In cancer therapy, enhanced thymidine uptake by the salvage pathway can bypass dTMP depletion, thereby conferring resistance to thymidylate synthase inhibition. We investigated whether sequential combination therapy of capecitabine and trifluridine/tipiracil (TAS-102) could synergistically enhance antitumor efficacy in colon cancer xenograft models. We also examined 3'-deoxy-3′-[18F]fluorothymidine ([18F]FLT) PET as a means to predict therapeutic response to a sequential combination of capecitabine and trifluridine/tipiracil. [3H]FLT uptake after 5-fluorouracil treatment in vitro and [18F]FLT uptake after capecitabine (360 mg/kg/day) in athymic nude mice (Balb/c-nu) with xenografts (n = 10–12 per group) were measured using eight human colon cancer cell lines. We determined the synergistic effects of sequential combinations of 5-fluorouracil and trifluridine in vitro as well as the sequential combination of oral capecitabine (30–360 mg/kg) and trifluridine/tipiracil (trifluridine 75 or 150 mg/kg with tipiracil) in six xenograft models (n = 6–10 per group). We observed significant increases in [3H]FLT uptake in all cell lines and [18F]FLT uptake in five xenograft models after 5-fluorouracil and capecitabine treatment, respectively. Increased [18F]FLT uptake after capecitabine followed by extinction of uptake correlated strongly with tumor growth inhibition (ρ = −0.81, P = 0.02). The effects of these combinations were synergistic in vitro. A synergy for sequential capecitabine and trifluridine/tipiracil was found only in mouse xenograft models showing increased [18F]FLT uptake after capecitabine. Our results suggest that the sequential combination of capecitabine and trifluridine/tipiracil is synergistic in tumors with an activated salvage pathway after capecitabine treatment in mice, and [18F]FLT PET imaging may predict the response to capecitabine and the synergistic antitumor efficacy of a sequential combination of capecitabine and trifluridine/tipiracil. Cancer Res; 77(24); 7120–30. ©2017 AACR.

Capecitabine is an orally administered prodrug of 5-fluorouracil (1). Capecitabine is sequentially converted to 5-fluorouracil by enzymatic processes and yields a substantially higher concentration of 5-fluorouracil in tumors than in normal tissues. Capecitabine is recommended as one of the initial chemotherapy regimens for advanced or metastatic colon cancer. Nonetheless, responses to capecitabine remain mostly in the form of a partial response and stable disease, with only a modest impact on survival (2, 3). In addition, frequent treatment-limiting adverse effects are typically managed by dosage reduction, despite a lack of relevant evidence from well-designed randomized trials (4).

Trifluridine/tipiracil (TAS-102) is an orally administered combination drug composed of a thymidine-based nucleic acid analogue (trifluridine), and a thymidine phosphorylase (TP) inhibitor (tipiracil hydrochloride), which prevents trifluridine from degrading as a result of TP (5). Trifluridine enters the cell by nucleoside transporter, where it is then converted to its monophosphate form by thymidine kinase 1 (TK1), and finally to a triphosphate form (6, 7). Trifluridine monophosphate exerts its cytocidal action by inhibiting thymidylate synthase (TS). In addition, the incorporation of trifluridine into DNA is thought to be an important factor that renders trifluridine active against tumors refractory to 5-fluorouracil (8). In patients with prior treated metastatic colorectal cancer, trifluridine/tipiracil was shown to prolong median overall survival (9), and has been approved as one of the later-line therapies (10).

Capecitabine exerts its anticancer effects through inhibition of TS (1). However, interference in endogenous thymidine synthesis by TS leads to an increase in TK1 levels (11, 12) and redistribution of nucleoside transporters (13). Enhanced thymidine uptake by the salvage pathway can bypass dTMP depletion, thereby conferring resistance to TS inhibition (14). Meanwhile, the same process may impart an improvement in the therapeutic index of anticancer nucleosides (15). It was previously shown that combining a nucleoside analogue with agents that increase nucleoside transport expression at the cell surface has the potential for increasing cell death (14, 16). For example, an increase in equilibrative nucleoside transporter 1 (ENT1) by TS inhibitors augmented the effect of gemcitabine (16). A phase III study in patients with advanced pancreatic cancer demonstrated that gemcitabine plus capecitabine improved progression-free survival compared with gemcitabine alone (17).

Since capecitabine and trifluridine/tipiracil have proved effective single agents in the treatment of advanced colon cancer and their modalities of action and toxicity profiles are somewhat different, a combination of the two drugs is of interest. Indeed, a capecitabine and trifluridine/tipiracil combination could be synergistic if capecitabine increases the number of nucleoside transporters, thereby acting as a potentiating mechanism. In addition, the cytotoxicity of trifluridine may be more potentiated by an increase in TK1 and a reduction of the cellular dTTP by capecitabine (18).

In this study, we investigated whether a sequential combination of capecitabine and trifluridine/tipiracil enhanced antitumor efficacy in colon cancer xenograft models. We hypothesized that combination therapy would be effective in xenograft models with an enhanced salvage pathway after TS inhibition. We undertook a combination of in vitro and in vivo studies using human colon cancer cell lines after 5-fluorouracil (in vitro) and capecitabine (in vivo) treatments, to obtain insights into activation of the salvage pathway in relation to synergistic antitumor efficacy. In addition, in relation to synergy, we examined TS, TP, and dihydropyrimidine dehydrogenase (DPD) as they are determinants of 5-fluorouracil and capecitabine sensitivity. We also performed 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) PET in mice with colon cancer xenografts, to investigate whether an increase in [18F]FLT uptake after TS inhibition (19, 20) could be used to predict the response to a sequential combination of capecitabine and trifluridine/tipiracil.

Cancer cell lines, cell culture, and viability assay

The colon cancer cell lines HT29, SW620, DLD-1, RKO, HCT116, and LOVO were purchased on October 8, 2012, and COLO205 was purchased on January 23, 2007, from the ATCC. HCT8 cells were obtained from the Korean Cell Line Bank on September 1, 2014. The last authentications were performed in July 2016 and July 2017, respectively, using short tandem repeat analysis, as described in 2012 in the ANSI Standard (ANS-0002) by the ATCC Standards Development Organization and Reid and colleagues (21). HCT8, SW620, DLD-1, HCT116, and LOVO are KRAS-mutated cell lines. Cell lines were selected to include the entire range of tumor response to capecitabine (22–24). Cell lines were tested to confirm the absence of mycoplasma contamination using an e-Myco Mycoplasma PCR detection Kit (iNtRON Biotechnology). The last date the cells were tested was in November 2016. The experiment was conducted using cells with a passage number of <8. Cell lines were cultured in RPMI1640 containing FBS (10%), l-glutamine (2 mmol/L), penicillin (50 IU/mL), and streptomycin (50 μg/mL), at 37°C in a humidified 5% CO2 incubator.

Cells were plated in fresh media in 96-well plates at a concentration of 2 × 103 cells per well. Twenty-four hours after seeding, the cells were washed and cultured for a further 72 hours in fresh media containing 5-fluorouracil (Sigma), trifluridine (Tocris), or 5-fluorouracil plus trifluridine. For combination therapy, the 5-fluorouracil treatment was performed first, for 16 hours, and then trifluridine was added for 8 hours. This sequential combination treatment of 5-fluorouracil and trifluridine was repeated for 72 hours. Cell viability was examined using a CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. Luminescence was measured using an EnSpire 2300 Multilabel Reader (PerkinElmer). The IC50 was defined as the concentration of the drug that resulted in a 50% decrease in cell viability in comparison with the untreated cells.

Reverse transcription real-time PCR

Reverse transcription real-time PCR (RT-PCR) was performed to detect the mRNA expression of ENT1, TK1, TS, TP, and DPD amplified by Blend Taq-Plus (Toyobo) and a SYBR Green Real-time PCR Master Mix (Toyobo), with an ABI7900HT Fast Real-Time PCR system (Applied Biosystems; ref. 12). The PCR primer sequences are shown in Supplementary Table S1.

[3H]FLT uptake, ENT1, and TK1 measurement

Exponentially growing cells were exposed to 5-fluorouracil for 24 hours. The media was then removed and replaced with 1 mL of fresh medium containing 370 Bq [methy-3H]FLT (148 GBq/mmol; Moravek Biochemicals), and the cells were then further incubated for 2 hours (25). [3H]FLT uptake was calculated as pmol/mg protein.

The number of [3H]-S-(p-nitrobenzyl)-6-thioinosine-binding sites/cell was determined for ENT1 measurement, as previously described (26). Colon cancer cells were incubated for 30 minutes in Hank's Buffered Salt Solution containing 10 nmol/L [3H]-S-(p-nitrobenzyl)-6-thioinosine (Moravek Biochemicals).

TK1 activity was measured as previously described (12). The cytosolic fraction of each sample was mixed with a reaction buffer containing 10 μmol/L [Methyl-3H]-thymidine (740 GBq/mmol; PerkinElmer). Protein content was measured using Pierce BCA Protein Assay reagent (Thermo Scientific).

Capecitabine treatment and [18F]FLT PET in mouse xenograft models

The antitumor activity of capecitabine (Selleckchem) was assessed in eight human colon cancer xenografts (HCT8, HT29, SW620, DLD-1, RKO, COLO205, HCT116, and LOVO). The research protocol was approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Science (registration no. 2015-02-055). Athymic nude mice (Balb/c-nu, male, 5–6 weeks old, 20–25 g) were purchased from the Japan Shizuoka Laboratory Center. Mice were maintained in accordance with the Institutional Animal Care and Use Committee guidelines of the Asan Institute for Life Science. Mice were inoculated with exponentially growing human colon cancer cells (1 × 107/0.2 mL) into the flank. Over the following 10 to 14 days, tumors were allowed to reach a volume of 100 to 150 mm3. Tumor volume was calculated as length × width × height × π/6. Mice were randomly allocated to vehicle control or capecitabine groups. At least 10 mice were studied per group. Capecitabine (360 mg/kg/day) was given orally for 21 days to mice bearing the xenografts. The capecitabine dose was equivalent to two-third of the maximum tolerated dose, on the basis of the results of a previous study on the antitumor activity of capecitabine (22).

Mice with xenografts underwent [18F]FLT PET three times, before capecitabine treatment (day 0), 24 hours after commencing the capecitabine treatment (day 2), and during the capecitabine treatment (day 4, before the fourth dose), using a microPET Focus 120 system (Concorde/Siemens). Static PET scans were acquired 110 minutes after a tail vein injection of 3.7 MBq (0.1 mCi) of [18F]FLT for 10 minutes (27). The standardized uptake value (SUV) of each tumor was calculated as previously reported (27), using the following formula: SUV = (tumor radio activity in the tumor volume of interest measured as Bq/cc × body weight) / injected radioactivity. Tumor volumes of interest were constructed by creating a cylindrical volume with a diameter of 3.0 mm and a length of three slices and centering it on the location of the maximum pixel value of the tumor regions.

Mice were observed daily to check survival and measure their body weight. The percent tumor growth inhibition (%TGI) was measured after 21 days of treatment according to the following equation: %TGI = (1 – Tt/Tc) × 100, where Tt and Tc are the relative tumor growth of the drug-treated and control groups, respectively. The investigators measuring %TGI were blinded to the treatment regimen. Tumors with %TGI ≥50% were considered as sensitive to antitumor treatment, and tumors with %TGI <50% were considered as resistant (28).

Ex vivo TK1 activity, RT-PCR, and IHC analysis of mouse xenograft models

Capecitabine (0–700 mg/kg/day) was given orally to mice bearing HCT8, HT29, and HCT116 xenografts (n = 3 to 7 per treatment). On day 4, tumors were excised, and TK1 activity was measured as previously described (12).

Ex vivo RT-PCR and immunohistochemistry studies were performed in mice with xenografts (n = 10 per cell line). Excised xenograft tumor tissues were homogenized by Tissue Lyser II QIAzol Lysis Reagent (Qiagen), and the RNA extraction process was performed according to the manufacturer's instructions. Two micrograms of total RNA from each sample was transcribed in mixtures containing AMV reverse transcriptase, dNTP, Oligo dT, and RNAsin (Promega). The resulting cDNAs were used for RT-PCR analysis.

IHC analysis was performed as described previously (26). The slides were stained with ENT1 (Abcam) and TP (Thermo Scientific) antibody, and counter-stained with hematoxylin. The number of cells was counted using inForm analysis software (PerkinElmer Applied Biosystems).

Sequential combination therapy of capecitabine and trifluridine/tipiracil in xenograft models

Trifluridine/tipiracil was provided by Taiho Pharmaceutical. The antitumor activity of trifluridine/tipiracil in combination with capecitabine was assessed in mice with capecitabine-resistant xenografts. Mice were randomly assigned to seven groups: treatment with vehicle, capecitabine alone (180 or 360 mg/kg), trifluridine/tipiracil alone (trifluridine 75 or 150 mg/kg/day with equivalent tipiracil), and combinations of capecitabine and trifluridine/tipiracil (capecitabine 180 mg/kg plus trifluridine 75 mg/kg with tipiracil; or capecitabine 360 mg/kg plus trifluridine 150 mg/kg with tipiracil). Mice with capecitabine-sensitive xenografts were treated with low-dose combinations: capecitabine 30 or 60 mg/kg alone, trifluridine/tipiracil alone (trifluridine 75 or 150 mg/kg with tipiracil), and combinations of capecitabine and trifluridine/tipiracil (capecitabine 30 mg/kg plus trifluridine 75 or 150 mg/kg with tipiracil; or capecitabine 60 mg/kg plus trifluridine 75 or 150 mg/kg with tipiracil). At least seven mice were studied per group. The daily doses of capecitabine and trifluridine/tipiracil were orally administered once a day. In the combination group, capecitabine was given daily from day 1, with trifluridine/tipiracil being administered daily from day 3, at a time interval of 8 hours after the capecitabine administration. The %TGI was measured after 28 days of treatment by investigators who were blinded to the treatment.

Statistical analysis

Data are expressed as mean ± SD unless specified otherwise. A value of P < 0.05 was considered statistically significant. The Kruskal–Wallis test was used to determine differences between the eight cell lines. Correlations between two variables were assessed using the Spearman rank correlation coefficient (ρ). A t test was used to compare two sample means when the samples were independent. Multiple comparison adjustments were made using the Bonferroni method. Repeated measures ANOVA was conducted to compare the effect of treatments on [18F]FLT PET in mice. The numbers of mice used for the capecitabine treatment and [18F]FLT PET were determined according to our previous data and a power calculation (27, 29). Statistical analyses were performed using IBM SPSS Statistics Version 21.0 for Windows (SPSS Inc.).

For drug combination studies and quantification of their in vitro synergy, combination indices (CIs) at different effect levels, or the Fa (fraction affected), were computed using CalcuSyn software (Biosoft) according to the Chou–Talalay method (30). The dose–effect curves were generated for the drug combination and each drug alone. CI values of <1, 1, and >1 indicate synergistic, additive, and antagonistic effects, respectively. For determination of the presence or absence of synergy in animals, the fractional tumor volume (FTV) method was used (31). The expected FTV (i.e., the product of FTV values for monotherapies) of the combination was divided by the observed FTV of the combination, yielding a ratio that indicates the nature of the interaction, with >1 indicating synergy and <1 indicating a less than additive effect. A minimum of six animals were used in each group.

Anticancer effect of 5-fluorouracil against colon cancer cell lines

We first determined the IC50 values of human colon cancer cell lines. A dose-dependent inhibition of cell viability was observed with 5-fluorouracil (Supplementary Fig. S1). The IC50 values ranged from 7.6 μmol/L to over 100 μmol/L (Supplementary Table S2). These results indicate that our colon cancer cell lines had varying sensitivity to 5-fluorouracil, and were suitable for studying the response to 5-fluorouracil or capecitabine.

5-Fluorouracil-induced changes in [3H]FLT uptake, ENT1, and TK1

Next, colon cancer cells were grown for 24 hours, and exposed to 0, 1, 3, 10, or 30 μmol/L concentrations of 5-fluorouracil for 24 hours. We examined 5-fluorouracil-induced changes in [3H]FLT uptake, TK1, and ENT1. All eight cell lines showed a significant increase in [3H]FLT uptake, which depended on the concentration of 5-fluorouracil (P < 0.05; Fig. 1A). Likewise, ENT1 increased significantly after 5-fluorouracil treatment in most cell lines (P < 0.05; Fig. 1B), except for DLD-1. TK1 activity was also significantly increased in five cell lines after 5-fluorouracil treatment (P < 0.05; Fig. 1C), but not in the HCT8, SW620, and LOVO cell lines.

Figure 1.

A, 5-Fluorouracil treatment for 24 hours induced changes in [3H]FLT uptake. Experiments were performed independently at least five times. B and C, 5-Fluorouracil treatment for 24 hours induced changes in ENT1 (B) and TK1 (C) activity of human colon cell lines. Experiments were performed independently at least three times with three technical replicates. P, Kruskal–Wallis test. [3H]NBTI, [3H]-S-(p-nitrobenzyl)-6-thioinosine.

Figure 1.

A, 5-Fluorouracil treatment for 24 hours induced changes in [3H]FLT uptake. Experiments were performed independently at least five times. B and C, 5-Fluorouracil treatment for 24 hours induced changes in ENT1 (B) and TK1 (C) activity of human colon cell lines. Experiments were performed independently at least three times with three technical replicates. P, Kruskal–Wallis test. [3H]NBTI, [3H]-S-(p-nitrobenzyl)-6-thioinosine.

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We also examined mRNA expression of 5-fluorouracil metabolic enzymes and a transporter. RT-PCR of eight cell lines showed that ENT1, TK1, and TS expression was apparent in all colon cancer cell lines (Supplementary Fig. S2A and S2B). However, the cancer cell lines showed highly variable mRNA expression of TP and DPD. We could not detect expression of TP in HCT8, HT29, SW620, DLD-1, and COLO205 cells, nor expression of DPD in HCT8, DLD-1, RKO, and COLO205 cells.

Correlations between anticancer effect of 5-fluorouracil, metabolic enzymes, and transporter

We assessed 5-fluorouracil metabolic enzymes and transporter that are potentially predictive of the sensitivity to 5-fluorouracil. There were no correlations between IC50 and mRNA expression of ENT1, TK1, TS, TP, and DPD (Supplementary Fig. S3A), although the correlation with TP mRNA expression approached statistical significance (ρ = −0.65, P = 0.08). The IC50 of 5-fluorouracil was negatively correlated with the baseline ENT1 binding sites (ρ = −0.86, P = 0.01), but was not correlated with [3H]FLT uptake, TK1 activity, and fold increase in [3H]FLT uptake, TK1, and nucleoside transporter (P > 0.10; Supplementary Fig. S3B, data not shown for TK1 and ENT1).

Antitumor efficacy of capecitabine in human colon cancer xenografts and correlation with [18F]FLT PET, metabolic enzymes, and 5-fluorouracil transporter

Capecitabine was well tolerated as mice exhibited no signs of weight loss or death. Three xenografts (COLO205, HCT116, and LOVO) were sensitive to capecitabine, but the rest were resistant with a %TGI of <50% (Fig. 2). Capecitabine sensitivity of human xenografts in mice correlated with the IC50 of 5-fluorouracil in vitro (ρ = −0.76, P = 0.03; Supplementary Fig. S4).

Figure 2.

Growth curve of human colon cancer xenografts in nude mice treated with orally administered vehicle or capecitabine at 360 mg/kg (n = 10–12 per cell line).

Figure 2.

Growth curve of human colon cancer xenografts in nude mice treated with orally administered vehicle or capecitabine at 360 mg/kg (n = 10–12 per cell line).

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Capecitabine-resistant SW620 and DLD-1 xenografts showed no significant differences in the SUV of [18F]FLT between the vehicle- and capecitabine-treated groups (Fig. 3A–C). The other xenografts showed significantly different SUV values between the two groups (P < 0.05). Serial [18F]FLT PET images after capecitabine treatment revealed no significant change in SUV in capecitabine-resistant HCT8, SW620, and DLD-1 xenografts (Fig. 3A and C). However, there was a significant increase in the SUV in all capecitabine-sensitive xenografts and capecitabine-resistant HT29 and RKO xenografts. It was noted that an increased [18F]FLT uptake on day 2 was followed by a distinguishable drop in [18F]FLT uptake on day 4 in the capecitabine-sensitive xenografts (P < 0.05), while a continuing [18F]FLT uptake on day 4 was observed in the capecitabine-resistant HT29 and RKO xenografts (Fig. 3A and C). Of the SUV and SUV ratio values of [18F]FLT uptake, only the ratios of SUV on day 4 and day 2 had a strong negative correlation with %TGI (ρ = −0.81, P = 0.02; Fig. 3D). The maximum SUV and ratio values showed the same results (Supplementary Fig. S5).

Figure 3.

[18F]FLT PET following vehicle or capecitabine treatment and correlation with antitumor efficacy of capecitabine (%TGI) in human colon cancer xenografts. A, Representative baseline, day 2, and day 4 images of [18F]FLT PET following capecitabine treatment at 360 mg/kg in HCT8, HT29, and HCT116 xenografts. B and C, SUV values of [18F]FLT PET (n = 10–12 per cell line). V, vehicle; C, capecitabine; P, t test and repeated measures ANOVA; *, **, ***, P <0.05, <0.01, and <0.001 (day 0 vs. day 2; and day 2 vs. day 4), respectively, obtained using ANOVA for the effect of time on SUV after capecitabine treatment. D, Correlation between %TGI by capecitabine and SUV values. The SUV ratio (day 4/day 2) correlated significantly with %TGI, but no other SUV values showed a significant correlation. P, Spearman correlation.

Figure 3.

[18F]FLT PET following vehicle or capecitabine treatment and correlation with antitumor efficacy of capecitabine (%TGI) in human colon cancer xenografts. A, Representative baseline, day 2, and day 4 images of [18F]FLT PET following capecitabine treatment at 360 mg/kg in HCT8, HT29, and HCT116 xenografts. B and C, SUV values of [18F]FLT PET (n = 10–12 per cell line). V, vehicle; C, capecitabine; P, t test and repeated measures ANOVA; *, **, ***, P <0.05, <0.01, and <0.001 (day 0 vs. day 2; and day 2 vs. day 4), respectively, obtained using ANOVA for the effect of time on SUV after capecitabine treatment. D, Correlation between %TGI by capecitabine and SUV values. The SUV ratio (day 4/day 2) correlated significantly with %TGI, but no other SUV values showed a significant correlation. P, Spearman correlation.

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Ex vivo TK1 activity analysis in HCT8 xenografts showed no significant difference on day 4 between the vehicle- and capecitabine-treated groups (Supplementary Fig. S6). However, TK1 activity increased in a dose-dependent manner on day 4 in HT29 xenografts (P = 0.0008), and decreased on day 4 in HCT116 (P = 0.03).

We assessed whether there was any correlation of ex vivo and [18F]FLT PET markers with the antitumor efficacy of capecitabine to identify a predictive marker. The baseline ex vivo mRNA expression and immunohistochemical staining of ENT1 and TP were similar to those of in vitro cells (Supplementary Fig. S2B, S7A–S7C, and S8A–S8C). However, we observed positive TP protein expression occurring differentially to mRNA expression in the HT29 xenograft. Of the 5-fluorouracil metabolic enzymes or transporter, only TP appeared to be related to %TGI, although the relationship was not statistically significant (Supplementary Fig. S8A–S8C and S9). Increased [18F]FLT uptake after capecitabine was not correlated with baseline mRNA expression of ENT1, TK1, TS, TP, and DPD. However, an increased [18F]FLT uptake was found in all xenografts with positive TP expression.

In vitro synergistic antitumor efficacy of 5-fluorouracil and trifluridine against human colon cancer cells

To investigate the effect of the combination of 5-fluorouracil and trifluridine in vitro, eight colon cancer cell lines were treated with 5-fluorouracil (1.25 to 80 μmol/L), trifluridine (6.25 to 400 μmol/L), or sequential 5-fluorouracil and trifluridine at a constant ratio combination. The combination ratio was determined according to the plasma trifluridine level (5) and the dosing schedule for trifluridine/tipiracil (7). The median effect dose of 5-fluorouracil was within the dose range studied in all cell lines. The linear correlation coefficient of the plot was 0.94 to 0.99. However, the median effect dose of trifluridine was less than 100 μmol/L in only RKO and LOVO cells, whereas those of the other cell lines were above 400 μmol/L. The linear correlation coefficient ranged from 0.63 to 0.99. We found no correlation of trifluridine sensitivity with metabolic enzymes and transporters.

Fa-CI plots illustrating the effects of fixed drug ratio combinations are depicted in Fig. 4. The combinations had synergistic effects in all cell lines (CI <1). Synergistic antitumor efficacy in all cell lines with an increased [3H]FLT uptake after the administration of 5-fluorouracil supports the idea that synergistic efficacy may be related to 5-fluorouracil-induced activation of the salvage pathway. The extent of synergism differed according to the cell line and combination variant. In most cell lines, except for COLO205, the combination had stronger synergy at lower doses than at higher doses. However, at the high effect levels (Fa >0.8), synergistic interactions were still observed in 5-fluorouracil-sensitive COLO205, HCT116, and LOVO, and resistant HT29 and DLD-1, cell lines.

Figure 4.

Growth inhibition of human colon cancer cell lines by the combination of 5-fluorouracil and trifluridine. The Fa-CI plot was generated using the method of Chou and Talalay (30). At least three independent experiments were performed, each of which was performed in triplicate.

Figure 4.

Growth inhibition of human colon cancer cell lines by the combination of 5-fluorouracil and trifluridine. The Fa-CI plot was generated using the method of Chou and Talalay (30). At least three independent experiments were performed, each of which was performed in triplicate.

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In vivo synergistic antitumor efficacy of capecitabine and trifluridine/tipiracil against human colon cancer xenografts

We next examined whether the sequential combination of capecitabine and trifluridine/tipiracil enhanced antitumor efficacy in mice with both capecitabine-resistant (HCT8, HT29, SW620, and DLD-1) and capecitabine-sensitive (HCT116 and LOVO) xenografts. Of 233 mice treated with capecitabine or trifluridine/tipiracil, two mice (one with capecitabine at 180 mg/kg, and the other with capecitabine at 360 mg/kg plus trifluridine 150 mg/kg) died unexpectedly during treatment. In addition, one mouse with capecitabine at 60 mg/kg and one with capecitabine at 360 mg/kg had tumor tissue damage that may possibly have been due to self-mutilation or cannibalism. Finally, 229 mice (at least six mice per xenograft model) were analyzed. Twenty-eight days after treatment, significant weight loss of over 10% was observed in three xenograft models treated with capecitabine at 360 mg/kg plus trifluridine at 150 mg/kg (HCT8, 10.7%; HT29, 10.5%; DLD-1, 12.1%), but not in the others.

The combination therapy showed significantly greater tumor growth inhibition than the vehicle group (P < 0.05, Fig. 5) in three capecitabine-resistant xenograft models: HCT8, HT29, and DLD-1. Marginal synergism was noted in the HCT8 xenograft, but the combination was indeed as effective as capecitabine monotherapy (growth volume difference: 1.1, P = 0.65; Fig. 5). However, only the HT29 xenograft, which had an increased [18F]FLT uptake after capecitabine, showed moderate synergistic efficacy with an observed FTV of <0.5 (%TGI = 51.1%). Capecitabine-sensitive HCT116 and LOVO xenografts, which had an increased [18F]FLT uptake after capecitabine, also showed moderate synergistic antitumor efficacy of low dose capecitabine and trifluridine/tipiracil (Fig. 6). Xenografts with positive mRNA expression or immunohistochemical staining of TP showed synergistic efficacy, but no xenografts with negative TP expression showed synergistic efficacy.

Figure 5.

Inhibitory effect of the capecitabine and trifluridine/tipiracil combination on capecitabine-resistant human colon cancer xenografts in mice (n = 6–10 mice per group). The mean value was plotted without error bars for clarity. P, t test between vehicle and treated mice with Bonferroni adjustment. P values of ≤0.1 in comparison with the vehicle control are shown. C, capecitabine; mpk, mg/kg; E-FTV, expected FTV; O-FTV, observed FTV; T, trifluridine/tipiracil.

Figure 5.

Inhibitory effect of the capecitabine and trifluridine/tipiracil combination on capecitabine-resistant human colon cancer xenografts in mice (n = 6–10 mice per group). The mean value was plotted without error bars for clarity. P, t test between vehicle and treated mice with Bonferroni adjustment. P values of ≤0.1 in comparison with the vehicle control are shown. C, capecitabine; mpk, mg/kg; E-FTV, expected FTV; O-FTV, observed FTV; T, trifluridine/tipiracil.

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Figure 6.

Tumor growth kinetics of capecitabine-sensitive HCT116 and LOVO xenografts after a low dose of capecitabine and trifluridine/tipiracil combination (n = 7–8 mice per group). The mean value was plotted without error bars for clarity. P, t test between vehicle and treated mice with Bonferroni adjustment. P values of ≤0.1 in comparison with the vehicle control are shown. C, capecitabine; mpk, mg/kg; E-FTV, expected FTV; O-FTV, observed FTV; T, trifluridine/tipiracil.

Figure 6.

Tumor growth kinetics of capecitabine-sensitive HCT116 and LOVO xenografts after a low dose of capecitabine and trifluridine/tipiracil combination (n = 7–8 mice per group). The mean value was plotted without error bars for clarity. P, t test between vehicle and treated mice with Bonferroni adjustment. P values of ≤0.1 in comparison with the vehicle control are shown. C, capecitabine; mpk, mg/kg; E-FTV, expected FTV; O-FTV, observed FTV; T, trifluridine/tipiracil.

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In this study, all in vitro cell lines showed a significant increase in [3H]FLT uptake after 5-fluorouracil treatment. We also found synergistic antitumor efficacy for a sequential combination of 5-fluorouracil and trifluridine in all cell lines. In vivo [18F]FLT imaging in xenograft models showed an increased [18F]FLT uptake after capecitabine treatment in both the capecitabine-sensitive COLO205, HCT116, and LOVO xenografts, and the capecitabine-resistant HT29 and RKO xenografts, but not in the capecitabine-resistant HCT8, SW620, and DLD-1 xenografts. We observed synergistic efficacy of the capecitabine and trifluridine/tipiracil combination in only those xenografts that demonstrated an increased [18F]FLT uptake after capecitabine. Our results indicate that activation of the salvage pathway after TS inhibition may be associated with a synergistic response to a sequential combination of capecitabine and trifluridine/tipiracil. [18F]FLT PET of patients on capecitabine medication could possibly be used to select those who would be likely to demonstrate synergistic effects.

The antitumor effect of 5-fluorouracil is caused by irreversible binding of the active metabolite fluorodeoxyuridine triphosphate to TS, together with 5,10-methylene tetrahydrofolate (1), which results in irreversible modification of TS. The duration of TS inhibition would be sustained for the enhancement of sequentially administered trifluridine (32). Nonetheless, increasing levels of dUTP pooling after TS inhibition may theoretically lead to decreased incorporation of trifluridine into DNA (5). Trifluridine also has TS-inhibitory activity. This may explain our finding of a slight to moderate synergism at high 5-fluorouracil and trifluridine concentrations (high Fa), but stronger synergism at low Fa. However, synergistic interactions at low effect levels may be crucial, because, at the most, only low effect levels of intratumoral 5-fluorouracil or trifluridine would be achievable in humans (5, 33), and these concentrations are within the levels of tolerable toxicity in vivo. Despite the presence of high dUTP pools, a greater degree of incorporation of trifluridine into DNA may be achieved by the high catalytic efficiency of TK1 for trifluridine (34), the refractoriness to cleavage by DNA glycosylases (8), and no dephosphorylation of the triphosphate form of trifluridine by dUTPase (35). Trifluridine and its cellular derivatives may be less prone to pathways that would otherwise reduce their ability to be incorporated into DNA (36).

In mice, the thymidine level in plasma is about an order of magnitude greater than that in humans. A high level of trifluridine is needed to obtain antitumor efficacy in mice (7). Therefore, in humans with much lower plasma thymidine, a stronger synergy might be expected. In addition, TP, which is the final enzymatic enzyme responsible for conversion of capecitabine to the active drug, is present at high levels in tumor cells compared with healthy tissue. This will theoretically allow for selective activation of capecitabine in tumor and less synergistic toxicity of sequentially administered trifluridine/tipiracil (2). Positive TP expression may also predict the synergy, as synergistic antitumor efficacy and activation of the salvage pathway were observed in all xenografts with positive TP expression. One concern is over the potential effect of tipiracil on TP and capecitabine metabolism. To avoid any antagonistic effects, an interval of at least 8 hours may be required between sequential administration (33, 37).

Our results suggest that the TP expression level may be an indicator of the antitumor activity of 5-fluorouracil and capecitabine. TP is responsible for the conversion of 5-fluorouracil to fluorodeoxyuridine, which can then be converted to the active metabolite fluorodeoxyuridine monophosphate. In previous studies on human cancer xenograft models, tumor susceptibility to capecitabine correlated directly with TP activity and inversely with DPD activity (22, 24). Tumors with high TP activity may respond better to capecitabine owing to its novel mechanism of activation (38, 39). In addition, the molecular consequences of TS inhibition can also determine the response to capecitabine. Evidence suggests that overexpression of TS (40), treatment-induced induction of TS (41), enhanced base-excision repair machinery (42), and overexpression of UTPase (43) may also be putative determinants of resistance to fluoropyrimidine. Although further studies with randomized designs may validate the role of TP or other markers, any single biomarker is unlikely to be sufficient to predict the outcome of capecitabine treatment, as capecitabine metabolism is extensive, with a complexity of multifactorial mechanisms of resistance (44). For patient management, it may be more clinically relevant to assess the pharmacodynamics of tumor-specific TS inhibition.

In vitro kinetic studies on cancer cells showed that TS inhibition first induces S-phase arrest within 12 hours, preceding the induction of DNA double-strand breaks (45, 46). In the following 12 to 24 hours after TS inhibition, cancer cells express proliferating cell nuclear antigen and maintain the ability to incorporate nucleoside precursors into DNA, implying the capacity for DNA synthesis (46, 47). After 24 hours of TS inhibition, apoptotic cell death occurs with the appearance of a sub-G1 peak, and thereafter, DNA fragmentation and apoptosis become dominant up to 48 to 72 hours (48, 49). In this study, serial [18F]FLT PET in capecitabine-sensitive xenograft models showed an increased [18F]FLT uptake on day 2, followed by a significant decrease on day 4. This observation is most probably related to a pharmacodynamic effect of TS inhibition on dTTP depletion, activation of the salvage pathway, and subsequent decreased cell viability as indicated by ex vivo TK1 activity data (Supplementary Fig. S6). Imaging at later time points is likely to show a tailing out of the increased [18F]FLT uptake because of the increasing number of nonviable tumor cells in tumor tissue with a positive response to TS inhibition (50). A strong correlation between [18F]FLT uptake and %TGI, despite only eight animal models having been studied, indicates the robustness of our results. [18F]FLT PET may be a useful tool for early evaluation of capecitabine response.

There are limitations to this study. First, we did not study synergistic toxicity to any great extent. Because of the high level of thymidine, the toxicity of a sequential combination may be underestimated in mice models. However, synergistic toxicity of trifluridine/tipiracil is not likely to occur, because the pharmacodynamics of capecitabine and enhanced trifluridine uptake may be largely restricted to tumors, as shown in a previous [18F]FLT PET study (20). Second, we used only colon cancer cell lines and a mouse model. To make generalizations, we need to study various kinds of cancer models. Third, we did not evaluate the effect of TP level on trifluridine metabolism in tumors. High TP expression may influence the sensitivity to trifluridine. However, we administered trifluridine combined with a TP inhibitor. In this study, all xenografts with high TP expression showed a synergistic effect of sequentially administered trifluridine/tipiracil. Finally, two or three [18F]FLT PET studies may be needed to precisely predict the response to capecitabine, and these may not be clinically appropriate. However, one [18F]FLT PET study at least 3 days after capecitabine medication may be enough to predict the synergy of the combination therapy. More studies are needed to confirm the ability of the single [18F]FLT PET to predict the synergy.

Implications for clinical practice are that patients who would be treated with capecitabine or trifluridine/tipiracil monotherapy for advanced or metastatic colorectal cancer may alternatively be treated with capecitabine and trifluridine/tipiracil in combination. Those patients who exhibit disease progression after capecitabine or trifluridine/tipiracil, or who do not tolerate intensive monotherapy, may be especially appropriate candidates. The pathways responsible for the production of biomass components essential for cell division are significantly upregulated in many types of tumor (2). Therefore, the synergistic efficacy may be applicable to other cancers. Any TS inhibitors with a primary target mechanism of TS inhibition may be combined with trifluridine/tipiracil.

In conclusion, in mice bearing human colon cancer xenografts, the sequential combination of capecitabine and trifluridine/tipiracil is synergistic in tumors with an activated salvage pathway after capecitabine. [18F]FLT PET can predict the response to capecitabine, as well as the synergistic antitumor efficacy of a sequential combination of capecitabine and trifluridine/tipiracil. This study provides insights into the quantitative measurement of [18F]FLT uptake as a valuable biomarker for selecting patients who are likely to benefit from TS inhibition and combination therapy. Further studies are needed to prove the synergistic interaction of a sequential combination of capecitabine and trifluridine/tipiracil in humans, in terms of toxicity in addition to its effectiveness. The role of [18F]FLT imaging in selecting patients for sequential combination therapy should also be investigated.

No potential conflicts of interest were disclosed.

Conception and design: S.-Y. Kim, S.J. Oh, D.H. Moon

Development of methodology: S.-Y. Kim, S.J. Oh, S.J. Lee, D.H. Moon

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.-Y. Kim, J.H. Jung, H.J. Lee, H. Soh, S.J. Lee, S.J. Oh, J.H. Lee

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.-Y. Kim, J.H. Jung, S.Y. Chae, J.H. Lee, Y.S. Hong, D.H. Moon

Writing, review, and/or revision of the manuscript: S.-Y. Kim, J.H. Jung, H.J. Lee, H. Soh, S. Ju Lee, S.Y. Chae, J.H. Lee, S.J. Lee, Y.S. Hong, T.W. Kim, D.H. Moon

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.-Y. Kim

Study supervision: D.H. Moon

We acknowledge the help of Taiho Pharmaceutical by providing trifluridine/tipiracil for in vivo studies.

This research was supported by the Korea Health Technology R&D Project (HI06C0868 to D.H. Moon); and the Radiation Technology Development Program (NRF-2016M2A2A7A03913219 to D.H. Moon).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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