therapeutic efficacy vs the single agents in 22Rv1 xenografts and the lack of overlapping monotherapy toxicity reported in the clinic supports clinical investigation in men with PCa. Citation Format: Caroline Foxton, Bart Cornelissen, Edward O'Neill, Bradley Waldron, Freja Pretzmann, Rikke Veggerby Grønlund, Mathias Wikke Hallund, Daniel J. Stevens. Evaluation of a synergistic drug combination with 177Lu-rhPSMA-10.1 for prostate cancer: Results of an in vitro screen and in vivo proof of concept study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 694.

Abstract Background Peptide receptor radionuclide therapy (PRRT) uses [177Lu]Lu-[DOTA0-Tyr3]octreotate ([177Lu]Lu-DOTA-TATE) to treat patients with neuroendocrine tumours (NETs) overexpressing the somatostatin receptor 2A (SSTR2A). It has shown significant short-term improvements in survival and symptom alleviation, but there remains room for improvement. Here, we investigated whether combining [177Lu]Lu-DOTA-TATE with chemotherapeutics enhanced the in vitro therapeutic efficacy of [177Lu]Lu-DOTA-TATE. Results Transfected human osteosarcoma (U2OS + SSTR2A, high SSTR2A expression) and pancreatic NET (BON1 + STTR2A, medium SSTR2A expression) cells were subjected to hydroxyurea, gemcitabine or triapine for 24 h at 37oC and 5% CO2. Cells were then recovered for 4 h prior to a 24-hour incubation with 0.7–1.03 MBq [177Lu]Lu-DOTA-TATE (25 nM) for uptake and metabolic viability studies. Incubation of U2OS + SSTR2A cells with hydroxyurea, gemcitabine, and triapine enhanced uptake of [177Lu]Lu-DOTA-TATE from 0.2 ± 0.1 in untreated cells to 0.4 ± 0.1, 1.1 ± 0.2, and 0.9 ± 0.2 Bq/cell in U2OS + SSTR2A cells, respectively. Cell viability post treatment with [177Lu]Lu-DOTA-TATE in cells pre-treated with chemotherapeutics was decreased compared to cells treated with [177Lu]Lu-DOTA-TATE monotherapy. For example, the viability of U2OS + SSTR2A cells incubated with [177Lu]Lu-DOTA-TATE decreased from 59.5 ± 22.3% to 18.8 ± 5.2% when pre-treated with hydroxyurea. Control conditions showed no reduced metabolic viability. Cells were also harvested to assess cell cycle progression, SSTR2A expression, and cell size by flow cytometry. Chemotherapeutics increased SSTR2A expression and cell size in U2OS + SSTR2A and BON1 + STTR2A cells. The S-phase sub-population of asynchronous U2OS + SSTR2A cell cultures was increased from 45.5 ± 3.3% to 84.8 ± 2.5%, 85.9 ± 1.9%, and 86.6 ± 2.2% when treated with hydroxyurea, gemcitabine, and triapine, respectively. Conclusions Hydroxyurea, gemcitabine and triapine all increased cell size, SSTR2A expression, and [177Lu]Lu-DOTA-TATE uptake, whilst reducing cell metabolic viability in U2OS + SSTR2A cells when compared to [177Lu]Lu-DOTA-TATE monotherapy. Further investigations could transform patient care and positively increase outcomes for patients treated with [177Lu]Lu-DOTA-TATE.