Integration of Bulk RNA and Single‐Cell Analyses Reveal Distinct Expression Patterns of Anoikis‐Related Genes and the Immunosuppressive Role of NQO1 + Macrophages in Hepatocellular Carcinoma

ABSTRACT Anoikis resistance plays a crucial role in the proliferation, metastasis, and invasion of hepatocellular carcinoma (HCC). However, the key genes involved remain to be identified. This study aimed to investigate the prognostic value and impact of anoikis‐related genes (ARGs) on the immunosuppressive microenvironment in HCC patients through the integration of bulk RNA and single‐cell RNA sequencing (scRNA‐seq) bioinformatic analysis. An anoikis‐related gene risk score model (ARGRS) comprising 11 ARGs was established via machine learning. scRNA‐seq was performed to assess the heterogeneity of ARGs in HCC. In vitro experiments were conducted to investigate the effects of NAD(P)H: quinone oxidoreductase 1 (NQO1) on the polarization, phenotype, and function of macrophages. Bioinformatics analysis demonstrated that ARGRS had perfect efficiency in predicting the prognosis of HCC patients and that ARGs potentially play a role in maintaining the invasion and metastasis of malignant cells. Notably, NQO1 + macrophages presented features consistent with alternatively activated macrophages (M2) and displayed a powerful immunosuppressive effect, particularly in close interaction with T cells within the tumor immune microenvironment. Moreover, inhibition of NQO1 expression via dicoumarol resulted in reduced expression of the M2‐associated markers CD206 and CD163, as well as the immunosuppressive cytokines IL‐10 and TGF‐β. Strikingly, this treatment effectively mitigated the immunosuppressive impact of macrophages on T cells. Collectively, ARGs are closely associated with the poor prognosis of HCC patients, and NQO1 + macrophages may have an immunosuppressive effect on HCC, suggesting that intervention in anoikis may represent a potential strategy for HCC treatment.