Brain ECM-mimetic hydrogel platform to investigate cellular and extracellular dynamics in the early metastatic niche of triple-negative breast cancer

Abstract

Brain metastasis of triple negative breast cancer (TNBC) rapidly progresses, causing severe neurological decline with a median survival of less than 6 months. This tragic disease is often difficult to identify with sufficient time for treatment and is exacerbated by a lack of effective pharmacological intervention. Replicating the biochemical and mechanical properties of the pre-metastatic niche in vitro is a critical step in expediting the development of new therapeutics. However, a high fidelity and reproducible model system is needed. In an effort to develop a model to simulate the adhesion signaling and cell to cell communication present in the pre-metastatic brain, we produced and characterized an in vitro, PEG based hydrogel and co-culture method to evaluate the cellular and extracellular dynamics of early cancer colonization. ☐ To quantify the influence of a brain-mimetic microenvironment on brain metastatic TNBC, we encapsulated and cultured the TNBC cell line, MDA-MB-231 (P231), and its braintropic subline MDA-MB-231-BrM2a-831 (BrM2a), in three premetastatic niches: a highly cell adhesive and highly cell degradable permissive niche, a highly adhesive but less degradable niche, and a non-adhesive but highly degradable niche. To mimic brain ECM, we functionalized the adhesive formulations with a brain-mimetic peptide cocktail and compared the cell responses to a ‘generic’ RGDS-only functionalization. This suite of conditions allowed us to investigate the influence of integrin-mediated adhesion, cell-mediated degradation, and cell type on the fate of P231s and BrM2as. By quantifying viability, viable cell density, apoptosis, proliferation, and morphology we demonstrate that brain-mimetic adhesion has little to no impact on P231 phenotype, but the BrM2as display reduced viable cell density, reduced proliferation, and a higher proportion of both spherical clusters and spherical individual cells compared to the generic RGDS-functionalized niches. ☐ With the demonstrated impact of brain-mimetic adhesion on brain preferential cells, we implemented the brain-mimetic functionalization to characterize the cellular response of cell native to the central nervous system. Primary human astrocytes and iPSC derived neural progenitor cells (NPC) were encapsulated and evaluated. In the astrocytes these metrics show modest differences between the brain-mimetic and RGDS functionalizations with reduced apoptosis in the brain permissive formulation. Both permissive formulations showed high viability and a larger portion of elongated cells than the adhesion restricted formulation. This shows that in monoculture, the astrocytes require ECM adhesion signaling to survive well but the diversity of the brain mimetic functionalization aids in reducing apoptosis. The iPSC derived NPCs were encapsulated in the brain mimetic permissive formulation as either single cells or spheres and differentiated toward forebrain neurons. Both seeding conditions produce clusters with robust neurite/axonal extension with the enlarged size of the spheres allowing extension across the surface of the hydrogel and the single cell encapsulated samples show projections that are more subdued in length but far more numerous. This demonstrates the compatibility of the brain-mimetic permissive formulation to cultivate complex neuronal cultures and further replicate the environment of the brain. ☐ Having characterized the baseline response of astrocytes and BrM2as, we proceeded to evaluate them in coculture. Similar to the astrocyte mono-culture, the difference between the brain-mimetic and RGDS functionalization was slight, with the brain permissive formulation producing a greater proportion of elongated clusters. To closer examine the protein expression differences induced by these culture conditions, proteomics analysis was performed. This analysis revealed coculture samples encapsulated in the brain mimetic permissive formulation have significant changes to structural and regulatory ECM expression as well as secreted immune signaling factors. Taken together these studies present a well characterized brain mimetic hydrogel platform to cultivate central nervous system cells and provide a tool to investigate brain metastatic cancer in a tissue relevant context. Future implementation of this platform could be to model other cancers that frequently metastasize to the brain and investigate the mechanisms involved in cancer progression and response of the native cell population.