卡博替尼联合PD-L1单抗对黑色素瘤小鼠移植瘤的抑制作用机制
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摘要
【目的】探讨卡博替尼联合PD-L1单抗对恶性黑色素瘤小鼠皮下移植瘤生长的影响及作用机制。【方法】利用体外培养的小鼠黑色素瘤细胞B16-F10建立小鼠皮下移植瘤模型,成瘤后将小鼠随机分为盐水对照组、溶剂对照组、PD-L1单抗组、卡博替尼组和卡博替尼联合PD-L1单抗组(联合组)。观察肿瘤生长情况及每2 d测量一次肿瘤体积。肿瘤体积达到2 000 mm~3或是组间差距有显著统计学差异时定义为研究终点。达到研究终点后处死小鼠取材,流式细胞学检测肿瘤组织中浸润免疫细胞情况,包括CD4~+、CD8~+T淋巴细胞及髓源性抑制细胞(MDSC)。另将体外培养的B16-F10细胞分别经不同药物处理后,行流式细胞术检测细胞凋亡情况;Western blot检测AKT、p-AKT、mTOR和p-mTOR蛋白表达水平。【结果】B16-F10黑色素瘤移植瘤模型显示,PD-L1单抗组没有明显抑瘤作用,卡博替尼组与联合组均有明显抑瘤效应,联合组抑瘤效果较卡博替尼组更为显著,差异具有统计学意义(P=0.001 5)。在体外实验中,药物处理24及48 h后流式细胞术检测B16-F10细胞凋亡,结果显示联合组与卡博替尼组相比,B16-F10细胞凋亡比例均显著增高,差异具有统计学意义(24 h:P=0.003 5;48 h:P=0.002 9)。Western blot检测显示联合组及卡博替尼组对AKT和mTOR蛋白水平无明显影响,但均可下调p-AKT和p-mTOR蛋白水平,联合组作用更为显著。【结论】卡博替尼联合PD-L1单抗有协同抗肿瘤作用,这种协同抗肿瘤作用机制可能是通过促进B16-F10细胞凋亡及抑制AKT/mTOR通路所致。
【Objective】The aim of this study was to investigate the effect and mechanism of cabozantinib combinedwith anti-PD-L1 antibody on the growth of subcutaneous transplanted malignant melanoma in mice.【Methods】Establishedmouse subcutaneous xenograft model using mouse melanoma cell line B16-F10,and then randomly divided into fivegroups:saline control group,vehicle control group,anti PD-L1 antibody group,cabozantinib group,cabozantinib incombined with anti-PD-L1 antibody group(combination group).Tumor growth was observed and tumor volume wasmeasured every 2 days.The research endpoint was defined as when the tumor volume reached 2 000 mm~3or the differencebetween the groups was statistically significant.Then the mice were sacrificed and tissue samples were taken at theendpoint of the study.Infiltrating immune cells including CD4~+,CD8~+T lymphocytes and myelogenous suppressor cells(MDSC)were detected by flow cytometry.In addition,B16-F10 cells cultured in vitro were treated with different drugs,the apoptosis was detected by flow cytometry,and the protein expressions of AKT,p-AKT,mTOR and p-mTOR weredetected by western blot assay.【Results】B16-F10 melanoma xenograft model showed that anti-PD-L1 antibody group had no obvious antitumor effect,while both cabozantinib group and combination group produced significant antitumor effect,and the combination group had more obvious antitumor effect compared to cabozantinib group(P=0.001 5).B16-F10 cells were treated with different drugs in vitro,and the apoptosis rate of the combination group was significantly higher than that of cabozantinib group at 24 h and 48 h,respectively(24 h:P=0.003 5;48 h:P=0.002 9).Western blot assay showed that the combination group and cabozantinib group had no significant effect on the protein expression of AKT and mTOR,but both could reduce their phosphorylation levels,and the combination group was more remarkable.【Conclusion】Cabozantinib in combined with anti-PD-L1 antibody had synergistic anti-tumor effect,which might be achieved by promoting B16-F10 cells apoptosis and inhibiting of AKT/mTOR pathway.
【Objective】The aim of this study was to investigate the effect and mechanism of cabozantinib combinedwith anti-PD-L1 antibody on the growth of subcutaneous transplanted malignant melanoma in mice.【Methods】Establishedmouse subcutaneous xenograft model using mouse melanoma cell line B16-F10,and then randomly divided into fivegroups:saline control group,vehicle control group,anti PD-L1 antibody group,cabozantinib group,cabozantinib incombined with anti-PD-L1 antibody group(combination group).Tumor growth was observed and tumor volume wasmeasured every 2 days.The research endpoint was defined as when the tumor volume reached 2 000 mm~3or the differencebetween the groups was statistically significant.Then the mice were sacrificed and tissue samples were taken at theendpoint of the study.Infiltrating immune cells including CD4~+,CD8~+T lymphocytes and myelogenous suppressor cells(MDSC)were detected by flow cytometry.In addition,B16-F10 cells cultured in vitro were treated with different drugs,the apoptosis was detected by flow cytometry,and the protein expressions of AKT,p-AKT,mTOR and p-mTOR weredetected by western blot assay.【Results】B16-F10 melanoma xenograft model showed that anti-PD-L1 antibody group had no obvious antitumor effect,while both cabozantinib group and combination group produced significant antitumor effect,and the combination group had more obvious antitumor effect compared to cabozantinib group(P=0.001 5).B16-F10 cells were treated with different drugs in vitro,and the apoptosis rate of the combination group was significantly higher than that of cabozantinib group at 24 h and 48 h,respectively(24 h:P=0.003 5;48 h:P=0.002 9).Western blot assay showed that the combination group and cabozantinib group had no significant effect on the protein expression of AKT and mTOR,but both could reduce their phosphorylation levels,and the combination group was more remarkable.【Conclusion】Cabozantinib in combined with anti-PD-L1 antibody had synergistic anti-tumor effect,which might be achieved by promoting B16-F10 cells apoptosis and inhibiting of AKT/mTOR pathway.
引文
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[16]Mittendorf EA,Philips AV,Meric-Bernstam F,et al.PD-L1 expression in triple-negative breast cancer[J].Cancer Immunol Res,2014,2(4):361-370.
[2]Garbe C,Eigentler TK,Keilholz U,et al.Systematic review of medical treatment in melanoma:current status and future prospects[J].Oncologist,2011,16(1):5-24.
[3]Topalian SL,Sznol M,McDermott DF,et al.Survival,durable tumor remission,and long-term safety in patients with advanced melanoma receiving nivolumab[J].J Clin Oncol,2014,32(10):1020-1030.
[4]Robert C,Schachter J,Long GV,et al.Pembrolizumab versus Ipilimumab in advanced melanoma[J].N Engl J Med,2015,372(26):2521-2532.
[5]Hamid O,Robert C,Daud A,et al.Safety and tumor responses with lambrolizumab(anti-PD-1)in melanoma[J].N Engl J Med,2013,369(2):134-144.
[6]Schachter J,Ribas A,Long GV,et al.Pembrolizumab versus ipilimumab for advanced melanoma:final overall survival results of a multicentre,randomised,open-label phase 3 study(KEYNOTE-006)[J].The Lancet,2017,390(10105):1853-1862.
[7]Rosenberg JE,Hoffman-Censits J,Powles T,et al.Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy:asingle-arm,multicentre,phase2trial[J].The Lancet,2016,387(10031):1909-1920.
[8]Fehrenbacher L,Spira A,Ballinger M,et al.Atezolizumab versus docetaxel for patients with previously treated non-small-cell lung cancer(POP-LAR):a multicentre,open-label,phase 2 randomised controlled trial[J].The Lancet,2016,387(10030):1837-1846.
[9]Chapman PB,Hauschild A,Robert C,et al.Improved survival with vemurafenib in melanoma with BRAF V600E mutation[J].N Engl J Med,2011,364(26):2507-2516.
[10]Sosman JA,Kim KB,Schuchter L,et al.Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib[J].N Engl J Med,2012,366(8):707-714.
[11]Cooper ZA,Juneja VR,Sage PT,et al.Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade[J].Cancer Immunol Res,2014,2(7):643-654.
[12]Davies H,Bignell GR,Cox C,et al.Mutations of the BRAF gene in human cancer[J].Nature,2002,417(6892):949-954.
[13]Si L,Kong Y,Xu X,et al.Prevalence of BRAFV600E mutation in Chinese melanoma patients:large scale analysis of BRAF and NRAS mutations in a 432-case cohort[J].Eur J Cancer,2012,48(1):94-100.
[14]Lu X,Horner JW,Paul E,et al.Effective combinatorial immunotherapy for castration-resistant prostate cancer[J].Nature,2017,543(7647):728-732.
[15]Song E-K,Tai WM,Messersmith WA,et al.Potent antitumor activity of cabozantinib,a c-METand VEGFR2 inhibitor,in a colorectal cancer patient-derived tumor explant model[J].Int J Cancer,2015,136(8):1967-1975.
[16]Mittendorf EA,Philips AV,Meric-Bernstam F,et al.PD-L1 expression in triple-negative breast cancer[J].Cancer Immunol Res,2014,2(4):361-370.