Abstract 2963: CD44+/CD133+ colorectal cancer stem cells are sensitive to trifluridine
Authors: Tsunekuni Kenta , Konno Masamitsu , Koseki Jun , Asai Ayumu , Matsuoka Kazuaki , Takechi Teiji , Doki Yuichiro , Mori Masaki , Ishii Hideshi 
Published: August 2019

Abstract
Background: Cancer stem cells (CSCs) are involved in disease recurrence, metastases, and therapeutic resistance. However, anticancer agents that target CSCs are not currently available. Trifluridine (FTD)/tipiracil (TPI), a novel oral antitumor drug, was approved for the treatment of patients with metastatic colorectal cancer who had been previously treated with anti-vascular endothelial growth factors; anti-epidermal growth factor receptors (in patients with ras wild-type genes); or fluoropyrimidine-, oxaliplatin-, irinotecan-based chemotherapies. FTD/TPI improves overall survival, and FTD is the key component of FTD/TPI. Here, we demonstrated the efficacy of FTD against CD44 and CD133 high-expressing (CD44+CD133+) colorectal cancer cells that possess CSC-like properties.
Method: CD44+CD133+ and other populations of DLD-1, a colorectal cell line, were separated by fluorescence-activated cell sorting (FACS). The sphere-forming activity of each population, and anti- sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells, were measured. In addition, DLD-1 and HCT-116, two colorectal cell lines, were treated with FTD for 5 months. The CSC markers CD44 and CD133 were measured by FACS, and the cells' in vitro and in vivo tumor-initiating properties were evaluated.
Results: CD44+CD133+ DLD-1 cells formed significantly more spheres than did CD44-CD133- cells (sphere ratio CD44+CD133+/CD44-CD133- = 4.0) and CD44+CD133- cells (sphere ratio CD44+CD133+/CD44+CD133- = 1.7). In the in vitro proliferation assay, CD44+CD133+ cells had greater 5-FU resistance, but not higher FTD resistance, than did unsorted cells. In addition, treatment of CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) disrupted sphere formation, which was superior to the effect of treatment with 1 µM 5-FU. The inhibition rates for FTD and 5-FU were 59.5% and 14.3%, respectively. DLD-1 and HCT-116 cell lines treated with FTD for 5 months had smaller CD44+CD133+ populations and lower sphere-forming activity than did untreated cell lines; population decline rates were 93.8% and 74.7%, respectively. Furthermore, 5 months of in vitro exposure to FTD reduced in vivo tumor formation potential in both cell lines. These results suggest that FTD is effective against CSC-like cells, and that treatment with FTD might reduce CSC-like populations.
Conclusion: FTD had a direct effect on the sphere-forming activity of CSC-like CD44+CD133+ cells that was superior to that of treatment with 5-FU. Its effectiveness against CSC-like CD44+CD133+ cells suggests that FTD might be useful for CSC-targeted chemotherapy in tumors that highly express CD44 and CD133.
Citation Format: Kenta Tsunekuni, Masamitsu Konno, Jun Koseki, Ayumu Asai, Kazuaki Matsuoka, Teiji Takechi, Yuichiro Doki, Masaki Mori, Hideshi Ishii. CD44+/CD133+ colorectal cancer stem cells are sensitive to trifluridine [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 2963.