A novel vasculogenic mimicry-related nomogram predicts prognosis in hepatocellular carcinoma

Abstract

Objective: Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has a poor prognosis. Vasculogenic mimicry (VM) is an angiogenic process associated with the growth and spread of malignant tumors. In this study, we aim to create a VM-related, gene-based prediction model to evaluate the prognosis and immune infiltration in HCC patients.

Materials and methods: A total of 364 patients from the TCGA database and 242 patients from the GEO database with complete clinical information and transcriptome sequencing data were enrolled in this study. LASSO Cox regression analysis was performed to identify VM-related hub genes. Biological process (BP), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and gene set enrichment analysis (GSEA) were applied to analyze the biological function of the hub genes. The predictive significance of the related gene signature was confirmed in the GSE14520 cohort. RT-PCR and CD31/E-cadherin immunofluorescence staining were applied to elucidate that the VM score can reflect the degree of vasculogenic mimicry within tumors.

Results: This study found that VM-related genes were enriched in the proteoglycans in the cancer pathway and the VEGF signaling pathway. A predictive signature based on five genes (MAPK3, MAPK1, VEGFA, NOTCH1, and TGFB1) was identified as an independent risk factor for HCC patient prognosis. GSEA revealed that genes that positively correlated with the signature were enriched in the "NOTCH signaling pathway," which is activated during angiogenesis. Additionally, CIBERSORTx analysis showed that higher expression of the VM score was associated with immune infiltration of naïve CD4⁺ T cells in HCC. Pearson correlation analysis revealed a positive link between an increased VM score and inhibitory immunological checkpoints (HVEM and PD-1). Furthermore, in vivo experiments have confirmed that the VM score can effectively reflect the degree of vasculogenic mimicry in hepatocellular carcinoma tissue. The nomogram that utilized the VM score and TNM stage to predict the survival probability of individual HCC patients was satisfactory.

Conclusion: The VM score and nomogram constructed to predict the survival probability of HCC patients achieved satisfactory outcomes in this study. The relationship between the biological function of the VM score and immune infiltration could potentially serve as a target for tumor therapy in liver cancer.

Keywords: hepatocellular carcinoma; immune checkpoints; immune infiltration; prognosis prediction; vasculogenic mimicry.

FIGURE 1

Expression and function of 24 VM-related genes in hepatocellular carcinoma. (a) Expression of VM-related genes between tumor and normal tissues. (b) GO and KEGG enrichment analyses of VM-related genes. (c) PPI network of VM-related genes.

FIGURE 2

Development of the VM score. (a) Forest plot of the univariate analysis of VM-related genes and their coefficients. (b–g) KM analysis of overall survival in groups with high expression of hub VM-related genes and low expression of hub VM-related genes in the TCGA dataset.

FIGURE 3

Association between the VM score and clinical characteristics of HCC. (a, b) Landscape of VM score-related clinical characteristics of HCC in training and test groups of the TCGA dataset. (c, d) KM analysis of overall survival in high and low VM score groups based on the TCGA dataset. (e, f) Differences in the VM score based on clinical characteristics, including status, TNM stage, and gender. The VM score was significantly increased in HCC patients with poor prognoses, those with a higher TNM stage, and in female patients in the training group but not in the test group.

FIGURE 4

Relationship between the VM score and clinical characteristics in the GEO dataset. (a) KM analysis of overall survival in high and low VM score groups in GSE14520. (b) All patients were divided into high and low VM score groups based on the best cutoff value in the GEO dataset. (c) Heatmap of VM score-related clinical characteristics in the validation group. (d) Different distributions of the VM score based on different clinical characteristics; the VM score was significantly increased in HCC patients with poor prognosis, those with higher TNM stage, and female patients in the validation group.

FIGURE 5

Biological functions associated with the VM score. (a, b) Gene set enrichment analysis results of the enrichment pathways associated with the VM score in the validation group. (c–e) Mostly related biological processes, cellular components, and molecular functions to the VM score in the validation group. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the VM score in the validation group.

FIGURE 6

Correlation between the VM score and immune cell infiltration and checkpoints. (a) Infiltration of 22 types of immune cells in high and low VM score groups based on TCGA and GEO datasets. *, P < 0.05; **, P < 0.01; and ****, P < 0.0001. (b) Correlation of the VM score with the expression level of naïve CD4⁺ T cells. (c) Correlation of the VM score with immune cell infiltration based on CIBERSORTx. (d) Spearman’s rank correlation between the VM score and inhibitory immune checkpoints. The color of the band represents the R-value.

FIGURE 7

Individualized prediction model based on the VM score for HCC patients. (a) Nomogram for HCC patients. (b–c) Time-dependent ROC curves of the nomogram in the TCGA and GEO datasets. (d–e) Calibration curves of the nomogram. The plots show the comparison between predicted and actual OS for 1-, 3-, and 5-year survival probabilities in TCGA and GEO datasets.

FIGURE 8

Validation of the relationship between the VM score and the degree of vasculogenic mimicry. (a) mRNA levels (relative quantification) of VM-related genes across eight samples. (b) Segregation into groups A and B based on the median VM score. There exists a statistically significant difference in VM scores between the two groups. (c) CD31/E-cadherin double staining for vasculogenic mimicry. The white arrow indicates tubular structures resembling VM. The lumens are surrounded by tumor tissue without endothelial cells. (d–e) CD31/E-cadherin double immunofluorescence staining of VM in tumor tissues in groups A and B. (f). The statistical difference was confirmed using the Mann-Whitney U test. Data are expressed as means.