Goldstein Lee J, Chen Haiying, Bauer Richard J, Bauer Stephen M, Velazquez Omaida C
Hospital of the University of Pennsylvania, Philadelphia 19104, USA.
Surgery. 2005 Sep;138(3):439-49. doi: 10.1016/j.surg.2005.06.031.
We previously reported that fibroblasts induce human microvascular endothelial cells (HMVECs) to differentiate from monolayer to capillarylike morphology. We now test the hypothesis that fibroblasts modulate angiogenesis in melanoma cells.
We tested 12 human melanoma lines (2 radial growth phase (RGP), 3 vertical growth phase (VGP), and 7 metastatic (MM)) for ability to induce HMVECs to invade/migrate into collagen and form capillarylike networks. HMVEC monolayers were overlaid with 3-dimensional collagen gels embedded with melanoma cells alone (M), fibroblasts alone (F), or a 1:1 mixture of the 2 cells (M+F). After 5 days, gels were removed, fixed, and HMVEC networks were quantified by von Willebrand's factor (vWF) immunofluorescence. The influence of soluble factors on HMVEC invasion/migration into collagen was assessed with the use of acellular 3-D collagen gels overlaid on HMVEC monolayers, cultured with conditioned media (CM) derived from monolayers of M, F, or M+F. Angiogenic growth factors involved in the observed invasion/migration were identified with the use of a RayBio Cytokine Antibody Array (RayBiotech, Norcross, Ga).
Cell line-specific variability in melanoma-supported angiogenesis was observed only when in combination with fibroblasts (analysis of variance [ANOVA], P < .01). Melanoma plus fibroblasts uniformly resulted in a significantly higher angiogenic response than melanoma alone (P < .05). One vertical growth phase and one metastatic melanoma line, while weakly angiogenic alone, induced significantly higher angiogenesis than either fibroblast or melanoma alone (P < .05) when combined with fibroblasts. CM from M or M+F induced significantly less HMVEC invasion/migration into collagen than CM from fibroblasts alone. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 were identified as significantly elevated in the media derived from M+F cultures, compared with either cell type alone.
To our knowledge, this is the first report demonstrating that melanoma-supported angiogenesis in collagen is more significantly influenced by normal skin-derived fibroblasts than by the intrinsic biology of the melanoma cell type. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 are implicated as potential paracrine factors regulating this observed effect.
我们之前报道过,成纤维细胞可诱导人微血管内皮细胞(HMVECs)从单层形态分化为毛细血管样形态。我们现在检验成纤维细胞调节黑色素瘤细胞中血管生成的这一假说。
我们检测了12种人黑色素瘤细胞系(2种径向生长期(RGP)、3种垂直生长期(VGP)和7种转移期(MM))诱导HMVECs侵入/迁移至胶原蛋白并形成毛细血管样网络的能力。HMVEC单层细胞上覆盖三维胶原蛋白凝胶,其中单独包埋黑色素瘤细胞(M)、单独包埋成纤维细胞(F)或两种细胞的1:1混合物(M+F)。5天后,移除凝胶并固定,通过血管性血友病因子(vWF)免疫荧光对HMVEC网络进行定量分析。利用覆盖在HMVEC单层细胞上的无细胞三维胶原蛋白凝胶,并用源自M、F或M+F单层细胞的条件培养基(CM)进行培养,评估可溶性因子对HMVEC侵入/迁移至胶原蛋白的影响。使用RayBio细胞因子抗体阵列(RayBiotech,诺克罗斯,佐治亚州)鉴定参与观察到的侵入/迁移过程的血管生成生长因子。
仅在与成纤维细胞联合时,才观察到黑色素瘤支持的血管生成存在细胞系特异性差异(方差分析[ANOVA],P < 0.01)。黑色素瘤加而成纤维细胞始终导致比单独黑色素瘤显著更高的血管生成反应(P < 0.05)。一种垂直生长期和一种转移期黑色素瘤细胞系,虽然单独时血管生成能力较弱,但与成纤维细胞联合时诱导的血管生成显著高于单独的成纤维细胞或黑色素瘤(P < 0.05)。来自M或M+F的CM诱导HMVEC侵入/迁移至胶原蛋白的能力显著低于单独来自成纤维细胞的CM。与单独的任何一种细胞类型相比,白细胞介素8、单核细胞趋化蛋白-1和金属蛋白酶组织抑制剂-2在源自M+F培养物的培养基中显著升高。
据我们所知,这是首次报道表明,在胶原蛋白中黑色素瘤支持的血管生成受正常皮肤来源的成纤维细胞影响比受黑色素瘤细胞类型的内在生物学特性影响更显著。白细胞介素8、单核细胞趋化蛋白-1和金属蛋白酶组织抑制剂-2被认为是调节这一观察到的效应的潜在旁分泌因子。