Supplementary Material for: Evaluation of a novel combination therapy, based on trifluridine/tipiracil and fruquintinib, against colorectal cancer

Introduction: Trifluridine/tipiracil hydrochloride (FTD/TPI, Lonsurf®) is an oral antineoplastic agent that has been approved as a 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.

引言：三氟尿苷/替吡嘧啶盐酸盐（Trifluridine/tipiracil hydrochloride, FTD/TPI, Lonsurf®）是一款口服抗肿瘤药物，已获批用于结直肠癌的晚期化疗。其主要作用机制为引发肿瘤DNA功能异常，包括DNA链断裂及DNA复制能力下降。呋喹替尼（fruquintinib, ELUNATE®）是一种新型激酶抑制剂，可选择性抑制血管内皮生长因子受体-1、-2及-3。本研究旨在体内评估FTD/TPI与呋喹替尼联合治疗的抗肿瘤活性。
方法：采用两种人结直肠癌裸小鼠移植瘤模型，对比单药给药与联合给药的抗肿瘤增效效应。FTD/TPI给药剂量为200 mg/kg，以7天为一个给药周期，每日口服给药连续5天，随后休息2天。针对SW48和HCT 116荷瘤模型，呋喹替尼的给药方案分别为连续口服2周和3周，剂量均为10 mg/kg。给药结束后，通过CD31免疫组化分析评估肿瘤微血管密度。
结果：在两种模型中，FTD/TPI与呋喹替尼单药均能显著抑制肿瘤生长，且联合治疗的抗肿瘤活性显著优于任一单药治疗组。所有组别均未观察到体重下降幅度超过20%的情况。组织化学分析显示，FTD/TPI治疗组可见细胞核增大、异常核分裂及核碎裂现象。经FTD/TPI与呋喹替尼联合治疗的HCT 116肿瘤，其微血管密度显著低于对照组。
结论：FTD/TPI与呋喹替尼联合疗法有望成为结直肠癌的潜在治疗选择。