Cathepsin-B driven regulation of fibroblast dynamics and matrix reorganization in the breast cancer microenvironment

Cancer-Associated Fibroblasts (CAFs) are pivotal components of the tumor microenvironment, exerting significant influence on cancer progression, particularly through their profound impact on extracellular matrix (ECM) dynamics. CAFs are major contributors to fibrillar collagen deposition and reorganization, processes that induce ECM stiffness and linear alignment, thereby fostering tumor growth, cellular invasion, and metastasis.

Our research highlights cathepsin B (CTSB), a cysteine proteinase, as a key regulator in this context. We observed a significant upregulation of CTSB expression in human breast cancer, correlating with a worse patient prognosis. Myeloid cells and myCAFs were identified as primary cellular sources of CTSB within the breast tumor stroma. Leveraging a 3D fibrillar type I collagen spheroid model, which was specifically devised to optimize CAF migratory behavior and closely mimic the native collagen architecture observed in vivo, we uncovered that intracellular CTSB proteolytic activity is critical for CAF invasion. Ctsb-deficient in MMTV-PymT-derived CAFs (BKO) displayed dramatically impaired invasive capacity, characterized by reduced cellular protrusions and increased cell death. This impairment was quantified using a tailored 3D image analysis pipeline that computes detailed morphometric parameters from primary 3D images.

Furthermore, using confocal reflectance microscopy and an advanced 3D analysis of collagen fiber density and orientation, Ctsb deficiency profoundly disrupted CAF-mediated collagen matrix remodeling, evidenced by reduced fiber density and a failure to form the invasion-promoting radial collagen bundles typically induced by wild-type CAFs. Mechanistically, BKO CAFs showed a compromised ability to generate traction forces and extend stable cytoplasmic protrusions on collagen, favoring cell-cell aggregation over cell-collagen interactions. This impairment was associated with disorganized and less persistent CAF migration, directly correlating with a significant disruption of the perinuclear actin cap, a known orchestrator of cell migration and nuclear shape. These findings underscore the central role of intracellular CTSB in dictating CAF-driven ECM remodeling and migratory behavior.