Tumor-expressed B7-H3 promotes vasculogenic mimicry formation rather than angiogenesis in non-small cell lung cancer.

Abstract

Vasculogenic mimicry (VM), an alternative microvascular circulation independent ofangiogenesis, is formed by aggressive cancer cells. Tumor-expressedB7-H3 has been reported to promote VM formation in hepatocellular carcinoma and modulate angiogenesis in breast cancer and colorectal cancer. However, its effects on VM generation and angiogenesis in non-small cell Lung cancer (NSCLC) remained to be elucidated. CRISPR/Cas9-mediated B7-H3 knockout (KO)was conducted in NSCLC A549 and H3255 cells. The expression of VM-related proteins, including vascular endothelial (VE)-cadherin and matrix metalloproteinase 14 (MMP14), and the secretion of vascular endothelial growth factor (VEGF) were measured by western blotting and chemiluminescence assay in both B7-H3 KO and mock-edited A549 and H3255 cells. To examine VM formation, a three-dimensional (3D)culture model was used for B7-H3 KO and mock A549 and H3255 cells. For in vivo analysis, xenograft mice models were established using B7-H3 KO and mock-edited A549 cells, and immunohistochemical (CD31) and histochemical (periodic acid-Schiff, PAS) double staining were performed to identify VM and endothelial vessels in tumor tissues. Finally, specific signaling inhibitors were used to analyze B7-H3-induced signaling pathway responsible for VE-cadherin and MMP14 expression and VM generation. Higher expression of B7-H3 was associated with a worse prognosis and moreadvanced T-category in NSCLC. CRISPR/Cas9-mediated B7-H3 KOin A549 and H3255 cells led to decreased expression of VE-cadherin and MMP14; however,the secretion of VEGFby the two cell lines remained unchanged. In the3D cell culture model, bothB7-H3 KO A549 and H3255cells showed a significant reduction in the formation of capillary-like tubular structures compared to mock-edited cells. In thein vivo xenograft model, mock-edited A549 cells formed excessive PAS+ CD31-VM channels, while B7-H3 KO restrained VM formation in the xenograft tumors. However, no significant differenceswere found in CD31+ endothelial vessels between xenografts formed by B7-H3 KO and mock-edited A549 cells. Finally, we analyzed the signaling pathway responsible for B7-H3-induced VM formation and found that selective inhibition of the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT) hyperactivation by LY294002 was associated with decreased expression of MMP14 and VE-cadherin, and in vitro VM formation by both A549 and H3255 cells. Tumor-expressed B7-H3 acts via PI3K/AKT signaling pathway to promote VM formation by NSCLC cells while bears no effects on angiogenesis in NSCLC.