Dose-Dependent Cytotoxic Profiling of Astatine-211 for Targeted Alpha Therapy

Dose-Dependent Cytotoxic Profiling of Astatine-211 for Targeted Alpha Therapy | 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-Dependent Cytotoxic Profiling of Astatine-211 for Targeted Alpha Therapy Jumpei Ueno, Masayuki Takamatsu, Koki Mayusumi, Atsushi Shimoyama, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7832223/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Journal of Radioanalytical and Nuclear Chemistry → Version 1 posted You are reading this latest preprint version Abstract Alpha particle therapy represents a promising cancer treatment approach, although the fundamental biological effects of alpha irradiation remain poorly characterized. We investigated in vitro cytotoxic effects of free astatine-211 (²¹¹At) using cell viability and proliferation assays across multiple cancer cell lines (LLC, HeLa, and AD293). The results demonstrated dose- and time-dependent cytotoxic effects, with IC₅₀ values between 0.125–0.25 MBq/mL for all cell lines after 72-hour exposure. Proliferation assays revealed that ²¹¹At progressively inhibited cell division in a dose-dependent manner, with complete cell cycle arrest at 0.25 MBq/mL. These findings establish quantitative methodologies for evaluating ²¹¹At-mediated α-particle cytotoxicity and suggest threshold-dependent cellular responses warranting further investigation. astatine-211 targeted alpha therapy (TAT) dose-dependent cytotoxicity cell viability assay proliferation assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Astatine-211 (²¹¹At) is an α-emitting radionuclide that has emerged as a particularly promising candidate for targeted alpha therapy (TAT) due to its favorable radiophysical and radiobiological characteristics (Fig. 2 a,b).[ 1 , 2 ] The high linear energy transfer (LET) of approximately 80 keV/µm and short particle range (< 70 µm) of α-particles enable precise cytotoxic effects on tumor cells while minimizing damage to the surrounding healthy tissues.[ 3 , 4 ] With a half-life of 7.2 hours, ²¹¹At provides an optimal balance for clinical applications—sufficiently long to accommodate radiopharmaceutical synthesis, quality control, and transportation, yet short enough to minimize long-term radiation exposure.[ 2 ] two branches: 41.8% undergoes direct α-particle emission to ²⁰⁷Bi, while 58.2% decays via electron capture to short-lived ²¹¹Po (t₁ / ₂ = 0.516 s), which subsequently emits an α-particle to stable ²⁰⁷Pb. This branched decay scheme ensures 100% α-particle emission per ²¹¹At decay while minimizing concerns about long-lived radioactive daughters, as the ²⁰⁷Bi produced has a 31.6-year half-life, meaning negligible decay occurs during typical treatment timeframes.[ 5 ] From a production perspective, ²¹¹At offers practical benefits for clinical implementation, as it can be efficiently produced by cyclotron irradiation of bismuth targets.[ 6 , 7 ] This production route is independent of nuclear reactors and isotopically enriched materials, enabling a scalable and sustainable supply chain for clinical use. As a halogen element, ²¹¹At shares chemical properties with iodine, enabling direct covalent bonding to carrier molecules without the need for bulky chelators that that may influence pharmacokinetics.[ 8 ] Clinical development of ²¹¹At-based radiopharmaceuticals is advancing rapidly worldwide. Currently, investigator-initiated clinical trials are underway globally, including phase I studies at Osaka University Hospital evaluating [ 211 At]NaAt for patients with differentiated thyroid cancer refractory to conventional ¹³¹I therapy, and [ 211 At]PSMA-5 for castration-resistant prostate cancer.[ 9 , 10 ] Recent results from the first-in-human study of [²¹¹At]NaAt demonstrated good tolerability and preliminary efficacy in patients with radioactive iodine-refractory differentiated thyroid cancer, with high accumulation observed in target lesions. These trials represent pioneering efforts in establishing the clinical utility of ²¹¹At-based TAT for cancer treatment. However, the fundamental properties of astatine, particularly its biological and chemical behavior, remain inadequately characterized due to the absence of stable isotopes. This knowledge gap has significantly hindered the development of quantitative methodologies for evaluating ²¹¹At efficacy, preventing direct comparisons with conventional anticancer therapeutics. Unlike conventional drugs that maintain stable molecular structures, ²¹¹At undergoes radioactive decay with a half-life of 7.2 hours, necessitating specialized analytical approaches that account for this unique decay characteristic. Additionally, the high-LET α-particles from ²¹¹At decay induce complex DNA double-strand breaks and activate DNA damage response pathways including Ataxia Telangiectasia Mutated (ATM) kinase, leading to cell cycle arrest and apoptosis through mechanisms distinct from conventional chemotherapy.[ 11 ] Consequently, the intrinsic therapeutic potency of ²¹¹At has not been comprehensively assessed using standardized metrics. Given these fundamental challenges, we have established validated protocols and methodologies to quantitatively evaluate ²¹¹At -mediated α-particle cytotoxicity in vitro , incorporating appropriate corrections for radioactive decay kinetics. Our research investigates the cellular effects of astatine exposure by systematically examining dose-response relationships and temporal effects through comprehensive analysis of cell viability and cell cycle progression parameters. Herein, we report the results of our investigation into the dose- and time-dependent effects of various ²¹¹At concentrations, approached through quantitative measurements of cell survival rates and cell division progression dynamics. Method Astatine-211 (At) was produced via the Bi(α,2n)At nuclear reaction and subsequently purified using the dry distillation method.[ 1 , 12 ] The isolated astatine was employed to treat cultured cells. Three days following astatine exposure, cell viability was assessed using the WST-8 cell counting kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions.[ 13 , 14 ] To evaluate the proliferative activity of the cells after astatine treatment, a cell proliferation assay was performed following established protocols.[ 15 ] Results and discussion 2.1. Cell viability assay Evaluation of 211 At-mediated cytotoxicity was first conducted using Lewis lung carcinoma (LLC) cells (Fig. 2 a,b).[ 16 ] Cells were exposed to a range of initial activities of astatine, and the nuclide was removed at 2, 4, 6, or 72 hours; cell viability at 72 hours after initial exposure was then quantified using a WST-8 assay (Cell Counting Kit-8).[ 13 , 14 ] Because astatine is a short-lived radionuclide, the cumulative delivered dose depends on exposure duration. Normalizing the total emitted radiation until complete decay to 100%, the relative integrated radiation delivered at the removal time points, calculated from the half-life, was 22.7%, 36.2%, 47.4%, and 99.9%, respectively.[ 1 , 17 , 18 ] Across all removal time points, viability decreased as a function of the initial activity (15.6 kBq/mL. – 2 MBq/mL, dose-dependent effect). In addition, longer exposure times resulted in lower viability (time-dependent effect).[ 11 ] In wells with continuous 72-hour exposure, the threshold initial activity yielding approximately 50% cell number (LD 50 by viability readout) fell between 0.125 MBq/mL and 0.25 MBq/mL. Next, the same evaluation was performed using HeLa cells as a representative carcinoma line (Fig. 2 c),[ 19 ] with exposure durations set at 2 h and 72 h. As with LLC cells, both initial activity–dependent and exposure time–dependent decreases in viability were observed. A similar experiment using AD293 cells also showed concordant trends with LLC and HeLa (Fig. 2 d).[ 20 ] Notably, in both additional cell lines, the initial activity producing approximately 50% cell number likewise fell between 0.125 MBq/mL and 0.25 MBq/mL. These findings are consistent with the dominant contribution of α-particle–induced DNA double-strand breaks to 211 At-mediated cytotoxicity, which is relatively insensitive to variations in cell morphology or cytoplasmic composition, thereby yielding comparable effective activity ranges across distinct cell types.[ 18 ] Conversely, in LLC and HeLa cells, viability plateaued at activities ≥ 0.25 MBq/mL, and complete eradication was not achieved. To elucidate the fate of the residual population at higher activities, subsequent analyses focused on the phenotype and status of the surviving cells. 2.2. Proliferation assay The effects of radiation on cells extend beyond direct cytotoxicity to encompass disruption of normal cellular activities, particularly cell division, which is highly sensitive to nuclear damage.[ 21 ] To investigate the impact of ²¹¹At -mediated injury on cell division, we employed a proliferation assay to assess the progression of cell division following ²¹¹At exposure. The assay principle involves initial labeling of cells with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), followed by culture maintenance. During cell division, the amount of fluorescent dye per cell decreases, and this reduction is repeated with each successive division cycle. Thus, measurement of cellular fluorescence intensity provides a direct readout of cell division progression (Fig. 3 ).[ 22 ] In untreated control cells, the peak corresponding to high fluorescence intensity gradually shifted toward lower intensity values over time, ultimately approaching the detection limit, confirming that this assay system could effectively monitor temporal changes associated with cell division (Fig. 4 a). In contrast, cells treated with an initial ²¹¹At activity of 15.6 kBq/mL exhibited a peak pattern largely similar to untreated controls, with only subtle changes in the proportion of the fluorescence intensity peak observed on Day 2 (Fig. 4 b). As the initial activity increased to 62.5 kBq/mL, the high-intensity fluorescence peak on Day 1 became more prominent, suggesting dose-dependent slowing or temporary arrest of cell division (Fig. 4 c). Furthermore, cells treated with ²¹¹At at 0.25 MBq/mL maintained a single fluorescence peak throughout the observation period, indicating complete cell division arrest (Fig. 4 d). This trend correlated well with the results obtained from the cell viability assay at high activity ranges. Comparative analysis of measurements at each time point was performed by overlaying the results (Fig. 4 e-g).[ 23 ] At all time points (Days 1–3), the 0.25 MBq/mL condition uniquely displayed a single peak indicative of arrested cell division. In contrast, cells treated with ²¹¹At at 15.6 kBq/mL and 62.5 kBq/mL showed biphasic peak patterns at 1- and 2-days post-treatment compared to untreated controls, suggesting delayed cell division. However, fluorescence ultimately decreased below the detection limit. Whether this biphasic pattern reflects radiation-induced cellular polarization or inherent variability in cell cycle phases at the experimental onset remains unclear. We anticipate that clearer trends could be obtained by synchronizing cell cycles through methods such as serum starvation or temporary treatment with cell cycle inhibitors during cell preparation.[ 24 , 25 ] These results demonstrate that cell cycle progression is dose-dependently delayed by 211 At exposure, and when this delay reaches a threshold level, cells exhibit apparent cell division arrest. Conclusion This study investigated the in vitro cytotoxic activity of free astatine and confirmed its concentration-dependent effects on cell viability. The methodological approaches employed in this investigation provide a foundation for developing quantitative comparative frameworks to assess the efficacy of astatine-based therapeutics. Furthermore, proliferation assays revealed the impact of ²¹¹At -mediated cytotoxicity on cell division dynamics. These findings quantitatively demonstrate astatine’s capacity to impair cell division progression and suggest a correlation with cell viability assay results, including evidence of discontinuous effects at specific threshold concentrations. The observed threshold-dependent responses indicate that astatine possesses additional unexplored properties that warrant further investigation. These results contribute to our understanding of astatine’s biological effects and establish validated methodologies for future studies aimed at optimizing ²¹¹At -based targeted alpha therapy applications. Materials and Experimental Methods 4.1. Materials and Reagents LLC cells were kindly provided by Prof. Fei Yu (Tongji University, School of Medicine, CN). HeLa were purchased from Japanese Collection of Research Bioresources. AD293 were purchased from Agilent Technologies (CA, USA). 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, CCK-8 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). 4.2. Cell culture 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. 4.3. Cell viability assay Cells were 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. 211 At at various concentrations in medium were added to it and the plate incubated at 37˚C under 5% CO 2 atmosphere. After the specified time had elapsed, the wells were washed twice in the medium to remove 211 At. After incubating for 3 days following the addition of 211 At, 10 µL of CCK-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. 4.4. Proliferation assay Cells were plated in a 6 well microplate (2 mL /well) at a density of 2.0×10 5 cells /well. The microplate was incubated for 24 h at 37˚C under 5% CO 2 atmosphere. After washing twice with PBS, add 1 mL of 1 µM CFSE in PBS and incubate for 30 minutes. After washing twice in medium, 211 At diluted in 2 mL of medium was added. On days 1, 2, and 3, cells were detached using trypsin/EDTA and measured by flow cytometer (Invitrogen, AFC2). Data processing was done with FlowJo version 10.10.0. Abbreviations TAT Targeted Alpha Therapy LET Linear Energy Transfer DNA Deoxyribonucleic Acid PSMA Prostate–Specific Membrane Antigen ATM Ataxia Telangiectasia Mutated kinase LLC Lewis Lung Carcinoma CFSE Carboxyfluorescein Succinimidyl Ester DMEM Dulbecco’s Modified Eagle Medium RPMI Roswell Park Memorial Institute Medium FBS Fetal Bovine Serum PBS Phosphate–Buffered Saline WST 8–Water–Soluble Tetrazolium Salt–8 CCK 8–Cell Counting Kit–8 Declarations Conflicts of Interest The authors declare no conflict of interest. Funding Declaration This work is supported by Japan Society for the Promotion of Science KAKENHI (JP20H05675, JP24K23086, JP25H00006) and Fukushima Institute for Research, Education and Innovation (JPFR25040302), Japan Science and Technology Agency -CREST (JPMJCR20R3) and Uehara Memorial Foundation. Author Contribution Conceptualization, M.T., J.U. and K.M.; cellular experiments, J.U., K.M.; chemical analysis and calculation M.T., J.U., K.M and S.N.; writing, M.T., J.U. astatine production and purification, K.O., M.M. and A.T.; supervision, M.T., A.S., K.F. and K.K.; project administration, F.L.G. and J.C.; and funding acquisition, T.W. and K.F.; All authors have read and agreed to the published version of the manuscript. Acknowledgement We are grateful to all members in Institute for Radiation Sciences in The University of Osaka for collaboration in this work. References Zalutsky MR, Pruszynski M (2011) Astatine-211: production and availability. 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Mol Biol Cell 13:1977–2000 Cooper S (2003) Rethinking synchronization of mammalian cells for cell cycle analysis. Cell Mol Life Sci 60:1099–1106 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Journal of Radioanalytical and Nuclear Chemistry → 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. 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-7832223","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":536169600,"identity":"d570b988-3890-46fd-8f11-4af6abb2c5bc","order_by":0,"name":"Jumpei Ueno","email":"","orcid":"","institution":"Osaka University","correspondingAuthor":false,"prefix":"","firstName":"Jumpei","middleName":"","lastName":"Ueno","suffix":""},{"id":536169601,"identity":"7468205b-2c52-4045-9099-30ded39f9d75","order_by":1,"name":"Masayuki 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18:17:07","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":71135,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7832223/v1/fa95589c2f8a6d5d05ac5713.html"},{"id":94986841,"identity":"60dabebb-69fb-4099-8da8-556209ce090a","added_by":"auto","created_at":"2025-11-03 07:00:52","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":278700,"visible":true,"origin":"","legend":"\u003cp\u003eAlpha-emitter and Astatine-211.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7832223/v1/6df0605a5e0109f3fbb3e8c6.jpeg"},{"id":94885520,"identity":"47f84f96-3223-4662-ae51-fdadf41ecd6c","added_by":"auto","created_at":"2025-10-31 18:17:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":466402,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability assay utilizing WST-8 kit. a) overview of WST-8 kit, b) relative Live cell count (%) in case of LLC treating with 2, 4, 6, 72 h, c) relative Live cell count (%) in case of HeLa cell treating with 2, 72 h, d) relative Live cell count (%) in case of AD293 cell treating with 2, 72 h. *normalized by 0 Bq/mL in 72h, respectively.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7832223/v1/48e2e452ae1b31edd8ee36f9.jpeg"},{"id":94885517,"identity":"508017c2-fdeb-4e9e-a54e-600d6b8f9138","added_by":"auto","created_at":"2025-10-31 18:17:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":132477,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of proliferation assay. CFSE: Carboxyfluorescein succinimidyl ester\u003c/p\u003e","description":"","filename":"floatimage317.png","url":"https://assets-eu.researchsquare.com/files/rs-7832223/v1/5d33ab991784fde5f3e3ce5a.png"},{"id":94987273,"identity":"7052d15b-5a8a-47eb-b001-45bd44ab830a","added_by":"auto","created_at":"2025-11-03 07:01:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":194590,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescent intensity through flowcytometry on cell proliferation assays. a-d) result using 0, 15.6, 62.5, 250 kBq/ml, e-g) marge the result on each radioactivity at the 1, 2, 3 Day respectively.\u003c/p\u003e","description":"","filename":"floatimage46.png","url":"https://assets-eu.researchsquare.com/files/rs-7832223/v1/13d4c427f4e44f87f4c26711.png"},{"id":99545261,"identity":"80a0fa1c-ea54-45ca-a44e-a28d0f0d2a3e","added_by":"auto","created_at":"2026-01-05 16:04:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1532660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7832223/v1/4f9fdee1-3e74-45c8-a2e2-cbfbf04218db.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dose-Dependent Cytotoxic Profiling of Astatine-211 for Targeted Alpha Therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAstatine-211 (\u0026sup2;\u0026sup1;\u0026sup1;At) is an α-emitting radionuclide that has emerged as a particularly promising candidate for targeted alpha therapy (TAT) due to its favorable radiophysical and radiobiological characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b).[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] The high linear energy transfer (LET) of approximately 80 keV/\u0026micro;m and short particle range (\u0026lt;\u0026thinsp;70 \u0026micro;m) of α-particles enable precise cytotoxic effects on tumor cells while minimizing damage to the surrounding healthy tissues.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] With a half-life of 7.2 hours, \u0026sup2;\u0026sup1;\u0026sup1;At provides an optimal balance for clinical applications\u0026mdash;sufficiently long to accommodate radiopharmaceutical synthesis, quality control, and transportation, yet short enough to minimize long-term radiation exposure.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] two branches: 41.8% undergoes direct α-particle emission to \u0026sup2;⁰⁷Bi, while 58.2% decays via electron capture to short-lived \u0026sup2;\u0026sup1;\u0026sup1;Po (t₁\u003csub\u003e/\u003c/sub\u003e₂ = 0.516 s), which subsequently emits an α-particle to stable \u0026sup2;⁰⁷Pb. This branched decay scheme ensures 100% α-particle emission per \u0026sup2;\u0026sup1;\u0026sup1;At decay while minimizing concerns about long-lived radioactive daughters, as the \u0026sup2;⁰⁷Bi produced has a 31.6-year half-life, meaning negligible decay occurs during typical treatment timeframes.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] From a production perspective, \u0026sup2;\u0026sup1;\u0026sup1;At offers practical benefits for clinical implementation, as it can be efficiently produced by cyclotron irradiation of bismuth targets.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] This production route is independent of nuclear reactors and isotopically enriched materials, enabling a scalable and sustainable supply chain for clinical use. As a halogen element, \u0026sup2;\u0026sup1;\u0026sup1;At shares chemical properties with iodine, enabling direct covalent bonding to carrier molecules without the need for bulky chelators that that may influence pharmacokinetics.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eClinical development of \u0026sup2;\u0026sup1;\u0026sup1;At-based radiopharmaceuticals is advancing rapidly worldwide. Currently, investigator-initiated clinical trials are underway globally, including phase I studies at Osaka University Hospital evaluating [\u003csup\u003e211\u003c/sup\u003eAt]NaAt for patients with differentiated thyroid cancer refractory to conventional \u0026sup1;\u0026sup3;\u0026sup1;I therapy, and [\u003csup\u003e211\u003c/sup\u003eAt]PSMA-5 for castration-resistant prostate cancer.[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Recent results from the first-in-human study of [\u0026sup2;\u0026sup1;\u0026sup1;At]NaAt demonstrated good tolerability and preliminary efficacy in patients with radioactive iodine-refractory differentiated thyroid cancer, with high accumulation observed in target lesions. These trials represent pioneering efforts in establishing the clinical utility of \u0026sup2;\u0026sup1;\u0026sup1;At-based TAT for cancer treatment. However, the fundamental properties of astatine, particularly its biological and chemical behavior, remain inadequately characterized due to the absence of stable isotopes. This knowledge gap has significantly hindered the development of quantitative methodologies for evaluating \u0026sup2;\u0026sup1;\u0026sup1;At efficacy, preventing direct comparisons with conventional anticancer therapeutics. Unlike conventional drugs that maintain stable molecular structures, \u0026sup2;\u0026sup1;\u0026sup1;At undergoes radioactive decay with a half-life of 7.2 hours, necessitating specialized analytical approaches that account for this unique decay characteristic. Additionally, the high-LET α-particles from \u0026sup2;\u0026sup1;\u0026sup1;At decay induce complex DNA double-strand breaks and activate DNA damage response pathways including Ataxia Telangiectasia Mutated (ATM) kinase, leading to cell cycle arrest and apoptosis through mechanisms distinct from conventional chemotherapy.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] Consequently, the intrinsic therapeutic potency of \u0026sup2;\u0026sup1;\u0026sup1;At has not been comprehensively assessed using standardized metrics. Given these fundamental challenges, we have established validated protocols and methodologies to quantitatively evaluate \u0026sup2;\u0026sup1;\u0026sup1;At -mediated α-particle cytotoxicity \u003cem\u003ein vitro\u003c/em\u003e, incorporating appropriate corrections for radioactive decay kinetics. Our research investigates the cellular effects of astatine exposure by systematically examining dose-response relationships and temporal effects through comprehensive analysis of cell viability and cell cycle progression parameters.\u003c/p\u003e\u003cp\u003eHerein, we report the results of our investigation into the dose- and time-dependent effects of various \u0026sup2;\u0026sup1;\u0026sup1;At concentrations, approached through quantitative measurements of cell survival rates and cell division progression dynamics.\u003c/p\u003e"},{"header":"Method","content":"\u003cp\u003eAstatine-211 (At) was produced via the Bi(α,2n)At nuclear reaction and subsequently purified using the dry distillation method.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] The isolated astatine was employed to treat cultured cells. Three days following astatine exposure, cell viability was assessed using the WST-8 cell counting kit (Dojindo, Kumamoto, Japan) according to the manufacturer\u0026rsquo;s instructions.[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] To evaluate the proliferative activity of the cells after astatine treatment, a cell proliferation assay was performed following established protocols.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cell viability assay\u003c/h2\u003e\u003cp\u003eEvaluation of \u003csup\u003e211\u003c/sup\u003eAt-mediated cytotoxicity was first conducted using Lewis lung carcinoma (LLC) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b).[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] Cells were exposed to a range of initial activities of astatine, and the nuclide was removed at 2, 4, 6, or 72 hours; cell viability at 72 hours after initial exposure was then quantified using a WST-8 assay (Cell Counting Kit-8).[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Because astatine is a short-lived radionuclide, the cumulative delivered dose depends on exposure duration. Normalizing the total emitted radiation until complete decay to 100%, the relative integrated radiation delivered at the removal time points, calculated from the half-life, was 22.7%, 36.2%, 47.4%, and 99.9%, respectively.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] Across all removal time points, viability decreased as a function of the initial activity (15.6 kBq/mL. \u0026ndash; 2 MBq/mL, dose-dependent effect). In addition, longer exposure times resulted in lower viability (time-dependent effect).[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] In wells with continuous 72-hour exposure, the threshold initial activity yielding approximately 50% cell number (LD\u003csub\u003e50\u003c/sub\u003e by viability readout) fell between 0.125 MBq/mL and 0.25 MBq/mL.\u003c/p\u003e\u003cp\u003eNext, the same evaluation was performed using HeLa cells as a representative carcinoma line (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec),[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] with exposure durations set at 2 h and 72 h. As with LLC cells, both initial activity\u0026ndash;dependent and exposure time\u0026ndash;dependent decreases in viability were observed. A similar experiment using AD293 cells also showed concordant trends with LLC and HeLa (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Notably, in both additional cell lines, the initial activity producing approximately 50% cell number likewise fell between 0.125 MBq/mL and 0.25 MBq/mL. These findings are consistent with the dominant contribution of α-particle\u0026ndash;induced DNA double-strand breaks to \u003csup\u003e211\u003c/sup\u003eAt-mediated cytotoxicity, which is relatively insensitive to variations in cell morphology or cytoplasmic composition, thereby yielding comparable effective activity ranges across distinct cell types.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] Conversely, in LLC and HeLa cells, viability plateaued at activities\u0026thinsp;\u0026ge;\u0026thinsp;0.25 MBq/mL, and complete eradication was not achieved. To elucidate the fate of the residual population at higher activities, subsequent analyses focused on the phenotype and status of the surviving cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Proliferation assay\u003c/h2\u003e\u003cp\u003eThe effects of radiation on cells extend beyond direct cytotoxicity to encompass disruption of normal cellular activities, particularly cell division, which is highly sensitive to nuclear damage.[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] To investigate the impact of \u0026sup2;\u0026sup1;\u0026sup1;At -mediated injury on cell division, we employed a proliferation assay to assess the progression of cell division following \u0026sup2;\u0026sup1;\u0026sup1;At exposure. The assay principle involves initial labeling of cells with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), followed by culture maintenance. During cell division, the amount of fluorescent dye per cell decreases, and this reduction is repeated with each successive division cycle. Thus, measurement of cellular fluorescence intensity provides a direct readout of cell division progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] In untreated control cells, the peak corresponding to high fluorescence intensity gradually shifted toward lower intensity values over time, ultimately approaching the detection limit, confirming that this assay system could effectively monitor temporal changes associated with cell division (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, cells treated with an initial \u0026sup2;\u0026sup1;\u0026sup1;At activity of 15.6 kBq/mL exhibited a peak pattern largely similar to untreated controls, with only subtle changes in the proportion of the fluorescence intensity peak observed on Day 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). As the initial activity increased to 62.5 kBq/mL, the high-intensity fluorescence peak on Day 1 became more prominent, suggesting dose-dependent slowing or temporary arrest of cell division (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Furthermore, cells treated with \u0026sup2;\u0026sup1;\u0026sup1;At at 0.25 MBq/mL maintained a single fluorescence peak throughout the observation period, indicating complete cell division arrest (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). This trend correlated well with the results obtained from the cell viability assay at high activity ranges. Comparative analysis of measurements at each time point was performed by overlaying the results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-g).[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] At all time points (Days 1\u0026ndash;3), the 0.25 MBq/mL condition uniquely displayed a single peak indicative of arrested cell division. In contrast, cells treated with \u0026sup2;\u0026sup1;\u0026sup1;At at 15.6 kBq/mL and 62.5 kBq/mL showed biphasic peak patterns at 1- and 2-days post-treatment compared to untreated controls, suggesting delayed cell division. However, fluorescence ultimately decreased below the detection limit. Whether this biphasic pattern reflects radiation-induced cellular polarization or inherent variability in cell cycle phases at the experimental onset remains unclear. We anticipate that clearer trends could be obtained by synchronizing cell cycles through methods such as serum starvation or temporary treatment with cell cycle inhibitors during cell preparation.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] These results demonstrate that cell cycle progression is dose-dependently delayed by \u003csup\u003e211\u003c/sup\u003eAt exposure, and when this delay reaches a threshold level, cells exhibit apparent cell division arrest.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study investigated the \u003cem\u003ein vitro\u003c/em\u003e cytotoxic activity of free astatine and confirmed its concentration-dependent effects on cell viability. The methodological approaches employed in this investigation provide a foundation for developing quantitative comparative frameworks to assess the efficacy of astatine-based therapeutics. Furthermore, proliferation assays revealed the impact of \u0026sup2;\u0026sup1;\u0026sup1;At -mediated cytotoxicity on cell division dynamics. These findings quantitatively demonstrate astatine\u0026rsquo;s capacity to impair cell division progression and suggest a correlation with cell viability assay results, including evidence of discontinuous effects at specific threshold concentrations. The observed threshold-dependent responses indicate that astatine possesses additional unexplored properties that warrant further investigation. These results contribute to our understanding of astatine\u0026rsquo;s biological effects and establish validated methodologies for future studies aimed at optimizing \u0026sup2;\u0026sup1;\u0026sup1;At -based targeted alpha therapy applications.\u003c/p\u003e"},{"header":"Materials and Experimental Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Materials and Reagents\u003c/h2\u003e\u003cp\u003eLLC cells were kindly provided by Prof. Fei Yu (Tongji University, School of Medicine, CN). HeLa were purchased from Japanese Collection of Research Bioresources. AD293 were purchased from Agilent Technologies (CA, USA). 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, CCK-8 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).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Cell culture\u003c/h2\u003e\u003cp\u003eLLC 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.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Cell viability assay\u003c/h2\u003e\u003cp\u003eCells were 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. \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 the specified time had elapsed, the wells were washed twice in the medium to remove \u003csup\u003e211\u003c/sup\u003eAt. After incubating for 3 days following the addition of \u003csup\u003e211\u003c/sup\u003eAt, 10 \u0026micro;L of CCK-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.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Proliferation assay\u003c/h2\u003e\u003cp\u003eCells were plated in a 6 well microplate (2 mL /well) at a density of 2.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells /well. The microplate was incubated for 24 h at 37˚C under 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. After washing twice with PBS, add 1 mL of 1 \u0026micro;M CFSE in PBS and incubate for 30 minutes. After washing twice in medium, \u003csup\u003e211\u003c/sup\u003eAt diluted in 2 mL of medium was added. On days 1, 2, and 3, cells were detached using trypsin/EDTA and measured by flow cytometer (Invitrogen, AFC2). Data processing was done with FlowJo version 10.10.0.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTAT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTargeted Alpha Therapy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLET\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLinear Energy Transfer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDeoxyribonucleic Acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePSMA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProstate\u0026ndash;Specific Membrane Antigen\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eATM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAtaxia Telangiectasia Mutated kinase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLLC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLewis Lung Carcinoma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCFSE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCarboxyfluorescein Succinimidyl Ester\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRPMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRoswell Park Memorial Institute Medium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFetal Bovine Serum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhosphate\u0026ndash;Buffered Saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e8\u0026ndash;Water\u0026ndash;Soluble Tetrazolium Salt\u0026ndash;8\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCCK\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e8\u0026ndash;Cell Counting Kit\u0026ndash;8\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding Declaration\u003c/h2\u003e\n\u003cp\u003eThis work is supported by Japan Society for the Promotion of Science KAKENHI (JP20H05675, JP24K23086, JP25H00006) and Fukushima Institute for Research, Education and Innovation (JPFR25040302), Japan Science and Technology Agency -CREST (JPMJCR20R3) and Uehara Memorial Foundation.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization, M.T., J.U. and K.M.; cellular experiments, J.U., K.M.; chemical analysis and calculation M.T., J.U., K.M and S.N.; writing, M.T., J.U. astatine production and purification, K.O., M.M. and A.T.; supervision, M.T., A.S., K.F. and K.K.; project administration, F.L.G. and J.C.; and funding acquisition, T.W. and K.F.; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe are grateful to all members in Institute for Radiation Sciences in The University of Osaka for collaboration in this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZalutsky MR, Pruszynski M (2011) Astatine-211: production and availability. Curr Radiopharm 4:177\u0026ndash;185\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlbertsson P, B\u0026auml;ck T, Bergmark K et al (2022) Astatine-211 based radionuclide therapy: Current clinical trial landscape. Front Med (Lausanne) 9:1076210\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePoty S, Francesconi LC, McDevitt MR et al (2018) Α-emitters for radiotherapy: From basic radiochemistry to clinical studies\u0026mdash;part 1. J Nucl Med 59:878\u0026ndash;884\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeter R, Sandmaier BM, Dion MP et al (2022) Small-scale (sub-organ and cellular level) alpha-particle dosimetry methods using an iQID digital autoradiography imaging system. Sci Rep 12:17934\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi F, Yang Y, Liao J, Liu N (2022) Recent progress of astatine-211 in endoradiotherapy: Great advances from fundamental properties to targeted radiopharmaceuticals. Chin Chem Lett 33:3325\u0026ndash;3338\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHenriksen G, Breist\u0026oslash;l K, Bruland \u0026Oslash;S et al (2002) Significant antitumor effect from bone-seeking, alpha-particle-emitting (223)Ra demonstrated in an experimental skeletal metastases model. Cancer Res 62:3120\u0026ndash;3125\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLindegren S, B\u0026auml;ck T, Jensen HJ (2001) Dry-distillation of astatine-211 from irradiated bismuth targets: a time-saving procedure with high recovery yields. Appl Radiat Isot 55:157\u0026ndash;160\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao J, Li M, Yin J et al (2024) The different strategies for the radiolabeling of [211At]-astatinated radiopharmaceuticals. Pharmaceutics 16:738\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatabe T, Kaneda-Nakashima K, Liu Y et al (2019) Enhancement of 211At uptake via the sodium iodide symporter by the addition of ascorbic acid in targeted α-therapy of thyroid cancer. J Nucl Med 60:1301\u0026ndash;1307\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWatabe T, Naka S, Shirakami Y et al (2025) Development of PSMA-targeted alpha therapy using [211At]PSMA-5. Semin Nucl Med. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1053/j.semnuclmed.2025.09.005\u003c/span\u003e\u003cspan address=\"10.1053/j.semnuclmed.2025.09.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePouget J-P, Constanzo J (2021) Revisiting the radiobiology of targeted alpha therapy. Front Med (Lausanne) 8:692436\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDry-distillation of astatine-211 from irradiated bismuth targets: a practical and efficient method. Applied Radiation and Isotopes\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChamchoy K, Pakotiprapha D, Pumirat P et al (2019) Application of WST-8 based colorimetric NAD(P)H detection for quantitative dehydrogenase assays. BMC Biochem 20:4\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTominaga H, Ishiyama M, Ohseto F et al (1999) A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal Commun 36:47\u0026ndash;50\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdan A, Alizada G, Kiraz Y et al (2017) Flow cytometry: basic principles and applications. Crit Rev Biotechnol 37:163\u0026ndash;176\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBertram JS, Janik P (1980) Establishment of a cloned line of Lewis Lung Carcinoma cells adapted to cell culture. Cancer Lett 11:63\u0026ndash;73\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSgouros G, Hobbs RF, Song H (2011) Modelling and dosimetry for alpha-particle therapy. Curr Radiopharm 4:261\u0026ndash;265\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSgouros G, Roeske JC, McDevitt MR et al (2010) MIRD Pamphlet 22 (abridged): radiobiology and dosimetry of alpha-particle emitters for targeted radionuclide therapy. J Nucl Med 51:311\u0026ndash;328\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScherer WF, Syverton JT, Gey GO (1953) Studies on the propagation \u003cem\u003ein vitro\u003c/em\u003e of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med 97:695\u0026ndash;710\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGraham FL, Smiley J, Russell WC, Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36:59\u0026ndash;74\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHall EJ, Giaccia AJ (2018) Radiobiology for the Radiologist, 8th edn. Wolters Kluwer Health, Baltimore, MD\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eQuah BJC, Warren HS, Parish CR (2007) Monitoring lymphocyte proliferation i\u003cem\u003en vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc 2:2049\u0026ndash;2056\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHawkins ED, Hommel M, Turner ML et al (2007) Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat Protoc 2:2057\u0026ndash;2067\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWhitfield ML, Sherlock G, Saldanha AJ et al (2002) Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol Biol Cell 13:1977\u0026ndash;2000\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCooper S (2003) Rethinking synchronization of mammalian cells for cell cycle analysis. Cell Mol Life Sci 60:1099\u0026ndash;1106\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":true,"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":"astatine-211, targeted alpha therapy (TAT), dose-dependent cytotoxicity, cell viability assay, proliferation assay","lastPublishedDoi":"10.21203/rs.3.rs-7832223/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7832223/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlpha particle therapy represents a promising cancer treatment approach, although the fundamental biological effects of alpha irradiation remain poorly characterized. We investigated \u003cem\u003ein vitro\u003c/em\u003e cytotoxic effects of free astatine-211 (\u0026sup2;\u0026sup1;\u0026sup1;At) using cell viability and proliferation assays across multiple cancer cell lines (LLC, HeLa, and AD293). The results demonstrated dose- and time-dependent cytotoxic effects, with IC₅₀ values between 0.125\u0026ndash;0.25 MBq/mL for all cell lines after 72-hour exposure. Proliferation assays revealed that \u0026sup2;\u0026sup1;\u0026sup1;At progressively inhibited cell division in a dose-dependent manner, with complete cell cycle arrest at 0.25 MBq/mL. These findings establish quantitative methodologies for evaluating \u0026sup2;\u0026sup1;\u0026sup1;At-mediated α-particle cytotoxicity and suggest threshold-dependent cellular responses warranting further investigation.\u003c/p\u003e","manuscriptTitle":"Dose-Dependent Cytotoxic Profiling of Astatine-211 for Targeted Alpha Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 18:17:02","doi":"10.21203/rs.3.rs-7832223/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":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T15:59:54+00:00","versionOfRecord":{"articleIdentity":"rs-7832223","link":"https://doi.org/10.1007/s10967-025-10671-5","journal":{"identity":"journal-of-radioanalytical-and-nuclear-chemistry","isVorOnly":false,"title":"Journal of Radioanalytical and Nuclear Chemistry"},"publishedOn":"2026-01-02 15:57:32","publishedOnDateReadable":"January 2nd, 2026"},"versionCreatedAt":"2025-10-31 18:17:02","video":"","vorDoi":"10.1007/s10967-025-10671-5","vorDoiUrl":"https://doi.org/10.1007/s10967-025-10671-5","workflowStages":[]},"version":"v1","identity":"rs-7832223","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7832223","identity":"rs-7832223","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}