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ABSTRACTNeural stem cells (NSCs) are multipotent cells of the central nervous system (CNS) capable of self‐renewal, differentiation, and responding to and shaping the surrounding microenvironment. Their continuous crosstalk with surrounding CNS cells is a key component of their therapeutic potential, particularly in tissue repair and regeneration. Communication in the CNS relies on complementary mechanisms, including connexins (Cxs)‐based intercellular communication, to maintain homeostasis and coordinate responses to physiological and pathological stimuli. Itaconate, an endogenous shunt product of the tricarboxylic acid cycle, functions as an immunometabolite involved in inflammation and oxidative stress and has recently been implicated in neuroimmune modulation. Although itaconate influences several signalling cascades and is exchanged between cells and/or released into the extracellular milieu, its effects on Cxs expression in NSCs and whether the modulation of Cxs expression profile represents a driving factor in shaping cell fate remain unclear. Here, we investigated the effect of dimethyl itaconate, a cell‐permeable esterified itaconate derivative, on the expression profile of Cxs in NSCs and its potential to modulate NSCs fate and differentiation. We found that dimethyl itaconate modulates Cxs expression in NSCs, increasing Cx36 levels, and promotes NSCs differentiation toward a neuronal phenotype, while inhibition of Cxs‐based channels with carbenoxolone or mefloquine abolishes these dimethyl itaconate‐induced effects. Collectively, these findings highlight a regulatory role for cell‐permeable itaconate and contribute to the understanding of intercellular communication in the CNS microenvironment, providing insights into potential therapeutic strategies for CNS repair and regeneration. The immunometabolite itaconate modulates the connexin profile of neural stem cells, and it upregulates Cx36‐based channels, promoting the differentiation toward a neuronal phenotype. These effects were abolished by connexin blockade, supporting a role for itaconate in the neuroimmune axis in inflammatory and neurodegenerative disorders.

SummaryDiffuse midline glioma (DMG) is a devastating pediatric brain tumor. The oncolytic adenovirus Delta-24-RGD has shown promising efficacy and safety in DMG patients but is not yet curative. Thus, we hypothesized that activating dendritic cells (DCs) through the CD40 costimulatory receptor could increase antigen presentation and enhance the anti-tumor effect of the virus, resulting in long-term responses. This study shows that the intratumoral co-administration of Delta-24-RGD and a CD40 agonistic antibody is well tolerated and induces long-term anti-tumor immunity, including complete responses (up to 40%) in DMG preclinical models. Mechanistic studies revealed that this therapy increased tumor-proliferating T lymphocytes and proinflammatory myeloid cells, including mature DCs with superior tumor antigen uptake capacity. Moreover, the lack of cross-presenting DCs and the prevention of DC recruitment into the tumor abolish the Delta-24-RGD+anti-CD40 anti-DMG effect. This approach shows potential for combining virotherapy with activating antigen-presenting cells in these challenging tumors. Graphical abstract Highlights•CD40 agonism boosts Delta-24-RGD efficacy in pediatric DMG models•cDC1s are essential for the long-lasting anti-tumor effect of Delta-24-RGD and anti-CD40•Microglia and tumor macrophages contribute to the cDC1 recruitment following the therapy Labiano et al. show that co-administration of the Delta-24-RGD virus and a CD40 agonistic antibody is safe, yielding complete responses in DMG-bearing mice. The combination relies on type 1 conventional dendritic cells (cDC1s), which remodel the tumor microenvironment into a proinflammatory landscape, fostering long-term anti-tumor immunity against this challenging tumor.

Insulin-like growth factor (IGF) is involved in regulating many processes during neural development, and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g., via inhibiting Wnt/β-catenin signaling). In the present study, neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and β-catenin. Phosphorylation levels of Akt, Erk1, 2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of β-catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1, 2 and p38 were not significantly changed in IGFBP4-treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway.