The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation

Figures

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  Figure 1. High ACE2 expression was linked to increased VM and better prognosis in NSCLC. (A) Typical image of ACE2, VE-cadherin, EphA2 protein expression and CD34/PAS double staining in TMA tissues. Case B1 had massive CD34−/PAS+ VM (yellow arrows) lined by ACE2, VE-cadherin and EphA2 high expressing tumor cells. Case G1 had abundant CD34+/PAS− MVs (black arrows) with ACE2, VE-cadherin and EphA2 low expressing tumor cells. (B) Kaplan-Meier analysis of OS in NSCLC patients with ACE2 low or high expression. P = 0.044.

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  Figure 2. ACE2-induced better outcome in NSCLC patients might be attributed to less vessels and more VM formation. (A, C, E, G) Typical tissue images of each group stained with ACE2 or CD34/PAS. Yellow arrows: CD34−/PAS+ VMs; black arrows: CD34+/PAS− MV. (B, D, F, H) Kaplan–Meier analysis of OS in each group.

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  Figure 3. Human ACE2 was stably overexpressed in A549-ACE2-OE cells. (A) Schematic representation of pLenti6.3-MCS/V5 DEST. (B) pLenti6.3-ACE2 expression vector was detected by PCR. (C) Fluorescence of EGFP in A549-ACE2-OE cells (left) and parental cells (right) was determined by fluorescence microscopy. (D) RT-PCR experiment of ACE2 mRNA level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, *p < 0.05. (E) Western blot analysis of ACE2 expression level in A549-ACE2-OE cells and parental cells. (F) Quantification of ACE2 expression level in A549-ACE2-OE cells and parental cells. Mean ± SD, n = 3, ***p < 0.001.

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  Figure 4. Tube formation ability of A549 cells was improved with ACE inhibitory state. (A) Morphologies of a panel of A549-ACE2-OE cells, A549-NC cells, and A549-NC cells treated with ACEI (1, 5, and 10 nM/L) were shown as sheet-like and thread-like cell types in 2D culture, which were outlined partly with yellow lines. Representative images were shown above. Upper: white light; lower: fluorescence. (B) Quantification of sheet-like or thread-like cells and pebble-like cells in three groups, representatively, Mean ± SD, n = 3, **p < 0.01. (C) Images of both above cell lines grown in 3D Matrix gel. A 10 nM/L ACEI dilution was performed. Representative images were shown above. Yellow arrows point out the free cancer cells escaping from tube wall and isolated segments. Upper: white light; lower: fluorescence. (D, E) Images of 3D culture were applied to determine average number of nodes, branches, isolated segments, meshes and mean mesh area in those groups, per field, Mean ± SD, n = 3, **p < 0.01, *p < 0.05. ns, no significance.

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  Figure 5. VM formation was increased, and vasculature was lessened due to inhibition of RAS in vivo. (A) Growth curve of allograft tumors of A549-ACE2-OE cells, A549-NC cells with or without ACEI treatment, Mean ± SD, n = 3, *p < 0.05. ns, no significance. (B) Weight of resected tumors, Mean ± SD, n = 3, **p < 0.01, *p < 0.05. (C) Continuous sections of allograft tumor tissues stained with PAS, CD34, VE-cadherin, or EphA2 immunohistochemical stain. Black arrow points out a typical MV (CD34+/PAS−); yellow arrows point out typical VM (CD34−/PAS+). (D) Quantification of MV and VM in different groups, Mean ± SD, n = 3, per field, ***p < 0.001, **p < 0.01. (E) Quantification of VE-cadherin and EphA2 mean optical density in three groups, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01.

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  Figure 6. VE-cadherin and EphA2 expression was upregulated in A549 cells and NSCLC tissues with impaired local RAS status. (A, B) RT-PCR experiment of VE-cadherin and EphA2 mRNA level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, ***p < 0.001. (C–E) Western blot analysis and quantification of VE-cadherin and EphA2 expression level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, ***p < 0.001. (F, G) Linear regressions of VM number and VE-cadherin (P < 0.0001) or EphA2 (P = 0.0108) score in TMA. (H) Typical tissue images of both groups stained with VE-cadherin, ACE2 or CD34/PAS. Case F13 with ACE2 low status was provided with rambling VM covered by tumor cells which only expressed VE-cadherin in nuclei; case D6 with ACE2 high status had ordered VM lined by tumor cells expressing VE-cadherin in both nuclei and cytomembranes. Red arrow: VE-cadherin membrane expression.

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  Figure 7. PI3K/AKT, p38MAPK, and HIF1-a were inactivated, and Nodal/Notch4 pathway was activated in A549-ACE2-OE cell model. (A–E) Western blot analysis and quantification of AKT, p-AKT, p38, and p-p38 expression level in A549-ACE2-OE cells and negative control, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01. (F, G) Immunoflurescence assay and quantification of HIF1-a mean optical density in A549-ACE2-OE cells and negative control, Mean ± SD, n = 4, *p < 0.05. (H, I) Western blot analysis and quantification of Nodal and Notch4 expression level in A549-ACE2-OE cells and negative control, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01.