The role of hyaluronan and the mechanical properties of cancer-associated fibroblasts in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is highly aggressive and lacks treatment targets. The high stroma content in TNBC correlates with poor prognosis. As the key non-cancerous cell in the tumour microenvironment (TME), cancer-associated fibroblasts (CAFs) are responsible for secreting the components and remodelling the extracellular matrix (ECM) in the TME. The role of CAFs in altering cancer progression by regulating ECM mechanical properties remains poorly understood.
This work aimed to observe the effects of primary CAFs on a TNBC cell line, compare these effects with primary NFs, and focus on mechanical changes. I characterised these fibroblasts using immunofluorescence, traction force microscopy, and migration assays. The CAFs showed a more elongated morphology, higher contractile force, and a more migratory phenotype. By establishing 2D and 3D co-culture methods and combining live-cell imaging with immunofluorescence, I studied interactions between primary fibroblasts and the TNBC cell line, as well as the effects of the key ECM component hyaluronan (HA) on TNBC cells. My results showed that CAFs secreted higher levels of HA into the cells and ECM, and that TNBC cells proliferated more than NFs in 3D co-culture; this effect was reduced after decreasing Hyaluronan Synthase 2 (HAS2) production. The oncogenic transcriptional co-regulator YAP was more active in TNBC cells in 2D CAF co-culture. Finally, I observed that ECM secreted by CAFs was stiffer than that secreted by NFs, and that HAS knockdown in CAFs reduced ECM stiffness. Taken together, these observations support my hypothesis that HA plays an important role in CAF function in the TNBC ECM and in TNBC cell growth, potentially via nuclear translocation of YAP. This work also highlights the importance of mechanical signalling and ECM properties, offering a new perspective on CAFs and HA as potential targets for future TNBC therapies.