Fig 2: Disruption of PTPRZ1 potently inhibits GSC tumour growth and prolongs the animal survival.(a–d) The bioluminescent images (a,c) and quantification (b,d) of xenografts derived from T4121 GSCs (a,b) or T0912 GSCs (c,d) expressing shPTPRZ1 or shNT control. Data are shown as means±s.e.m., n=5, *P<0.05, **P<0.01, ANOVA test. (e,f) The representative IHC images (left panel) and the quantification (right panel) of Ki67 expression in xenografts derived from T4121 GSCs (e) or T0912 GSCs (f) expressing shPTPRZ1 or shNT control. Xenografts were collected at 28 days (T4121) or 56 days (T0912) after tumour implantation. Data are shown as means±s.d., n=5, **P<0.01, ANOVA test. Scale bar represents 25 µm. (g,h) Kaplan–Meier survival curves of mice implanted with T4121 GSCs (g) or T0912 GSCs (h) expressing shPTPRZ1 or shNT control. Disruption of PTPRZ1 significantly extends the survival of mice bearing GSC-derived xenografts. n=5, P<0.01, log-rank test.

Fig 3: The PTN receptor PTPRZ1 is preferentially expressed by GSCs.(a) Bivariate correlation analyses showing a positive correlation of PTN and PTPRZ1 expression in human GBMs from the TCGA database (https://tcga-data.nci.nih.gov/tcga). The expressions of PTN and PTPRZ1 in human GBMs were obtained from gene-profiling data from the TCGA database. n=541, P<0.001, Pearson's r test. (b) Expression heatmap of top 10 upregulated genes in GSC lines (n=27) relative to NSTC lines (n=36) from GEO profiles (GEO: GDS3885). Candidate genes enriched in GSCs were identified when the average gene expression ratio of GSCs/NSTCs was =3.0-fold. Heatmap was visualized using Cluster/Java Treeview. (c) Immunoblot analysis showing preferential expression of PTPRZ1 and the GSC marker SOX2 in GSCs relative to matched NSTCs derived from human GBMs. a-Tubulin was used for normalization. (d) Immunofluorescence of PTPRZ1 (in green) in GSCs or NSTCs from T0912 GBM. Scale bar represents 50 µm. (e–g) Co-immunofluorescent staining of PTPRZ1 (in green) and the GSC marker SOX2 (in red, e), OLIG2 (in red, f) and CD133 (in red, g) in human GBMs. PTPRZ1 is preferentially expressed in GBM cells expressing GSC markers. Scale bar represents 25 µm. (h–j) Co-immunofluorescent staining of PTPRZ1 (in green) and the GSC marker SOX2 (in red, h), OLIG2 (in red, i) or CD133 (in red, j) in GSC tumourspheres. Co-enrichment of PTPRZ1 with the GSC markers was observed in GSC tumourspheres. Scale bar represents 25 µm. DAPI, 4,6-diamidino-2-phenylindole.

Fig 4: The PTN–PTPRZ1 signalling axis is critical for GSC maintenance.(a,b) The cell viability assay (a) and in vitro limiting dilution assay (b) of GSCs treated with rhPTN showing that rhPTN facilitated GSC proliferation and self-renewal. Data are shown as means±s.d., n=5, **P<0.01, Student's t-test (a); n=10, *P<0.05, likelihood ratio test (b). (c,d) The cell viability assay (c) and in vitro limiting dilution assay (d) of GSCs expressing shPTPRZ1 or shNT in combination of rhPTN treatment. Disruption of PTPRZ1 compromised the promoting role of PTN on GSC proliferation (c) and self-renewal (d). Data are shown as means±s.d., n=5, **P<0.01, Student's t-test (c); n=10, *P<0.05, **P<0.01, likelihood ratio test (d). (e–g) Tumoursphere formation assay of GSCs expressing shPTPRZ1 or shNT in combination with the treatment of rhPTN. The representative images of GSC tumourspheres (e) and the quantification of numbers (f) and diameter (g) of the GSC tumourspheres showing that silencing PTPRZ1 expression significantly compromises rhPTN-promoted GSC tumoursphere formation ability. Scale bar represents 100 µm. Data are shown as means±s.d., n=5, *P<0.05, **P<0.01, ANOVA test. (h) Immunoblot analyses of PTPRZ1, phospho-AKT (p-Ser473) and total AKT in GSCs expressing shPTPRZ1 or shNT in combination with rhPTN stimulation.

Fig 5: PTN–PTPRZ1 signalling activates the Fyn–AKT pathway in GSCs.(a,b) Hierarchical clustering analysis (a) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (b) of differentially expressed genes between the PTPRZ1-silencing GSCs and control GSCs (fold change =2.0). Cluster analyses were performed and visualized using the Cluster/Java Treeview. Pathway enrichment significance is presented as a P value with log2 transformation, Fisher's exact test and ?2-test. (c) Immunoblot analyses of phospho-AKT (p-Ser473) and total AKT in GSCs (T387 and T0912), showing that rhPTN stimulation markedly increases AKT-activating phosphorylation, while the anti-PTPRZ1 antibody treatment compromises rhPTN-stimulated AKT activation in GSCs. Cells were pretreated with anti-PTPRZ1 antibody or control IgG for 1 h followed by rhPTN treatment for 20 min. (d) Co-immunoprecipitation of PTPRZ1 with the Fyn-specific antibody from T387 and T0912 GSC cell lysates. Precipitation with normal rabbit IgG was used as a negative control. PTPRZ1 binds to Fyn in GSCs. (e) Immunoblot analyses of p-SFK (Tyr416) and Fyn in T387 GSCs, showing that rhPTN stimulation markedly increases activating phosphorylation of SFK (p-Tyr416), while the anti-PTPRZ1 antibody treatment largely abrogates SFK activation in GSCs. (f) Co-immunoprecipitation of p-SFK (p-Tyr416) with the Fyn-specific antibody in T387 GSCs. Phosphorylated Fyn, as represented by immunoprecipitated p-SFK with Fyn antibody, was increased after rhPTN exposure, and was compromised by anti-PTPRZ1 antibody. Precipitation with rabbit IgG was used as a negative control.