ALOX5 contributes to glioma progression by promoting 5-HETE-mediated immunosuppressive M2 polarization and PD-L1 expression of glioma-associated microglia/macrophages.

BACKGROUND: Oxylipin metabolism plays an essential role in glioma progression and immune modulation in the tumor microenvironment. Lipid metabolic reprogramming has been linked to macrophage remodeling, while the understanding of oxylipins and their catalyzed enzymes lipoxygenases in the regulation of glioma-associated microglia/macrophages (GAMs) remains largely unexplored. METHODS: To explore the pathophysiological relevance of oxylipin in human glioma, we performed Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) analysis in human glioma and non-tumor brain tissues. To comprehensively investigate the role of arachidonate lipoxygenase 5 (ALOX5) in glioma, we performed in vivo bioluminescent imaging, immunofluorescence staining and flow cytometry analysis on tumors from orthotopic glioma-bearing mice. We developed an ALOX5-targeted nanobody, and tested its anti-glioma efficacy of combination therapy with α-programmed cell death protein-1 (PD-1). RESULTS: In this study, we found that ALOX5 and its oxylipin 5-hydroxyeicosatetraenoic acid (5-HETE) are upregulated in glioma, accumulating programmed death-ligand 1 (PD-L1)+ M2-GAMs and orchestrating an immunosuppressive tumor microenvironment. Mechanistically, 5-HETE derived from ALOX5-overexpressing glioma cells, promotes GAMs migration, PD-L1 expression, and M2 polarization by facilitating nuclear translocation of nuclear factor erythroid 2-related factor 2. Additionally, a nanobody targeting ALOX5 is developed that markedly suppresses 5-HETE efflux from glioma cells, attenuates M2 polarization of GAMs, and consequently ameliorates glioma progression. Furthermore, the combination therapy of the ALOX5-targeted nanobody plus α-PD-1 exhibits superior anti-glioma efficacy. CONCLUSIONS: Our findings reveal a pivotal role of the ALOX5/5-HETE axis in regulating GAMs and highlight the ALOX5-targeted nanobody as a potential therapeutic agent, which could potentiate immune checkpoint therapy for glioma.

Immunized Microspheres Engineered Hydrogel Membrane for Reprogramming Macrophage and Mucosal Repair.

The "double-edged sword" effect of macrophages under the influence of different microenvironments determines the outcome and prognosis of tissue injury. Accurate and stable reprogramming macrophages (Mφ) are the key to rapid wound healing. In this study, an immunized microsphere-engineered GelMA hydrogel membrane is constructed for oral mucosa treatment. The nanoporous poly(lactide-co-glycolide) (PLGA) microsphere drug delivery system combined with the photo-cross-linkable hydrogel is used to release the soybean lecithin (SL)and IL-4 complexes (SL/IL-4) sustainedly. In this way, it is realized effective wound fit, improvement of drug encapsulation, and stable triphasic release of interleukin-4 (IL-4). In both in vivo and in vitro experiments, it is demonstrated that the hydrogel membrane can reprogram macrophages in the microenvironment into M2Mφ anti-inflammatory types, thereby inhibiting the local excessive inflammatory response. Meanwhile, high levels of platelet-derived growth factor (PDGF) secreted by M2Mφ macrophages enhanced neovascular maturation by 5.7-fold, which assisted in achieving rapid healing of oral mucosa. These findings suggest that the immuno-engineered hydrogel membrane system can re-modulating the biological effects of Mφ, and potentiating the maturation of neovascularization, ultimately achieving the rapid repair of mucosal tissue. This new strategy is expected to be a safe and promising immunomodulatory biomimetic material for clinical translation.

USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4.

BACKGROUND: Glioblastomas (GBMs) are grade IV central nervous system tumors characterized by a poor prognosis and a short median overall survival. Effective induction of GBM cell death is difficult because the GBM cell population is genetically unstable, resistant to chemotherapy and highly angiogenic. In recent studies, ubiquitin-specific protease 7 (USP7) is shown to scavenge ubiquitin from oncogenic protein substrates, so effective inhibition of USP7 may be a potential key treatment for GBM. METHODS: Immunohistochemistry and western blotting were used to detect the expression of USP7 in GBM tissues. In vitro apoptosis assay of USP7 inhibition was performed by western blotting, immunofluorescence, and flow cytometry. Anti-apoptotic substrates of USP7 were defined by Co-IP and TMT proteomics. Western blotting and IP were used to verify the relationship between USP7 and its substrate. In an in vivo experiment using an intracranial xenograft model in nude mice was constructed to assess the therapeutic effect of target USP7. RESULTS: Immunohistochemistry and western blotting confirmed that USP7 was significantly upregulated in glioblastoma samples. In in vitro experiments, inhibition of USP7 in GBM induced significant apoptosis. Co-IP and TMT proteomics identified a key anti-apoptotic substrate of USP7, ADP-ribosylation factor 4 (ARF4). Western blotting and IP confirmed that USP7 interacted directly with ARF4 and catalyzed the removal of the K48-linked polyubiquitinated chain that binded to ARF4. In addition, in vivo experiments revealed that USP7 inhibition significantly suppressed tumor growth and promoted the expression of apoptotic genes. CONCLUSIONS: Targeted inhibition of USP7 enhances the ubiquitination of ARF4 and ultimately mediates the apoptosis of GBM cells. In a clinical sense, P5091 as a novel specific inhibitor of USP7 may be an effective approach for the treatment of GBM.

Dual PLK1 and STAT3 inhibition promotes glioblastoma cells apoptosis through MYC.

Glioblastoma (GBM) is the deadliest primary brain tumor that is highly resistant to current treatments. Polo-like kinase 1 (PLK1) and signal transducer and activator of transcription 3 (STAT3) are highly expressed in gliomas, especially GBM. Previous studies have shown reciprocal activation between PLK1 and STAT3 and that they regulate the same pools of MYC downstream. We have demonstrated that PLK1 and STAT3 levels are elevated in gliomas compared with those in normal brain tissues, and high expression of both PLK1 and STAT3 is associated with poor prognosis in TCGA. Moreover, there was direct or indirect reciprocal regulation between PLK1 and STAT3. Furthermore, we found that PLK1 and STAT3 can regulate the same pools of MYC downstream. Compared to monotherapy, combined treatment of glioma cells with PLK1 and STAT3 inhibitors, BI2536 and Stattic, respectively, showed lower expression of MYC, synergistic induction of cell invasion and apoptosis in vitro, and tumor inhibition in xenografts. PLK1 and STAT3 were able to directly regulate the expression of MYC and induce apoptosis of glioma cells through the regulation of MYC. These findings may help develop a therapeutic strategy for dual inhibition of PLK1 and STAT3 against the tumorigenesis of glioma.