Abstract Purpose: Targeted alpha therapy (TAT) represents an emerging treatment approach attempting to deliver systemic radiation selectively to cancer cells and bypassing mechanisms of acquired resistance while minimizing systemic toxic effects. Radium-223 dichloride (Ra-223) is a first-in-class TAT shown to prolong survival in patients with metastatic castration-resistant prostate cancer and bone metastases. Yet, the understanding of the biological effects of α-particle irradiation is still limited. Here, we used a novel in vitro assay designed to assess alpha irradiation specific effects to investigate the biological effects of Ra-223 in various cancer cell lines. Furthermore, the combination of α-particle irradiation with DNA repair inhibitors was tested. Methods: The following cancer cell lines were used: lung (H460), ovarian (OVCAR-3, COV362, COV644, ES2) and prostate (22Rv1, LNCaP). The effects of different radiation doses and exposure times of Ra-223 on various biological parameters were analyzed using a Transwell® system where cells grow on a 10 µm membrane located on top of an underlying Ra-223 coating. Results: After 4 hours of exposure to α-particle irradiation (5, 10 and 20 kBq/3.8 cm2), a dose-dependent induction of DNA double strand breaks (DSB) as assessed by 53BP1 positivity was observed. Exposure to α-particle irradiation (10 and 20 kBq/3.8 cm2) for 1 hour dose-dependently induced DNA damage in a comet assay. Exposure to α-particle irradiation (5 kBq/3.8 cm2) for 1, 4 and 8 hours time-dependently reduced the surviving fraction in the subsequent colony formation assay. The frequency of micronuclei formation was increased in an activity- and time-dependent manner after α-particle exposure at 5, 10, 20, 40 kBq/3.8 cm2 for 1, 4 and 8 hours. In the same assay, the number of dead cells was significantly increased after an 8-hour exposure in all dose groups tested. Pre-incubation with the ATR inhibitor BAY 1895344 (5 nM for 2 hours prior to radiation) synergistically enhanced micronuclei formation (< 5% vs 12% vs 42% in ATR inhibitor only vs α-particle irradiation only vs ATR inhibitor + α-particle irradiation, respectively). Conclusion: Using several cancer cell lines, we demonstrated that short term exposure to α-particle irradiation in the range of 1 to 8 hours is sufficient to induce DNA damage including DNA DSB which leads to cellular damage and cell death. Synergistic effects were observed when Ra-223 was combined with the ATR inhibitor BAY 1895344 . Citation Format: Kristina Bannik, Sabine Zitzmann-Kolbe, Arne Scholz, Sabrina Jarke, Marco Jarzombek, Andreas Sutter, Gerhard Siemeister, Dominik Mumberg. Radium-223 α-particle radiation: Characterization of the in vitro effects on cancer cells in monotherapy and in combination with DNA repair inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3927.