背景:透明细胞肾细胞癌(ccRCC)是最常见的肾癌亚型。大规模代谢组学数据将代谢改变与肾癌的发病机制和进展相关,并将线粒体活性与部分患者的不良生存率相关。这项研究的目的是确定靶向线粒体-溶酶体相互作用是否可能是使用患者来源的类器官作为药物反应的化身的新型治疗方法。
方法:RNAseq数据分析和免疫组织化学用于显示透明细胞癌中嘌呤能受体4(P2XR4)的过表达。海马实验,免疫荧光和荧光细胞分选用于证明P2XR4调节线粒体活性和自由基氧物种的平衡。药物抑制剂和基因沉默促进溶酶体损伤,线粒体钙超载和细胞死亡通过坏死和凋亡。最后,我们建立了患者来源的类器官和鼠异种移植模型,通过成像药物筛选研究P2XR4抑制的抗肿瘤作用,活力测定和免疫组织化学。
结果:我们的数据表明,在表达P2XR4的ccRCC细胞亚群中,氧代磷酸化是肿瘤源性ATP的主要来源,对肿瘤能量代谢和线粒体活性具有重要影响。由药物抑制或P2XR4沉默引起的延长的线粒体衰竭与氧自由基种类增加有关。线粒体通透性的变化(即,过渡孔复合物的开口,膜电位的耗散,和钙超载)。有趣的是,在异种移植模型中,患者来源的类器官中线粒体活性较高与对P2XR4抑制和肿瘤减少的敏感性较高相关.
结论:总体而言,我们的研究结果表明,P2XR4抑制引起的溶酶体完整性和线粒体活性之间的失衡可能代表了对一部分肾癌患者的新治疗策略,个体化的类器官可能有助于预测药物疗效.
BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer. Large-scale metabolomic data have associated metabolic alterations with the pathogenesis and progression of renal carcinoma and have correlated mitochondrial activity with poor survival in a subset of patients. The aim of this study was to determine whether targeting mitochondria-lysosome interaction could be a novel therapeutic approach using patient-derived organoids as avatar for drug response.
METHODS: RNAseq data analysis and immunohistochemistry were used to show overexpression of Purinergic receptor 4 (P2XR4) in clear cell carcinomas. Seahorse experiments, immunofluorescence and fluorescence cell sorting were used to demonstrate that P2XR4 regulates mitochondrial activity and the balance of radical oxygen species. Pharmacological inhibitors and genetic silencing promoted lysosomal damage, calcium overload in mitochondria and cell death via both necrosis and apoptosis. Finally, we established patient-derived organoids and murine xenograft models to investigate the antitumor effect of P2XR4 inhibition using imaging drug screening, viability assay and immunohistochemistry.
RESULTS: Our data suggest that oxo-phosphorylation is the main source of tumor-derived ATP in a subset of ccRCC cells expressing P2XR4, which exerts a critical impact on tumor energy metabolism and mitochondrial activity. Prolonged mitochondrial failure induced by pharmacological inhibition or P2XR4 silencing was associated with increased oxygen radical species, changes in mitochondrial permeability (i.e., opening of the transition pore complex, dissipation of membrane potential, and calcium overload). Interestingly, higher mitochondrial activity in patient derived organoids was associated with greater sensitivity to P2XR4 inhibition and tumor reduction in a xenograft model.
CONCLUSIONS: Overall, our results suggest that the perturbed balance between lysosomal integrity and mitochondrial activity induced by P2XR4 inhibition may represent a new therapeutic strategy for a subset of patients with renal carcinoma and that individualized organoids may be help to predict drug efficacy.