Department of Laboratory Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei, China.
Research Center for Translational Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei, China.
J Cancer Res Clin Oncol. 2023 Sep;149(11):8729-8741. doi: 10.1007/s00432-023-04790-3. Epub 2023 May 2.
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.
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 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.
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.
血管生成拟态（VM）是一种不依赖血管生成的替代性微血管循环，由侵袭性癌细胞形成。已报道肿瘤表达的 B7-H3 可促进肝癌中的 VM 形成，并调节乳腺癌和结直肠癌中的血管生成。然而，其在非小细胞肺癌（NSCLC）中对 VM 生成和血管生成的影响仍有待阐明。
在 NSCLC A549 和 H3255 细胞中使用 CRISPR/Cas9 介导的 B7-H3 敲除（KO）。通过 Western blot 和化学发光测定法在 B7-H3 KO 和模拟编辑的 A549 和 H3255 细胞中测量 VM 相关蛋白（包括血管内皮（VE）-钙粘蛋白和基质金属蛋白酶 14（MMP14））的表达和血管内皮生长因子（VEGF）的分泌。为了研究 VM 形成，使用 B7-H3 KO 和模拟 A549 和 H3255 细胞的三维（3D）培养模型。为了进行体内分析，使用 B7-H3 KO 和模拟编辑的 A549 细胞建立异种移植小鼠模型，并进行免疫组织化学（CD31）和组织化学（过碘酸希夫，PAS）双重染色，以鉴定肿瘤组织中的 VM 和内皮血管。最后，使用特异性信号抑制剂分析 B7-H3 诱导的信号通路，该通路负责 VE-cadherin 和 MMP14 表达和 VM 生成。
B7-H3 的高表达与 NSCLC 中较差的预后和更晚期的 T 分期相关。CRISPR/Cas9 介导的 B7-H3 KO 在 A549 和 H3255 细胞中导致 VE-cadherin 和 MMP14 的表达下调；然而，两种细胞系的 VEGF 分泌保持不变。在 3D 细胞培养模型中，与模拟编辑的细胞相比，B7-H3 KO 的 A549 和 H3255 细胞形成的毛细血管样管状结构明显减少。在体内异种移植模型中，模拟编辑的 A549 细胞形成了过多的 PAS-CD31VM 通道，而 B7-H3 KO 抑制了异种移植肿瘤中的 VM 形成。然而，B7-H3 KO 和模拟编辑的 A549 细胞形成的异种移植物中的 CD31 内皮血管之间没有发现明显差异。最后，我们分析了负责 B7-H3 诱导的 VM 形成的信号通路，发现选择性抑制磷酸肌醇 3-激酶（PI3K）/蛋白激酶 B（AKT）的过度激活通过 LY294002 与 MMP14 和 VE-cadherin 的表达降低以及 A549 和 H3255 细胞的体外 VM 形成有关。
肿瘤表达的 B7-H3 通过 PI3K/AKT 信号通路发挥作用，促进 NSCLC 细胞的 VM 形成，而对 NSCLC 中的血管生成没有影响。
