Platelet Activation in Ovarian Cancer Ascites: Assessment of GPIIb/IIIa and PF4 in Small Extracellular Vesicles by Nano-Flow Cytometry Analysis.
Platelet Activation in Ovarian Cancer Ascites: Assessment of GPIIb/IIIa and PF4 in Small Extracellular Vesicles by Nano-Flow Cytometry Analysis.
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血小板在凝血和纤溶过程中发挥着关键作用，但最近的文献也表明，它们在免疫反应、癌症进展和转移中发挥着重要作用。在血小板激活的过程中，小的细胞外小泡(EVS)被释放。腹水是晚期卵巢癌患者腹膜内形成的一种液体。这项研究分析了从晚期卵巢癌患者腹水中分离的小EVS人群中血小板标志物GPIIb/IIIa和PF4的表达。血小板来源的小EVS的百分比与血小板分布宽度/血清中血小板计数(PDW/PLT)呈正相关，PDW/PLT是反映血小板活化的替代指标。总体而言，我们提出了一种方法，可以在未来的研究中帮助确定腹水中血小板激活程度与疾病状态之间的相关性。在卵巢癌中，腹水代表了血小板在疾病进展中发挥作用的微环境。我们的目标是开发一种检测腹水中血小板活化的方法。采用聚乙二醇沉淀法从12例卵巢上皮性癌患者腹水中浓缩40~200 nm的胞外小泡(EV)，经标记和超速离心后用纳米流式细胞仪进行单颗粒表型分析。原子力显微镜单粒子纳米力学分析表明，析出粒子的尺寸和机械硬度分布不均。用针对血小板标志物GPIIb/IIIa和PF4的抗体对样本进行荧光标记，显示2.6%至18.16%的颗粒对生物标志物和EV膜标记呈阳性。单颗粒表型分析使我们能够量化非EV颗粒的总数、小EV的数量和血小板衍生的小EV的数量，提供独立于腹水量的血小板激活评估。血小板源EVS百分率与血清中血小板分布宽度/血小板计数(PDW/PLT)呈正相关。总体而言，我们提出了一种高通量的方法，有助于在未来的研究中确定腹水中血小板激活的程度与疾病状态之间的相关性。
Platelets play a critical role in coagulation and fibrinolysis processes, but recent literature also indicates their central involvement in immune response, cancer progression and metastasis. During platelet activation, small extracellular vesicles (EVs) are released. The ascites are a fluid developing in the peritoneum of ovarian cancer patients in an advanced stage. This study analysed the expression of platelet markers GPIIb/IIIa and PF4 in small-EVs populations isolated from the ascitic fluid of patients with advanced ovarian cancer. The percentage of platelet-derived small-EVs was positively correlated with platelet distribution width to platelet count in sera (PDW/PLT), a surrogate indicator of platelet activation. Overall, we presented a method that can be helpful in future studies to determine the correlation between the extent of platelet activation in ascites and disease status. In ovarian cancer, ascites represent the microenvironment in which the platelets extravasate to play their role in the disease progression. We aimed to develop an assay to measure ascites’ platelet activation. We enriched small extracellular vesicles (EVs) (40–200 nm) from ascites of high-grade epithelial ovarian cancer patients (n = 12) using precipitation with polyethylene glycol, and we conducted single-particle phenotyping analysis by nano-flow cytometry after labelling and ultra-centrifugation. Atomic force microscopy single-particle nanomechanical analysis showed heterogeneous distributions in the size of the precipitated particles and their mechanical stiffness. Samples were fluorescently labelled with antibodies specific to the platelet markers GPIIb/IIIa and PF4, showing 2.6 to 18.16% of all particles stained positive for the biomarkers and, simultaneously, the EV membrane labelling. Single-particle phenotyping analysis allowed us to quantify the total number of non-EV particles, the number of small-EVs and the number of platelet-derived small-EVs, providing a platelet activation assessment independent of the ascites volume. The percentage of platelet-derived small-EVs was positively correlated with platelet distribution width to platelet count in sera (PDW/PLT). Overall, we presented a high-throughput method that can be helpful in future studies to determine the correlation between the extent of platelet activation in ascites and disease status.
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