Second Department of Pathology, University of Brescia and Spedali Civili, Brescia, Italy.
Lab Invest. 2012 Sep;92(9):1297-309. doi: 10.1038/labinvest.2012.94. Epub 2012 Jun 25.
Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-β1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-β1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-β1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-β1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-β1 with anti-TGF-β1 antibodies or with Ly-364947 (a specific inhibitor TGF-β1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-β1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.
肝细胞癌 (HCC) 是一种非常血管生成和恶性的癌症。由于 HCC 浸润血管的异常结构组织，常规化疗效果不佳。在之前的工作中，我们证明 HCC 血管生成是由转化生长因子β-1(TGF-β1)/CD105 轴驱动的，刺激肝源性微血管内皮细胞 (Ld-MECs) 迁移。由于 TGF-β1 也会影响壁细胞 (MCs) 的募集和成熟，我们想知道它是否会导致 HCC 诱导的血管异常。对 12 名患者的 HCC 和相邻非肿瘤性肝脏 (nNL) 活检进行了免疫组织化学分析，检测血管生成标记物 CD105、TGF-β1、CD44 和血管内皮生长因子-a (VEGFa) 以及 MC 标记物 NG2、α-平滑肌肌动蛋白 (αSMA) 和神经细胞黏附分子 (NCAM)。还通过免疫细胞化学检测了从同一肝活检中分离的 HCC 衍生基质细胞 (HCC-StCs) 和 nNL 衍生基质细胞 (nNL-StCs) 中的相同标记物。通过 ELISA 分析基质细胞释放的血管生成因子，并通过粘附试验分析基质细胞与 Ld-MECs 的相互作用。与 nNL 相比，HCC 活检显示血管生成标记物和 αSMA 增加，αSMA 定位于血管中。相比之下，NG2 和 NCAM 主要定位于肿瘤细胞中，而不存在于血管和基质中。培养的 HCC-StCs 显示 NG2、αSMA 和 NCAM 的表达水平较低。它们还表现出释放血管生成因子的能力较低，并且与 Ld-MECs 粘附的能力也较低。用 TGF-β1 或 HepG2(人肝癌细胞系)衍生的条件培养基 (CM) 处理 HCC-StCs 和 nNL-StCs 后，NCAM 表达下调，而抗 NCAM 抗体显著减少了基质细胞与 Ld-MECs 的粘附。进一步用抗 TGF-β1 抗体或 Ly-364947(一种特异性 TGF-β1 受体抑制剂)阻断 TGF-β1，分别部分恢复了 Ld-MECs 的粘附和 NCAM 表达。TGF-β1 通过下调 NCAM 表达，影响 HCC-StCs 与 Ld-MECs 的相互作用，从而导致 HCC 诱导的血管改变。
