To deeply understand the antitumor mechanism of EH@Ag2S under US irradiation and immunotherapy at the biomolecular level, RNA-sequencing (RNA-seq) technique analysis of tumor tissues after treatment was performed. Tumors in the PBS group, EH@Ag2S and EH@Ag2S +US were served as control. Key genes related to the PD-1/PD-L1 pathway were analyzed. For the EH@Ag2S +US group, it down-regulated the PDCD1 gene, as well as other immune-related genes such as IDO1 and TP53, etc. (Fig. ). But for the EH@Ag2S group, these results were not very obvious, we inferred that Ag2S could generate ROS under US irradiation, thereby regulating the immune microenvironment, resulting in changes in the release of immune-related factors to exert anti-tumor effects. Genes involved in oxidative stress (Fig. ), such as SESN2, SLC7A11, and PRDX1, were significantly up-regulated by synergistic SDT and immune checkpoint blockade treatment. For tumor suppression and immune response processes, we mainly focused on genes related to tumor necrosis and apoptosis. The genes associated with tumor necrosis and apoptosis were up-regulated in EH@Ag2S +US groups, such as TNFSF4, TNFSF10 and PSMB2, etc. (Fig. ). Then, to understand the molecular pathways affected by EH@Ag2S +US group, Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to identify molecular pathways and describe biological processes in transcriptional analysis data. As shown in Fig. and Fig. , we can clearly observe that the altered pathways are related with TNF signaling pathway, p53 signaling pathway and chemical carcinogenesis-reactive oxygen species in EH@Ag2S +US group. These are signals for tumor apoptosis. These results all indicated the potential of the EH@Ag2S +US group in terms of immune activation and anti-tumor. (E) KEGG analysis of significantly altered genes.