背景:酪氨酸激酶抑制剂(TKI)最初被证明是肾细胞癌(RCC)的有效治疗方法。然而,在中位治疗时间为14个月后,绝大多数患者产生耐药性。这项研究分析了针对外泌体赋予耐药性的替比法尼(Tipi)+舒尼替尼的联合治疗。
方法:786-O,786-O-SR(舒尼替尼耐药),A498,A498-SR,Caki-2,Caki-2-SR,培养293T细胞。使用差异超速离心收集外来体。细胞增殖,JurkatT细胞免疫测定,和免疫印迹分析用于下游分析。
结果:SR外泌体处理对免疫细胞显示出细胞毒性作用。与舒尼替尼敏感(SS)外泌体相比,这种细胞毒性作用与SR外泌体上PD-L1的表达增加有关。此外,Tipi处理下调源自SR细胞系的外泌体上的PD-L1表达。Tipi下调外泌体中PD-L1的能力在患者中具有重要应用。从患有RCC的患者收集的外泌体显示比没有RCC的受试者增加的PD-L1表达。接下来,然后在Tipi治疗后比较外泌体浓度,所有SS细胞系显示更大的减少。在免疫印迹分析中,293T细胞显示Alix的剂量依赖性增加,nSMase或Rab27a均无变化。相反,所有SS和SR细胞系在所有三种标志物中显示出减少。在对所有SS和SR细胞系进行48小时处理的细胞增殖后,药物组合显示出降低肿瘤生长的协同能力。
结论:Tipifarnib减弱了转运(ESCRT)依赖性和ESCRT非依赖性途径所需的内体分选复合物所需的外泌体内体分选复合物,从而阻断外泌体生物发生和分泌以及下调SS和SR细胞上的PD-L1。
BACKGROUND: Tyrosine kinase inhibitors (TKI) were initially demonstrated as an efficacious treatment for renal cell carcinoma (RCC). However, after a median treatment length of 14 months, a vast majority of patients develop resistance. This study analyzed a combination therapy of
tipifarnib (Tipi) + sunitinib that targeted exosome-conferred drug resistance.
METHODS: 786-O, 786-O-SR (sunitinib resistant), A498, A498-SR, Caki-2, Caki-2-SR, and 293T cells were cultured. Exosomes were collected using differential ultracentrifugation. Cell proliferation, Jurkat T cell immune assay, and immunoblot analysis were used for downstream analysis.
RESULTS: SR exosomes treatment displayed a cytotoxic effect on immune cells. This cytotoxic effect was associated with increased expression of PD-L1 on SR exosomes when compared to sunitinib-sensitive (SS) exosomes. Additionally, Tipi treatment downregulated PD-L1 expression on exosomes derived from SR cell lines. Tipi\'s ability to downregulate PD-L1 in exosomes has a significant application within patients. Exosomes collected from patients with RCC showed increased PD-L1 expression over subjects without RCC. Next, exosome concentrations were then compared after Tipi treatment, with all SS cell lines displaying an even greater reduction. On immunoblot assay, 293T cells showed a dose-dependent increase in Alix with no change in either nSMase or Rab27a. Conversely, all the SS and SR cell lines displayed a decrease in all three markers. After a cell proliferation employed a 48-h treatment on all SS and SR cell lines, the drug combination displayed synergistic ability to decrease tumor growth.
CONCLUSIONS: Tipifarnib attenuates both the exosome endosomal sorting complex required for endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways, thereby blocking exosome biogenesis and secretion as well as downregulating PD-L1 on SS and SR cells.