Tumor cell-derived LC3B⁺extracellular vesicles mediate the crosstalk between tumor microenvironment and immunotherapy efficacy in hepatocellular carcinoma via the HSP90-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 HSP90 alpha in peripheral blood of liver cancer patients. We correlated the levels of plasma IL-6, IL-8 with LC3B(+) EVs carrying HSP90 alpha 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 HSP90 alpha, 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 HSP90 alpha 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 HSP90 alpha. 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 HSP90 alpha 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 HSP90 alpha. Targeting HSP90 alpha 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.