Introduction: Trifluridine/tipiracil hydrochloride (FTD/TPI, Lonsurf®) is an oral antineoplastic agent that has been approved as late-stage chemotherapy for colorectal cancer. Its major mechanism of action is the dysfunction of tumoral DNA including DNA strand breaks and decreased replication. Fruquintinib (ELUNATE®) is a novel kinase inhibitor that selectively inhibits the vascular endothelial growth factor receptor-1, -2, and -3. In this study, we evaluated the antitumor activity of combination therapy with FTD/TPI and fruquintinib in vivo. Methods: The enhancement of the antitumor effects with FTD/TPI and fruquintinib combination, compared to the single drugs given alone was evaluated using two human colorectal cancer xenografts in nude mouse models. FTD/TPI (200 mg/kg) was orally administered for 5 consecutive days followed by 2 days of rest in a 7-day period. Fruquintinib (10 mg/kg) was orally administered consecutively for 2 and 3 weeks in SW48 and HCT 116 tumor-bearing models, respectively. After treatment with these agents, the microvessel density was evaluated by CD31 immunohistochemical analyses. Results: In both models, FTD/TPI and fruquintinib significantly inhibited tumor growth, and the activity of the combined treatment was significantly superior to that of either monotherapy. Body weight loss of greater than 20% was not observed in any group. A histochemical analysis showed nuclei enlargement, abnormal mitosis, and karyorrhexis in the FTD/TPI treatment group. The microvessel density in the HCT 116 tumors treated with FTD/TPI and fruquintinib was significantly lower than that in the control group. Conclusion: The combination of FTD/TPI and fruquintinib could be a promising treatment option for colorectal cancer.

Keywords:
Trifluridine/tipiracil, Fruquintinib, Colorectal cancer, Combination therapy, Pathological analysis

Colorectal cancer is the third most common cancer in eastern Asia, with an age-standardized incidence of 25.9 per 100,000, and is the 4th leading cause of cancer-related deaths, with an age-standardized mortality rate of 11.8 per 100,000 in 2020 [1]. In results reported in China, the incidence was 23.9 per 100,000, and it was the 5th leading cause of cancer-related death, with an age-standardized mortality rate of 12.0 per 100,000 in 2020 [2].

Current treatments for unresectable metastatic colorectal cancer include systemic chemotherapeutic agents such as fluoropyrimidines [3], irinotecan hydrochloride (CPT-11) [4, 5], oxaliplatin and fluoropyrimidines [6], as well as targeted agents such as bevacizumab, a monoclonal antibody for vascular endothelial growth factor-A [6] and cetuximab, a monoclonal antibody for epidermal growth factor receptor [5]. Consequently, the survival of patients with unresectable metastatic colorectal cancer has improved.

Trifluridine/tipiracil hydrochloride (FTD/TPI, Lonsurf®) consists of an antineoplastic thymidine analogue, FTD, and a thymidine phosphorylase inhibitor, TPI, at a molar ratio 1:0.5 [7, 8]. FTD/TPI has been approved for late-stage chemotherapy for colorectal cancer [9, 10] and gastric cancer [11] after standard chemotherapy. The triphosphate form of FTD is incorporated into DNA in tumor cells and induces DNA dysfunctions, including DNA strand breaks and decreased replication [12‒14]. When FTD is administered orally, it is rapidly degraded to its inactive form by thymidine phosphorylase in the intestines and liver (first-pass effect). TPI inhibits the degradation of FTD, allowing an adequate plasma concentration of orally administered FTD to be maintained, potentiating its antitumor activity [15].

To achieve higher therapeutic effects of FTD/TPI in patients with refractory metastatic colorectal cancer, combination therapy with CPT-11 [16, 17], oxaliplatin [18], or bevacizumab [17, 19] has been reported. To attain long-term therapy, oral administration, rather than an intravenous route, would be a useful option for patients.

Fruquintinib (ELUNATE®) is a novel and specific inhibitor for vascular endothelial growth factor receptor (VEGFR) kinases 1, 2, and 3, which contribute to angiogenesis, and is available as an oral formulation [20]. In a phase 3 trial FRESCO (Clinical Trials. gov. identifier: NCT02314819), fruquintinib improved both overall and progression-free survival in patients with metastatic colorectal cancer; it was approved in China for metastatic colorectal cancer previously treated with fluorouracil-based standard chemotherapy in 2018 [21] and received fast-track authorization from the FDA in 2020. Based on the initial trial, FRESCO-2 (Clinical Trials. gov. identifier: NCT04322539) was conducted as a global phase 3 trial investigating the efficacies and safety of fruquintinib in metastatic colorectal cancer in patients enrolled in the USA, Europe, Australia, and Japan [22]. In the present manuscript, combination chemotherapy using FTD/TPI and fruquintinib was evaluated using preclinical models. The results suggest that combination therapy could be a promising new option for patients with colorectal cancer.

Chemicals

FTD and TPI were synthesized at YUKI GOSEI KOGYO, LTD. (Tokyo, Japan) and Taiho Pharmaceutical., Co., Ltd. (Tokyo, Japan), respectively. Fruquintinib was purchased from Med­ChemExpress (Monmouth Junction, NJ). Hydroxypropyl methylcellulose TC-5 (HPMC) and carboxymethyl cellulose sodium salt (CMC-Na) were purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan) and Wako Pure Chemicals Co., Ltd. (Osaka, Japan), respectively. Fetal bovine serum, Dulbecco’s Modified Eagle Medium, and Matrigel® (matrix basement membrane, phenol red-free) were purchased from HyClone Laboratories (Logan, UT), Sigma-Aldrich Japan Co., LLC (Tokyo, Japan), and Corning Discovery Labware (Bedford, MA), respectively.

FTD/TPI was prepared by mixing FTD and TPI at a molar ratio of 1:0.5 in 0.5% (w/v) HPMC. The dose of FTD/TPI was expressed according to the amount of FTD. FTD/TPI solution was proven to be stable for 7 days at 4°C; therefore, the solution was prepared once a week. Fruquintinib was suspended in 0.5% (w/v) CMC-Na just before administration.

Tumor Cell Lines

The human colorectal cancer cell line SW48, derived from Dukes’ type C, grade IV, colon adenocarcinoma, and colon carcinoma, and HCT 116 were purchased from American Type Culture Collection (Rockville, MD). SW48 and HCT 116 cells were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 95% air and 5% CO2. SW48 and HCT 116 were previously authenticated using short tandem repeat analyses for 9 loci in 2017 and 2015, respectively, and were proven to have database agreements of 94% and 100%, respectively.

Antibodies

Anti-CD31 rabbit polyclonal antibody (ab28364) was obtained from abcam® (Cambridge, UK). Anti-bromo-deoxyuridine (BrdU) mouse monoclonal IgG1 antibody (Clone 3D4) was purchased from BD Biosciences (Tokyo, Japan). Horseradish peroxidase (HRPO)-conjugated anti-rabbit IgG goat antibody Fab’ (Histofine® simple stain MAX-PO [R]) and HRPO-conjugated anti-mouse IgG goat antibody Fab’ (Histofine® simple stain MAX-PO [M]) were purchased from NICHIREI BIOSCIENCES INC. (Tokyo, Japan).

In vivo Antitumor Activity

Male nude mice (BALB/cAJcl-nu/nu) were purchased from CLEA Japan Inc. (Tokyo, Japan) and were housed under specific pathogen-free conditions with food and water provided ad libitum. After the animals had been placed in quarantine for 5 days, they were subcutaneously implanted with human tumor cells (SW48: 4 × 106 cells/body in 50% Matrigel®/PBS [23] and HCT 116: 2 × 106 cells/body in PBS). To evaluate the antitumor activity, the mice were randomized into six groups. On Day 0 according to their tumor volumes once the mean tumor volume had reached about 306–550 mm3 for SW48 or 108–291 mm3 for HCT 116. The number of mice per group was set at 6, which was thought to be sufficient to evaluate the antitumor effect based on previous experiments [24]. FTD/TPI was administered on days 1–5, 8–12, and 15–19 for the HCT 116-xenograft model and on days 1–5, 8–12, and 15 for the SW48-xenograft model, respectively, according to the reported effective dose and schedule [25, 26]. Fruquintinib was orally administered at the reported effective dose of 10 mg/kg [20] for 15 (SW48-xenograft model) or 21 (HCT 116-xenograft model) consecutive days. The clinical dose of fruquintinib is 5 mg/body/day, which is equivalent to 4 mg/kg for mice based on the AUC level [27]. Therefore, the dose levels of fruquintinib were reasonable. The administered volume was 5 mL/kg for all compounds. The tumor diameters were measured twice a week, and the tumor volume was estimated using the following formula: 0.5 × length × width2. To evaluate the tolerability of treatment, the body weight change (BWC) was calculated using the following formula: BWC (%) = ((body weight on measured day) – (body weight on day 0)) / (body weigth on day 0) × 100 (%). In cases in which the mean body weight loss was more than 20% or in which toxic death was observed as a result of the treatment, the treatment was designated as intolerable [28].

Immunohistochemistry

HCT 116 and SW48 xenografts were collected on days 22 and 15, respectively. The tissues were fixed in 10% phosphate buffered formalin for 1 day and then embedded in paraffin. Tumor tissue sections were prepared at a thickness of 3 μm from paraffin-embedded blocks. Hematoxylin and eosin (H&E) staining was done according to the standard conventional method. The microvessels in the xenografts were stained immunohistochemically using anti-CD31 rabbit polyclonal antibody, while FTD incorporation in the tumor cell nuclei was stained using anti-BrdU mouse monoclonal antibody (Clone 3D4), which reportedly recognizes phosphorylated FTD in nuclei [29].

Whole Slide Image Analysis and Pathological Analysis

For immunohistochemical analysis slides were scanned for whole slide image using the digital slide scanner, NanoZoomer ver. 3.3.3 (Hamamatsu Photonics, Hamamatsu, Japan). To evaluate the CD31-positive area in tumor tissues and FTD incorporation in tumor nuclei, the sections were deparaffinized and subjected to antigen retrieval in succinate buffer (pH 6.1; Agilent Technologies Japan, Ltd., Tokyo Japan). The sections were incubated with anti-CD31 (dilution, 1:50) and anti-BrdU antibody (dilution, 1:100) for 60 min at room temperature and treated with hydrogen peroxide, followed by the secondary antibody (HRPO-conjugated anti-rabbit IgG or anti-mouse IgG) for 30 min (room temperature); the secondary antibody was visualized using 3, 3′-diaminobenzidine tetrahydrochloride. The sections were counterstained with Mayer’s hematoxylin [29, 30]. For a negative control, one section of the control was stained without application of the primary antibody. The HALO AI issue classifier software module (version 3.2; Indica Labs, Albuquerque, NM) was adapted to determine the CD31-positive area (µm2) within the tumor tissue area after excluding areas of necrosis [31].

Statistical Methods

The significance of the differences in the mean tumor volume between the treated and control groups was analyzed using logarithmically transformed values and the Dunnett test. The combination effect of FTD/TPI and fruquintinib was analyzed according to the closed testing procedure using the Aspin-Welch t test [32]. The CD31-positive area rate was analyzed using a non-parametrical Dunnett test. The statistical analyses were performed using SAS system release 9.4 (SAS Institute Inc. Japan, Tokyo Japan) using EXSUS, version 10, running on Windows 10 (CAC Croit Corporation, Osaka, Japan).

Increased Antitumor Activity of Combined FTD/TPI and Fruquintinib in Human Colorectal Cancer in vivo

The tumor volume changes of HCT 116 xenografts in treated mice are shown in Figure 1a. The tumor volumes on day 22 in the FTD/TPI (200 mg/kg) and fruquintinib (10 mg/kg) monotherapy groups were significantly lower than that in the control group. In the FTD/TPI and fruquintinib combination therapy group, the antitumor effects were significantly superior to those seen for each monotherapy (Fig. 1a). Since one of the six mice treated with FTD/TPI alone was severely injured as a result of fighting, its data were excluded from the analysis. The tumor volume changes of SW48 xenografts in treated mice are shown in Figure 1b. According to the guidelines [28], the treatment was terminated on day 15 when tumor burden reached greater than 10% of body weight. The tumor volumes on day 15 in the FTD/TPI and fruquintinib monotherapy groups were significantly lower than that in the control group. In the FTD/TPI and fruquintinib combination therapy group, the antitumor effects were significantly superior to those seen for each monotherapy (Fig. 1b). Since one of the six mice treated with fruquintinib alone was severely injured as a result of fighting, its data were excluded from the analysis.

Fig. 1.
Tumor volume of xenografted HCT 116 (a) and SW48 (b) human colorectal tumors. Mice were randomized according to tumor volume on day 0. For mice with HCT 116 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15–19 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–21 at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). For mice with SW48 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–15, at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). Values are the mean and SE. *, **: p< 0.05 and 0.01, respectively versus Control, Dunnett test. ##p< 0.01 versus FTD/TPI alone, Aspin-Welch ttest. §p< 0.05 versus fruquintinib alone, Aspin-Welch ttest.
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Tumor volume of xenografted HCT 116 (a) and SW48 (b) human colorectal tumors. Mice were randomized according to tumor volume on day 0. For mice with HCT 116 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15–19 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–21 at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). For mice with SW48 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–15, at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). Values are the mean and SE. *, **: p< 0.05 and 0.01, respectively versus Control, Dunnett test. ##p< 0.01 versus FTD/TPI alone, Aspin-Welch ttest. §p< 0.05 versus fruquintinib alone, Aspin-Welch ttest.

Fig. 1.
Tumor volume of xenografted HCT 116 (a) and SW48 (b) human colorectal tumors. Mice were randomized according to tumor volume on day 0. For mice with HCT 116 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15–19 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–21 at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). For mice with SW48 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–15, at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). Values are the mean and SE. *, **: p< 0.05 and 0.01, respectively versus Control, Dunnett test. ##p< 0.01 versus FTD/TPI alone, Aspin-Welch ttest. §p< 0.05 versus fruquintinib alone, Aspin-Welch ttest.
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Tumor volume of xenografted HCT 116 (a) and SW48 (b) human colorectal tumors. Mice were randomized according to tumor volume on day 0. For mice with HCT 116 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15–19 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–21 at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). For mice with SW48 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–15, at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). Values are the mean and SE. *, **: p< 0.05 and 0.01, respectively versus Control, Dunnett test. ##p< 0.01 versus FTD/TPI alone, Aspin-Welch ttest. §p< 0.05 versus fruquintinib alone, Aspin-Welch ttest.

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Tolerability of Combination Therapy Using FTD/TPI and Fruquintinib

The BWCs from day 0 to 22 in nude mice with HCT 116 xenografts and from day 0 to 15 in mice with SW48 xenografts are shown in Figure 2a, b, respectively. The body weight loss did not exceed 20% in any of the groups; therefore, the combined treatment with FTD/TPI and fruquintinib was designated as tolerable.

Fig. 2
Body weight changes of nude mice with HCT 116 (a) and SW48 (b) xenografts. Mice were randomized according to tumor volume on day 0. For mice with HCT 116 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15–19 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–21 at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). For mice with SW48 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–15, at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). Values are the mean and SE.
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Body weight changes of nude mice with HCT 116 (a) and SW48 (b) xenografts. Mice were randomized according to tumor volume on day 0. For mice with HCT 116 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15–19 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–21 at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). For mice with SW48 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–15, at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). Values are the mean and SE.

Fig. 2
Body weight changes of nude mice with HCT 116 (a) and SW48 (b) xenografts. Mice were randomized according to tumor volume on day 0. For mice with HCT 116 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15–19 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–21 at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). For mice with SW48 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–15, at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). Values are the mean and SE.
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Body weight changes of nude mice with HCT 116 (a) and SW48 (b) xenografts. Mice were randomized according to tumor volume on day 0. For mice with HCT 116 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15–19 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–21 at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). For mice with SW48 xenografts, control mice (○) were not treated; mice treated with FTD/TPI alone (days 1–5, 8–12, and 15 at 200 mg/kg, p.o., •), fruquintinib alone (days 1–15, at 10 mg/kg, p.o., □) or FTD/TPI plus fruquintinib (■). Values are the mean and SE.

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Abnormal Nuclear Morphologies Induced by FTD

H&E staining of HCT 116 (upper part) and SW48 (lower part) tumor tissues are shown in Fig. 3a–d. In both the control (Fig. 3a) and fruquintinib monotherapy (Fig. 3c) groups, no nuclei abnormality was observed both in HCT 116 (upper part) and SW48 (lower part). In FTD/TPI monotherapy group (Fig. 3b), swollen nuclei, condensed chromosomes, and karyorrhexis were observed in HCT 116 (upper part) and SW48 tumors (lower part). In the FTD/TPI and fruquintinib combined treatment group (Fig. 3d), almost the same pathological changes were observed in HCT 116 (upper part) and SW48 tumors (lower part).

Fig. 3.
Typical images of hematoxylin-and-eosin-stained tissue in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The white triangles (Δ) indicate swollen nuclei, and the black triangles (▴) indicate condensed chromosomes and karyorrhexis. The original magnification was ×400; the scale bar indicates 100 μm.
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Typical images of hematoxylin-and-eosin-stained tissue in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The white triangles (Δ) indicate swollen nuclei, and the black triangles (▴) indicate condensed chromosomes and karyorrhexis. The original magnification was ×400; the scale bar indicates 100 μm.

Fig. 3.
Typical images of hematoxylin-and-eosin-stained tissue in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The white triangles (Δ) indicate swollen nuclei, and the black triangles (▴) indicate condensed chromosomes and karyorrhexis. The original magnification was ×400; the scale bar indicates 100 μm.
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Typical images of hematoxylin-and-eosin-stained tissue in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The white triangles (Δ) indicate swollen nuclei, and the black triangles (▴) indicate condensed chromosomes and karyorrhexis. The original magnification was ×400; the scale bar indicates 100 μm.

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FTD Incorporation in Nuclei of Tumor Cells

The incorporation of FTD in tumor tissue nuclei was visualized by using anti-BrdU antibody on HCT 116 (upper part) and SW48 (lower part) tumor tissues, as shown in Fig. 4a–d. In the control (Fig. 4a) and fruquintinib monotherapy groups (Fig. 4c), tumor tissue was not stained except for connective tissue in the tumor which contains mouse IgG in common HCT 116 and SW48. FTD was similarly detected in tumor cell nuclei of HCT 116 treated with FTD/TPI alone (Fig. 4b, upper part) and FD/TPI plus fruquintinib (Fig. 4d, upper part). And FTD was detected in SW48 tumor cell nuclei treated with FTD/TPI alone (Fig. 4b, lower part) and in combination with fruquintinib (Fig. 4d, lower part).

Fig. 4.
Typical images of immunohistochemical visualization of FTD using anti-BrdU antibody in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The original magnification was ×400; and the scale bar indicates 100 μm.
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Typical images of immunohistochemical visualization of FTD using anti-BrdU antibody in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The original magnification was ×400; and the scale bar indicates 100 μm.

Fig. 4.
Typical images of immunohistochemical visualization of FTD using anti-BrdU antibody in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The original magnification was ×400; and the scale bar indicates 100 μm.
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Typical images of immunohistochemical visualization of FTD using anti-BrdU antibody in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The original magnification was ×400; and the scale bar indicates 100 μm.

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Increased Antiangiogenic Activity of Fruquintinib in Combination with FTD/TPI

Microvessels were visualized as brown using an anti-CD31 antibody, and typical CD31-positive images of HCT 116 (upper part) and SW48 (lower part) are shown in Fig. 5a–d. CD31-positive area of SW48 (Fig. 5a–d, lower part) was lower than that of HCT 116 (Fig. 5a–d, upper part). Serial section of HCT 116 solid tumor and adjacent normal tissue was stained with anti-CD31 antibody and H&E. Tumor tissue itself was not stained brown using anti-CD31 primary antibody (online suppl. Fig. S1; for all online suppl. material, see www.karger.com/doi/10.1159/000528867).

Fig. 5.
Typical images of immunohistochemical visualization of tumor vessels using anti-CD31 antibody in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The original magnification was approximately ×200, and the scale bar indicates 250 μm.
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Typical images of immunohistochemical visualization of tumor vessels using anti-CD31 antibody in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The original magnification was approximately ×200, and the scale bar indicates 250 μm.

Fig. 5.
Typical images of immunohistochemical visualization of tumor vessels using anti-CD31 antibody in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The original magnification was approximately ×200, and the scale bar indicates 250 μm.
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Typical images of immunohistochemical visualization of tumor vessels using anti-CD31 antibody in HCT 116 (upper part) and SW48 (lower part) xenografts. a Control. bFTD/TPI. c Fruquintinib. dFTD/TPI plus fruquintinib. The original magnification was approximately ×200, and the scale bar indicates 250 μm.

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The ratio of the necrotic area to the total tumor area in SW48 xenografts was obviously higher than that in HCT 116 xenografts whose volume was almost equivalent to that in SW48 xenografts, macroscopically by H&E staining (online suppl. Fig. S2). Therefore, we abandoned to analyze the ratio of the CD31-positive area in SW48 tumor tissues quantitatively, and we performed the quantitative pathological analysis only in HCT116 tumors. In the fruquintinib monotherapy group, the CD31-positive area in HCT 116 viable tumor tissue tended to be smaller than that in the control, but the change was not significant. In the combination therapy group, the CD31-positive area in viable tumor tissue was significantly smaller than that in the control (Fig. 6).

Fig. 6.
Proportion of CD31-positive area in HCT 116 xenografts. Values are the mean and SE *p< 0.05 versus Control using the Dunnett test.
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Proportion of CD31-positive area in HCT 116 xenografts. Values are the mean and SE *p< 0.05 versus Control using the Dunnett test.

Fig. 6.
Proportion of CD31-positive area in HCT 116 xenografts. Values are the mean and SE *p< 0.05 versus Control using the Dunnett test.
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Proportion of CD31-positive area in HCT 116 xenografts. Values are the mean and SE *p< 0.05 versus Control using the Dunnett test.

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The treatment of unresectable or metastatic colorectal cancer requires chemotherapy. For several decades, fluoropyrimidine-based combination therapy has been recognized as the standard treatment regimen; however, new candidates for use after fluoropyrimidine-based regimens have been anticipated. FTD/TPI is reportedly active against tumors with acquired resistance to fluoropyrimidine [7] and has consequently been approved in more than 30 nations including the USA, EU, and Japan. To achieve an even higher clinical efficacy, clinical trials of FTD/TPI plus other chemotherapeutic agents, such as CPT-11 [16, 17], oxaliplatin [18], and the anti-vascular endothelial growth factor-A monoclonal antibody bevacizumab [17, 19], are presently in progress. Fruquintinib is a novel selective VEGFR inhibitor that has been approved in China; its major antitumor mechanism originates from its antiangiogenesis activity [20]. In the present study, we showed that the novel combination of FTD/TPI and fruquintinib was significantly superior to each monotherapy in mice with subcutaneous colorectal tumor xenografts.

To clarify the mechanisms of the enhanced antitumor activity achieved by the combination of FTD/TPI and fruquintinib, we performed histopathological analyses using tumor tissues collected on the day after the last administration. As mentioned above, fruquintinib is an antiangiogenic drug. Therefore, the CD31-positive area was evaluated using HCT 116 xenografts (Fig. 5, 6), being CD31 a typical angiogenesis marker expressed by endothelial cells [33]. In tumor xenograft, cancer cells did not react with the anti-CD31 antibody. The CD31-positive area in the SW48 xenografts could not be evaluated quantitatively, because smaller than that of HCT 116, as shown in online Suppl. Figure S2. The CD31-positive area in viable tumor tissue tended to be smaller after fruquintinib monotherapy, although the difference relative to the control group was not significant. In the combination therapy group, however, the microvessel area was significantly lower. The antiangiogenic effects of the combination therapy might contribute to the enhanced antitumor activity seen in the HCT 116 xenografts. Using anti-BrdU antibody, FTD was detected in tumor nuclei, it was reported that FTD is persisted in tumor nucleus longer than normal cell [30]. FTD is supposed to induce DNA dysfunction after incorporation into DNA [7, 12]. After treatment with FTD/TPI in the presence of fruquintinib, FTD was shown to be incorporated into DNA by immunohistochemical. Therefore, fruquintinib seemed to give little effect for FTD incorporation into DNA.

When bevacizumab and FTD/TPI were administered in combination, bevacizumab normalized the microvessels in tumors [34], and this phenomenon was suggested to lead to the increased FTD content in tumors treated with FTD/TPI, contributing to the therapeutic efficacy [24]. Hence, fruquintinib is expected to have similar effects, since it also inhibits VEGFR signals. However, the levels of swollen nuclei, chromosome mitosis, and karyorrhexis in tumor cells treated with FTD/TPI monotherapy and FTD/TPI and fruquintinib combination therapy were almost the same in the present study. Further studies are needed to clarify the mechanism of the enhanced antitumor activity of the combination therapy since the mechanisms by which FTD/TPI and fruquintinib contribute to antitumor activity are entirely different and do not compete with each other. Body weight loss was not increased in the combination therapy group, compared with the monotherapy groups. However, these data alone cannot confirm that the combination therapy is well tolerated because the clinical dose-limiting toxicity of FTD/TPI is myelosuppression, while that of fruquintinib is proteinuria and hand-foot syndrome. Measures corresponding to these adverse events were not evaluated in the present experiment; thus, the toxicity profiles should be evaluated in further detail in future studies.

Generally, an increased adherence and long-term treatment are essential to improve efficacy, compared with the efficacy of existing drugs. Patients tend to prefer oral administration to intravenous treatments [35]; therefore, oral administration is a suitable option for long-term treatment to improve therapeutic efficacy and minimize hospitalization. Since both FTD/TPI and fruquintinib are orally available drugs, this combination therapy is likely to have a high adherence. In addition, Guan et al. performed a cost-effectiveness analysis comparing fruquintinib to oral regorafenib, which is approved for the treatment of metastatic colorectal cancer after the failure of fluoropyrimidine-based chemotherapy [36]. These support the use of FTD/TPI and fruquintinib combination therapy as a treatment option for patients with colorectal cancer that is not only more effective but also less expensive than existing treatment options.

Our findings suggest that a novel combination therapy using FTD/TPI, an antineoplastic nucleoside, and fruquintinib, an antiangiogenic agent, might be a highly promising therapy for refractory advanced or metastatic colorectal cancer.

All papers must contain the following statements after the main body of the text and before the reference list.

We would like to thank Mr. Masaaki Abe for their expert technical assistance and meaningful advices. We would like to thank International Medical Information Center (www.imic.or.jp) for editing English correction.

This study protocol was reviewed and approved according to the guidelines for endpoints in animal study proposals by NIH [25] and with the approval of the Institutional Animal Care and Use Committee of Taiho Pharmaceutical Co., Ltd. (Approval number, 20TC11).

All authors are employee of Taiho Pharmaceutical Co., Ltd., and this study was sponsored by Taiho Pharmaceutical Co., Ltd. All authors have no other conflicts of interest to declare.

This study was sponsored by Taiho Pharmaceutical Co., Ltd.

Mamoru Nukatsuka: provided the original conceptual design of this study; Akio Fujioka: carried out the experiments and collected the data; Gotaro Tanaka: handled histological analysis; Hideki Nagase and Hiroaki Hayashi: participated in the interpretation of the results and manuscript writing and revision.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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