Programmable microfluidic modeling of the glioblastoma immune microenvironment recapitulates temporally regulated tumor–immune signaling

BACKGROUND: Glioblastoma (GBM) progression is driven by intricate interactions between neoplastic cells and immune populations within the tumor microenvironment (TME), yet the temporal organization of these processes remains insufficiently defined. While tumor-associated macrophages (TAMs), composed of microglia and monocyte-derived macrophages, constitute a major immune population in GBM, microglia play a prominent role in shaping local immunosuppressive niches, thereby limiting cytotoxic lymphocyte infiltration. However, static co-culture models constrain interrogation of dynamic tumor-immune signaling. METHODS: We utilized a programmable multi-inlet microfluidic platform that allows sequential, order-specific introduction of immune cells into GBM constructs, enabling controlled interrogation of MG- and natural killer (NK)-mediated interactions under defined temporal conditions. Bulk transcriptomic profiling and single-cell analyses were used to characterize tumor- and immune-associated pathways elicited by distinct temporal configurations. RESULTS: In triculture constructs containing identical populations of GBM cells, microglia, and NK cells, MG-first delivery preferentially activated STAT3-associated immunoregulatory transcriptional programs, whereas NK-first delivery induced cytotoxic and interferon-related gene expression profiles. IL12A expression was selectively increased under NK-first conditions and correlated with immune-active GBM states in patient datasets. STAT3 inhibition reduced MG-associated suppression, increased IL12A expression, and enhanced the response to temozolomide. CONCLUSIONS: This programmable spheroid platform not only recapitulates hallmark features of the GBM immune microenvironment but also uncovers temporally regulated pathways arising from sequential cellular interactions. By enabling controlled reconstruction of dynamic tumor-immune signaling, this system offers a versatile strategy for mechanistic investigation and provides a foundation for evaluating immune-modulatory approaches and microenvironment-targeted interventions in GBM and other malignancies.