Vascular mimicry (VM) describes the phenomenon that tumor cells but not endothelial cells form vascular-like channels, which provide blood perfusion for tumor tissues. VM is associated with tumor growth, metastasis and worse survival of different, cancers. The mechanisms of VM formation remain largely unknown. We showed that the conditioned medium of cancer-associated fibroblast (CM-CAF) promoted tumor cells to form capillary-like structure in vitro. Consistently, co-implantation of CAFs with tumor cells significantly enhanced VM formation in mouse xenografts, and higher amount of CAFs was found in VM+ human HCC tissues compared to V- ones. However, the CM-CAF-promoted VM formation was attenuated when TGF-beta or SDF1 signaling was abrogated. Similar to CM-CAF, recombinant TGF-beta 1 and SDF1 induced VM formation. We further disclosed that the CAF-secreted TGF-beta and SDF1 enhanced the expression of VE-cadherin, MMP2 and laminin5 gamma 2 via TGF-beta R1 and CXCR4 in tumor cells, thereby promoted VM formation. Moreover, tumor cells with high activity of self-sustaining TGF-beta signaling displayed strong capability of VM formation. Subsequent investigations showed that miR-101, which was down-regulated in both tumor cells and CAFs, suppressed the CAF-promoted VM formation in vitro and in vivo. Gain- and loss-of-function analyses revealed that miR-101 attenuated TGF-beta signaling transduction by targeting TGF-beta R1 and Smad2 in tumor cells, and simultaneously abrogated SDF1 signaling by suppressing SDF1 expression in CAFs and inhibiting VE-cadherin expression in tumor cells. Our findings suggest that the miR-101-TGF-beta/SDF1-VE-cadheriniMMP2/LAMC2 networks regulate VM formation and represent the potential targets for cancer therapy. (C) 2016 Elsevier Ireland Ltd. All rights reserved.