Bioengineered Lymphatic Vessels in Synthetic Matrices to Study Breast Cancer Cell Functions

Lymphatic vessels play an important role in pathologies, such as cardiovascular diseases or metastasizing cancers. However, mechanisms involved in cancer metastasis through lymphatic vessels remain largely unknown. In this study, we engineer 3D lymphatic microvessels by co-culturing human lymphatic endothelial cells (hLECs) with human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) in synthetic and fully defined enzymatically crosslinked poly(ethylene glycol) (TG-PEG) hydrogels. By tuning the stiffness of the TG-PEG scaffolds, varying seeding densities of hLECs and hBM-MSCs, and stimulation with lymphangiogenic factors, we identify suitable culture conditions for lymphatic network formation. Immunocytochemistry-based evaluations reveal the formation of lymphatic structures consisting of hLECs that are ensheathed with basement membrane extracellular matrix components and hBM-MSCs, which express perivascular-specific markers. Finally, the addition of non-invasive MCF-7 and invasive MDA-MB-231 breast cancer cells to the forming lymphatic structures shows a clear negative influence of cancer cells on the length of lymphatic networks. Moreover, we observe a closer association of invasive than of non-invasive cancer cells with the lymphatic networks, which can be inhibited by blocking of CCL21. Our findings present a promising approach to investigate the cancer microenvironment, as our model can serve as a tool to study physiological lymphangiogenesis and pathological phenomena in vitro.