Inhibition of extracellular matrix protein fibulin-3 reduces immunosuppressive signaling and increases macrophage activation in glioblastoma

Glioblastoma (GBM) tumors remain a challenge for immunotherapy owing to their heterogeneous and immunologically cold properties. GBM cells change the composition of the neural extracellular matrix (ECM), affecting the mobility, survival, and function of immune cells such as tumor-associated microglia and infiltrated macrophages (TAMs). The ECM protein EFEMP1/fibulin-3 is a pericellular component uniquely upregulated in GBM compared to normal brain, which promotes tumor growth and invasion. Here, we demonstrate that fibulin-3 indirectly modulates TAM behavior and can be targeted to restore innate immune responses. Intracranial tumors initiated by fibulin-3-deficient GBM cells showed increased presence of TAMs with decreased immunosuppression markers (Arginase, CD206), whereas the opposite effects were observed in tumors overexpressing fibulin-3. In silico analyses revealed a positive correlation between fibulin-3 and an immunosuppressive signature that was validated in GBM stem cells (GSCs) and in vivo. We further demonstrated that fibulin-3 regulates the expression of immunosuppressive signals (CSF-1, TGF-β1, and CD47) by autocrine activation of NF-κB signaling. Immunosuppressive signals were downregulated by knockdown of fibulin-3 in GSCs and by inhibition of this protein using an anti-fibulin-3 antibody. Myeloid cells exhibited higher phagocytic activity and killing of GBM cells in presence of this antibody. Furthermore, locoregional delivery of anti-fibulin-3 in mice with intracranial GBM increased the presence of pro-inflammatory TAMs, thereby reducing tumor viability. Our findings show that anti-fibulin-3 approaches, which affect the ECM surrounding tumor and immune cells, can diminish immunosuppression in GBM and boost innate immune responses against the tumor.