Article Figures & Data
Figures
- Figure 1
Downregulation of circTMEM56 is associated with a poor radiotherapy (RT) response and prognosis in patients with liver cancer. (A) t-SNE diagram of CD45+ major tumor-infiltrating immune cell types in patients with and without abscopal effects (AEs). (B) CD8+ T cell level and number of dendritic cells (DCs) in the blood of patients with and without AEs. (C) IFN-β level in the blood of patients with and without AEs. (D) Peripheral blood CD141+ DCs (cDC1 subset) levels in AE-positive versus AE-negative patient cohorts. (E) Heat maps of circRNA expression in tumor tissues of liver cancer patients with and without AEs after RT. (F) Differentially expressed circRNAs identified in tumor tissues and plasma exosomes of patients with and without AEs. (G) qPCR results showing circTMEM56 transcribed using primers from cDNA. (H) Schematic diagram of circTMEM56. Sanger sequencing revealed back-splicing sites. (I) qPCR results showing significantly higher circTMEM56 expression in hepatocellular carcinoma (HCC) tissues of patients with AEs compared to patients without AEs. (J) qPCR results showing significantly higher circTMEM56 expression in the blood of patients with AEs compared to patients without AEs. (K) In situ hybridization results indicating significantly higher expression of circTMEM56 in HCC tissues of patients with AEs compared to patients without AEs. (L) Variations in circTEME56 expression in 30 HCC tissues and adjacent tissues. (M) In situ hybridization results showing significantly lower circTMEM56 expression in HCC tissues compared to para-cancer tissues. (N) Kaplan-Meier analysis of overall survival (OS) and progression-free survival (PFS) in 209 HCC patients was performed based on circTEME56 expression. *P < 0.05; **P < 0.01; ***P < 0.001.
- Figure 2
Exosome circTMEM56 from hepatocellular carcinoma (HCC) cells is associated with the radiotherapy (RT) effect. (A) Representative image of exosomes extracted from patient blood using ultracentrifugation. Exosome markers are detected on exosomes enriched in patient blood. (B) circTMEM56 expression in patient blood with an abscopal effect (AE) is significantly higher than patients without AEs. (C) circTMEM56 levels in the blood of 76 patients with HCC after RT analyzed by qPCR. (D) Interferon-beta (IFN-β) levels in the blood of 76 patients with HCC after RT analyzed by ELISA. (E) Number of cDC1s in the blood of 76 patients with HCC after RT. (F) Correlation analysis showing a positive relationship between circTMEM56 and the IFN-β level. (G) Correlation analysis showing a positive relationship between circTMEM56 and the number of cDC1s. (H) Representative images of circTMEM56 and CD141 staining in HCC. (I) Correlation analysis showing a positive relationship between circTMEM56 and the IFN-β level in 209 patients with HCC. ***P < 0.001.
- Figure 3
circTMEM56 increases the production of type I IFN (IFN-I) from dendritic cells (DCs) and mediates an anti-tumor immune response. (A) circTMEM56 level measured by qPCR in 7 different hepatocellular carcinoma (HCC) cell lines. (B) Efficacy of circTMEM56 overexpression and interference analyzed by qRT-PCR. (C) dsDNA content in the cell supernatant following different radiation doses. (D) Extracellular dsDNA content detected by immunofluorescence after different radiation doses. (E) Effects of circTMEM56 overexpression and knockdown on dsDNA release from hepatocellular carcinoma cells after radiation. (F) Effects of circTMEM56 overexpression and knockdown on IFNB1 mRNA levels in hepatocellular carcinoma cells after radiation. (G) IFNB1 mRNA levels in DCs after co-culturing with irradiated liver cancer cells. (H) Number of DCs after co-culturing with irradiated liver cancer cells. (I) IFN-β concentration in the supernatant after co-culturing of DCs with irradiated hepatocellular carcinoma cells. (J) Changes in IFNB1 mRNA levels in DCs after co-culturing with irradiated HCCs with or without GW4869. (K) Number of DCs after co-culturing with irradiated hepatocellular carcinoma cells with or without GW4869. ***P < 0.001.
- Figure 4
circTMEM56 amplifies radiotherapy (RT)-induced anti-tumor immune response through the miR-136-5p/STING1 axis in dendritic cells (DCs). (A) RIP was performed for circRNA in DCs using a circTMEM56 probe and a negative control (NC) probe. (B) Putative binding sites of miR-136-5p with respect to circTMEM56 predicated via StarBase v3.0. (C) Luciferase activity of pLG3-circTMEM56 in 293T cells after co-transfection with miR-136-5p. (D) circTMEM56 levels in the streptavidin-captured fractions of the 293T cell lysates after transfection with biotinylated miR-136-5p or the NC. (E) FISH analysis in hepatocellular carcinoma (HCC) showing co-localization of circTMEM56 with miR-136-5p in the cytoplasm. (F) A graph showing overlap of differentiated proteins. (G) GO and KEGG analyses were performed. (H) Levels of STING and CXCL10 protein expression in DCs with varying levels of circTMEM56 expression detected by western blot analysis. (I) Putative binding site of miR-136-5p on STING via StarBase v3.0. (J) Luciferase activity of pLG3-STING in 293T cells co-transfected with miR-136-5p. (K) STING mRNA levels in DCs expressed by different miR-136-5p or circTMEM56. (L) Interferon-β (IFN-β) levels in DC supernatant detected after co-culturing with irradiated HCC cells with different levels of miR-136-5p or circTMEM56 expression. (M) IFN-β levels in DCs supernatant after co-culturing with irradiated HCC cells and varying levels of STING expression. ***P < 0.001.
- Figure 5
Elevated exosome circTMEM56 specifically targets dendritic cells (DCs) and promotes activation of cGAS-STING pathway. (A) Relative levels of miR-136-5p in THP-1, macrophages, and cDC1 detected by qPCR. (B) Levels of STING expression in THP-1, M2 macrophages, and cDC1 cultured in conditioned medium and detected by qPCR. (C) Interferon-β (IFN-β) levels in the supernatant of THP-1 cells after co-culturing with irradiated liver cancer cells and the number of CD8+ T cells detected after co-culturing with CD8+ T cells. (D) IFN-β levels in the supernatant of M2 macrophages after co-culturing with irradiated liver cancer cells and the number of CD8+ T cells detected after co-culturing with CD8+ T cells. (E) Activation of STING detected by western blot at different radiation doses and time points. (F) Western blot analysis of STING pathway activation in DCs cultured in different conditioned media. (G) Western blot analysis of STING pathway activation in DCs cultured with different miR-136-5p levels in different conditioned media. (H) Western blot analysis to detect STING pathway activation in DCs with different levels of STING expression cultured in different conditioned media. (I) Representative images of CD8, CD141, and p-STING staining in HCC tissues. ***P < 0.001.
- Figure 6
Exosome circTMEM56 improves the radiotherapy (RT)-induced anti-tumor immunity efficacy in vivo. (A) Representative CT images of the abdomen of mice with corresponding quantification. (B) Representative images of CD8 and CD11c staining in hepatocellular carcinoma (HCC). (C) Representative CT images of the abdomen of mice with corresponding quantification. (D) Representative images of CD8 and CD11c staining in HCC. (E) Representative photographs of the Hepa1-6 subcutaneous tumor model, imaged using the IVIS Imaging System. *P < 0.05; ***P < 0.001.
- Figure 7
Working model: circTMEM56 regulates cGAS-STING signaling pathway in DCs. miR-136-5p in the cytoplasm exerts an inhibitory effect on the STING activation before radiation (left panel). Under normal conditions STING is activated when DCs detect dsDNA released by tumor cells. Once activated STING moves from the endoplasmic reticulum to the Golgi apparatus, where STING interacts with TBK1 on the Golgi membrane. However, the inhibitory action of miR-136-5p impairs STING activity and prevents STING translocation and interaction with TBK1, thereby disrupting IRF3 phosphorylation and leading to suppression of downstream IFN signaling. Consequently, the STING signaling pathway remains in a relatively inactive state, limiting immune activation and impairing IFN-mediated responses. Radiation induces the upregulation of circTMEM56 in the cytoplasm (right panel), which acts as a ceRNA to sponge miR-136-5p, thereby reducing the inhibitory effects on STING signaling. Consequently, activation of STING leads to enhanced recruitment of TBK1 and phosphorylation of IRF3. Phosphorylated IRF3 translocates into the nucleus, where phosphorylated IRF3 binds to specific promoter regions of DNA and promotes the expression of IFN. This radiation-induced enhancement of STING activation suggests a potential mechanism for improving anti-tumor immunity through regulation of circRNA-miRNA interactions. circTMEM56, circular RNA TMEM56; ER, endoplasmic reticulum; IFN, interferon; IRF3, interferon regulatory factor 3; miR-136-5p, microRNA-136-5p; STING, Stimulator of Interferon Genes; TBK1, TANK-binding kinase 1.
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