From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion

Abstract

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM–ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM–ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.

ECM-transmitted shear stress induces apoptotic cell extrusion in early breast gland development

“…On day 4 of development, spheroids appeared poorly developed with unorganized cell clusters and thin BM scaffold. This thin BM was frequently penetrated by actin protrusions ( Figure 5A' , white arrows), as reported previously ( Eschenbruch et al, 2021 ). Basoapical cell polarization was reached partially at day 11 and fully at day 18, at which partial lumen clearance was regularly observed.…”

“…We found that the basal cell layer of spheroids at day 3–4 formed short actin microspikes penetrating the thin BM. Moreover, it was shown that these ECM probing microspikes form focal adhesions to the BM and the ECM, especially in the early developmental stages of spheroids with low polarization ( Eschenbruch et al, 2021 ). Considering actin-rich protrusions as the prevailing mechanism for ECM-mechanosensation, we hypothesize a shear strain-induced opening of PIEZO-1 ion channels as one of the primary sensing mechanisms.…”

Matrix Metalloproteinases Shape the Tumor Microenvironment in Cancer Progression

“…Invadosomes also have a dynamic life cycle [ 282 ]. Cells of breast acini probe the ECM with non-proteolytic actin microspikes, which, as functional precursors of invadopodia, widen pores in the collagen IV meshwork with force-transmitting focal adhesions and initiate BM penetration [ 372 ]. In cells disseminating into the ECM, such non-proteolytic microspikes are then converted into proteolytically active invadopodia containing MMP-14 [ 372 ].…”

“…Cells of breast acini probe the ECM with non-proteolytic actin microspikes, which, as functional precursors of invadopodia, widen pores in the collagen IV meshwork with force-transmitting focal adhesions and initiate BM penetration [ 372 ]. In cells disseminating into the ECM, such non-proteolytic microspikes are then converted into proteolytically active invadopodia containing MMP-14 [ 372 ]. The formation of invadopodia is stimulated by ECM rigidity, degradation of ECM molecules, and growth factor signaling [ 361 , 373 , 374 , 375 , 376 , 377 ].…”

Effects of Nanopillars and Surface Coating on Dynamic Traction Force

“…19 These results agreed with previous observations that more actin-rich protrusions from cell edges generated larger contractile forces during cell migration. 49…”