Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini

Abstract

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs’ barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.

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

“…To examine the formation of actin-based cell protrusions (CP) and their function for BM invasion, we used MCF10A-derived breast cell acini with an endogenously formed BM scaffold. This cell model has been widely used to study normal breast gland morphogenesis [33] and the invasion-promoting role of immature BM mechanics [6]. We adopted acini with either a low-developed BM (ld-BM, 11 days old) or a highly-developed BM (hd-BM, 22 days old) from the latter work.…”

“…A sample transfer onto cover slips was mandatory to capture the dynamic process of CP formation and retraction with high microscopic resolution. Besides, such stiff substrate stimulation was purposely used in this work to trigger the invasive transition of benign breast acini, as previously described [6].…”

“…Finally, we tested how the switch from MS to SF cells contributes to physical BM disruption. Placing non-invasive MCF10A breast acini onto tumor-like elastomeric substrates (12 kPa) induces the invasive transition characterized by local BM disruption and cell dissemination [6]. Quantitative cell traction force microscopy was applied to measure cell force-based BM stress at the acinus-BM-substrate interface.…”

“…Matured BM scaffolds act as physical barriers capable of sustaining physiological mechanical loads in homeostatic microenvironments [4,5]. In cancer progression, the tumorous extracellular matrix (ECM), characterized by oncogenic EGFR signaling and increased local stiffness, induces cell-force generation, finally fueling BM disruption [6]. Invasive cells undergo epithelial to mesenchymal transitions (EMT) programs with a fundamental reorganization of actin cytoskeletal structures.…”

Fibronectin: Molecular Structure, Fibrillar Structure, and Mechanochemical Signaling

“…On a mesocellular level, microcontact printing, microfluidics and microfabrication have been combined to study the role of FN fibrils in transmitting forces, using either cell-derived fibrils [19,45] or artificially derived fibers [65,96], but these studies do not specifically investigate FN fibril mechanics. In the interest of measuring cell-substrate forces, some techniques such as atomic force microscopy (AFM) [103], (astigmatic) traction force microscopy [104], elastic resonator interference stress microscopy (ERISM) [105], or hex dot microcontact printing [106] tend not to focus directly on ECM response to deformation, where often FN, if involved, may only be considered for facilitating substrate attachment or is subjected to the survey only at the molecular or domain constitution [97].…”