Distinct Transcriptional and Migratory Programs Are Associated with Vasculogenic Mimicry Heterogeneity in Triple-Negative Breast Cancer

Background: Vasculogenic mimicry (VM) is a tumor-driven vascularization strategy in which aggressive cancer cells form perfusable, endothelium-independent channels that support tumor growth, metastasis, and therapy resistance. VM is prevalent in triple-negative breast cancer (TNBC), but within this group of tumors, VM heterogeneity is underexplored. Likewise, VM competence and its relationship to classical endothelial angiogenesis (EA) remain incompletely understood. Methods: Here, as a proof of concept, we combine functional analysis of three molecularly distinct TNBC cell lines with a panel-wide DepMap transcriptomic survey to characterize VM heterogeneity. Results: Using an in vitro tube formation assay, we show that the VM-competent TNBC cell lines MDA-MB-231 and MDA-MB-231-4175 form robust 3D vessel-like networks in a matrigel matrix, whereas the VM-incompetent line MDA-MB-468 does not. As a control, we use an immortalized endothelial cell line, 3B-11, that forms classical EA vessel-like networks. Moreover, we visualize VM (Laminin-5+) and EA (CD31+) markers in vessel-like networks of VM-competent TNBC xenografts using immunohistochemical staining and show that while they are distinctly labeled, they can also coexist to form mosaic-like vessels. Then, we use DepMap-based transcription profiles and reveal that VM competence is associated with a distinct signature. Interestingly, VM and EA transcription profiles partially overlap, yet they also remain transcriptionally distinct, with inferred mechanistic divergence, with VM being more associated with cancer cell stemness (CSC), epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodeling programs and EA being more associated with vessel strength. In addition, VM-competent TNBC cells display migration patterns and transcriptomic features consistent with endothelial-like mechanosensitivity. Conclusions: Together, these findings indicate that VM is a distinct, heterogeneous, and therapy-relevant state in TNBC that complements classical angiogenesis. Finally, the mechanistic distinction between VM and EA programs made here will motivate future studies on dual-targeting strategies that inhibit both vascularization processes while also motivating future studies on VM for precision treatment in TNBC.