Extracellular vesicles as dynamic surrogates for drug transporter expression. A new tool to personalize pharmacotherapy?

P-糖蛋白(P-gp/ABCB1)、多药耐药相关蛋白2(MRP2/ABCC2)和乳腺癌耐药蛋白(BCRP/ABCG2)属于ABC转运蛋白超家族(ATP结合盒)，在药物动力学中起重要作用。它们在肝、肾、肠和血脑屏障等具有高度药理相关性的器官和组织中表达。此外，它们还解释了癌细胞对化疗药物的耐药性。ABC转运蛋白在人群中的表达是高度可变的，可以受到联合用药、饮食和激素等因素的调节。通过这种方式，可以预期个体之间以及在个体寿命期间的药物清除量会有显著的差异。一般来说，较高的转运蛋白表达会导致较高的药物清除率和治疗失败。较低的转运蛋白表达导致较低的药物清除率和毒性。因此，对ABC转运蛋白表达的个体化估计可以帮助调整治疗，以避免暴露过多和暴露不足的情况。到目前为止，重复提取活检组织将是测量ABC转运蛋白在相关组织中表达的唯一选择。然而，由于其固有的侵袭性，这是不能常规进行的。细胞外小泡(EV)是由有机体的所有细胞类型释放的纳米颗粒。EV货物由细胞蛋白质、RNA和脂类组成，这些成分的阵列代表了起源细胞的指纹。来自血液中广泛组织的EVS的存在，加上尿液中存在肾脏EVS，将使临床实践中很容易获得这些纳米颗粒。该项目的总体目标是研究ABC转运体-货物在EVS中作为转运体在正常和肿瘤细胞中表达的动态替代物的潜力，从而研究其药物排泄能力。我们将建立、优化和验证一种超灵敏的UPLC-MS/MS方法，用于绝对和同时定量EVS和来源细胞中的P-gp、MRP2和BCRP。本实验将用于分析在基础和诱导条件下转运蛋白在EVS中的表达与细胞中表达和活性的相关性。在肿瘤细胞中，转运体在EVS中的表达与化疗药物耐药性之间的相关性也将被研究。最后，将开发一种从多细胞模型(血-脑-屏障有机物)中分离细胞特异性EVS的方法，以阐明EVS在更复杂的系统中估计细胞特异性转运蛋白表达的潜力。总之，使用EVS来估计转运体的表达可以允许根据患者的药物清除能力对患者进行分层，从而调整药物治疗，最终防止毒性或治疗失败的情况。

The P-glycoprotein (P-gp/ABCB1), the multidrug resistance-associated protein 2 (MRP2/ABCC2) and the breast cancer resistance protein (BCRP/ABCG2) belong to the superfamily of ABC transporters (ATP binding cassette) and play a major role in drug pharmacokinetics. They are expressed in organs and tissues of high pharmacological relevance such as the liver, the kidneys, the intestine and the blood-brain barrier. In addition, they account for the resistance towards chemotherapeutic agents in cancer cells. The expression of ABC transporters is highly variable within the population and can be regulated by coadministered drugs, the diet and hormones, among other factors. This way, significant differences in the drug clearance between individuals as well as during the lifespan of an individual can be expected. In general, higher transporter expression leads to higher drug clearance and therapy failure. Lower transporter expression leads to lower drug clearance and toxicity. Thus, the individualized estimation of the expression of ABC transporters could aid to adjust the therapy to avoid cases of over- and under-exposure. So far, repeated extraction of biopsies would be the only option to measure ABC transporter expression in relevant tissues. However, due to its intrinsic invasive character, this cannot be performed routinely.Extracellular vesicles (EVs) are nanoparticles released by all cell types of the organism. The EV cargo consists of cellular proteins, RNA, and lipids, the array of these components representing a fingerprint of the cell of origin. The presence of EVs from a wide range of tissues in the blood, plus the presence of kidney EVs in the urine, would allow for an easy access to these nanoparticles in the clinical practice. The general aim of this project is to investigate the potential of the ABC transporter-cargo in EVs as a dynamic surrogate for the transporter expression in normal and tumoral cells and thus, for their drug excretion capacity. We will develop, optimize and validate an ultrasensitive UPLC-MS/MS assay for the absolute and simultaneous quantification of P-gp, MRP2 and BCRP in EVs and the cells of origin. This assay will be applied to analyze the correlation between the transporter expression in EVs and the expression and activity in the cells both under basal and inducing conditions. In tumor cells, the correlation between the transporter expression in EVs and the resistance to chemotherapeutic agents will also be investigated. Finally, a method to isolate cell-specific EVs from a multicellular model (blood-brain-barrier organoids) will be developed in order to elucidate the potential of EVs to estimate the cell-specific transporter expression in a more complex system. Altogether, the use of EVs to estimate the transporter expression could allow to stratify the patients based on their drug clearance capacity and, thus, adjust pharmacotherapy and, ultimately, prevent situations of toxicity or therapy failure.