Evaluation of Plasma LC3B+extracellular Vesicles As a Potential Novel Diagnostic Marker for Hepatocellular Carcinoma.

Background: Circulating extracellular vesicles (EVs) are recognized as a promising source of cancer biomarkers. We previously reported that tumor cell-released autophagosomes, a new subgroup of EVs expressing the mature autophagosome-specific marker LC3B (LC3B(+) EVs), are critical modulators of host anti-tumor immunity. This study aimed to assess the level of plasma LC3B(+) EVs and the correlation with clinical outcomes in liver cancer patients. Methods: The plasma and ascites samples were obtained from patients with liver cancer, non-malignant liver disease, and healthy controls. EVs were isolated by differential centrifugation and characterized using flow cytometry, nanoparticle tracking analysis, transmission electron microscopy, and western blotting. Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic efficacy of plasma LC3B(+) EVs or HSP90 alpha(+)LC3B+ EVs from liver cancer patients. The relationship between the expression levels of HSP90AA1 or MAP1LC3B and survival were analyzed using patient data from the TCGA database. The correlation between HSP90 alpha in LC3B+ EVs and PD-1(high)CD8(+) exhausted T cells from the ascites and peripheral blood of liver cancer patients was also evaluated. Results: The EVs preparation from liver cancer patients contained LC3B(+) EVs expressing epithelial tumor cell adhesion molecules (EpCAM), indicating that these LC3B(+) EVs originated from epithelial tumor cells. The levels of plasma LC3B(+) EVs and HSP90 alpha(+)LC3B(+) EVs in liver cancer patients were significantly higher than in nonmalignant liver disease patients and healthy controls. The expression of HSP90 alpha in plasma LC3B(+) EVs (AUC 0.9595, sensitivity 86.00%, specificity 96.67%) accurately differentiated liver cancer patients from non-liver cancer controls. Additionally, a significant decrease in the levels of plasma LC3B(+) EVs and HSP90 alpha(+)LC3B(+) EVs was found post-surgery in each patient, and high expression of HSP90AA1 or MAP1LC3B in the tumor tissue correlated with significantly worse survival compared to those with low expression. We also observed that the level of LC3B(+) EVs and HSP90 alpha(+)LC3B(+) EVs positively correlated with the PD-1(high)CD8(+) exhausted T cells in liver cancer patients. Human CD8(+) T cells treated with purified LC3B(+) EVs in vitro exhibited a dose-dependent increase in the percentage of PD-1(+)CD8(+) T cells, whereas the production of IFN-gamma was decreased. Conclusions: We demonstrated that isolation and detection of plasma LC3B(+) EVs carrying bioactive molecules is an effective diagnostic marker of liver cancer, and may also be used as a potential marker for immune monitoring and predicting prognosis clinically.