Aberrant variations in angiogenesis have been observed in tumor tissues with abnormal stiffness of extracellular matrix (ECM). However, it remains largely unclear how ECM stiffness influences tumor angiogenesis. Numerous studies have reported that vascular endothelial growth factor-A (VEGF-A) released from tumor cells plays crucial roles in angiogenesis. Hence, we demonstrated the role of ECM stiffness in VEGF-A release from neuroblastoma (NB) cells and the underlying mechanisms. Based on 17 NB clinical samples, a negative correlation was observed between the length of blood vessels and stiffness of NB tissues. In vitro, an ECM stiffness of 30 kPa repressed the secretion of VEGF₁₆₅ from NB cells which subsequently inhibited the tube formation of human umbilical vein endothelial cells (HUVECs). Knocked down VEGF₁₆₅ in NB cells or blocked VEGF₁₆₅ with neutralizing antibodies both repressed the tube formation of HUVECs. Specifically, 30 kPa ECM stiffness repressed the expression and nuclear accumulation of Yes-associated protein (YAP) to regulate the expression of Serine/Arginine Splicing Factor 1 (SRSF1) via Runt-related transcription factor 2 (RUNX2), which may then subsequently induce the expression and secretion of VEGF₁₆₅ in NB tumor cells. Through implantation of 3D col-Tgels with different stiffness into nude mice, the inhibitory effect of 30 kPa on NB angiogenesis was confirmed in vivo. Furthermore, we found that the inhibitory effect of 30 kPa stiffness on NB angiogenesis was reversed by YAP overexpression, suggesting the important role of YAP in NB angiogenesis regulated by ECM stiffness. Overall, our work not only showed a regulatory effect of ECM stiffness on NB angiogenesis, but also revealed a new signaling axis, YAP-RUNX2-SRSF1, that mediates angiogenesis by regulating the expression and secretion of VEGF₁₆₅ from NB cells. ECM stiffness and the potential molecules revealed in the present study may be new therapeutic targets for NB angiogenesis.

在细胞外基质 (ECM) 异常僵硬的肿瘤组织中观察到血管生成的异常变化。然而，目前尚不清楚 ECM 刚度如何影响肿瘤血管生成。许多研究报道，从肿瘤细胞释放的血管内皮生长因子-A (VEGF-A) 在血管生成中起着至关重要的作用。因此，我们证明了 ECM 刚度在神经母细胞瘤 (NB) 细胞释放 VEGF-A 中的作用及其潜在机制。基于17个NB临床样本，观察到血管长度与NB组织硬度呈负相关。在体外，30 kPa 的 ECM 刚度抑制了 VEGF _165的分泌来自 NB 细胞，其随后抑制人脐静脉内皮细胞 (HUVEC) 的管形成。敲低NB 细胞中的 VEGF 165 或用中和抗体阻断 VEGF ₁₆₅都抑制了 HUVEC 的管形成_。具体来说，30 kPa ECM 刚度抑制 Yes 相关蛋白 (YAP) 的表达和核积累，以通过 Runt 相关转录因子 2 (RUNX2) 调节丝氨酸/精氨酸剪接因子 1 (SRSF1) 的表达，然后可能诱导VEGF _{165的表达和分泌}在 NB 肿瘤细胞中。通过将不同刚度的 3D col-Tgels 植入裸鼠体内，在体内证实了 30 kPa 对 NB 血管生成的抑制作用。此外，我们发现 YAP 过表达逆转了 30 kPa 刚度对 NB 血管生成的抑制作用，这表明 YAP 在 ECM 刚度调节的 NB 血管生成中的重要作用。总的来说，我们的工作不仅显示了 ECM 刚度对 NB 血管生成的调节作用，而且还揭示了一个新的信号轴 YAP-RUNX2-SRSF1，它通过调节NB 细胞中 VEGF _{165的表达和分泌来介导血管生成。}本研究中揭示的 ECM 刚度和潜在分子可能是 NB 血管生成的新治疗靶点。