Tumor cell-derived LC3B+extracellular vesicles mediate the crosstalk between tumor microenvironment and immunotherapy efficacy in hepatocellular carcinoma via the HSP90α-IL-6/IL-8 signaling axis.

BACKGROUND: Inflammatory factors are being recognized as critical modulators of host antitumor immunity in liver cancer. We have previously shown that tumor cell-released LC3B positive extracellular vesicles (LC3B+ EVs) are responsible for malignant progression by dampening antitumor immunity. However, the relationship between LC3B+ EVs and inflammatory factors in the regulation of the liver cancer microenvironment remains unclear. METHODS: Flow cytometry analyses were performed to examine the panel of 12 cytokines, the main source of positive cytokines, and plasma LC3B+ EVs carrying HSP90α in peripheral blood of liver cancer patients. We correlated the levels of plasma IL-6, IL-8 with LC3B+ EVs carrying HSP90α and with prognosis. In vitro culture of healthy donor leukocytes with liver cancer-derived LC3B+ EVs was performed to evaluate the potential effect of blocking HSP90α, IL-6 or IL-8 alone or in combination with PD-1 inhibitor on CD8+ T cell function. We also investigated the potential associations of MAP1LC3B, HSP90AA1, IL6 or IL8 with immunotherapy efficacy using the TCGA databases. RESULTS: In liver cancer patients, plasma IL-6 and IL-8 levels were significantly higher than in healthy controls and associated with poor clinical outcome. In peripheral blood, levels of plasma LC3B+ EVs carrying HSP90α were significantly elevated in HCC patients and positively associated with IL-6 and IL-8 levels, which are predominantly secreted by monocytes and neutrophils. Moreover, LC3B+ EVs from human liver cancer cells promoted the secretion of IL-6 and IL-8 by leukocytes through HSP90α. Besides, we show that the cytokines IL-6 and IL-8 secreted by LC3B+ EVs-induced leukocytes were involved in the inhibition of CD8+ T-cell function, while blockade of the HSP90α on the LC3B+ EVs, IL-6, or IL-8 could enhance anti-PD-1-induced T cell reinvigoration. Finally, patients who received anti-PD-1/PD-L1 immunotherapy with high MAP1LC3B, HSP90AA1, IL6, or IL8 expression had a lower immunotherapy efficacy. CONCLUSIONS: Our data suggest that liver cancer-derived LC3B+ EVs promote a pro-oncogenic inflammatory microenvironment by carrying membrane-bound HSP90α. Targeting HSP90α on the LC3B+ EVs, IL-6, or IL-8 may synergize with anti-PD-1 treatment to enhance the CD8+ T-cell functions, which may provide novel combination strategies in the clinic for the treatment of liver cancer.

Interferon-α induces differentiation of cancer stem cells and immunosuppression in hepatocellular carcinoma by upregulating CXCL8 secretion.

Interferon-alpha (IFN-α) is widely used in the clinical treatment of patients with chronic hepatitis B and hepatocellular carcinoma (HCC). However, high levels of CXCL8 are associated with resistance to IFN-α therapy and poorer prognosis in advanced cancers. In this study, we investigated whether IFN-α could directly induce the production of CXCL8 in HCC cells and whether CXCL8 could antagonize the antitumor activity of IFN-α. We found that IFN-α not only upregulated the expression of the inducible genes CXCL9, CXCL10, CXCL11 and PD-L1, but also significantly stimulated CXCL8 secretion in HCC cells. Mechanically, IFN-α induces CXCL8 expression by activating the AKT and JNK pathways. In addition, our results demonstrate that IFN-α exposure significantly increases the differentiation of HCC stem cells, but this effect is reversed by the addition of the CXCL8 receptor CXCR1/2 inhibitor Reparixin and STAT3 inhibitor Stattic. Besides, our study reveals that the cytokine CXCL8 secreted by IFN-α-induced HCC cells inhibits T-cell function. Conversely, inhibition of CXCL8 promotes TNF-α and IFN-γ secretion by T cells. Finally, liver cancer patients who received anti-PD-1/PD-L1 immunotherapy with high CXCL8 expression had a lower immunotherapy efficacy. Overall, our findings clarify that IFN-α triggers immunosuppression and cancer stem cell differentiation in hepatocellular carcinoma by upregulating CXCL8 secretion. This discovery provides a novel approach to enhance the effectiveness of HCC treatment in the future.

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 HSP90α+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 HSP90α in LC3B+ EVs and PD-1highCD8+ 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 HSP90α+LC3B+ EVs in liver cancer patients were significantly higher than in non-malignant liver disease patients and healthy controls. The expression of HSP90α 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 HSP90α+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 HSP90α+LC3B+ EVs positively correlated with the PD-1highCD8+ 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-γ 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.