南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (10): 1448-1456.doi: 10.12122/j.issn.1673-4254.2020.10.10

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帕那替尼对内源性表达FGFR2-CCDC6融合蛋白的人胆管癌异种移植小鼠的抗肿瘤作用

吴天宇，蒋晓青，徐 斌，王 宇   

  • 出版日期:2020-10-20 发布日期:2020-10-20

Ponatinib inhibits growth of patient-derived xenograft of cholangiocarcinoma expressing FGFR2-CCDC6 fusion protein in nude mice

  • Online:2020-10-20 Published:2020-10-20

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摘要： 目的 探讨FGFR抑制剂帕那替尼对内源性表达FGFR2-CCDC6融合蛋白的新型胆管癌患者异种移植小鼠模型的抗肿瘤作用。方法 通过一个晚期肝内胆管细胞癌（iCCA）患者的肺转移瘤组织建立了一个表达了FGFR2-CCDC6融合蛋白异种移植（PDX）小鼠模型。将PDX小鼠模型分为4组（每组10只，帕那替尼组9只可供评估）。对照组（柠檬酸盐缓冲液）、帕那替尼组（20 mg/kg，溶于柠檬酸盐溶液中）、吉西他滨（50 mg/kg，每周腹腔注射）+顺铂（2.5 mg/kg，每周腹腔注射）组、帕那替尼联合吉西他滨+顺铂组（上述等剂量），用免疫组织化学染色对p-FGFR、p-FRS2、p-AKT、p-ERK、CD31、Ki-67进行染色评估其表达。通过裂解半胱天冬酶-3（CC3）染色和TUNEL染色试验评估细胞凋亡。Western blot 检测FGFR2、p-FGFR、AKT、p-AKT、ERK、pERK、FRS2和p-FRS2的表达。结果 与对照组相比，帕那替尼组小鼠肿瘤生长体积明显减小（P<0.0001），Ki-67染色显示肿瘤细胞增殖减少，裂解半胱天冬酶-3染色和TUNEL染色试验显示肿瘤细胞凋亡显著增加。蛋白质印迹和IHC显示，FGFR及其下游信号标记FRS2、AKT和ERK的磷酸化均降低。与对照组相比，吉西他滨+顺铂组显著抑制肿瘤生长（P<0.0001），且明显比单独使用帕那替尼更有效（P<0.0001）。但没有降低FGFR或下游信号分子FRS2、AKT和ERK的磷酸化。结论 FGFR抑制剂帕那替尼在含有FGFR2融合的胆管癌肿瘤中调节FGFR信号传导、抑制细胞增殖和诱导凋亡的能力，可作为FGFR2融合的胆管癌患者的治疗选择方案。

关键词: 肝内胆管细胞癌, FGFR2, 帕那替尼, 吉西他滨, 顺铂

Abstract: Objective To investigate the antitumor effect of ponatinib on the growth of cholangiocarcinoma xenograft derived from a clinical patient in a mouse model expressing FGFR2-CCDC6 fusion protein. Methods Lung metastatic tumor tissue was collected from a patient with advanced intrahepatic cholangiocarcinoma and implanted subcutaneously a NOD/SCID/Il2rg-knockout (NSG) mouse. The tumor tissues were harvested and transplanted in nude mice to establish mouse models bearing patient-derived xenograft (PDX) of cholangiocarcinoma expressing FGFR2-CCDC6 fusion protein. The PDX mouse models were divided into 4 groups for treatment with citrate buffer (control group), intragastric administration of 20 mg/kg ponatinib dissolved in citrate buffer (ponatinib group), weekly intraperitoneal injections of 50 mg/kg gemcitabine and 2.5 mg/kg cisplatin (gemcitabine group), or ponatinib combined with gemcitabine and cisplatin at the same doses (10 mice in each group, and 9 mice were evaluated in ponatinib group). The expressions of p-FGFR, p-FRS2, p-AKT, p-ERK, CD31, and Ki-67 in the xenografts were evaluated with immunohistochemistry, and cell apoptosis was analyzed with cleaved caspase-3 (CC3) staining and TUNEL staining. Western blotting was used to detect the expressions of FGFR2, p-FGFR, AKT, p-AKT, ERK, p-ERK, FRS2 and p-FRS2 in the tumor tissues. Results Compared with those in the control group, the mice in ponatinib group showed a significantly reduced tumor volume (P<0.0001) and suppressed tumor cell proliferation with significantly increased cell apoptosis. Western blotting and immunohistochemistry revealed obviously lowered phosphorylation level of FGFR and its downstream signal markers FRS2, AKT and ERK in the xenografts from ponatinib-treated mice. Gemcitabine treatment combined with cisplatin more effectively inhibited tumor growth than ponatinib alone (P<0.0001) but did not further decrease the phosphorylation levels of FGFR or its downstream signaling molecules FRS2, AKT and ERK. Conclusion Ponatinib can regulate FGFR signaling to inhibit the proliferation and induce apoptosis of tumor cells in mice bearing patient-derived cholangiocarcinoma xenograft with FGFR2 fusion. FGFR inhibitor can serve as a treatment option for patients with cholangiocarcinoma with FGFR2 fusion.

Key words: intrahepatic cholangiocarcinoma, FGFR2, ponatinib, gemcitabine, cisplatin