Protein-modified hyaluronic acid hydrogels to elucidate microenvironment-mediated chemotherapy response in triple...

Brain metastatic breast cancer (BMBC) remains a major clinical challenge, in part, due to the development of drug resistance. However, the mechanisms associated with response to therapy remain poorly understood. In this work, we developed hyaluronic acid (HA)-based hydrogels functionalized with tunable concentrations of laminin-1 (LM1) or tenascin-C (Tn-C) to study how extracellular matrix (ECM) protein composition regulates triple negative BMBC drug response. The successful incorporation of LM1 and Tn-C onto HA hydrogels was verified by quantifying protein presence using a micro-BCA assay. To confirm their impact on cell behavior, MDA-MB-231Br triple negative BMBC cells were seeded onto the protein-modified hydrogels, and it was observed that the cells displayed distinct morphological changes and cell spreading as a function of varying protein concentration. Next, cells were cultured on HA hydrogels with low-to-high concentrations of LM1 or Tn-C and subsequently exposed to paclitaxel, a chemotherapy drug. At low ECM protein concentration, cells exhibited a drug-insensitive phenotype, while higher concentrations promoted proliferation accompanied by enhanced drug responsiveness, as measured by proliferation (EdU, Ki67) and apoptosis (cleaved caspase-3) assays. Mechanistic studies revealed that BMBC cells engage the protein-modified HA hydrogel through β1 integrin, and blocking this receptor significantly suppressed proliferation and increased paclitaxel sensitivity. Further, analysis of downstream signaling showed that β1 integrin blockade reduced phosphorylated ERK (p-ERK) levels, implicating the β1 integrin-ERK pathway as a mediator of ECM-driven drug response. In sum, this study demonstrates that protein-modified HA hydrogels provide a versatile platform to study the mechanisms of ECM-mediated drug response in triple negative BMBC.