To investigate the effects of hypoxia on the transcriptional phenotype and ultrastructure of tumor-associated macrophages (TAMs) in glioma.

CD14⁺ monocytes were isolated from healthy human peripheral blood samples collected from the Blood Bank of the First Affiliated Hospital of Army Medical University, and the cells were induced to differentiate into TAMs through co-culture with glioma cell-conditioned medium. Hypoxic TAM models were established using varying concentrations of cobalt chloride hexahydrate (CoCl₂, 50~400 μmol/L) or hypoxic conditions (1%, 5%, 10%O₂) for 48 h, while normoxic TAM models (21%O₂) served as controls. RT-qPCR and transcriptome sequencing were employed to analyze transcriptional changes in TAMs under normoxic and hypoxic conditions. Gene set enrichment analysis (GSEA) was applied to compare the differences in angiogenesis, glycolysis and other hypoxia-responsive pathways between the 2 conditions. Transmission electron microscopy (TEM) or immunofluorescence staining was conducted to assess the ultrastructural alterations in cytoskeleton, endoplasmic reticulum (ER), and mitochondria in normoxic and hypoxic TAMs (1%O₂).

Hypoxic TAMs exhibited up-regulated transcription of hypoxia-responsive markers (oxygen transport, glycolysis, pro-angiogenesis), with the effects correlating with hypoxia severity (P<0.05). GSEA revealed significant up-regulation of hypoxia, angiogenesis regulation, glycolysis and gluconeogenesis, and starvation stress pathways, alongside down-regulation of innate immunity, macrophage activation, cytoskeleton, and protein maturation pathways in hypoxic TAMs (P<0.05). TEM and immunofluorescence staining demonstrated obvious ultrastructure changes, including disrupted cytoskeletal organization, shortened rough ER with reduced ribosomes, mitochondrial swelling with cristae damage, and diminished ER-mitochondria contacts in hypoxic TAMs.

CoCl₂ and hypoxia induce a hypoxic transcriptional phenotype in TAMs, which may potentially associated with ultrastructural remodeling of the cytoskeleton, ER, and mitochondria.

To explore the effect of glioma stem cells with high expression of protein tyrosin phosphatase receptor type Z1 (PTPRZ1) on the phenotypic polarization and phagocytosis of tumor-associated macrophages and its regulatory mechanism.

GSCs and non-stem tumor cells (NSTCs) were screened out from human glioblastoma (GBM) specimens using flow cytometry, and the PTPRZ1 expression in paired GSCs and NSTCs were detected. Human peripheral blood mononuclear cells (PBMC)-derived CD14⁺ monocytes were exposed to the conditioned medium from glioma cells or recombinant chemokine C-C motif ligand 20 (CCL20) for TAM polarization. Stable PTPRZ1 knockout GSCs (PTPRZ1-KO GSCs) were constructed using CRISPR/Cas9. TAM phagocytosis to GSCs, NSTCs, PTPRZ1-Control GSCs (PTPRZ1-Ctrl GSCs) and PTPRZ1-KO GSCs and the expression of immunosuppressive phenotype (M2) polarization marker CD163 were examined using flow cytometry. Differentially expressed genes (DEGs) between paired GSCs and NSTCs were determined using a bulk RNA-sequencing dataset (GSE54791) from Gene Expression Omnibus (GEO). A gene set informing worse outcome of patients with GBM was generated using The Cancer Genome Atlas (TCGA)-GBM cohort. By intersecting the aforementioned gene set with the gene set that encodes for humanmembrance proteins, the PTPRZ1 gene is obtained. Gene set enrichment analysis (GSEA) was used for pathway enrichment analysis to compare the differentially regulated pathways between GBMs with high or low PTPRZ1 expression. Bulk RNA sequencing, qRT-PCR and Western blotting were used to identify the DEGs between PTPRZ1-KO GSCs and PTPRZ1-Ctrl GSCs.

GSCs were more capable of escaping from TAM phagocytosis than NSTCs (P<0.05) and had specifically up-regulated PTPRZ1 expression. PTPRZ1-KO significantly suppressed GSCs escaping from TAM phagocytosis (P<0.01). GBMs with high PTPRZ1 expression showed significant inhibition of pathways mediating phagocytosis (P<0.05). The expression of CCL20 as a M2 TAM polarization chemokine was significantly down-regulated in PTPRZ1-KO GSCs (P<0.05). Treatment with recombinant CCL20 up-regulated the expression of CD163 as a M2 TAM marker in TAM.

PTPRZ1⁺ GSCs mediate M2 TAM polarization and inhibit TAM phagocytosis, which may be related to the up-regulation of CCL20 in PTPRZ1⁺ GSCs.

To prepare a monoclonal antibody targeting protein tyrosine phosphatase receptor-type Z polypeptide1（PTPRZ 1）, to investigate the potential to disrupt the pleiotrophin（PTN）-PTPRZ 1 paracrine signaling axis, and to evaluate the inhibitory effects on the self-renewal, migration, and invasion of glioma stem cells.

The extracellular domain of PTPRZ 1（amino acids 26-300）served as the antigen to immunize female BALB/c mice aged 8 to 10 weeks. Following a 2-week immunization period, serum samples were collected via tail vein puncture, centrifuged to separate, and the antibody titer was quantified by ELISA. Subsequently, B cell fusion and positive hybridoma cell selection were conducted to isolate murine monoclonal antibodies targeting PTPRZ 1. Antibody clones with the highest affinity were identified through specialized affinity assays. Human glioma stem cells were infected with sgRNA-PTPRZ 1 lentivirus for PTPRZ 1 gene silencing, and the changes in the expression of PTPRZ 1 at mRNA level were analyzed using RT-qPCR. Western blotting was employed to confirm the antibodies' specificity for PTPRZ 1. The experiments on the functions and mechanisms of glioma stem cells were divided into 4 groups: the control group, the human recombinant PTN-treated group, the mouse anti-PTPRZ 1 antibody（clone 2F10）-treated group, and the combination treatment of human recombinant PTN and mouse anti-PTPRZ 1 antibody（clone 2F10）group. The expression of PTPRZ 1 downstream signaling pathways in glioma stem cells was examined via Western blotting. Clonogenic, migration, and invasion assays were utilized to determine the effects on self-renewal, migration, and invasion capabilities of glioma stem cells.

Five murine monoclonal antibodies targeting PTPRZ 1 were successfully generated, with clone 2F10 displaying the highest binding affinity that was found to be effective for detecting PTPRZ 1 protein expression. Compared with the control group, treatment with human recombinant PTN significantly increased ERK phosphorylation downstream of PTPRZ 1 in glioma stem cells, thereby enhancing their self-renewal（P<0.01）, migration（P<0.01）, and invasion（P<0.05）. The application of mouse anti-PTPRZ 1 antibody（2F10）effectively blocked PTN-mediated ERK phosphorylation, resulting in a significant reduction in self-renewal（P<0.01）, migration（P<0.01）, and invasion（P<0.05）abilities of glioma stem cells.

The mouse anti-PTPRZ 1 monoclonal antibody, clone 2F10, efficiently targets and recognizes the PTPRZ 1 protein in glioma stem cells, impeding PTN-mediated processes essential for their self-renewal, migration, and invasion.