IntroductionBreast cancer remains one of the leading causes of cancer-related mortality among women globally, with brain metastasis significantly worsening patient prognosis. Brain metastatic breast cancer (BMBC) presents unique challenges, particularly due to the phenomenon of metastatic dormancy, where disseminated tumor cells (DTCs) enter a quiescent state, evading therapeutic interventions and potentially leading to late relapse1. Dormancy is characterized by the persistence of cancer cells in a non-proliferative but viable state. These dormant cells exhibit remarkable plasticity, allowing them to adapt to various microenvironments, survive therapeutic stress, and resist conventional and targeted therapies2,3,4. The resistance of dormant cells to treatment is largely due to their non-cycling nature, as most cytotoxic therapies target rapidly dividing cells5. Understanding the mechanisms underlying dormancy-associated resistance to treatment is crucial for developing more effective treatments.DTCs can be maintained either as independent dormant cells—a state known as cellular dormancy—or as small clusters of cells where a balance between proliferation and cell death leads to a constant tumor size, referred to as tumor mass dormancy6. These DTCs can form micrometastatic lesions whose size does not increase due to this equilibrium, allowing the tumor to remain undetected and exhibit resistance to therapies targeting rapidly dividing cells. Dormancy in the micrometastasis is influenced by several factors, such as alterations in the extracellular matrix (ECM) and signaling pathways such as p38 and ERK, which regulate the balance between dormancy and proliferation7,8. ECM, a major component of the tumor microenvironment (TME), differs significantly between brain and breast tissues9. These differences in ECM composition and structure can profoundly affect the behavior of dormant micrometastasis. These interactions not only influence their survival but also determine their eventual reactivation and proliferation under specific microenvironmental conditions. In the context of dormancy, the biophysical and biochemical characteristics of the ECM—such as stiffness and cell-scaffold interactions—are critical in maintaining the dormant state10. Consequently, studying ECM-induced dormancy has become a significant area of interest. While traditional in vivo models have provided valuable insights into the mechanisms of BMBC and dormancy, they often lack the ability to precisely tune the TME. This limitation, coupled with the significant challenge of BMBC resistance to therapy, underscores the need for in vitro models to investigate the effects of drugs on dormant BMBC micrometastasis and elucidate the mechanisms underlying their responses.Dormant cancer cells resist drugs through a complex interplay of cellular adaptations, microenvironmental influences, and molecular mechanisms. Known mechanisms of drug resistance in cancer cells include: failure of cytotoxic therapies designed to target proliferative cells to affect dormant cells4; engagement with the ECM activating integrin signaling pathways that promote survival11; maintenance of cells in dormancy enhancing their ability to withstand stress12; and elevated efflux of chemotherapeutic drugs from cancer cells leading to lower drug accumulation13. However, the specific mechanisms through which this resistance is sustained in the context of dormant BMBC require further investigation. Understanding the mechanisms of drug resistance associated with ECM-induced dormancy could potentially benefit the development of effective therapies for BMBC.To this end, Kondapaneni et al., previously demonstrated that a biomimetic hyaluronan (HA) hydrogel model induced dormancy in BMBC spheroids, while a free suspension culture promoted proliferation14. However, the ability of the model to study dormancy-associated drug resistance and the associated mechanisms was not explored. In this study, we aimed to address this gap by comparing the drug response of dormant and proliferating three-dimensional (3D) BMBC spheroids, wherein we hypothesized that HA hydrogel-induced dormant spheroids would exhibit resistance to therapy compared to proliferative spheroids cultured in suspension. We employed Paclitaxel (PTX) as a chemotherapy (for the triple negative breast cancer subtype) and Lapatinib (LAP) as a targeted therapy (for the human epidermal growth factor receptor 2 (HER2) subtype)15,16,17. We evaluated cell proliferation and apoptosis, as well as the percentage of cells positive for phosphorylated-ERK and phosphorylated-p38 in BMBC spheroids following drug treatment. Additionally, we investigated if modulating the culture environment reverses therapy response in BMBC spheroids. Finally, we focused on the p38 signaling pathway, which has been linked to both dormancy and drug resistance18,19, to assess its role in modulating drug responses in dormant spheroids.ResultsMorphological characterization of spheroids on HA hydrogel and suspension culture in response to treatmentBMBC cell spheroids were prepared and treated for 72 h in suspension culture to determine cell viability as a function of drug concentration. Drug concentrations ranging from 25 nM to 50 µM were tested for PTX for the MDA-MB-231Br cell line (Fig. S1), while LAP at concentrations from 10 to 5000 nM was tested for the BT474Br3 cell line (Fig. S2). The lowest concentration for both the drugs at which cell viability was reduced to