Dose-Time-Dependent Cellular Cytotoxicity of Astatine-211-Labeled FAP-Targeting Radiopharmaceuticals

Dose-Time-Dependent Cellular Cytotoxicity of Astatine-211-Labeled FAP-Targeting Radiopharmaceuticals | 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 Dose-Time-Dependent Cellular Cytotoxicity of Astatine-211-Labeled FAP-Targeting Radiopharmaceuticals Masayuki Takamatsu, Jumpei Ueno, Taketo Toda, Atsushi Shimoyama, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8476643/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fibroblast activation protein (FAP), which is overexpressed in cancer-associated fibroblasts (CAFs) is an attractive stromal target for a-therapy. We previously reported moderate anti-tumor effects of ²¹¹At-labeled FAP inhibitors (FAPI) in a PANC-1 xenograft mouse model, where insufficient tumor accumulation and the absence of FAP expression in tumor cells limited therapeutic efficacy. In this study, we conducted in vitro experiments using FAP-negative PANC-1 cells to elucidate the cytotoxicity of ²¹¹At-labeled FAPI in comparison with free 211 At. We found that, whereas free 211 At showed only limited cytotoxicity, ²¹¹At-labeled FAPI exerted significant cytotoxic effects. Systematic variation of activity concentrations and exposure durations revealed that cytotoxicity depended primarily on sustained pericellular exposure rather than instantaneous dose. These findings demonstrate that effective cytotoxicity of CAF-targeted ²¹¹At radiopharmaceuticals can be achieved without direct tumor cell targeting and provide a mechanistic basis for optimizing stromal-targeted a -therapy toward clinical translation. radiopharmaceutical astatine-211 α-emitter cytotoxic effect Fibroblast activation protein (FAP) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Fibroblast activation protein (FAP) is highly expressed in cancer-associated fibroblasts across many epithelial tumors, while its expression in normal tissues is limited.[1] FAP-targeted radiotheranostics using FAP inhibitors (FAPIs) are therefore attracting increasing attention.[2] Various FAPIs that have been developed bind to the catalytic site of FAP via the pyrrolidine ring and nitrile warhead, while the quinoline moiety forms a cation–π interaction with Arg123, resulting in efficient inhibition of FAP.[3, 4] Among these, 68 Ga- and 18 F-labeled FAPI tracers have rapidly advanced in clinical imaging, consistently demonstrating high tumor-to-background contrast, rapid tumor uptake, and favorable pharmacokinetics. Early clinical PET/CT studies using FAPI derivatives such as FAPI-02, FAPI-04, FAPI-46, and FAPI-74 have confirmed clear visualization of a wide range of tumors, and further optimization continues with the development of dimeric and PEG-modified variants as well as automated radiosynthesis protocols.[2] Collectively, these findings establish FAPI-based PET imaging as a clinically feasible platform for FAP-targeted radiotheranostics. While diagnostics using FAPI-based PET tracers are advancing rapidly, both a and b radiation therapy are under development, each with its own unique requirements. For β-emitters such as 177 Lu, achieving a high tumor-to-normal tissue ratio and sufficient radiation dose delivery through cross-fire effects can result in meaningful tumor control. Indeed, 177 Lu -FAPI-46 demonstrated marked antitumor activity in xenograft mice at 30 MBq.[5] In contrast, a-particle therapy relies on very short path lengths and high linear energy transfer (LET); therefore, therapeutic efficacy depends more critically on sufficient tumor uptake and residence time at the target site. Consistent with this concept, 225 Ac-labeled FAPI-04 has shown moderate antitumor activity even at low administered radioactivity.[2, 3] From the perspective of clinical feasibility, we focused on 211 At as an a-emitter with 7.2 h half-life and a simple decay scheme producing only 207 Bi and 207 Pb daughters. These characteristics make 211 At-labeled agents attractive candidates for clinical translation by potentially reducing hospitalization and improving cancer patients' quality of life.[6] Our group developed various 211 At-labeled candidates: [ 211 At]NaAt, 211 At-PA, 211 At-PSMA, 211 At-AAMT, and 211 At-AuNP [7–13]. Based on these considerations, we previously synthesized a series of ²¹¹At-labeled FAPI derivatives and evaluated their antitumor efficacy and biodistribution in mouse models (Figure 1).[14–16] In PANC-1 xenograft mice, 211 At-FAPI1 and 211 At-FAPI5 (Figure 1) significantly inhibited tumor growth without notable toxicity, as reflected by stable body weights and normal behavior.[15] Biodistribution studies revealed modest tumor uptake, with 211 At-FAPI5 showing 1.24 %ID/g at 1 h and 1.48 %ID/g at 3 h, whereas 211 At-FAPI1 achieved higher uptake values of 2.15 %ID/g and 3.04 %ID/g at the corresponding time points (Figure 1). Although 211 At-FAPI1 exhibited slightly superior tumor retention and antitumor activity, tumor regression was incomplete and regrowth occurred after treatment. Uptake in the thyroid and stomach, organs susceptible to free halogen accumulation, suggested partial in vivo cleavage of the carbon–astatine bond.[17] In a subsequent study, we investigated the effect of linker length by comparing 211 At-FAPI1 with 211 At-FAPI2 in BxPC-3 xenograft models.[26] Although the longer linker improved tumor retention, no appreciable enhancement in therapeutic efficacy was observed, and tumor regrowth occurred in all mice approximately one month after treatment. These findings indicate that neither linker modification nor the administered activity (1 MBq/mouse) was sufficient to achieve durable tumor control, underscoring the need to clarify the intrinsic pharmacological efficacy of 211 At-FAPI. In particular, the contribution of stromal targeting to therapeutic effect remains poorly understood. Because many pancreatic cancers lack FAP expression in tumor cells themselves, an important question is whether CAF-targeted a-therapy can induce sufficient cytotoxicity in FAP-negative cancer cells, and whether instantaneous dose or sustained pericellular exposure is the dominant determinant of efficacy. Herein, we addressed these questions through detailed in-vitro studies using FAP-negative PANC-1 cells. We compared the cytotoxicity of 211 At-labeled FAPI with that of free 211 At and systematically varied both activity concentration and exposure duration. We found that 211 At-FAPI exerted marked cytotoxicity despite the absence of FAP expression in tumor cells, and that cytotoxicity depended primarily on sustained pericellular exposure rather than instantaneous activity concentration. These results provide a mechanistic basis for the in-vivo observations of limited yet measurable therapeutic efficacy and offer guidance for optimizing stromal-targeted a-therapy toward clinical translation. Method The experimental protocol for the 211 At studies has been described in our previous reports,[ 15 , 16 ] and is summarized briefly below. 211 At was produced via the 209 Bi(α, 2n) 211 At reaction and purified by dry distillation. FAPI precursors bearing a dihydroxyboryl groups (B-FAPI) were labeled with 211 At via substitution reactions, yielding derivatives shown in Fig. 1 ( 211 At-FAPI1, 211 At-FAPI5) with radiochemical yields (RCYs) ranging from 15% to 100% and purities exceeding 78%.[ 14 ] Biodistribution studies were performed in mice to evaluate tumor and organ uptake of 211 At-FAPI1 and 211 At-FAPI5 at 1 and 3 hours post-injection. Therapeutic efficacy was evaluated in PANC-1 xenograft mice by monitoring tumor growth and body weight in the control and treatment groups for four weeks. The viability of LLC and PANC-1 cell to free 211 At was evaluated three days after 211 At exposure using the WST-8 cell counting kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. The cytotoxicity of 211 At-FAPI1 to PANC-was examined using a standard protocol of colony forming assay. Result and Discussion 2.1 Cellular cytotoxicity with free 211 At solution Cell viability assays were performed using Lewis lung carcinoma (LLC) and pancreatic cancer (PANC-1) cells (Figure 2). In the cell experiments with LLC, consistent with our previous reports, free 211 At solution exhibited dose-dependent cytotoxicity. However, when cell experiments were conducted with PANC-1, cell viability did not decrease at an initial dose of 10 kBq/mL. Furthermore, when the dose was increased to 100 and 1000 kBq/mL, a slight decrease in cell viability was observed; however, strong cytotoxicity was not detected. These results indicate that 211 At alone has limited cytotoxic effects on PANC-1 cells. 2.2 Cellular cytotoxicity with B-FAPI1 and 211 At-FAPI1 Based on our previous study, we focused on FAPI1, which exhibited the highest antitumor efficacy, and evaluated its cytotoxicity against PANC-1 cells by a colony-forming assay. In this study, we employed a labeling strategy utilizing a boronic acid-containing precursor (B-FAPI) for astatine radiolabeling. No cytotoxicity of B-FAPI1 was observed across a wide concentration range from 0.09 nM to 17 nM (Figure 3). In subsequent biochemical experiments, the concentration of unreacted boron precursor that needs to be considered is at most approximately 10 nM. Thus, we reconfirmed that toxicity from the precursor would not affect the outcomes of in vitro experiments described below. The cytotoxicity of 211 At-FAPI1 toward PANC-1 cells was evaluated (Figure 4). The incubation times with the radiolabeled compound were set to 1, 2, and 24 h. After 1 h of treatment, no clear decrease in cell survival was observed. In contrast, 2 h of treatment at a dose of 150 kBq resulted in a significant reduction in cell survival. When the treatment time was further prolonged to 24 h, a decrease in cell survival was observed at doses as low as 6 kBq, and a dose-dependent reduction in cell survival was demonstrated. In comparison with the ²¹¹At solution described above (Figure 2), much stronger cytotoxicity was observed. This result can be attributed to the ²¹¹At-labeled FAPI conjugate being more readily incorporated into cell membranes and intracellular compartments than free ²¹¹At. It is generally known that FAP is expressed in cancer-associated fibroblasts (CAFs) within the tumor stroma, whereas PANC-1 pancreatic cancer cells do not express FAP.[18, 19] Therefore, FAP-mediated ligand recognition of ²¹¹At-FAPI is not responsible for the observed results, suggesting that ²¹¹At-FAPI1 enters the cell membrane primarily via passive diffusion. Given the similar chemical properties of iodine and astatine, the experimentally determined Log P (2.51) and tPSA (133 Ų) calculated for the iodinated analog using ChemDraw Professional (Ver. 25.0.2.7, revvity Signals; default settings) suggest that ²¹¹At-labeled FAPI1 possesses sufficient membrane permeability. In exerting cytotoxic effects on cells, molecules that readily permeate membranes approach the nucleus relatively more closely and are more likely to inflict extensive DNA damage. Our recent data indicate that the a-particle exposure rate correlates with cell viability.[20] Consistent with this, the present study demonstrated that cell viability varied markedly with exposure duration. Notably, short-term exposure to a high activity concentration (150 kBq, 2 h) produced a comparable level of cytotoxicity to long-term exposure to a lower concentration (6 kBq, 24 h). These findings suggest that cytotoxicity is influenced not only by the administered activity but also by the effective a-particle exposure rate and duration of pericellular retention. Although these conclusions are based on in vitro data, they provide a mechanistic basis for understanding the therapeutic effects observed in vivo and may inform the optimization of stromal-targeted α-therapy. Conclusions In this study, we elucidated the cellular cytotoxicity of free ²¹¹At and ²¹¹At-labelled FAPI1. ²¹¹At- FAPI1 exhibited stronger cytotoxicity than 211 At solution owing to its sufficient membrane permeability. These results are consistent with our previously reported antitumor effects in mouse models. Importantly, systematic variation of activity concentration and exposure duration suggested that cell viability was not determined by administered activity alone but rather by the rate of α-particle exposure. Consistently, short-term exposure to a high activity concentration resulted in a similar level of cytotoxicity as long-term exposure to a lower concentration, indicating that the α-particle exposure rate correlates closely with cell viability. Overall, this work offers preliminary insight into the pharmacological behavior of 211 At-FAPI and may contribute to the future development of FAP-targeted α-particle therapies. Materials and Experimental Methods The synthesis procedures for the precursors are described below. We established a synthetic methodology to secure intellectual property. Radiolabeled products were prepared using the following methodology: 4.1. Synthesis of 211 At-FAPI Compounds The protocol in animal experiment has been shown in our previous report,[ 15 ] also briefly shown in below. 211 At was obtained from QST (Takasaki, Japan) and RIKEN (Wako, Japan) via a short-lived radioisotope supply platform. It was produced using the 209 Bi (α, 2n) 211 At reaction and separated from the Bi target by dry distillation, then dissolved in pure water. B-FAPIs were synthesized as precursors for 211 At labeling according to published procedures. 211 At-FAPI1 and 211 At-FAPI5 were synthesized by reacting 10 µL aqueous B-FAPI (1 mg/mL), 10 µL NaHCO 3 (7% w/v), 90 µL H 2 O, 2–19 µL 211 At (0.28–1.01 MBq), and 30 µL KI (0.1 mol/L) in a polypropylene tube at room temperature, then heated at 50°C or 80°C for 45 min. Radiochemical yields were 100% for 211 At-FAPI1 and 90% for 211 At-FAPI5. Products were purified using an Oasis HLB column and the radiochemical purities after purification were 100% ( 211 At-FAPI1) and 95% ( 211 At-FAPI5). For in vivo experiments, 5.6–14.1 MBq of 211 At-FAPI1 and 5.0–17.6 MBq of 211 At -FAPI5 were prepared. 4.2. Preparation of Animals The animals were prepared according to previously published protocol,[ 15 , 21 ] also briefly shown in below. PANC-1 cells were obtained from ATCC and male BALB/c nude mice from Japan SLC Inc. (Hamamatsu, Japan). Cells were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37°C in 5% CO 2 . Tumor xenografts were established by subcutaneous injection of 1 × 10 7 cells in 0.2 mL medium: Matrigel (1:1, BD Biosciences) into nude mice. Mice with tumors > 50 mm 3 were used for experiments. This study was approved by the Osaka University Graduate School of Science Animal Care and Use Committee (No. 2020-02-0) and conducted according to The University of Osaka animal experimentation regulations. 4.3. Materials and Reagents D-MEM (High Glucose) with L-Glutamine, Phenol Red and Sodium Pyruvate and RPMI-1640 with L-Glutamine and Phenol Red were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan), 96 well, WST-8 reagent kit were purchased from DOJINDO LABORATORIES (Kumamoto, Japan), CFSE were purchased from Biotium (CA, USA). 6 well and 96 well microplates were purchased from AGC TECHNO GLASS Co., Ltd. (Shizuoka, Japan). LLC cells were kindly provided by Prof. Fei Yu (Tongji University, School of Medicine, CN). HeLa were purchased from Japanese Collection of Research Bioresources. LLC were grown in RPMI-1640 supplemented with 10% FBS and 1% penicillin–streptomycin solution (FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan). HeLa and AD293 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin solution (FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan). Cells were incubated at 37˚C under 5% CO 2 atmosphere. Human pancreatic cancer PANC-1 were purchased from Japanese Collection of Research Bioresources. PANC-1 cells were cultured in Dulbecco’s modified Eagle’s medium (D-MEM) high-glucose supplemented by 10% Fetal bovine serum (FBS) and 1% penicillin-streptomycin (×100), then incubated at 37°C in a 5% carbon dioxide atmosphere. 4.4. Cell viability assay by WST-8 assay LLC and PANC-1 cells were respectively plated in a 96 well microplate (100 µL /well) at a density of 2.5×10 4 cells /well. Background control wells (n = 3) containing the same volume of complete culture medium were included in each assay. The microplate was incubated for 24 h at 37˚C under 5% CO 2 atmosphere. non-labeled 211 At at various concentrations in medium were added to it and the plate incubated at 37˚C under 5% CO 2 atmosphere. After incubating for 3 days following the addition of 211 At, 10 µL of WST-8 reagent was added to each well, followed by incubation at 37˚C under 5% CO 2 atmosphere for 1 hour. The absorbance was measured using an infinite F50 (Tecan) at 450 nm. For each measurement value, subtract the average background measurement value and normalized by the untreated 211 At group. Data processing was done with GraphPad Prism. The results are expressed as the means ± the standard error(no mark: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control group. One-way ANOVA followed by Dunnett's test was performed using GraphPad Prism 9 software). 4.5. Cell viability assay by colony-forming assay The ability of the 211 At-FAPI1 to inhibit cancer cell proliferation was evaluated using a colony-forming assay. PANC-1 cells were seeded in 24-well plates at a density of 2,000 cells/well in 1 mL supplemented D-MEM high glucose and incubated overnight. The cells were treated in triplicate with various doses of the radionuclide for 1, 2 or 24 hours at 37°C in 5% CO 2 , washed with pre-warmed PBS, and the medium was changed to a fresh medium. The cells were incubated for 7 days, fixed, and stained with 0.5% crystal violet. The cells were observed by microscope, and the stained crystal violet was extracted using 0.05 M NaH 2 PO 4 in 50% ethanol for OD measurement at 570 nm, normalized to the control group. For each measurement value, subtract the average background measurement value and normalized by the untreated group. The results are expressed as the means ± the standard error. The number of PANC-1 by treated 1% EtOH colony controls was set to 100%. Values are expressed as mean ± SD, n = 3, and asterisks indicate statistical significance compared to controls (no mark: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control group. One-way ANOVA followed by Dunnett's test was performed using GraphPad Prism 9 software). Abbreviations FAP - Fibroblast activation protein FAPI - Fibroblast activation protein inhibitor PEG - Polyethylene glycol PIP - Piperazine PET - Positron emission tomography CT - Computed tomography RCY - Radiochemical yield %ID/g - Percent injected dose per gram LogP - Logarithm of partition coefficient tPSA - topological Polar surface area DOTA - 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid NOTA - 1,4,7-triazacyclononane-1,4,7-triacetic acid PSMA - Prostate-specific membrane antigen PA - Phenylalanine AAMT - Alpha-methyl Tyrosine AuNP - Gold nanoparticles PANC-1 - Human pancreatic carcinoma cell line Declarations Acknowledgements We are grateful to all members in Institute for Radiation Science in The University of Osaka for collaboration in this work. This work is supported by JSPS KAKENHI (JP20H05675, JP24K23086, JP25H00006) and F-REI (JPFR25040302), JST-CREST (JPMJCR20R3) and Uehara Memorial Foundation. Conflicts of Interest The authors declare no conflict of interest. References Luo Y, Fu H, Yu C (2025) Based on small molecules: development and application of fibroblast activation protein inhibitors radiopharmaceutical in tumor precision therapy. Front Pharmacol 16:1593380 Zhao L, Chen J, Pang Y et al (2022) Fibroblast activation protein-based theranostics in cancer research: A state-of-the-art review. Theranostics 12:1557–1569 Ma H, Ye T, Qu G et al (2025) Locoregional radionuclide therapy of glioblastoma with [211At]At-PDA-FAPI. Sci Rep 15:18248 Suzuki H, Kannaka K, Hirayama M et al (2024) In vivo stable 211At-labeled prostate-specific membrane antigen-targeted tracer using a neopentyl glycol structure. EJNMMI Radiopharm Chem 9:48 Zhang Z, Tao J, Qiu J et al (2023) From basic research to clinical application: targeting fibroblast activation protein for cancer diagnosis and treatment. 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J Nucl Med 65:52–58 Ueno J, Takamatsu M, Mayusumi K et al (2025) Dose-Dependent Cytotoxic Profiling of Astatine-211 for Targeted Alpha Therapy. Res Sq. https://doi.org/10.21203/rs.3.rs-7832223/v1 Kaneda-Nakashima K, Zhang Z, Manabe Y et al (2021) α-Emitting cancer therapy using 211 At-AAMT targeting LAT1. Cancer Sci 112:1132–1140 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-8476643","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":598545218,"identity":"2e7eaeb8-d945-45fa-b596-bd7b4ab7e744","order_by":0,"name":"Masayuki 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Haba","email":"","orcid":"","institution":"RIKEN","correspondingAuthor":false,"prefix":"","firstName":"Hiromitsu","middleName":"","lastName":"Haba","suffix":""},{"id":598545232,"identity":"d70f1111-ba70-41a8-92e7-bd282bba5482","order_by":14,"name":"Jens Cardinale","email":"","orcid":"","institution":"Düsseldorf University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jens","middleName":"","lastName":"Cardinale","suffix":""},{"id":598545233,"identity":"391efa96-f65c-4e8e-b134-16f79cdaff7a","order_by":15,"name":"Frederik Giesel","email":"","orcid":"","institution":"Düsseldorf University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Frederik","middleName":"","lastName":"Giesel","suffix":""},{"id":598545234,"identity":"ba3a17ce-37a6-41aa-9850-06ac746d85b8","order_by":16,"name":"Kazuko Kaneda-Nakashima","email":"","orcid":"","institution":"Osaka University","correspondingAuthor":false,"prefix":"","firstName":"Kazuko","middleName":"","lastName":"Kaneda-Nakashima","suffix":""},{"id":598545235,"identity":"007821a9-8db0-4fc9-bfca-90ad61c2de5d","order_by":17,"name":"Koichi Fukase","email":"","orcid":"","institution":"Osaka University","correspondingAuthor":false,"prefix":"","firstName":"Koichi","middleName":"","lastName":"Fukase","suffix":""}],"badges":[],"createdAt":"2025-12-30 02:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8476643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8476643/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103733331,"identity":"6a5145f2-5e83-4c6b-875e-aa8da15cb3e5","added_by":"auto","created_at":"2026-03-02 09:27:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":167974,"visible":true,"origin":"","legend":"\u003cp\u003eOur previous animal experiment utilizing \u003csup\u003e211\u003c/sup\u003eAt-labeled FAPIs (\u003csup\u003e211\u003c/sup\u003eAt-FAPI1 and \u003csup\u003e211\u003c/sup\u003eAt-FAPI5). Anti-tumor effect in PANC-1 xenograft mice after administration of \u003csup\u003e211\u003c/sup\u003eAt-FAPIs (approximately 1 MBq). (a) Body weight of mice. (b) Tumor sizes of the experimental mice. Means ± S.E. Filled squares are the control group, filled triangles are the \u003csup\u003e211\u003c/sup\u003eAt -FAPI1 group, and white circles are the \u003csup\u003e211\u003c/sup\u003eAt -FAPI5 group. * p \u0026lt; 0.05, ** p \u0026lt; 0.01. Biodistribution of\u0026nbsp;(c)\u0026nbsp;\u003csup\u003e211\u003c/sup\u003eAt-FAPI1 and\u0026nbsp;(d) \u003csup\u003e211\u003c/sup\u003eAt-FAPI5 at 1 h (blue) and 3 h (red) after injection (\u003csup\u003e211\u003c/sup\u003eAt-FAPI1 0.81 MBq and \u003csup\u003e211\u003c/sup\u003eAt-FAPI5 0.54 MBq). After the mice were dissected, the intensities were measured and the % ID/g were calculated. All data are shown as means ± SE. All groups consisted of three mice (% ID: % of injection dose). CC BY from. Aso A \u003cem\u003eet al.\u003c/em\u003e,\u003cem\u003e Int.J. Mol. Sci.\u003c/em\u003e\u0026nbsp;\u003cstrong\u003e2023\u003c/strong\u003e,\u0026nbsp;\u003cem\u003e24\u003c/em\u003e(10), 8701.[15]\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8476643/v1/27ae3a68607f48ac918111cf.png"},{"id":103733387,"identity":"3f4f2f77-3266-4db8-b999-1f072c8faa83","added_by":"auto","created_at":"2026-03-02 09:28:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":106284,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of cell viability by WST-8 assay following ²¹¹At solution treatment in a) LLC and b) PANC-1 cells.\u003cem\u003e \u003c/em\u003eThe number of LLC and PANC-1 by non-treated controls was set to 100%. Values are expressed as mean ± SD, n = 3, and asterisks indicate statistical significance compared to controls (no mark: no significance, *p\u0026lt;0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs. control group. One-way ANOVA followed by Dunnett's test was performed using GraphPad Prism 9 software).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8476643/v1/fbf35173fed677dbb86b5ead.png"},{"id":103733349,"identity":"053ec410-103b-4a4c-b033-34862d36229a","added_by":"auto","created_at":"2026-03-02 09:28:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120557,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of cell viability by colony forming assay with B-FAPI1(labeling precursor of \u003csup\u003e211\u003c/sup\u003eAt-FAPI1).\u003cem\u003e \u003c/em\u003eThe number of PANC-1 by treated 1% EtOH colony controls was set to 100%. Values are expressed as mean ± SD, n = 3, and asterisks indicate statistical significance compared to controls (no mark: no significance, *p\u0026lt;0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs. control group. One-way ANOVA followed by Dunnett's test was performed using GraphPad Prism 9 software).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8476643/v1/2ac445371168e2d72e896cf0.png"},{"id":103733191,"identity":"0c401e76-d804-4a19-8962-4056a30ad5f4","added_by":"auto","created_at":"2026-03-02 09:27:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":195042,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of cell viability by colony forming assay with \u003csup\u003e211\u003c/sup\u003eAt -FAPI1. Time in treatment of \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 is 1 h (blue), 2 h (purple), 24 h (pink). The number of PANC-1 by treated 1% EtOH colony controls was set to 100%. Values are expressed as mean ± SD, n = 3, and asterisks indicate statistical significance compared to controls (no mark: no significance, *p\u0026lt;0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs. control group. One-way ANOVA followed by Dunnett's test was performed using GraphPad Prism 9 software).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8476643/v1/3194630becf485b886843420.png"},{"id":103733655,"identity":"04f18c59-a4e0-447a-a587-2e295ce9f3d7","added_by":"auto","created_at":"2026-03-02 09:29:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1200661,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8476643/v1/a0fbe0a8-1cb0-48c5-bf9e-4b3e769f7aef.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dose-Time-Dependent Cellular Cytotoxicity of Astatine-211-Labeled FAP-Targeting Radiopharmaceuticals","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFibroblast activation protein (FAP) is highly expressed in cancer-associated fibroblasts across many epithelial tumors, while its expression in normal tissues is limited.[1] FAP-targeted radiotheranostics using FAP inhibitors (FAPIs) are therefore attracting increasing attention.[2] Various FAPIs that have been developed bind to the catalytic site of FAP via the pyrrolidine ring and nitrile warhead, while the quinoline moiety forms a cation\u0026ndash;\u0026pi; interaction with Arg123, resulting in efficient inhibition of FAP.[3, 4] Among these, \u003csup\u003e68\u003c/sup\u003eGa- and \u003csup\u003e18\u003c/sup\u003eF-labeled FAPI tracers have rapidly advanced in clinical imaging, consistently demonstrating high tumor-to-background contrast, rapid tumor uptake, and favorable pharmacokinetics. Early clinical PET/CT studies using FAPI derivatives such as FAPI-02, FAPI-04, FAPI-46, and FAPI-74 have confirmed clear visualization of a wide range of tumors, and further optimization continues with the development of dimeric and PEG-modified variants as well as automated radiosynthesis protocols.[2] Collectively, these findings establish FAPI-based PET imaging as a clinically feasible platform for FAP-targeted radiotheranostics.\u003c/p\u003e\n\u003cp\u003eWhile diagnostics using FAPI-based PET tracers are advancing rapidly, both\u0026nbsp;a\u0026nbsp;and\u0026nbsp;b\u0026nbsp;radiation therapy are under development, each with its own unique requirements. For \u0026beta;-emitters such as \u003csup\u003e177\u003c/sup\u003eLu, achieving a high tumor-to-normal tissue ratio and sufficient radiation dose delivery through cross-fire effects can result in meaningful tumor control. Indeed, \u003csup\u003e177\u003c/sup\u003eLu -FAPI-46 demonstrated marked antitumor activity in xenograft mice at 30 MBq.[5] In contrast,\u0026nbsp;a-particle therapy relies on very short path lengths and high linear energy transfer (LET); therefore, therapeutic efficacy depends more critically on sufficient tumor uptake and residence time at the target site. Consistent with this concept, \u003csup\u003e225\u003c/sup\u003eAc-labeled FAPI-04 has shown moderate antitumor activity even at low administered radioactivity.[2, 3]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom the perspective of clinical feasibility, we focused on \u003csup\u003e211\u003c/sup\u003eAt as an\u0026nbsp;a-emitter with 7.2 h half-life and a simple decay scheme producing only \u003csup\u003e207\u003c/sup\u003eBi and \u003csup\u003e207\u003c/sup\u003ePb daughters. These characteristics make \u003csup\u003e211\u003c/sup\u003eAt-labeled agents attractive candidates for clinical translation by potentially reducing hospitalization and improving cancer patients\u0026apos; quality of life.[6] Our group developed various \u003csup\u003e211\u003c/sup\u003eAt-labeled candidates: [\u003csup\u003e211\u003c/sup\u003eAt]NaAt, \u003csup\u003e211\u003c/sup\u003eAt-PA, \u003csup\u003e211\u003c/sup\u003eAt-PSMA, \u003csup\u003e211\u003c/sup\u003eAt-AAMT, and \u003csup\u003e211\u003c/sup\u003eAt-AuNP [7\u0026ndash;13]. Based on these considerations, we previously synthesized a series of \u0026nbsp;\u0026sup2;\u0026sup1;\u0026sup1;At-labeled FAPI derivatives and evaluated their antitumor efficacy and biodistribution in mouse models (Figure 1).[14\u0026ndash;16] In PANC-1 xenograft mice, \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 and \u003csup\u003e211\u003c/sup\u003eAt-FAPI5 (Figure 1) significantly inhibited tumor growth without notable toxicity, as reflected by stable body weights and normal behavior.[15] Biodistribution studies revealed modest tumor uptake, with \u003csup\u003e211\u003c/sup\u003eAt-FAPI5 showing 1.24 %ID/g at 1 h and 1.48 %ID/g at 3 h, whereas \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 achieved higher uptake values of 2.15 %ID/g and 3.04 %ID/g at the corresponding time points (Figure 1). Although \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 exhibited slightly superior tumor retention and antitumor activity, tumor regression was incomplete and regrowth occurred after treatment. Uptake in the thyroid and stomach, organs susceptible to free halogen accumulation, suggested partial \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003ecleavage of the carbon\u0026ndash;astatine bond.[17]\u003c/p\u003e\n\u003cp\u003eIn a subsequent study, we investigated the effect of linker length by comparing \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 with \u003csup\u003e211\u003c/sup\u003eAt-FAPI2 in BxPC-3 xenograft models.[26] Although the longer linker improved tumor retention, no appreciable enhancement in therapeutic efficacy was observed, and tumor regrowth occurred in all mice approximately one month after treatment. These findings indicate that neither linker modification nor the administered activity (1 MBq/mouse) was sufficient to achieve durable tumor control, underscoring the need to clarify the intrinsic pharmacological efficacy of \u003csup\u003e211\u003c/sup\u003eAt-FAPI.\u003c/p\u003e\n\u003cp\u003eIn particular, the contribution of stromal targeting to therapeutic effect remains poorly understood. Because many pancreatic cancers lack FAP expression in tumor cells themselves, an important question is whether CAF-targeted a-therapy can induce sufficient cytotoxicity in FAP-negative cancer cells, and whether instantaneous dose or sustained pericellular exposure is the dominant determinant of efficacy.\u003c/p\u003e\n\u003cp\u003eHerein, we addressed these questions through detailed \u003cem\u003ein-vitro\u003c/em\u003e studies using FAP-negative PANC-1 cells. We compared the cytotoxicity of \u003csup\u003e211\u003c/sup\u003eAt-labeled FAPI with that of free \u003csup\u003e211\u003c/sup\u003eAt and systematically varied both activity concentration and exposure duration. We found that \u003csup\u003e211\u003c/sup\u003eAt-FAPI exerted marked cytotoxicity despite the absence of FAP expression in tumor cells, and that cytotoxicity depended primarily on sustained pericellular exposure rather than instantaneous activity concentration. These results provide a mechanistic basis for the \u003cem\u003ein-vivo\u0026nbsp;\u003c/em\u003eobservations of limited yet measurable therapeutic efficacy and offer guidance for optimizing stromal-targeted a-therapy toward clinical translation.\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003eThe experimental protocol for the \u003csup\u003e211\u003c/sup\u003eAt studies has been described in our previous reports,[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and is summarized briefly below. \u003csup\u003e211\u003c/sup\u003eAt was produced via the \u003csup\u003e209\u003c/sup\u003eBi(α, 2n)\u003csup\u003e211\u003c/sup\u003eAt reaction and purified by dry distillation. FAPI precursors bearing a dihydroxyboryl groups (B-FAPI) were labeled with \u003csup\u003e211\u003c/sup\u003eAt via substitution reactions, yielding derivatives shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (\u003csup\u003e211\u003c/sup\u003eAt-FAPI1, \u003csup\u003e211\u003c/sup\u003eAt-FAPI5) with radiochemical yields (RCYs) ranging from 15% to 100% and purities exceeding 78%.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Biodistribution studies were performed in mice to evaluate tumor and organ uptake of \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 and \u003csup\u003e211\u003c/sup\u003eAt-FAPI5 at 1 and 3 hours post-injection. Therapeutic efficacy was evaluated in PANC-1 xenograft mice by monitoring tumor growth and body weight in the control and treatment groups for four weeks. The viability of LLC and PANC-1 cell to free \u003csup\u003e211\u003c/sup\u003eAt was evaluated three days after \u003csup\u003e211\u003c/sup\u003eAt exposure using the WST-8 cell counting kit (Dojindo, Kumamoto, Japan) according to the manufacturer\u0026rsquo;s instructions. The cytotoxicity of \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 to PANC-was examined using a standard protocol of colony forming assay.\u003c/p\u003e"},{"header":"Result and Discussion","content":"\u003cp\u003e2.1 Cellular cytotoxicity with free \u003csup\u003e211\u003c/sup\u003eAt solution\u003c/p\u003e\n\u003cp\u003eCell viability assays were performed using Lewis lung carcinoma (LLC) and pancreatic cancer (PANC-1) cells (Figure 2). In the cell experiments with LLC, consistent with our previous reports, free \u003csup\u003e211\u003c/sup\u003eAt solution exhibited dose-dependent cytotoxicity. However, when cell experiments were conducted with PANC-1, cell viability did not decrease at an initial dose of 10 kBq/mL. Furthermore, when the dose was increased to 100 and 1000 kBq/mL, a slight decrease in cell viability was observed; however, strong cytotoxicity was not detected. These results indicate that \u003csup\u003e211\u003c/sup\u003eAt alone has limited cytotoxic effects on PANC-1 cells.\u003c/p\u003e\n\u003cp\u003e2.2 Cellular cytotoxicity with B-FAPI1 and \u003csup\u003e211\u003c/sup\u003eAt-FAPI1\u003c/p\u003e\n\u003cp\u003eBased on our previous study, we focused on FAPI1, which exhibited the highest antitumor efficacy, and evaluated its cytotoxicity against PANC-1 cells by a colony-forming assay. In this study, we employed a labeling strategy utilizing a boronic acid-containing precursor (B-FAPI) for astatine radiolabeling. No cytotoxicity of B-FAPI1 was observed across a wide concentration range from 0.09 nM to 17 nM (Figure 3). In subsequent biochemical experiments, the concentration of unreacted boron precursor that needs to be considered is at most approximately 10 nM. Thus, we reconfirmed that toxicity from the precursor would not affect the outcomes of \u003cem\u003ein vitro\u003c/em\u003e experiments described below.\u003c/p\u003e\n\u003cp\u003eThe cytotoxicity of \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 toward PANC-1 cells was evaluated (Figure 4). The incubation times with the radiolabeled compound were set to 1, 2, and 24 h. After 1 h of treatment, no clear decrease in cell survival was observed. In contrast, 2 h of treatment at a dose of 150 kBq resulted in a significant reduction in cell survival. When the treatment time was further prolonged to 24 h, a decrease in cell survival was observed at doses as low as 6 kBq, and a dose-dependent reduction in cell survival was demonstrated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn comparison with the \u0026sup2;\u0026sup1;\u0026sup1;At solution described above (Figure 2), much stronger cytotoxicity was observed. This result can be attributed to the \u0026sup2;\u0026sup1;\u0026sup1;At-labeled FAPI conjugate being more readily incorporated into cell membranes and intracellular compartments than free \u0026sup2;\u0026sup1;\u0026sup1;At. It is generally known that FAP is expressed in cancer-associated fibroblasts (CAFs) within the tumor stroma, whereas PANC-1 pancreatic cancer cells do not express FAP.[18, 19] Therefore, FAP-mediated ligand recognition of \u0026sup2;\u0026sup1;\u0026sup1;At-FAPI is not responsible for the observed results, suggesting that \u0026sup2;\u0026sup1;\u0026sup1;At-FAPI1 enters the cell membrane primarily via passive diffusion. Given the similar chemical properties of iodine and astatine, the experimentally determined Log P (2.51) and tPSA (133 \u0026Aring;\u0026sup2;) calculated for the iodinated analog using ChemDraw Professional (Ver. 25.0.2.7, revvity Signals; default settings) suggest that \u0026sup2;\u0026sup1;\u0026sup1;At-labeled FAPI1 possesses sufficient membrane permeability. In exerting cytotoxic effects on cells, molecules that readily permeate membranes approach the nucleus relatively more closely and are more likely to inflict extensive DNA damage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur recent data indicate that the a-particle exposure rate correlates with cell viability.[20] Consistent with this, the present study demonstrated that cell viability varied markedly with exposure duration. Notably, short-term exposure to a high activity concentration (150 kBq, 2 h) produced a comparable level of cytotoxicity to long-term exposure to a lower concentration (6 kBq, 24 h). These findings suggest that cytotoxicity is influenced not only by the administered activity but also by the effective a-particle exposure rate and duration of pericellular retention. Although these conclusions are based on \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003edata, they provide a mechanistic basis for understanding the therapeutic effects observed in vivo and may inform the optimization of stromal-targeted \u0026alpha;-therapy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we elucidated the cellular cytotoxicity of free \u0026sup2;\u0026sup1;\u0026sup1;At and \u0026sup2;\u0026sup1;\u0026sup1;At-labelled FAPI1. \u0026sup2;\u0026sup1;\u0026sup1;At- FAPI1 exhibited stronger cytotoxicity than \u003csup\u003e211\u003c/sup\u003eAt solution owing to its sufficient membrane permeability. These results are consistent with our previously reported antitumor effects in mouse models. Importantly, systematic variation of activity concentration and exposure duration suggested that cell viability was not determined by administered activity alone but rather by the rate of α-particle exposure. Consistently, short-term exposure to a high activity concentration resulted in a similar level of cytotoxicity as long-term exposure to a lower concentration, indicating that the α-particle exposure rate correlates closely with cell viability. Overall, this work offers preliminary insight into the pharmacological behavior of \u003csup\u003e211\u003c/sup\u003eAt-FAPI and may contribute to the future development of FAP-targeted α-particle therapies.\u003c/p\u003e"},{"header":"Materials and Experimental Methods","content":"\u003cp\u003eThe synthesis procedures for the precursors are described below. We established a synthetic methodology to secure intellectual property. Radiolabeled products were prepared using the following methodology:\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Synthesis of \u003csup\u003e211\u003c/sup\u003eAt-FAPI Compounds\u003c/h2\u003e \u003cp\u003eThe protocol in animal experiment has been shown in our previous report,[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] also briefly shown in below. \u003csup\u003e211\u003c/sup\u003eAt was obtained from QST (Takasaki, Japan) and RIKEN (Wako, Japan) via a short-lived radioisotope supply platform. It was produced using the \u003csup\u003e209\u003c/sup\u003eBi (α, 2n) \u003csup\u003e211\u003c/sup\u003eAt reaction and separated from the Bi target by dry distillation, then dissolved in pure water. B-FAPIs were synthesized as precursors for \u003csup\u003e211\u003c/sup\u003eAt labeling according to published procedures. \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 and \u003csup\u003e211\u003c/sup\u003eAt-FAPI5 were synthesized by reacting 10 \u0026micro;L aqueous B-FAPI (1 mg/mL), 10 \u0026micro;L NaHCO\u003csub\u003e3\u003c/sub\u003e (7% w/v), 90 \u0026micro;L H\u003csub\u003e2\u003c/sub\u003eO, 2\u0026ndash;19 \u0026micro;L \u003csup\u003e211\u003c/sup\u003eAt (0.28\u0026ndash;1.01 MBq), and 30 \u0026micro;L KI (0.1 mol/L) in a polypropylene tube at room temperature, then heated at 50\u0026deg;C or 80\u0026deg;C for 45 min. Radiochemical yields were 100% for \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 and 90% for \u003csup\u003e211\u003c/sup\u003eAt-FAPI5. Products were purified using an Oasis HLB column and the radiochemical purities after purification were 100% (\u003csup\u003e211\u003c/sup\u003eAt-FAPI1) and 95% (\u003csup\u003e211\u003c/sup\u003eAt-FAPI5). For \u003cem\u003ein vivo\u003c/em\u003e experiments, 5.6\u0026ndash;14.1 MBq of \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 and 5.0\u0026ndash;17.6 MBq of \u003csup\u003e211\u003c/sup\u003eAt -FAPI5 were prepared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Preparation of Animals\u003c/h2\u003e \u003cp\u003eThe animals were prepared according to previously published protocol,[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] also briefly shown in below. PANC-1 cells were obtained from ATCC and male BALB/c nude mice from Japan SLC Inc. (Hamamatsu, Japan). Cells were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. Tumor xenografts were established by subcutaneous injection of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells in 0.2 mL medium: Matrigel (1:1, BD Biosciences) into nude mice. Mice with tumors\u0026thinsp;\u0026gt;\u0026thinsp;50 mm\u003csup\u003e3\u003c/sup\u003e were used for experiments. This study was approved by the Osaka University Graduate School of Science Animal Care and Use Committee (No. 2020-02-0) and conducted according to The University of Osaka animal experimentation regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Materials and Reagents\u003c/h2\u003e \u003cp\u003eD-MEM (High Glucose) with L-Glutamine, Phenol Red and Sodium Pyruvate and RPMI-1640 with L-Glutamine and Phenol Red were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan), 96 well, WST-8 reagent kit were purchased from DOJINDO LABORATORIES (Kumamoto, Japan), CFSE were purchased from Biotium (CA, USA). 6 well and 96 well microplates were purchased from AGC TECHNO GLASS Co., Ltd. (Shizuoka, Japan). LLC cells were kindly provided by Prof. Fei Yu (Tongji University, School of Medicine, CN). HeLa were purchased from Japanese Collection of Research Bioresources. LLC were grown in RPMI-1640 supplemented with 10% FBS and 1% penicillin\u0026ndash;streptomycin solution (FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan). HeLa and AD293 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin\u0026ndash;streptomycin solution (FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan). Cells were incubated at 37˚C under 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Human pancreatic cancer PANC-1 were purchased from Japanese Collection of Research Bioresources. PANC-1 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (D-MEM) high-glucose supplemented by 10% Fetal bovine serum (FBS) and 1% penicillin-streptomycin (\u0026times;100), then incubated at 37\u0026deg;C in a 5% carbon dioxide atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Cell viability assay by WST-8 assay\u003c/h2\u003e \u003cp\u003eLLC and PANC-1 cells were respectively plated in a 96 well microplate (100 \u0026micro;L /well) at a density of 2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells /well. Background control wells (n\u0026thinsp;=\u0026thinsp;3) containing the same volume of complete culture medium were included in each assay. The microplate was incubated for 24 h at 37˚C under 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. non-labeled \u003csup\u003e211\u003c/sup\u003eAt at various concentrations in medium were added to it and the plate incubated at 37˚C under 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. After incubating for 3 days following the addition of \u003csup\u003e211\u003c/sup\u003eAt, 10 \u0026micro;L of WST-8 reagent was added to each well, followed by incubation at 37˚C under 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for 1 hour. The absorbance was measured using an infinite F50 (Tecan) at 450 nm. For each measurement value, subtract the average background measurement value and normalized by the untreated \u003csup\u003e211\u003c/sup\u003eAt group. Data processing was done with GraphPad Prism. The results are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard error(no mark: no significance, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 vs. control group. One-way ANOVA followed by Dunnett's test was performed using GraphPad Prism 9 software).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Cell viability assay by colony-forming assay\u003c/h2\u003e \u003cp\u003eThe ability of the \u003csup\u003e211\u003c/sup\u003eAt-FAPI1 to inhibit cancer cell proliferation was evaluated using a colony-forming assay. PANC-1 cells were seeded in 24-well plates at a density of 2,000 cells/well in 1 mL supplemented D-MEM high glucose and incubated overnight. The cells were treated in triplicate with various doses of the radionuclide for 1, 2 or 24 hours at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e, washed with pre-warmed PBS, and the medium was changed to a fresh medium. The cells were incubated for 7 days, fixed, and stained with 0.5% crystal violet. The cells were observed by microscope, and the stained crystal violet was extracted using 0.05 M NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e in 50% ethanol for OD measurement at 570 nm, normalized to the control group. For each measurement value, subtract the average background measurement value and normalized by the untreated group. The results are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard error. The number of PANC-1 by treated 1% EtOH colony controls was set to 100%. Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;3, and asterisks indicate statistical significance compared to controls (no mark: no significance, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 vs. control group. One-way ANOVA followed by Dunnett's test was performed using GraphPad Prism 9 software).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eFAP - Fibroblast activation protein\u003c/p\u003e\n\u003cp\u003eFAPI - Fibroblast activation protein inhibitor\u003c/p\u003e\n\u003cp\u003ePEG - Polyethylene glycol\u003c/p\u003e\n\u003cp\u003ePIP - Piperazine\u003c/p\u003e\n\u003cp\u003ePET - Positron emission tomography\u003c/p\u003e\n\u003cp\u003eCT - Computed tomography\u003c/p\u003e\n\u003cp\u003eRCY - Radiochemical yield\u003c/p\u003e\n\u003cp\u003e%ID/g - Percent injected dose per gram\u003c/p\u003e\n\u003cp\u003eLogP - Logarithm of partition coefficient\u003c/p\u003e\n\u003cp\u003etPSA - topological Polar surface area\u003c/p\u003e\n\u003cp\u003eDOTA - 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid\u003c/p\u003e\n\u003cp\u003eNOTA - 1,4,7-triazacyclononane-1,4,7-triacetic acid\u003c/p\u003e\n\u003cp\u003ePSMA - Prostate-specific membrane antigen\u003c/p\u003e\n\u003cp\u003ePA - Phenylalanine\u003c/p\u003e\n\u003cp\u003eAAMT - Alpha-methyl Tyrosine\u003c/p\u003e\n\u003cp\u003eAuNP - Gold nanoparticles\u003c/p\u003e\n\u003cp\u003ePANC-1 - Human pancreatic carcinoma cell line\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to all members in Institute for Radiation Science in The University of Osaka for collaboration in this work. This work is supported by JSPS KAKENHI (JP20H05675, JP24K23086, JP25H00006) and F-REI (JPFR25040302), JST-CREST (JPMJCR20R3) and\u0026nbsp;Uehara Memorial Foundation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLuo Y, Fu H, Yu C (2025) Based on small molecules: development and application of fibroblast activation protein inhibitors radiopharmaceutical in tumor precision therapy. Front Pharmacol 16:1593380\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao L, Chen J, Pang Y et al (2022) Fibroblast activation protein-based theranostics in cancer research: A state-of-the-art review. 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Res Sq. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21203/rs.3.rs-7832223/v1\u003c/span\u003e\u003cspan address=\"10.21203/rs.3.rs-7832223/v1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaneda-Nakashima K, Zhang Z, Manabe Y et al (2021) α-Emitting cancer therapy using 211 At-AAMT targeting LAT1. Cancer Sci 112:1132\u0026ndash;1140\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"radiopharmaceutical, astatine-211, α-emitter, cytotoxic effect, Fibroblast activation protein (FAP)","lastPublishedDoi":"10.21203/rs.3.rs-8476643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8476643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFibroblast activation protein (FAP), which is overexpressed in cancer-associated fibroblasts (CAFs) is an attractive stromal target for a-therapy. We previously reported moderate anti-tumor effects of \u0026sup2;\u0026sup1;\u0026sup1;At-labeled FAP inhibitors (FAPI) in a PANC-1 xenograft mouse model, where insufficient tumor accumulation and the absence of FAP expression in tumor cells limited therapeutic efficacy.\u003c/p\u003e \u003cp\u003eIn this study, we conducted \u003cem\u003ein vitro\u003c/em\u003e experiments using FAP-negative PANC-1 cells to elucidate the cytotoxicity of \u0026sup2;\u0026sup1;\u0026sup1;At-labeled FAPI in comparison with free \u003csup\u003e211\u003c/sup\u003eAt. We found that, whereas free \u003csup\u003e211\u003c/sup\u003eAt showed only limited cytotoxicity, \u0026sup2;\u0026sup1;\u0026sup1;At-labeled FAPI exerted significant cytotoxic effects. Systematic variation of activity concentrations and exposure durations revealed that cytotoxicity depended primarily on sustained pericellular exposure rather than instantaneous dose. These findings demonstrate that effective cytotoxicity of CAF-targeted \u0026sup2;\u0026sup1;\u0026sup1;At radiopharmaceuticals can be achieved without direct tumor cell targeting and provide a mechanistic basis for optimizing stromal-targeted a -therapy toward clinical translation.\u003c/p\u003e","manuscriptTitle":"Dose-Time-Dependent Cellular Cytotoxicity of Astatine-211-Labeled FAP-Targeting Radiopharmaceuticals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 09:24:11","doi":"10.21203/rs.3.rs-8476643/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f32a232-c7c8-467b-b5f1-eede00817ff5","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T05:55:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 09:24:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8476643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8476643","identity":"rs-8476643","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}