Engineering the ECM to influence macrophage responses: early insights for immunomodulatory applications.

The immune system integrates environmental signals to regulate activation and maintain tissue homeostasis. Among these, the extracellular matrix (ECM) plays a pivotal role by influencing immune cell adhesion, migration, and differentiation. Macrophages, as first responders, are highly sensitive to ECM composition and architecture, which shape their inflammatory profile and function. Understanding how ECM properties affect macrophage response is crucial to design immunomodulatory therapies, especially for cancer or chronic inflammation. In this study, we examined how ECM-like substrates influence macrophage polarization in response to nanomaterial-induced inflammatory cues. We developed 3D scaffolds of denatured collagen (gelatin), functionalized with various concentrations of carbon nanotubes (CNTs), used as model pro-inflammatory fibers. Macrophages derived from Human THP-1 cells were cultured on these scaffolds for up to five days. Viability was preserved after 24 hours, but higher CNT concentrations and prolonged exposure triggered marked morphological changes, including increased filopodia and reduced spreading area, indicating cytoskeletal remodeling and altered activation. Electron microscopy confirmed that macrophages actively remodeled the ECM-like substrates, exposing embedded CNTs and facilitating their uptake. Flow cytometry and ROS assays showed a polarization shift toward a pro-inflammatory M1-like phenotype. To assess functional consequences, conditioned media from macrophages on CNT-loaded scaffolds was applied to glioblastoma spheroids, significantly reducing their growth and size, suggesting release of anti-tumoral factors. Overall, our results highlight how the ECM environment shapes macrophage responses to nanomaterials, affecting their activation and secretory profiles. This platform represents a biologically relevant model to study immune-nanomaterial interactions and may support the development of innovative strategies in cancer immunotherapy.