Human mammary 3D spheroid models uncover the role of filopodia in breaching the basement membrane to facilitate invasion
Basement membranes (BM) are thin, nanoporous sheets of specialized extracellular matrix (ECM) that line epithelial tissues. They are dynamic structures that serve multiple key functions, as evidenced by numerous diseases, including cancer progression, that are associated with their alterations. Our understanding of the BM and its communication with adjoining epithelial cells remains highly fragmented due to the BMs complex molecular architecture, the lack of molecular tools, limitations in utilizing high-resolution imaging techniques to BMs assembled on tissues, and the difficulty of assessing their functional contributions in vivo. Here, by combining multiple -omics analyses and advanced microscopy methodologies, we characterized the BM from two normal human mammary epithelial cell lines, MCF10 and HMLE, grown as spheroids in 3D matrices. Our findings indicate that the spheroids autonomously assemble a BM exhibiting all the molecular, structural, and biophysical characteristics of physiological BM. Using these minimalist model systems, we show for the first time that the laminins, perlecan, and the hemidesmosomes are all arranged along a shared porous lattice defined by the collagen IV molecular network. Next, we demonstrate that the invasion-promoting PSD4/EFA6B knockout, found in patients with breast cancer, decreases the expression of BM components and their assembly on the spheroid surface. We then show that invasive spheroids develop enlarged pores in the BM via filopodia-like plasma membrane extensions, which further expand in a protease-dependent manner, thereby facilitating the passage of invasive cells. Significance StatementThe basement membrane (BM) directs epithelial tissue architecture and behavior. How tumor cells breach this barrier during invasion remains poorly understood. Using minimalist 3D spheroid models mimicking physiological BM assembly, we reveal a novel infiltration mechanism. Filopodia-like protrusions perforate and widen BM pores. This facilitates cell dissemination through sequential protease-independent and -dependent steps.