"Triple-negative breast cancer (TNBC), a subtype of breast cancer, is characterized by rapid local invasive growth, metastasis, and chemotherapeutic resistance that strongly impacts mortality. Identifying and developing effective treatments for TNBC is necessary for improving patient outcomes. The Turley and collaborator’s laboratories have shown that high expression of a hyaluronan receptor RHAMM is linked to poor outcomes in TNBC cell subsets and is a critical factor in the experimental progression and metastasis of TNBC. RHAMM+/+ and RHAMM-/- MDA-MB-231 cells were seeded into 96-well tissue culture plates with media (2D culture) or Matrigel™ (3D culture) and grown for 4 and 7 days, respectively. Cells were exposed to 0.001-10µM doxorubicin or 0.0002-0.625µM trametinib 24 and 72 hours after seeding in 2D and 3D cultures, respectively and grown for an additional 72 hours. Spheroid size, metabolic activity and proliferation were quantified to identify RHAMM-dependent alterations with and without drug treatment. RHAMM-loss significantly reduces metabolic activity and proliferation of MDA-MB-231 cells in 2D and 3D cultures. RHAMM+/+ MDA-MB-231 cells are more sensitive than RHAMM-/- comparators to clinical chemotherapeutics doxorubicin and trametinib in 2D cultures. The presence of RHAMM does not alter sensitivity to doxorubicin or trametinib in 3D cultures. However, both RHAMM+/+ and RHAMM-/- MDA-MB-231 cells are more sensitive to trametinib in 3D cultures than in 2D cultures. Ongoing analyses will assess the ability of stapled RHAMM peptide mimetics, which block RHAMM signalling, to replicate the effect of RHAMM-loss on invasive growth and drug sensitivity in TNBC cell lines."