Glioblastoma Recurrence Correlates With NLGN3 Levels

ObjectiveGlioblastoma(GBM)is the most aggressive type of tumor in the central nervous system(CNS).Surgery is the main treatment for malignant gliomas.The average survival time of patients with postoperative radiotherapy or chemotherapy is 14 months.The recurrence of malignant gliomas in the short term after surgery is almost inevitable.Recent studies have found that NLGN3 is an environmental factor necessary for growth of GBMs.Therefore,we investigate in depth whether the level of NLGN3 in the brain is directly related to the recurrence of GBM after surgery.MethodsCollecting diagnostic imaging data of GBM patients in recent years.Collecting MRI images of representative malignant glioma patients.Collecting representative GBM patients surgically removed GBM tissue mass.Western blot was used to detect the expression of NLGN3 in normal human cortex(frontal lobe,temporal lobe,parietal lobe,occipital lobe)and deep region(basal ganglia,thalamus,corpus callosum).Western blot was used to detect the expression of NLGN3 in the cortex and core of normal C57BL/6 brain.The expression level of NLGN3 on the surface and fundus of GBM tissue was performed by Western blot.Immunofluorescence staining was used to determine the expression and level of NLGN3 in cortical and deep regions of normal human brain tissue,cortical and core regions of normal mouse brain tissue,and GBM tissues.Western blot was used to detect the level of phosphorylated AKT[p-AKT(S473)]in the surface and fundus of GBM tissues,and in normal cortical and normal deep tissues of GBM patients.The cell counts were used to count the number of U251 cells and U87-MG cells(both GBM cell lines)treated with different concentrations of NLGN3 at different time points.The levels of p-AKT(S473)in U251 cells and U87-MG cells treated with different concentrations of NLGN3 were detected by Western blot.Cell counting was used to count normal cell culture medium,cortical neuron culture medium(C-NCM),basal ganglion neuron culture medium(BG-NCM),cortical neuron culture medium + AD AM10 inhibitor,basal ganglion neuron culture medium + U251 cells treated with ADAM 10 inhibitor and The number of cells at different time points in U87-MG cells.Primary glioma cell culture were used to culture GBM cells from ten different GBM patients after surgery.Cell counting was used to count DMEM,C-NCM,BG-NCM,C-NCM + ADAM10i,BG-NCM + ADAM10i treated ex vivo cultured GBM cells at different time points.Results1)The primary area of tumors in malignant glioma patients is mostly cortex,including frontal,temporal,parietal,and occipital lobes;recurrences often occur in deep brain regions,including:basal ganglia,thalamus,corpus callosum.2)A follow-up study of patients with glioma patients found that as the time of relapse progressed,malignant gliomas grew deeper into the brain.3)In normal human brain tissue,NLGN3 expression levels in the cortex(frontal lobe,temporal lobe,parietal lobe,and occipital lobe)are higher than in deeper regions(basal ganglia,thalamus,corpus callosum).4)In normal C57BL/6 mouse brain tissue,the expression level of NLGN3 in the cortex is higher than that in the core region.5)The expression level of NLGN3 on the surface of malignant glioma tissues is lower than that of the underlying area.6)In malignant glioma tissue,the level of phosphorylated AKT in the surface layer is lower than the underlying area.7)In the normal brain tissue of malignant glioma patients,the phosphorylation of AKT in the deep region is higher than that in the cortical region.8)Cell counts were recorded.Under the same conditions,high concentrations of NLGN3 promoted faster growth of U251 and U87-MG cells.9)High concentrations of NLGN3 promote higher levels of phosphorylated AKT in U251 cells and U87-MG cells.10)C-NCM and BG-NCM can promote the growth of U251 cells and U87-MG cells;ADAM 10 inhibitor effectively inhibited the proliferation effects of C-NCM and BG-NCM for U251 cells and U87-MG cells.11)C-NCM and BG-NCM can promote the growth of in vitro cultured GBM cells;ADAM 10 inhibitor effectively inhibited the proliferation effects of C-NCM and BG-NCM for in vitro cultured GBM cells.Conclusion1)The primary area of human GBM is the cortex.The recurrent areas are mainly the basal ganglia,thalamus,and corpus callosum.Over time,the recurrent GBM will grow deeper in the brain.2)In normal human brain and normal C57BL/6 mouse brain,the level of NLGN3 in the cortical region is higher than in the deep brain region,whereas in the GBM patient,the NLGN3 level in the cortical region is lower than in the deep region,and the GBM patients cortical region have lower levels of phosphorylated AKT than deep brain region.3)High levels of NLGN3 can promote the growth of GBM cells and the activation of AKT.4)Normal mouse cortical neurons secrete more NLGN3 than basal ganglia neurons to promote the growth of GBM cells,and the treatment of ADAM10 inhibitors inhibits the release of NLGN3,thereby effectively inhibiting the growth of GBM cells induced by NLGN3 effect.