Journal: Cancer Science

Article Title: Multiple roles of single‐minded 2 in esophageal squamous cell carcinoma and its clinical implications

doi: 10.1111/cas.13531

Figure Lengend Snippet: SIM2 and ARNT cooperatively decrease PDPN‐positive tumor basal cells. A, Immunofluorescence showing colocalization of SIM2‐GFP and ARNT in the nuclei of TE8‐SIM2‐2 cells. B, Immunoprecipitation of SIM2‐GFP with anti‐green fluorescent protein (GFP) antibody 1 day after transient SIM2‐GFP transfection to TE8 cells followed by western blot with anti‐SIM2, ‐ ARNT, or‐α tubulin antibody. Input, unprecipitated extracts. IgG, control IP. C, Real‐time RT‐PCR of ARNT (left) and percentage of PDPN‐positive cells (right) in KYSE510 cells that were transfected with control siRNA (white column) or ARNT siRNA (red column) (n = 3, mean + SE; * P < .05). D, Real‐time RT‐PCR of SIM2 (left) and percentage of PDPN‐positive cells (right) in KYSE510 that were transfected with control siRNA (white column) or SIM2 siRNA (red column) (n = 3, mean + SE; * P < .05)

Article Snippet: Sections were blocked by 10% FBS in PBS, and reacted with primary antibodies against PDPN (1:40; Abnova, Taipei, Taiwan) at 4°C overnight, followed by incubation with EnVision + Dual Linl System‐HRP (Dako, Carpinteria, CA, USA).

Techniques: Immunofluorescence, Immunoprecipitation, Transfection, Western Blot, Control, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN is expressed in infiltrating gliomas and negatively correlates with survival. (A) Boxplots show increasing PDPN expression across increasing glioma grades in TCGA (n=601, ****p<0.0001). (B) Kaplan–Meier curves show that PDPN mRNA expression inversely correlates with OS across glioma grades in TCGA cohorts. Median OS for patients with PDPN-high and PDPN-low tumors was 14.4 and 94.5 months, respectively (n=601, p<0.0001). (C) Representative IHC examples of PDPN expression in FFPE tumor tissue using the M2A antibody in tumors. Subpanels (A, B) shows intense cytoplasmic staining in nearly all cells, while expression is undetectable in tumors in subpanels (C, D) . (D) Kaplan–Meier curves show that PDPN protein expression inversely correlates with OS among GBM WHO grade IV patients. Median OS for PDPN+ and PDPN− groups was 53.4 and 148.1 weeks, respectively (n=206, p<0.0001). IHC KM curves are shown as solid (PDPN+) and dashed (PDPN−) lines. (E) Kaplan–Meier curves show that PDPN protein expression inversely correlates with PFS among GBM WHO grade IV patients. Median PFS for PDPN+ and PDPN− groups were 21.0 and 34.6 weeks, respectively (n=206, p=0.03). (F) Kaplan–Meier curves show that PDPN protein expression inversely correlates with OS among grade II/III diffuse astrocytoma patients (n=93, p=0.003). (G) Radiation response is correlated with PDPN expression (n=16, *p=0.0326). (H) MRI examples of a radiation responder (A, B) and non-responder (C, D) , pre- and post-radiation (RT).

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques: Expressing, Staining

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: Cox multivariate survival analysis for GBM patients.

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques:

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: Cox multivariate survival analysis for diffuse astrocytoma patients.

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques:

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN+ glioma-stem-like cells form aggressive tumors in vivo and mark a stem-like radioresistant subpopulation of cells. (A) PDPN protein is expressed in seven of the nine GSC lines shown. (B) Flow cytometry of GSCs shows PDPN expression is pervasive and more prominent in these GSCs. (C) Kaplan–Meier survival analysis of GSC11 PDPN−/CD133+, PDPN+/CD133+, and PDPN+/CD133− FACS-sorted subpopulations orthotopically injected into the brains of immunocompromised mice reveal that mice harboring tumors from CD133+/PDPN+ cells had a median survival of 99 days (n=5), while mice that received CD133+/PDPN− cells did not succumb to tumor formation (n=3) (p=0.0136, log-rank test). (D) PDPN+ FACS-sorted subpopulations have a higher sphere formation ability (*p<0.005, multiple t-tests, PDPN+ vs . PDPN− groups). (E) PDPN+ FACS-sorted subpopulations have a higher surviving fraction of cells after 2 Gy of radiation (*p<0.005, multiple t-tests, PDPN+ vs . PDPN− groups). (F) Transcriptome analysis revealed distinct gene expression signatures in PDPN+ and PDPN− sorted GSCs (three different cell lines). (G) GSEA of differentially expressed genes (p<0.05) demonstrated significant enrichment of the mesenchymal subtype signature (NES=2.04, q=0.02) in PDPN+ populations.

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques: In Vivo, Flow Cytometry, Expressing, Injection, Gene Expression

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN knockdown sensitizes adherent glioma cell lines to radiation and reduces invasion, migration, and colony formation ability. (A) PDPN expression in adherent glioma cell lines and normal human astrocytes (NHA). (B) Western blot of PDPN knockdown in U87 cell lines. (C) Invasion and migration assays of U87 PDPN knockdown lines (*p<0.005, multiple t-tests, sh-neg vs . sh-PDPN). (D) Clonogenic radiation survival assay with PDPN knockdown lines, PDPN silencing sensitized U87 cells to in vitro radiation compared with controls. (E) Representative crystal violet stains after a colony formation assay with U87 PDPN knockdown lines. (F) Quantification of colony diameter and colony number after colony formation assay of control and PDPN shRNA cell lines (*p<0.005, two tailed t-test).

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques: Knockdown, Migration, Expressing, Western Blot, Clonogenic Cell Survival Assay, In Vitro, Colony Assay, Control, shRNA, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: PDPN marks a subset of aggressive and radiation-resistant glioblastoma cells

doi: 10.3389/fonc.2022.941657

Figure Lengend Snippet: PDPN knockdown in glioma stem-like cells leads to radiosensitization in vitro and slows intracranial tumor growth. (A) Western blot of GSC line 23 (GSC23) showing PDPN expression levels after exposure to doxycycline (doxy), indicating inducible PDPN knockdown. (B) qPCR validation of PDPN knockdown (*p<0.005, two-tailed t-test). (C) In vitro clonogenic survival assays revealed that PDPN silencing by the shRNA MD5 and MD7 was effective in sensitizing GSC23 spheres to radiation in a dose-dependent manner [0 Gy, not significant (ns); 2 Gy, *p<0.0001; 4 Gy, *p<0.0001]. (D) Kaplan–Meier survival analysis (n=5 per condition) of PDPN knockdown, with negative (shLuc and no doxy) controls (p=0.0018, log-rank test). (E) Time course IVIS imaging of tumors from days 1, 30 and 60. (F) Representative tumor histology and PDPN IHC of animal tumors at time of animal death.

Article Snippet: Upon blocking, PVDF membranes were incubated overnight with primary monoclonal antibodies targeting PDPN (clone NZ-1, 1:200, AngioBio) and vinculin (1:1,000, Abcam) at 4°C.

Techniques: Knockdown, In Vitro, Western Blot, Expressing, Biomarker Discovery, Two Tailed Test, shRNA, Imaging