Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients.
Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients.
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DOI：
10.3390/cancers12113297
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发表时间：
2020-11-07
期刊：
影响因子：
5.2
通讯作者：
Broekman MLD
中科院分区：
文献类型：
--
作者：
Maas SLN;van Solinge TS;Schnoor R;Yekula A;Senders JT;de Vrij J;Robe P;Carter BS;Balaj L;Arkesteijn GJA;Nolte-'t Hoen ENM;Broekman MLD
胶质母细胞瘤（GB）是一种中枢神经系统的恶性肿瘤，目前只能通过组织活检进行诊断。在这项研究中，我们能够分离出GB衍生的细胞外囊泡（EV）的血液中，患者接受5-氨基乙酰丙酸（5-ALA）手术前。这种分离是基于荧光原卟啉IX（PpIX）在这些EV中积累引起的荧光。我们发现这些EV含有各种GB相关的microRNA。虽然我们的技术在能够在临床上实施之前有许多方法需要改进，但这项研究表明，基于PpIX荧光检测和分析循环GB衍生的EV是可行的。在未来，我们的技术可以通过血液样本而不是脑活检来诊断和监测GB。背景：在胶质母细胞瘤（GB）中，组织是准确诊断和分型所必需的。组织可以通过切除或（立体定向）活检获得，但这些侵入性手术给患者带来风险。细胞外囊泡（EV）是含有miRNA、蛋白质和脂质的小的、细胞衍生的囊泡，并且是液体活检的可能候选物。可以在患者的血液中发现GB衍生的EV，但很难将它们与循环的非肿瘤EV区分开来。5-将氨基乙酰丙酸（5-ALA）口服给予GB患者以促进肿瘤可视化和最大切除，因为其代谢为在神经胶质瘤细胞中积累的荧光原卟啉IX（PpIX）。在这项研究中，我们评估了PpIX是否在GB衍生的EV中积累，以及这些EV是否可以被分离和表征以在GB中进行液体活检。方法：采用差速离心法从5-ALA处理的U87细胞条件培养液中分离EV。从健康对照组和接受5-ALA引导的GB手术的患者中收集并处理血液样品。高分辨率流式细胞术（hFC）能够检测和分选PpIX阳性EV，随后通过数字液滴PCR（ddPCR）对其进行分析。结果：PpIX阳性EV可以在条件细胞培养基中检测到，以及在5-ALA给药后的患者样本中检测到。通过使用hFC，我们可以分选PpIX阳性EV，用于ddPCR的进一步分析，这表明EV和GB相关miRNA的存在。结论：5-ALA诱导荧光法可从GB患者血浆中分离到GB源性EV。尽管仍存在许多挑战，但我们的研究结果显示了GB患者血液液体活检的新可能性。
In Glioblastoma (GB), a malignant tumor of the central nervous system, diagnosis can currently only be obtained via tissue biopsy. In this study we were able to isolate GB derived extracellular vesicles (EVs) in the blood, after patients received 5-aminolevulinic acid (5-ALA) before surgery. This isolation is based on fluorescence caused by the accumulation of fluorescent protoporphyrin IX (PpIX) in these EVs. We show that these EVs contain various GB-related micro RNAs. While there are many ways in which our technique needs to be improved before being able to be implemented in the clinic, this study shows that detecting and analyzing circulating GB-derived EVs based on PpIX fluorescence is feasible. In the future, our technique could be developed to diagnose and monitor GB via blood samples instead of a brain biopsy. Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.
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影响因子：
16.6
作者：
Clancy, James W.;Sedgwick, Alanna;Rosse, Carine;Muralidharan-Chari, Vandhana;Raposo, Graca;Method, Michael;Chavrier, Philippe;D'Souza-Schorey, Crislyn
通讯作者：
D'Souza-Schorey, Crislyn
影响因子：
2
作者：
Johnson, Derek R.;O'Neill, Brian P.;Hammack, Julie E.
通讯作者：
Hammack, Julie E.
影响因子：
120.7
作者：
Jakola, Asgeir S.;Myrmel, Kristin S.;Solheim, Ole
通讯作者：
Solheim, Ole
侵入性手术程序对原发性肿瘤生长和转移的潜在影响。
影响因子：
4
作者：
Alieva M;van Rheenen J;Broekman MLD
通讯作者：
Broekman MLD
DOI：
10.1016/s1011-1344(96)07358-7
发表时间：
1996-11-01
影响因子：
5.4
作者：
Krammer, B;Uberriegler, K
通讯作者：
Uberriegler, K