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

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Abstract

Background

Vasculogenic mimicry (VM), an alternative microvascular circulation independent of angiogenesis, is formed by aggressive cancer cells. Tumor-expressed B7-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.

Methods

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.

Results

Higher expression of B7-H3 was associated with a worse prognosis and more advanced T-category in NSCLC. CRISPR/Cas9-mediated B7-H3 KO in A549 and H3255 cells led to decreased expression of VE-cadherin and MMP14; however, the secretion of VEGF by the two cell lines remained unchanged. In the 3D cell culture model, both B7-H3 KO A549 and H3255 cells showed a significant reduction in the formation of capillary-like tubular structures compared to mock-edited cells. In the in 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 differences were 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.

Conclusions

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.

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Data availability

The datasets supporting the conclusions of this article are included within the article and its additional file.

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Funding

This study was supported by The Natural Science Foundation of Anhui Province (Grant No. 1808085MH229); Key Research and Development Program of Anhui Province (Grant No. 202004j07020027). Funder: Liwen Chen.

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Author notes

  1. Xingyu Fan and Junfeng Huang have contributed equally to this work.

Authors and Affiliations

  1. Department of Laboratory Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei, China

    Xingyu Fan, Junfeng Huang, Bingqi Hu, Jing Zhou & Liwen Chen

  2. Research Center for Translational Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei, China

    Xingyu Fan, Junfeng Huang, Bingqi Hu, Jing Zhou & Liwen Chen

Authors
  1. Xingyu Fan
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  2. Junfeng Huang
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  3. Bingqi Hu
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  4. Jing Zhou
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Contributions

XYF, JFH, and LWC designed the experiments. XYF, JFH and JZ performed in vitro experiments. XYF, BQH, and JZ performed in vivo experiments. JFH performed bioinformatics analysis. XYF drafted the manuscript. LWC supervised the research and edited the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Liwen Chen.

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The authors declare no competing interests.

Ethics statement

All animal studies were performed in compliance with the regulations of Anhui Medical University (Hefei, China) institutional animal care and conducted according to the Institutional Animal Care and Use Committee (IACUC) guidelines.

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Fan, X., Huang, J., Hu, B. et al. Tumor-expressed B7-H3 promotes vasculogenic mimicry formation rather than angiogenesis in non-small cell lung cancer. J Cancer Res Clin Oncol 149, 8729–8741 (2023). https://doi.org/10.1007/s00432-023-04790-3

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