Isolation and characterization of CD34+ blast-derived exosomes in acute myeloid leukemia.
Isolation and characterization of CD34+ blast-derived exosomes in acute myeloid leukemia.
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DOI：
10.1371/journal.pone.0103310
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Whiteside TL
中科院分区：
文献类型：
--
作者：
Hong CS;Muller L;Boyiadzis M;Whiteside TL
外来体是在所有生物流体中发现的膜结合囊泡。在诊断时收集的AML患者血浆相对于正常供体（ND）血浆含有升高的外泌体水平。AML外泌体的分子谱在治疗过程中发生变化，可以作为疾病进展或对治疗反应的衡量指标。然而，血浆含有来源于各种细胞类型的外来体的混合物。为了能够利用胚细胞来源的外泌体作为AML的生物标志物，我们开发了一种基于免疫亲和性的捕获方法，该方法利用了包被有抗CD 34抗体（Ab）的磁性微珠。该Ab对AML原始细胞的独特标志物CD 34具有特异性。使用来源于Kasumi-1 AML细胞培养物上清液的CD 34+外来体开发捕获程序。CD 34微珠的捕获能力显示与输入的外来体线性相关。10 uL等份的CD 34微珠能够捕获100- 1，000 uL AML血浆中存在的所有CD 34+外泌体。免疫捕获的CD 34+外泌体的水平与AML患者外周血中CD 34+原始细胞的百分比相关。免疫捕获的外泌体具有典型的杯状形态，通过透射电子显微镜，其分子货物是类似的亲本胚。这些外来体具有生物活性。在与自然杀伤（NK）细胞共孵育后，捕获的胚细胞来源的外泌体下调表面NKG 2D表达，而未捕获的外泌体降低NKp 46的表达水平。我们的数据提供了一个原理证明，即原始细胞衍生的外泌体可以从AML患者的血浆中定量回收，它们的分子谱概括了自体原始细胞的分子谱，并且它们保留了介导免疫抑制的能力。这些数据表明，免疫捕获的胚细胞来源的外泌体可能在未来的AML的诊断和/或预后中有用。
Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected at diagnosis contains elevated exosome levels relative to normal donor (ND) plasma. The molecular profile of AML exosomes changes in the course of therapy and may serve as a measure of disease progression or response to therapy. However, plasma contains a mix of exosomes derived from various cell types. To be able to utilize blast-derived exosomes as biomarkers for AML, we have developed an immunoaffinity-based capture method utilizing magnetic microbeads coated with anti-CD34 antibody (Ab). This Ab is specific for CD34, a unique marker of AML blasts. The capture procedure was developed using CD34+ exosomes derived from Kasumi-1 AML cell culture supernatants. The capture capacity of CD34microbeads was shown to linearly correlate with the input exosomes. A 10 uL aliquot of CD34 microbeads was able to capture all of CD34+ exosomes present in 100–1,000 uL of AML plasma. The levels of immunocaptured CD34+ exosomes correlated with the percentages of CD34+ blasts in the AML patients' peripheral blood. The immunocaptured exosomes had a typical cup-shaped morphology by transmission electron microscopy, and their molecular cargo was similar to that of parental blasts. These exosomes were biologically-active. Upon co-incubation with natural killer (NK) cells, captured blast-derived exosomes down-regulated surface NKG2D expression, while non-captured exosomes reduced expression levels of NKp46. Our data provide a proof-of-principle that blast-derived exosomes can be quantitatively recovered from AML patients' plasma, their molecular profile recapitulates that of autologous blasts and they retain the ability to mediate immune suppression. These data suggest that immunocaptured blast-derived exosomes might be useful in diagnosis and/or prognosis of AML in the future.
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