Innate immune cross-talk of glioblastoma pathophysiology: Understanding NLRP3 and NLRP12.

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Background: Glioblastoma (GBM), a grade IV astrocytoma, is a highly aggressive malignant brain tumor with a median survival of less than 14 months despite surgery, chemotherapy, and radiotherapy. Tumor progression is driven by a heterogeneous microenvironment composed of glioma stem cells, astrocytes, microglia, macrophages, and endothelial cells. Microglia and macrophages, the brain’s innate immune cells, contribute to tumor growth by sustaining a pro-tumorigenic inflammatory milieu. Nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) regulate inflammatory signaling in astrocytes and microglia. NLRP3 promotes inflammation through Caspase-1–mediated maturation of IL-1β and IL-18, inducing inflammatory responses and pyroptosis. In contrast, NLRP12 primarily suppresses inflammation by modulating NF-κB signaling. Both exhibit context-dependent tumor-promoting or suppressive roles in cancer. Although elevated NLRP12 expression correlates with poor survival in GBM, the underlying cellular mechanisms remain unclear. Methods: This study investigated the roles of NLRP3 and NLRP12 in GBM progression. Baseline expression was analyzed in established cell lines and patient-derived GBM cells using PCR, western blotting, and immunocytochemistry. Cell proliferation and migration were evaluated in wild-type, NLRP3 ^-/- , and NLRP12 ^-/- cells. 3D spheroids comprising GBM cells and astrocyte cells were generated to study tumor growth. 3D organoids derived from patient-derived tumor tissue were generated to examine expression patterns and tumor growth. Results: We identify markedly higher expression of NLRP12 and NLRP3 in astrocytes compared to GBM cells and demonstrate that modulating these receptors significantly influences tumor cell proliferation, survival, migration, and multicellular architecture. Using patient-derived samples and organoid models, we observe heterogeneous expression patterns, with elevated pro-inflammatory cytokine levels correlating with increased extracellular matrix deposition and distinct tumor morphological features. Conclusions: Heterocellular co-culture systems reveal a reciprocal, paracrine-dependent interaction in which GBM cells and astrocytes maintain tumor compactness in the absence of NLRP3 and NLRP12, underscoring their context-dependent roles. Pharmacological inhibition of NLRP3 with glyburide reduced inflammatory cytokine release in GBM cells but showed pro-inflammatory effects in astrocytes. Patient-derived cells displayed differential NLRP3 and NLRP12 expression compared to established cell lines, potentially contributing to intra- and inter-tumoral heterogeneity and variable therapeutic responses. Overall, NLRP3- and NLRP12-mediated signaling circuits represent integral components of the GBM microenvironment and promising therapeutic targets warranting further investigation.