64Cu-ATSM therapy targets regions with activated DNA repair and enrichment of CD133+ cells in an HT-29 tumor model: Sensitization with a nucleic acid antimetabolite

64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM) is a potential theranostic agent targeting the over-reduced state under hypoxia within tumors. Recent clinical Cu-ATSM positron emission tomography studies have revealed a correlation between uptake and poor prognosis; however, the reason is unclear. Here, using a human colon carcinoma HT-29 model, we demonstrated that the intratumoral 64Cu-ATSM high-uptake regions exhibited malignant characteristics, such as upregulated DNA repair and elevated %CD133+ cancer stem-like cells. Based on this evidence, we developed a strategy to enhance the efficacy of 64Cu-ATSM internal radiotherapy (IRT) by inhibiting DNA repair with a nucleic acid (NA) antimetabolite. The results of the analyses showed upregulation of pathways related to DNA repair along with NA incorporation (bromodeoxyuridine uptake) and elevation of %CD133+ cells in 64Cu-ATSM high-uptake regions. In an in vivo64Cu-ATSM treatment study, co-administration of an NA antimetabolite and 64Cu-ATSM synergistically inhibited tumor growth, with little toxicity, and effectively reduced %CD133+ cells. 64Cu-ATSM therapy targeted malignant tumor regions with activated DNA repair and high concentrations of CD133+ cells in the HT-29 model. NA antimetabolite co-administration can be an effective approach to enhance the therapeutic effect of 64Cu-ATSM IRT.