Abstract: Objective  To investigate the effect of hypoxia on the tumor microenvironment in hepatocellular carcinoma (HCC), providing the guidance for the diagnosis and treatment of HCC patients. Methods  Tissue samples and corresponding clinicopathological data of 116 HCC patients who underwent radical hepatectomy in Guangxi Medical University Cancer Hospital from May 2018 to July 2019 were collected. The hypoxia scores of  HCC patients were calculated based on the HALLMARK⁃HYPOXIA gene set and single⁃sample gene set enrichment analysis, and the scores were validated by using transcriptome sequencing data and immunohistochemical staining results detected by a deep learning model. The relationship between the hypoxia score and the prognostic and clinicopathological parameters was analyzed by Log⁃rank, univariable and multivariable Cox proportional hazards model, and chi⁃square test. The molecular features and tumor microenvironment heterogeneity of HCC patients with high hypoxia scores and low hypoxia scores were analyzed by using transcriptomics and proteomics techniques. Results 116 HCC patients were divided into a high hypoxia score group (n=58) and a low hypoxia score (n=58) group according to the hypoxia score. Compared with the low hypoxia score group, the high hypoxia score group showed a significantly higher expression level of hypoxia⁃inducible factor 1α (HIF⁃1α) (P<0.001), and the hypoxia score of HCC patients was positively correlated with the expression level of HIF⁃1α (r=0.672, P<0.001). The overall survival (P=0.001) and recurrence⁃free survival (P=0.006) of the patients in the high hypoxia score group were significantly lower than those in the low hypoxia score group, and the high hypoxia score was associated with higher Barcelona stage (P=0.040) and tumor recurrence rate (P=0.020) in HCC patients. High hypoxia score was an independent factor of poor prognosis in HCC patients (HR=2.074, 95%CI: 1.036-4.153, P=0.040). The high hypoxia score mainly enriched in the signaling pathways regulating HCC stem cells (including Wnt, Hedgehog, TGF⁃β and other signaling pathways) and the expression levels of liver cancer cells related markers (including CD24, CD44, CXCL12, EpCAM, ICAM1, KRT19, PROM1 and THY1) were up⁃regulated in this group. The cell compositions of tumor microenvironment in high and low hypoxia groups were heterogeneous, and the expression levels of HCC stem cell⁃associated markers (including CD54, CK19, CD34, OV6, CD13, and CD133) in the CD45-CD326+ tumor stem cell subsets and CD45+CD326+ malignant double positive cell subsets in the high hypoxia group were higher than those in the low hypoxia group. Conclusions The patients with high hypoxia scores have a poorer survival prognosis than those with low hypoxia scores, and have a tumor microenvironment with stronger tumor stem characteristics.

Key words: Hepatocellular carcinoma, Hypoxia, Hypoxia score, Tumor microenvironment, Cytometry by time?of?flight