TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine (FTD) 
and tipiracil hydrochloride (TPI). The compound improves overall survival of 
colorectal cancer (CRC) patients who are insensitive to standard chemotherapies. 
FTD possesses direct antitumor activity since it inhibits thymidylate synthase 
(TS) and is itself incorporated into DNA. However, the precise mechanisms 
underlying the incorporation into DNA and the inhibition of TS remain unclear. 
We found that FTD-dependent inhibition of TS was similar to that elicited by 
fluorodeoxyuridine (FdUrd), another clinically used nucleoside analog. However, 
washout experiments revealed that FTD-dependent inhibition of TS declined 
rapidly, whereas FdUrd activity persisted. The incorporation of FTD into DNA was 
significantly higher than that of other antitumor nucleosides. Additionally, 
orally administered FTD had increased antitumor activity and was incorporated 
into DNA more effectively than continuously infused FTD. When TAS-102 was 
administered, FTD gradually accumulated in tumor cell DNA, in a TPI-independent 
manner, and significantly delayed tumor growth and prolonged survival, compared 
to treatment with 5-FU derivatives. TAS-102 reduced the Ki-67-positive cell 
fraction, and swollen nuclei were observed in treated tumor tissue. The amount 
of FTD incorporation in DNA and the antitumor activity of TAS-102 in xenograft 
models were positively and significantly correlated. These results suggest that 
TAS-102 exerts its antitumor activity predominantly due to its DNA 
incorporation, rather than as a result of TS inhibition. The persistence of FTD 
in the DNA of tumor cells treated with TAS-102 may underlie its ability to 
prolong survival in cancer patients.