靶向结直肠癌的小白菊内酯脂质体纳米颗粒诱导程序性坏死并改善T细胞耗竭

Liposome nanoparticles for targeted delivery of parthenolide induce colorectal cancer necroptosis to ameliorate tumor-infiltrating T cell exhaustion in mice

目的研究小白菊内酯(PTL)诱导结直肠癌细胞程序性坏死,并通过调节T细胞耗竭抑制结直肠癌的发展的机制,并通过构建其靶向脂质体改进其临床应用。方法通过CCK8实验检测不同结直肠肿瘤细胞经PTL处理后的增殖能力。利用ROS与LDH检测分析PTL诱导MC38细胞死亡的方式并进行蛋白免疫印迹分析。将24只小鼠随机分成对照组(等量生理盐水)、低剂量PTL组(5 mg/kg)与高剂量PTL组(15 mg/kg),通过在小鼠腹股沟皮下注射MC38细胞构建皮下瘤模型,给药后检测各组小鼠皮下瘤的重量与CD8^(+)T细胞的浸润情况,分析CD8^(+)T细胞耗竭表型的变化。构建PTL脂质体并对其进行表征。通过在小鼠回盲部移植肿瘤构建结直肠癌原位移植瘤模型,将32只小鼠随机分成对照组(等量生理盐水)、PTL处理组(100μg/mL)、低剂量靶向脂质体TCP-1-PTL-LNPs组(100μg/mL)和高剂量TCP-1-PTL-LNPs组(200μg/mL),尾静脉注射给药,通过免疫组化分析CD8在肿瘤中的表达情况。结果多种结直肠癌细胞(SW480、DLD1、HCT116和MC38)在PTL作用下随着药物浓度梯度增加细胞活力逐渐下降。在MC38细胞中,这一效应可被抑制剂Nec-1拮抗,同时免疫蛋白印迹分析显示经PTL处理细胞出现RIP3和MLKL的磷酸化。在小鼠皮下瘤模型中,经流式细胞术分析,与对照组或低剂量组相比,高剂量PTL组出现了肿瘤CD3^(+)CD8^(+)T细胞的浸润增加(P<0.01),同时PD1hiTIM3^(+)T细胞比例显著增加(P<0.01),而PD1loTIM3-T细胞比例显著降低(P<0.01)。在小鼠结直肠癌原位移植瘤模型中,经过PTL治疗,免疫组化结果显示CD8表达较对照组明显增加,而使用了高浓度靶向脂质体治疗的肿瘤中CD8表达更进一步增加(P<0.01)。结论小白菊内酯通过诱导细胞程序性坏死、增加肿瘤CD8^(+)T细胞浸润,改善CD8^(+)T细胞耗竭,其靶向脂质体能提高其抗肿瘤效应。

Objective To investigate the mechanism of parthenolide for inducing necroptosis and ameliorating CD8^(+)T cell exhaustion in colorectal cancer(CRC)and construct liposome nanoparticles for targeted delivery of parthenolide.Methods The inhibitory effect of parthenolide on proliferation of different CRC cell lines was examined using CCK8 assay,and ROS LDH detection and Western blotting were used to analyze the cell death pathways.In a mouse model bearing subcutaneous MC38 cell xenografts,the effects of 5 and 15 mg/kg parthenolide on tumor growth and CD8^(+)T cell depletion were observed.In a mouse model bearing orthotopic CRC cell xenograft in the ileocecal region,free parthenolide(100μg/mL)or low(100μg/mL)and high doses(200μg/mL)of liposome nanoparticles loaded with parthenolide were injected via the tail vein,and the changes in CD8 expression in the xenografts were analyzed using immunohistochemistry.Results Treatment with parthenolide dose-dependently lowered the viability of the CRC cell lines SW480,DLD1,HCT116 and MC38 cells,and its effect was obviously antagonized by Nec-1.Immunoblotting analysis showed that parthenolide treatment resulted in increased RIP3 and MLKL phosphorylation in the CRC cells.In the mouse model bearing subcutaneous xenografts,parthenolide treatment at the high dose,but not at the low dose,significantly increased the number of infiltrating CD3^(+)CD8^(+)T cells and PD1hiTIM3^(+)T cell percentage(P<0.01)and lowered the percentage of PD1loTIM3-T cells in the tumor tissue(P<0.01).In the mouse models bearing orthotopic CRC xenograft,intravenous injection of the liposomes loaded with parthenolide,especially at the high dose,significantly increased CD8 expression in the tumor tissue(P<0.01).Conclusion Parthenolide induces necroptosis in CRC and increases infiltrating CD8^(+)T cells to ameliorate CD8^(+)T cell exhaustion in the tumor.Liposome nanoparticles for targeted delivery of parthenolide produce stronger,anti-tumor effect.