Vasculogenic mimicry in hepatocellular carcinoma contributes to portal vein invasion

Abstract

Portal vein invasion (PVI) is common in hepatocellular carcinoma (HCC) and largely contributes to tumor recurrence after radical tumor resection or liver transplantation. Vasculogenic mimicry (VM) was an independent vascular system lined with tumor cells and associated with poor prognosis of HCC. The present study was conducted to evaluate the relationship between VM and portal vein invasion. A total of 44 HCC cases receiving anatomic liver resection were included in the study and were divided into groups with and without PVI. The prevalence of VM in each group was examined by CD34-PAS dual staining. The regulatory molecules of VM formation such as Notch1, Vimentin and matrix metalloproteinases (MMPs) were investigated by immunohistochemical staining. Analysis was performed to explore the association of PVI, VM and the VM regulatory molecules. PVI was found in 40.91% (18/44) cases and VM was found in 38.64% (17/44) cases in total samples. The incidence of VM was 72.22% (13/18) in PVI group while it was 15.38% (4/26) in non-PVI group (P<0.001), VM formation was positively correlated with PVI (r=0.574, P<0.001). The VM forming regulatory molecules such as Notch1, Vimentin, MMP-2 and MMP-9 were found to be correlated with PVI in HCC patients. Taken together, our results suggested that VM formation, alone with its regulatory molecules, is the promoting factor of PVI in hepatocellular carcinoma.

Keywords: Notch1; hepatocellular carcinoma (HCC); portal vein invasion (PVI); vasculogenic mimicry (VM).

Figure 1. Prevalence of VM in HCC patients with and without PVI

A and B. PAS staining in HCC specimens. A: PAS staining in well to moderate differentiated samples. B: PAS staining in poorly differentiated samples. C and D. PAS-CD34 double-staining. C: specimen without VM. D: specimen with VM. Original magnification: 400×. E. Prevalence of VM in PVI and non-PVI groups. F. Presence of VM positively associated with PVI.

Figure 2. Immunohistochemical staining of Notch1 in HCC specimens and its association with PVI

A. Notch1 staining in HCC specimens with PVI, B. Notch1 staining in HCC specimens without PVI, C. Notch1 staining in adjacent non-tumor specimens. Original magnification: 400×. D. Different staining density and proportion of Notch1 in specimens of PVI, non-PVI and control (non-tumor) specimens. E. Spearman correlation analysis indicated a positive correlation between Notch1 expression and PVI.

Figure 3. Immunohistochemical staining of Vimentin in HCC specimens and its association with VM and PVI

A and B. E-cadherin staining in non-PVI and PVI samples. C and D. Negative and positive Vimentin staining in non-PVI and PVI samples. Original magnification was 400×. E. Incidence of positive vimentin staining in PVI and non-PVI patients. Chi-Square test was used. F. E-cadherin staining in Vimentin positive and negative samples; E-cadherin staining in non-PVI and PVI samples. T-test was used. G. Increased Vimentin positively associated with PVI. Spearman correlation was used. H. Decreased E-cadherin negatively associated with PVI. Spearman Correlation analysis was used.

Figure 4. Increased expressions of MMPs associated with VM and higher incidence of PVI

A and B. MMP-9 immunohistochemical staining in HCC with and without PVI, original magnification: 400×. C and D. MMP-2 immunohistochemical staining in HCC with and without PVI, original magnification: 400×. E and F. MMP-2, MMP-9 IHC score were correlated with Notch1. G and H. MMP-2 and MMP-9 expression positively correlated with VM. I: MMP-2 and MMP-9 significantly increased in PVI group compared with non-PVI group. J and K: MMP-2, MMP-9 IHC score were positively correlated with PVI.

Figure 5. Schematic diagram of summary of this study

Elevated Notch1 induces a cohort of tumor cells translate from epithelial to mesenchymal phenotype in HCC. During the process, Vimentin increases and E-cadherin decreases in cells with malignant phenotype, therefore migration and deattachment are enhanced; MMP-2 and MMP-9 increases, therefore ECM degradation and remolding are enhanced. These support the PVI development at the cellular level. Additionally, EMT induced VM aggravates vasoganglion abnormality inside tumor mass, connects portal vein and internal circulation of tumor mass, therefore offers pathways for tumor cells to metastasize into portal vein. These support the PVI development at the circulation level.