Koksal Ali Riza, Thevenot Paul, Aydin Yucel, Nunez Kelley, Sandow Tyler, Widmer Kyle, Nayak Leela, Scott John, Delk Molly, Moehlen Martin W, Cohen Ari J, Dash Srikanta
Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, USA.
Department of Gastroenterology and Hepatology, Tulane University Health Sciences Center, New Orleans, LA, USA.
J Hepatocell Carcinoma. 2022 Sep 7;9:959-972. doi: 10.2147/JHC.S376210. eCollection 2022.
HCC development in liver cirrhosis is associated with impaired autophagy leading to increased production of extracellular vesicles (EVs) including exosomes and microvesicles. The goal of the study is to determine which of these particles is primarily involved in releasing of HCC-specific biomarker glypican-3 (GPC3) when autophagy is impaired.
Streptavidin-coated magnetic beads were coupled with either biotinylated CD63 or Annexin A1 antibodies. Coupled beads were incubated with EVs isolated from either HCC culture or serum. EVs captured by immuno-magnetic beads were then stained with FITC or PE fluorescent-conjugated antibodies targeting exosomes (CD81), and microvesicles (ARF6). The percentage of GPC3 enrichment in the microvesicles and exosomes was quantified by flow cytometry. The impact of autophagy modulation on GPC3 enrichment in exosomes and microvesicles was assessed by treating cells with Torin 1 and Bafilomycin A1. For clinical validation, GPC3 content was quantified in microvesicles, and exosomes were isolated from the serum of patients with a recent HCC diagnosis.
The immune-magnetic bead assay distinguishes membrane-derived microvesicles from endosome-derived exosomes. The GPC3 expression was only seen in the CD63 beads group but not in the Annexin A1 beads group, confirming that in HCC, GPC3 is preferentially released through exosomes. Furthermore, we found that autophagy induction by Torin1 decreased GPC3-positive exosome secretion and decreased microvesicle release. Conversely, autophagy inhibition by Bafilomycin A1 increased the secretion of GPC3-positive exosomes. Serum analysis showed CD81+ve EVs were detected in exosomes and ARF6+ve vesicles were detected in microvesicles, suggesting that immunoaffinity assay is specific. The exosomal GPC3 enrichment was confirmed in isolated EVs from the serum of patients with HCC. The frequency of GPC3-positive exosomes was higher in patients with HCC (12.4%) compared to exosomes isolated from non-cirrhotic and healthy controls (3.7% and 1.3% respectively, p<0.001).
Our results show that GPC3 is enriched in the endolysosomal compartment and released in exosome fractions when autophagy is impaired.
肝硬化中肝癌的发展与自噬受损有关，导致包括外泌体和微囊泡在内的细胞外囊泡（EVs）产生增加。本研究的目的是确定当自噬受损时，这些颗粒中哪一种主要参与肝癌特异性生物标志物磷脂酰肌醇蛋白聚糖-3（GPC3）的释放。
将链霉亲和素包被的磁珠与生物素化的CD63或膜联蛋白A1抗体偶联。将偶联的磁珠与从肝癌培养物或血清中分离的EVs一起孵育。然后，用针对外泌体（CD81）和微囊泡（ARF6）的异硫氰酸荧光素（FITC）或藻红蛋白（PE）荧光共轭抗体对免疫磁珠捕获的EVs进行染色。通过流式细胞术对微囊泡和外泌体中GPC3富集的百分比进行定量。通过用托瑞米芬1和巴弗洛霉素A1处理细胞，评估自噬调节对外泌体和微囊泡中GPC3富集的影响。为了进行临床验证，对微囊泡中的GPC3含量进行定量，并从近期诊断为肝癌的患者血清中分离外泌体。
免疫磁珠分析可区分膜来源的微囊泡和内体来源的外泌体。GPC3表达仅在CD63磁珠组中可见，而在膜联蛋白A第1磁珠组中未见，这证实了在肝癌中，GPC3优先通过外泌体释放。此外，我们发现托瑞米芬1诱导自噬可减少GPC3阳性外泌体的分泌并减少微囊泡的释放。相反，巴弗洛霉素A1抑制自噬可增加GPC3阳性外泌体的分泌。血清分析显示，在外泌体中检测到CD81阳性EVs，在微囊泡中检测到ARF6阳性囊泡，这表明免疫亲和分析具有特异性。在肝癌患者血清中分离的EVs中证实了外泌体中GPC3的富集。与从非肝硬化和健康对照中分离的外泌体相比，肝癌患者中GPC3阳性外泌体的频率更高（分别为12.4%、非肝硬化患者为3.7%、健康对照为1.3%，p<0.001）。
我们的结果表明，当自噬受损时，GPC3在内溶酶体区室中富集并在外泌体部分中释放。
