The CEBPB+ glioblastoma subcluster specifically drives the formation of M2 tumor-associated macrophages to promote malignancy growth.

Rationale: The heterogeneity of tumor cells within the glioblastoma (GBM) microenvironment presents a complex challenge in curbing GBM progression. Understanding the specific mechanisms of interaction between different GBM cell subclusters and non-tumor cells is crucial. Methods: In this study, we utilized a comprehensive approach integrating glioma single-cell and spatial transcriptomics. This allowed us to examine the molecular interactions and spatial localization within GBM, focusing on a specific tumor cell subcluster, GBM subcluster 6, and M2-type tumor-associated macrophages (M2 TAMs). Results: Our analysis revealed a significant correlation between a specific tumor cell subcluster, GBM cluster 6, and M2-type TAMs. Further in vitro and in vivo experiments demonstrated the specific regulatory role of the CEBPB transcriptional network in GBM subcluster 6, which governs its tumorigenicity, recruitment of M2 TAMs, and polarization. This regulation involves molecules such as MCP1 for macrophage recruitment and the SPP1-Integrin αvß1-Akt signaling pathway for M2 polarization. Conclusion: Our findings not only deepen our understanding of the formation of M2 TAMs, particularly highlighting the differential roles played by heterogeneous cells within GBM in this process, but also provided new insights for effectively controlling the malignant progression of GBM.

Combinatorial Application of Papain and CD66B for Isolating Glioma- Associated Neutrophils.

BACKGROUND: Stromal cells in the tumor microenvironment play crucial roles in glioma development. Current methods for isolating tumor-associated stromal cells (such as neutrophils) are inefficient due to the conflict between tissue dissociation and cell surface protein protection, which hampers the research on patient-derived stromal cells. Our study aims to establish a novel method for isolating glioma-associated neutrophils (GANs). METHODS: To observe neutrophil-like polymorphonuclear cells, we performed Hematoxylin-Eosin staining on glioma tissues. For isolating single cells from glioma tissues, we evaluated the efficiency of tissue dissociation with FastPrep Grinder-mediated homogenization or proteases (trypsin or papain) digestion. To definite specific markers of GANs, fluorescence-activated cell sorting (FACS) and immunofluorescence staining were performed. FACS and Ficoll were performed for the separation of neutrophils from glioma tissue-derived single-cell or whole blood pool. To identify the isolated neutrophils, FACS and RT-PCR were carried out. RESULTS: Neutrophil-like cells were abundant in high-grade glioma tissues. Among the three tissue dissociation methods, papain digestion produced a 5.1-fold and 1.7-fold more living cells from glioma mass than physical trituration and trypsin digestion, respectively, and it preserved over 97% of neutrophil surface protein markers. CD66B could be adopted as a unique neutrophil surface protein marker for FACS sorting in glioma. Glioma-derived CD66B+ cells specifically expressed neutrophil marker genes. CONCLUSION: A combination of papain-mediated tissue dissociation and CD66B-mediated FACS sorting is an effective novel method for the isolation of GANs from glioma tissues.

Identification of Neural Progenitor Cell-associated Chemoradiotherapy Resistance Gene Set (ARL4C, MSN, TNFAIP6) for Prognosis of Glioma.

BACKGROUND: Glioma is the most common malignant intracranial tumor with high lethality. Despite surgery combined with chemoradiotherapy, the prognosis for patients with glioma remains poor. This is primarily due to acquired chemoradiotherapy resistance. Therefore, to improve the prognosis of glioma, further study into the mechanism of chemoradiotherapy resistance is needed. OBJECTIVE: This study aimed to (1) evaluate the prognosis of patients with glioma by using a prognostic risk score model constructed by chemoradiotherapy resistance genes, (2) provide new targets and directions for precise treatment of glioma, and (3) discuss the tumor heterogeneity of tumor cells. METHODS: According to therapy class and overall survival (OS), we identified 53 genes associated with glioma chemoradiotherapy resistance in The Cancer Genome Atlas Glioblastoma (TCGA GBM) database. Considering the important role of chemoradiotherapy resistance-related genes in the prognosis of glioma, we preliminarily screened and identified vital prognostic factors among these genes by using the Cox regression model of absolute contraction and selection operators in the TCGA GBM lower-grade glioma (TCGA GBMLGG) dataset. Next, the heterogeneity of the chemoradiotherapy resistance-associated genes in different glioma cells was revealed by single-cell sequencing in the GSE117891 cohort. RESULTS: A prognostic risk score model consisting of three genes (ARL4C, MSN, TNFAIP6) was constructed. The expression of this model was high in glioma neural progenitor cells (NPCs) and low in glioma oligodendrocytes. The OS rates were significantly lower in the high- vs. low-risk group. CONCLUSION: Our 3 gene risk score complements the current glioma diagnosis and provides a novel insight into chemoradiotherapy resistance mechanisms for the prognosis of patients with glioma.

Identification of a novel GJA3 mutation in a large Chinese family with congenital cataract using targeted exome sequencing.

Autosomal dominant congenital cataract (ADCC) is a clinically and genetically heterogeneous ocular disease in children that results in serious visual impairments or even blindness. Targeted exome sequencing (TES) is an efficient method used for genetic diagnoses of inherited diseases. In the present study, we used a custom-made TES panel to identify the genetic defect of a four-generation Chinese family with bilateral pulverulent nuclear cataracts. A novel heterozygous missense mutation c.443C>T (p. T148I) in GJA3 was identified. The results of the bioinformatic analysis showed that the mutation was deleterious to the structure and hemichannel function of Cx46 encoded by GJA3. Plasmids expressing wild-type and mutant human Cx46 were constructed and ectopically expressed in human lens epithelial cells (HLECs) or human embryonic kidney (HEK-293) cells. Fluorescent images indicated aggregated signals of mutant protein in the cytoplasm, and a higher protein level was also detected in T148I stable cell lines. In summary, we identified a novel mutation in GJA3 for ADCC, which provided molecular insights into the pathogenic mechanism of ADCC.

CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells.

Clustered interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL) locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

Molecular diagnosis of putative Stargardt disease by capture next generation sequencing.

Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis.