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Xianghua Shi,1,2,* Sansan Chen,3,* Yongjun Zhang,4 Weiwei Xie,1 Zhiming Hu,4 Hongwei Li,4 Jinlong Li,4 Zhongxin Zhou,5 Wanlong Tan11Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China; 2Department of Health Management, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China; 3Department of Urology, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, Guangdong, People’s Republic of China; 4Institute of Biotherapy, School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China; 5Department of Vascular Surgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, People’s Republic of China*These authors contributed equally to this work Introduction: Cancer stem cells (CSCs) are the main source of tumor resistance and recurrence. At present, the main treatment for patients with advanced or metastatic bladder cancer (BCa) is cisplatin-based combination chemotherapy. However, CSCs are not sensitive to DNA-damaging drugs due to their enhanced DNA damage response (DDR) activity.Materials and methods: Bladder cancer stem cell-like cells (BCSLCs) were obtained by treating UMUC3 cells with cisplatin. The characteristics of the BCSLCs were identified by qPCR, flow cytometry, scratch wound-healing assays, transwell assays, tumorigenic ability experiments, Edu assays and Western blot assays in vivo. After BCSLCs were treated with norcantharidin (NCTD), the expression of Cdc6 and activation of the ATR-Chk1 pathway were detected by Western blotting. A subcutaneous tumor model in nude mice was successfully established to assess the anti-tumor efficacy of NCTD and cisplatin either alone or in combination in vivo. The tumor tissues were detected by immunohistochemistry.Results: The derived BCSLCs showed higher expression of stemness markers, increased invasiveness, improved resistance to multiple chemotherapeutics, and higher tumorigenic capacity in vivo. The protein expression level of chromatin-binding Cdc6 was increased in BCSLCs; however, NCTD decreased the level of chromatin-binding Cdc6 and inhibited the activation of the ATR-Chk1 pathway, which ultimately led to reduction in DDR activity in BCSLCs. NCTD enhanced the killing effect of cisplatin on BCSLCs in vitro and vivo. NCTD combined with cisplatin enhanced cisplatin-induced DNA damage in BCSLCs.Conclusion: Long-term cisplatin treatment can enrich BCSLCs. However, NCTD enhanced the killing effect of cisplatin on BCSLCs in vitro and vivo. The mechanism is inhibiting the DDR activity by reducing the expression of chromatin-binding Cdc6.Keywords: bladder cancer stem-like cells, norcantharidin, DDR, Cdc6, cisplatin