Introduction

Trifluridine/tipiracil (FTD/TPI, also named TAS-102) is an orally administered anticancer drug that is approved worldwide for the treatment of metastatic colorectal cancer (mCRC) [1, 2] and heavily pretreated metastatic gastric cancer (mGC) [3, 4]. FTD is a fluorinated thymidine analog and the active cytotoxic component of FTD/TPI, while tipiracil is a thymidine phosphorylase inhibitor and inhibits FTD degradation in vivo [5]. FTD is transported into tumor cells via nucleoside transporters [6,7,8], then metabolized to FTD triphosphate through initial phosphorylation of FTD by thymidine kinase 1 [6, 9, 10] and incorporated into DNA during DNA replication [7, 11,12,13]. The cytotoxicity of FTD correlates with its incorporation into DNA [12]. Using commercially available antibodies against bromodeoxyuridine (BrdU), FTD incorporated into DNA can be detected using immunoblotting, immunohistochemistry, and fluorescence-activated cell sorting [7], which enables us to trace FTD spatiotemporally and quantitatively in clinical specimens. We previously reported that we successfully detected FTD in peripheral blood mononuclear cells (PBMCs) isolated from mCRC patients administered FTD/TPI [14]. Intriguingly, the percentage of FTD-positive PBMCs fluctuates according to the schedule of FTD/TPI administration [14]. Conversely, although FTD was detected in the xenograft tumors of FTD-administered mice [7], it has not been reported whether FTD can be detected in the tumor specimens of patients who were administered FTD/TPI, because surgery or biopsy is typically not performed for such patients. The temporal changes in FTD levels in the tumors of mCRC patients according to the schedule of FTD/TPI administration have also not been investigated. The purposes of this study were to detect FTD in the tumors of patients who were administered FTD/TPI and to investigate whether there is a difference in the turnover rate of FTD between tumors and bone marrow after the cessation of FTD/TPI treatment in a mouse model.

Materials and methods

Patients

Patients were diagnosed with mCRC from April 2016 to September 2018 and treated at Kyushu University Hospital. Patients’ information, including therapy, histopathology, and the interval between the last FTD/TPI treatment and the acquisition of a specimen, is summarized in Table 1. The Institutional Review Board of Kyushu University Hospital approved the study protocol (number 3–444), which conforms with the ethical guidelines of the 1975 Declaration of Helsinki.

Table 1 Clinical information of mCRC patients
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Mouse model of peritoneal tumor dissemination and drug treatment

All animal studies were performed according to the guidelines and with the approval of the Institutional Animal Care and Use Committee of Taiho Pharmaceutical Co., Ltd. Ethical approval was obtained prior to conducting the animal experiments. Five-week-old male nude mice (BALB/c nu/nu, 30 mice) were purchased from CLEA Japan, Inc. (Tokyo, Japan) and housed under specific pathogen-free conditions. Suspensions of human CRC HT-29 cells (2 × 107 cells/mouse) were injected into the peritoneal cavity [15], and FTD/TPI (oral, 200 mg/kg/day) was first administered 1 day later (day 1), and then administered once daily for 5 consecutive days, followed by 2 days without treatment. This protocol was followed for 5 weeks. Treatment was discontinued on day 33, and on days 39, 46, and 53, six mice of each the FTD/TPI treatment groups were killed and tumors disseminated in the peritoneum and bone marrow were excised. Control mice were orally administered vehicle (0.5% hydroxypropyl methylcellulose solution, 10 ml/kg) once daily for 5 consecutive days, followed by 2 days without treatment. This protocol was followed for 5 weeks and the mice were killed on day 33.

Immunohistochemical analysis

Clinical and mouse specimens were fixed with 10% formaldehyde and 4% paraformaldehyde, respectively, in PBS for 24 h and embedded in paraffin. To detect FTD in paraffin-embedded clinical specimens, immunohistochemical staining (IHC-p staining) was performed using a BrdU In-Situ Detection Kit (Cat# 550,803, BD Bioscience, Franklin Lakes, NJ) as previously described [7]. IHC-p staining with anti-Ki67 antibody (Clone: MTB-1; Cat#M7240, Dako, Santa Clara, CA), M30 Cytodeath™ (Cat#10,700, VLVbio, Nacka, Sweden), and anti-cleaved caspase 3 antibody (Cat#9664, Cell Signaling Technologies, Danvers, MA) was also performed [16]. Images were captured using a NanoZoomer (Hamamatsu Photonics K.K., Hamamatsu, Japan).

DNA slot-blot analysis

Genomic DNA was purified from the transplanted tumors and bone marrow of nude mice or FTD-treated HCT116 cells using the Gentra Puregene Tissue Kit or the Gentra Puregene Cell Kit (Cat#158,667 or #158,745, QIAGEN, Hilden, Germany), respectively. DNA (50 ng) was denatured in alkaline solution (0.1 N NaOH) for 5 min and slot-blotted onto Hybond-N+ blotting membrane (GE Healthcare, Chicago, IL) using a BIO-DOT SF apparatus (BioRad, Hercules, CA). The membranes were probed with anti-BrdU antibodies (3D4 and B44; BD Bioscience) and the signals were detected using a LAS4000mini (GE Healthcare). The signal intensity of slot-blot bands was measured using ImageJ software (NIH, Bethesda, MD). The signal intensity was normalized with that of genomic DNA purified from HCT-116 cells treated with 3 µM FTD for 4 h to calculate relative signal intensity.

Statistical analysis

Statistical analysis was performed using Prism software (GraphPad, San diego, CA). Dunnett’s test was applied for statistical analysis (Fig. 4c, d).

Results

Detection of FTD in tumors disseminated in the peritoneum of patients with mCRC after the cessation of FTD/TPI treatment

The first patient, who received two courses of FTD/TPI combined with panitumumab, developed peritoneal dissemination that was detected using computerized tomography (Fig. S1A). The tumor was surgically resected during laparotomy to treat gastrointestinal transit failure 28 days after the last administration of FTD/TPI (Fig. S1A). Hematoxylin and eosin (H&E) staining identified dispersed metastases (Fig. 1a) and IHC-p staining detected FTD-positive tumor cells representing 14.9% of the cell population (Fig. 1b, c). The tumor was positively stained with anti-Ki67 antibody (Fig. 1d) but not with M30 Cytodeath™ (Fig. 1e) or anti-cleaved caspase 3 antibody (Fig. 1f). These data indicate that FTD was incorporated into the proliferating tumor cells but did not induce apoptosis.

Fig. 1
figure 1

Peritoneal dissemination resected from a metastatic colorectal cancer (mCRC) patient 28 days after the cessation of FTD/TPI treatment. a Image of hematoxylin and eosin staining (H&E image). b Image of immunohistochemical staining of paraffin-embedded samples (IHC-p image) of FTD. c Higher magnification image of the dashed box in b. Higher magnification IHC images of Ki67 (d), M30 (e), and cleaved caspase 3 (f) near the area shown in c from serial sections. Scale bars = 50 μm

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Detection of FTD in liver metastasis after the cessation of FTD/TPI treatment

The second patient developed liver metastasis (Fig. S1B) and received three courses of FTD/TPI combined with bevacizumab. The shrunken liver metastasis was surgically resected 43 days after the final administration of FTD/TPI (Fig. S1B). H&E staining identified metastases in the liver, but there were a number of fissures in the surrounding tissue (Fig. 2a), which suggests tumor necrosis associated with chemotherapy. IHC-p staining detected FTD-positive tumor cells (1.3%; Fig. 2b, c) and the tumor was positively stained with anti-Ki67 antibody (Fig. 2d), M30 Cytodeath™ (Fig. 2e), and anti-cleaved caspase 3 antibody (Fig. 2f). These data indicate that FTD was incorporated into the proliferating tumor cells and induced apoptotic cell death.

Fig. 2
figure 2

Metastatic liver tissue resected from an mCRC patient 43 days after the cessation of FTD/TPI treatment. a H&E image. b IHC-p image of FTD. c Higher magnification image of the dashed box in b. Higher magnification IHC-p images of Ki67 (d), M30 (e), and cleaved caspase 3 (f) near the area shown in c from serial sections. Scale bars = 50 μm

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Another patient who developed liver metastasis received a similar regimen. The shrunken liver metastasis was surgically resected 35 days after the last administration of FTD/TPI. The tumor (Fig. S2A) or an FTD-positive tumor was undetectable (Fig. S2B and S2C), which suggests that chemotherapy eradicated the liver metastasis.

FTD is undetectable in the normal gastric mucosa shortly after the cessation of FTD/TPI treatment

The fourth patient received one course of FTD/TPI combined with bevacizumab. Endoscopic biopsy recovered the normal gastric mucosa 16 days after the last administration of FTD/TPI (Fig. S1C). IHC-p staining of the normal gastric mucosa (H&E staining; Fig. 3a) did not detect FTD-positive cells (Fig. 3b, c). The mucosa was positively stained with anti-Ki67 antibody (Fig. 3d), but not with M30 Cytodeath™ (Fig. 3e) or anti-cleaved caspase 3 antibody (Fig. 3f).

Fig. 3
figure 3

Normal gastric mucosa removed as a biopsy from an mCRC patient 16 days after the cessation of FTD/TPI treatment. a H&E image. b IHC-p image of FTD. c Higher magnification image of the dashed box in b Higher magnification IHC-p images of Ki67 (d), M30 (e), and cleaved caspase 3 (f) near the area shown in c from serial sections. Scale bars = 50 μm

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FTD persists longer in transplanted tumors than in bone marrow in a mouse model of peritoneal dissemination

Our findings from the clinical specimens showed that FTD persisted in human tumors for several weeks after the cessation of FTD/TPI treatment. Conversely, our previous study of mCRC patients treated with FTD/TPI showed that few FTD-positive PBMCs were detected 2 weeks after the cessation of treatment [14]. These results suggest that the turnover rate of FTD-positive cells differed between tumors and normal white blood cells. To evaluate the turnover rate of FTD-positive cells in tumors and normal tissues experimentally, we used a mouse model of peritoneal dissemination, in which a human CRC cell line, HT29, was engrafted [15]. These model mice were administered FTD/TPI for 5 weeks (FTD/TPI treatment ceased on day 33) to incorporate a significant amount of FTD into tissues, and disseminated tumors and bone marrow cells were obtained on days 33, 39, 46, and 53 (Fig. 4a). On the day of the last FTD/TPI administration (day 33), IHC-p staining showed that FTD was detected in both the tumors and bone marrow (Fig. 4b). However, at 6 days after the last FTD/TPI administration (day 39), FTD was detected in the tumors but at much lower levels in the bone marrow (Fig. 4b).

Fig. 4
figure 4

FTD incorporation and its turnover in tumors and bone marrow of a peritoneal dissemination mouse model treated with FTD/TPI. a Timeline of FTD/TPI treatment, b IHC-p staining of FTD on days 33 and 39 in tumors and bone marrow. Higher magnification images of the dashed boxes are also shown. Scale bar = 100 μm. c Slot-blot analysis of genomic DNA purified from tumors. Left, image of immunoblotting with anti-BrdU antibody, B44. Right, scatter plot of the relative signal intensity of FTD. The intensity of each band was normalized with that of HCT116 treated with 3 µM FTD for 4 h. d Slot-blot analysis of genomic DNA purified from bone marrow. Left, image of immunoblotting with anti-BrdU antibody, B44. Right, scatter plot of relative signal intensity of FTD: sample missing (vehicle #1 and #2). The middle bars indicate the median relative signal intensity of FTD. Statistical analysis was performed using Dunnett’s test with the vehicle group, *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant

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To quantitate FTD in the genomic DNA of tumors and bone marrow, we performed a slot-blot analysis using anti-BrdU antibody B44, because the signal intensity of this antibody exhibited a good linear correlation with the amount of FTD-containing genomic DNA (Supplementary Fig. 3A), while that of another antibody 3D4 did not (Supplementary Fig. 3B). In the tumors, the FTD signals declined gradually and were detected until 13 days after the cessation of FTD/TPI treatment (day 46, Fig. 4c). Conversely, in the bone marrow, the FTD signals declined rapidly and were not detected even at 6 days after the cessation of FTD/TPI treatment (day 39; Fig. 4d). These data support the idea that the turnover rates of FTD differed among tissues and that the FTD in tumors persisted longer than that in rapidly proliferating bone marrow cells.

Discussion

Here we report the detection of FTD in tumor tissues obtained from mCRC patients who were treated with FTD/TPI. To our knowledge, this is the first report of FTD detection in tumor specimens from patients. Spatiotemporal tracing of the active components of chemotherapeutic drugs helps predict their efficacy and safety. For that purpose, mass spectrometric imaging is a powerful tool for imaging the spatial distribution of unlabeled exogenous drugs [17]. However, cryosectioning and high-performance equipment are required. Conversely, FTD incorporation into DNA can be readily measured using a standard clinical IHC-p technique and commercially available anti-BrdU antibodies [7]. The convenience of this method may encourage its clinical application.

Intriguingly, we detected FTD in tumors obtained from patients several weeks after the cessation of FTD/TPI treatment (Figs. 1 and 2). This observation indicated that FTD persisted for several weeks in tumors even after FTD/TPI treatment was discontinued. We previously detected FTD in PBMCs isolated from mCRC patients with FTD/TPI treatment [14]. The percentages of FTD-positive PBMCs, however, fluctuated according to the schedule of FTD/TPI treatment, and only a few FTD-positive PBMCs were detected 2 weeks after the cessation of treatment [14]. These data suggest that FTD persists longer in tumor cells than in PBMCs, most of which are derived from bone marrow. This idea was supported by our experimental data using a mouse model of peritoneal dissemination (Fig. 4). However, the question remains as to the possible mechanism underlying the difference in the turnover rate of FTD between tumors and bone marrow-derived cells. FTD is incorporated into the DNA of both tumor cells and white blood cells in tumor-bearing nude mice in a dose-dependent manner [18]. Once FTD is incorporated into DNA it is slowly eliminated [11], and the elimination rate of FTD from the DNA of tumor cell lines is comparable with that of thymidine [6]. A possible mechanism that explains the rapid disappearance of FTD-positive PBMCs or bone marrow cells may be caused by their cell death and their replacement by a large number of naïve bone marrow-derived cells. Conversely, FTD-positive tumor cells may survive long-term with a suppressed proliferation rate (Fig. 1) or die by apoptosis and be eradicated (Figs. 2 and S2). In any case, the differential turnover rates of FTD-positive tumor cells and bone marrow-derived cells may explain the persistent tumor-specific cytotoxicity with tolerable hematological toxicity [2, 4]. In support of this notion, data acquired using a mouse xenograft model showed that antitumor effects persisted long after FTD/TPI treatment was discontinued [19].

In our analysis, FTD was not detected in normal gastric mucosa tissue obtained from an mCRC patient only 16 days after the cessation of FTD/TPI treatment (Fig. 3b, c). Although it is not known whether FTD was not incorporated into the DNA of cells in the gastric mucosa or that FTD-positive normal gastric cells were rapidly eliminated from the mucosa, limited FTD toxicity toward the gastric mucosa would be expected.

An inherent limitation of the present study was the small number of clinical samples; further studies are, therefore, required to confirm our findings. FTD/TPI is approved to treat patients with mCRC and mGC [2, 4]. Multiple ongoing clinical trials aim to develop novel therapeutic regimens containing FTD/TPI [20, 21]. A wide collection of data regarding the distribution of FTD in clinical specimens will provide valuable information about this drug and will contribute to the development of a surrogate marker of FTD/TPI treatment.

Conclusions

The data obtained from the tissue specimens of mCRC patients and from a xenograft mouse model with FTD/TPI treatment revealed that FTD persists longer in tumors than in highly proliferating bone marrow cells, which may cause a sustained antitumor effect with tolerable hematotoxicity in FTD/TPI treatment.