Abstract TTFields cause mitotic arrest and cell death by the disruption of spindle microtubule arrangement and interference with cytokinesis. Recently it was reported that TTFields application results in reduced clonogenicity of cells surviving treatment. We hypothesized that TTFields can impair chromosome segregation during mitosis thus leading to a long term effect on surviving cancer cells. A2780 human ovarian cancer cells were treated with TTFields for 24, 48 and 72 hours and the cytotoxic effect and clonogenic potential were determined. Abnormal mitotic figures were analyzed using fluorescence microscopy and cell karyotyping was performed using Giemsa staining and SKY analysis. TTFields application led to 36, 52 and 43% cell growth inhibition after 24, 48 and 72 hours, respectively. Clonogenic assays revealed that only 12.9% of cells treated with TTFields for 72 hours maintained their ability to form colonies after 2 weeks. Immunofluorescence images revealed a significant increase in the number of abnormal mitotic figures amongst treated cells. Karyotyping analysis following 24 and 48 hours of TTFields application revealed 38.5% and 53.8% abnormal segregation events, respectively. After 24 hours of treatment, the proportion of cells with reduced and increased chromosomes number was equal, however, following 48 hours of treatment the population with a reduced chromosome number disappeared. These results demonstrate that in addition to their cytotoxic effect leading to mitotic death, TTFields also lead to abnormal chromosome segregation. The surviving aneuploid cells are impaired, providing a possible explanation for the reduced clonogenic potential reported in cancer cells treated with TTFields. Citation Format: Rosa S. Schneiderman, Moshe Giladi, Yaara Porat, Mijal Munster, Eilon D. Kirson, Uri Weinberg, Yoram Palti. TTFields reduce cancer cell clonogenic potential through abnormal chromosome segregation during mitosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5521. doi:10.1158/1538-7445.AM2014-5521