Journal: bioRxiv

Article Title: Inhibition of HVEM suppresses invasion and growth of mesenchymal glioblastoma

doi: 10.64898/2026.01.16.699674

Figure Lengend Snippet: (A) Effects of an anti-human HVEM nanobody on invasion of mesenchymal GICs (U3031MG and U3054MG). Nanobodies were used at 1 μM. Fluorescence images with median invasion index in each group are demonstrated as representative images. Quantification data are shown as mean±SD (n=9 biological replicates; ** P <0.01, *** P <0.001; two-tailed unpaired Student’s t-test). (B, C) In vivo effects of the anti-human HVEM nanobody on tumor progression of mesenchymal GICs. Mesenchymal GICs were injected intracranially into nude mice (n=5 mice per group). At 105 days (U3031MG) or 91 days (U3054MG) after tumor engraftment, the tumor size was quantified by bioluminescence imaging, and the mice were divided into two groups such that the amount of luminescence was equalized. Then, the nanobodies were administrated intraperitoneally at 10 mg/kg/day every day. Bioluminescence was quantified (* P <0.05, ** P <0.01; two-tailed unpaired Student’s t-test) (B). Survival curves of mice bearing tumors (** P <0.01; two-tailed log-rank test) (C).

Article Snippet: Dynabeads His-Tag Isolation & Pulldown (#10104D, Thermo Fisher Scientific) to which His-tagged human HVEM protein (#H10334-H08H, Sino Biological) or His tag peptide (#3310-205, MBL) were coupled, were mixed with HA-tagged human APRIL protein (#5860-AP/CF, R&D SYSTEMS), and His-tagged proteins were eluted by following the manufacturer’s instructions.

Techniques: Fluorescence, Two Tailed Test, In Vivo, Injection, Imaging

Journal: bioRxiv

Article Title: Inhibition of HVEM suppresses invasion and growth of mesenchymal glioblastoma

doi: 10.64898/2026.01.16.699674

Figure Lengend Snippet: (A) A heatmap of differentially expressed genes encoding membrane proteins between mesenchymal and non-mesenchymal GBM from GSE72217. (B) Expression levels of TNFRSF14 among human neural stem cells (hNSCs) and 4 subtypes of human glioblastoma-initiating cells (GICs). GBM cell lines in the HGCC resource were analyzed for the expression of TNFRSF14 by quantitative real-time PCR. Data are shown as mean±SD (n=3 biological replicates). PN, proneural; NL, neural; CL, classical; MES, mesenchymal. (C) Expression levels of TNFRSF14 in normal brain and brain tumor tissues in The Cancer Genome Atlas (TCGA) dataset (* P <0.05, ** P <0.01, *** P <0.001; two-tailed Kruskal-Wallis test with Bonferroni’s correction). (D, E) Expression levels of TNFRSF14 in subtypes of GBM tissues in the TCGA dataset (*** P <0.001; two-tailed Kruskal-Wallis test). Brain tumor tissues with or without IDH mutations (D) and those with or without glioma CpG island methylator phenotype (G-CIMP) (E) were analyzed. (F) Kaplan-Meier plot of the survival of GBM patients in TCGA dataset. The patients were equally divided into two groups based on TNFRSF14 expression level (* P <0.05; two-tailed log-rank test).

Article Snippet: The antibodies were used as follows; PE-conjugated anti-TNFRSF14 antibody (#130-101-593, Miltenyi Biotec), PE-conjugated REA Control human IgG1 (#130-104-612, Miltenyi Biotec), anti-TNFSF13/APRIL (#MAB5860, R&D SYSTEMS), mouse IgG1 isotype control (#MAB002, R&D SYSTEMS), HRP-conjugated anti-His tag antibody (#D291-7, MBL), and HRP-conjugated anti-HA tag antibody (#M180-7, MBL).

Techniques: Membrane, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibition of HVEM suppresses invasion and growth of mesenchymal glioblastoma

doi: 10.64898/2026.01.16.699674

Figure Lengend Snippet: (A) Cell surface expression of HVEM in mesenchymal GBM cells upon genetic disruption. The TNFRSF14 gene was knocked down in mesenchymal GICs (U3031MG and U3054MG) by lentivirus-mediated shRNA, or edited in U3054MG cells by lentivirus-mediated CRISPR-Cas9 system (U3054MG-hCas9). Cell surface expression of HVEM was evaluated by flowcytometric analysis. (B) Growth curves of mesenchymal GICs expressing shRNA or combination of Cas9 and sgRNA. Data are shown as mean±SD (n=4 biological replicates; ** P <0.01, *** P <0.001; Tukey’s HSD test). (C) Analysis of proliferative ability by 5-ethynyl-2’-deoxyuridine (EdU) labeling of U3054MG cells. The incorporated EdU was visualized by using EdU-Click 647 kit (green) and nuclei were stained with DAPI (blue). Scale bar: 50 μm. (left). Quantification data of EdU-positive cells are shown as mean± SD (n=4 biological replicates; *** P <0.001; Tukey’s HSD test) (right). (D) Sphere formation ability of mesenchymal GICs expressing shRNA or combination of Cas9 and sgRNA. Sphere formation of mesenchymal GICs was evaluated by limiting dilution assay. Data are shown as mean±SD (n=3 independent experiments; ** P <0.01, *** P <0.001; two-way ANOVA with Bonferroni’s correction).

Article Snippet: The antibodies were used as follows; PE-conjugated anti-TNFRSF14 antibody (#130-101-593, Miltenyi Biotec), PE-conjugated REA Control human IgG1 (#130-104-612, Miltenyi Biotec), anti-TNFSF13/APRIL (#MAB5860, R&D SYSTEMS), mouse IgG1 isotype control (#MAB002, R&D SYSTEMS), HRP-conjugated anti-His tag antibody (#D291-7, MBL), and HRP-conjugated anti-HA tag antibody (#M180-7, MBL).

Techniques: Expressing, Disruption, shRNA, CRISPR, Labeling, Staining, Limiting Dilution Assay

Journal: bioRxiv

Article Title: Inhibition of HVEM suppresses invasion and growth of mesenchymal glioblastoma

doi: 10.64898/2026.01.16.699674

Figure Lengend Snippet: (A) In vivo bioluminescent imaging of mesenchymal GICs in which TNFRSF14 had been knocked down by shRNA, using the firefly luciferase method. Quantification data of bioluminescence are shown as mean±SD (n=8 mice per group; *** P <0.001; Tukey’s HSD test). (B) Survival curves of mice bearing tumors derived from shRNA-expressing mesenchymal GICs (n=8 mice per group; ** P <0.01, *** P <0.001; two-tailed log-rank test with Bonferroni’s correction).

Article Snippet: The antibodies were used as follows; PE-conjugated anti-TNFRSF14 antibody (#130-101-593, Miltenyi Biotec), PE-conjugated REA Control human IgG1 (#130-104-612, Miltenyi Biotec), anti-TNFSF13/APRIL (#MAB5860, R&D SYSTEMS), mouse IgG1 isotype control (#MAB002, R&D SYSTEMS), HRP-conjugated anti-His tag antibody (#D291-7, MBL), and HRP-conjugated anti-HA tag antibody (#M180-7, MBL).

Techniques: In Vivo, Imaging, shRNA, Luciferase, Derivative Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Inhibition of HVEM suppresses invasion and growth of mesenchymal glioblastoma

doi: 10.64898/2026.01.16.699674

Figure Lengend Snippet: (A) Cell surface expression of HVEM in non-mesenchymal GICs upon ectopic expression. Ectopic HVEM was transduced lentivirally into non-mesenchymal GICs (proneural U3047MG and classical U3017MG). Cell surface expression of HVEM was evaluated by flowcytometric analysis. (B) Growth curves of non-mesenchymal GICs expressing HVEM. Data are shown as mean±SD (n=3 biological replicates; * P <0.05, ** P <0.01, *** P <0.001; two-tailed unpaired Student’s t-test). (C) Sphere formation ability of non-mesenchymal GICs expressing HVEM was evaluated by limiting dilution assay. Data are shown as mean±SD (n=3 independent experiments; * P <0.05, *** P <0.001; two-way ANOVA). (D) Invasiveness of non-mesenchymal GICs expressing HVEM was evaluated by organotypic invasion assay. Fluorescence images with median invasion index in each group are demonstrated as representative images. Quantification data are shown as mean±SD (n=9 biological replicates; * P <0.05, *** P <0.001; two-tailed unpaired Student’s t-test). (E) In vivo bioluminescent imaging of non-mesenchymal GICs expressing HVEM and firefly luciferase. Quantification data of bioluminescence are shown as mean±SD (n=5 mice per group; * P <0.05, *** P <0.001; two-tailed unpaired Student’s t-test). (F) Survival curves of mice bearing tumors derived from HVEM-expressing non-mesenchymal GICs (n=5 mice per group; ** P <0.01; two-tailed log-rank test).

Article Snippet: The antibodies were used as follows; PE-conjugated anti-TNFRSF14 antibody (#130-101-593, Miltenyi Biotec), PE-conjugated REA Control human IgG1 (#130-104-612, Miltenyi Biotec), anti-TNFSF13/APRIL (#MAB5860, R&D SYSTEMS), mouse IgG1 isotype control (#MAB002, R&D SYSTEMS), HRP-conjugated anti-His tag antibody (#D291-7, MBL), and HRP-conjugated anti-HA tag antibody (#M180-7, MBL).

Techniques: Expressing, Two Tailed Test, Limiting Dilution Assay, Invasion Assay, Fluorescence, In Vivo, Imaging, Luciferase, Derivative Assay

Journal: bioRxiv

Article Title: Inhibition of HVEM suppresses invasion and growth of mesenchymal glioblastoma

doi: 10.64898/2026.01.16.699674

Figure Lengend Snippet: (A) Analysis of TCGA data set of the expression levels of genes encoding interactants of HVEM/TNFRSF14 in brain tumor tissue. NB, normal brain (* P <0.05, ** P <0.01, *** P <0.001; two-tailed Kruskal-Wallis test with Bonferroni’s correction). (B) Analysis of APRIL/TNFSF13 concentrations in mesenchymal and non-mesenchymal GICs by sandwich ELISA. (C) Physical interaction between recombinant HVEM and APRIL analyzed by pulldown assay. (D) NF-κB activity in mesenchymal GICs upon knockdown of HVEM / TNFRSF14 or APRIL / TNFSF13 by shRNA. (E) Growth curves of U3054MG cells expressing shRNA for APRIL / TNFSF13 . Data are shown as mean ±SD (n=4 biological replicates; ** P <0.01, *** P <0.001; Tukey’s HSD test). (F) Sphere formation of U3054MG cells expressing shRNA for APRIL / TNFSF13 . Data are shown as mean ± SD (n=3 independent experiments; * P <0.05, ** P <0.01, *** P <0.001; two-way ANOVA with Bonferroni’s correction). (G) Effect of a neutralizing antibody against APRIL on cell proliferation of mesenchymal GICs at day 7. Data are shown as mean±SD (n=3 biological replicates; ** P <0.01, *** P <0.001; Tukey’s HSD test). (H, I) Analysis of TCGA dataset of APRIL / TNFSF13 in GBM. The expression levels of APRIL / TNFSF13 correlates with lack of IDH1/2 mutations and the G-CIMP phenotype in GBM. (J) Kaplan-Meier plots of brain tumor patients in TCGA data set. The patients were equally divided into two groups based on expression level of TNFSF13 (encoding APRIL).

Article Snippet: The antibodies were used as follows; PE-conjugated anti-TNFRSF14 antibody (#130-101-593, Miltenyi Biotec), PE-conjugated REA Control human IgG1 (#130-104-612, Miltenyi Biotec), anti-TNFSF13/APRIL (#MAB5860, R&D SYSTEMS), mouse IgG1 isotype control (#MAB002, R&D SYSTEMS), HRP-conjugated anti-His tag antibody (#D291-7, MBL), and HRP-conjugated anti-HA tag antibody (#M180-7, MBL).

Techniques: Expressing, Two Tailed Test, Sandwich ELISA, Recombinant, Activity Assay, Knockdown, shRNA

Journal: bioRxiv

Article Title: Inhibition of HVEM suppresses invasion and growth of mesenchymal glioblastoma

doi: 10.64898/2026.01.16.699674

Figure Lengend Snippet: (A) Effects of an anti-human HVEM nanobody on invasion of mesenchymal GICs (U3031MG and U3054MG). Nanobodies were used at 1 μM. Fluorescence images with median invasion index in each group are demonstrated as representative images. Quantification data are shown as mean±SD (n=9 biological replicates; ** P <0.01, *** P <0.001; two-tailed unpaired Student’s t-test). (B, C) In vivo effects of the anti-human HVEM nanobody on tumor progression of mesenchymal GICs. Mesenchymal GICs were injected intracranially into nude mice (n=5 mice per group). At 105 days (U3031MG) or 91 days (U3054MG) after tumor engraftment, the tumor size was quantified by bioluminescence imaging, and the mice were divided into two groups such that the amount of luminescence was equalized. Then, the nanobodies were administrated intraperitoneally at 10 mg/kg/day every day. Bioluminescence was quantified (* P <0.05, ** P <0.01; two-tailed unpaired Student’s t-test) (B). Survival curves of mice bearing tumors (** P <0.01; two-tailed log-rank test) (C).

Article Snippet: The antibodies were used as follows; PE-conjugated anti-TNFRSF14 antibody (#130-101-593, Miltenyi Biotec), PE-conjugated REA Control human IgG1 (#130-104-612, Miltenyi Biotec), anti-TNFSF13/APRIL (#MAB5860, R&D SYSTEMS), mouse IgG1 isotype control (#MAB002, R&D SYSTEMS), HRP-conjugated anti-His tag antibody (#D291-7, MBL), and HRP-conjugated anti-HA tag antibody (#M180-7, MBL).

Techniques: Fluorescence, Two Tailed Test, In Vivo, Injection, Imaging

Journal: bioRxiv

Article Title: Inhibition of HVEM suppresses invasion and growth of mesenchymal glioblastoma

doi: 10.64898/2026.01.16.699674

Figure Lengend Snippet: (A, B) Effects of temozolomide on cell proliferation of HVEM/TNFRSF14 -inactivated mesenchymal GICs (A) or HVEM -overexpressing non-mesenchymal GICs (B). (C, D) Effects of erlotinib on cell proliferation of HVEM -inactivated mesenchymal GICs (C) or HVEM -overexpressing non-mesenchymal GICs (D). GICs were treated for 7 days with temozolomide or erlotinib (A-D). (E) Apoptosis was monitored by annexin V staining. HVEM -inactivated mesenchymal GICs (U3054MG-hCas9) were treated for 7 days with indicated concentrations of temozolomide or erlotinib. Quantification data of annexin V-positive cells are shown as mean±SD (n=3 biological replicates; * P <0.05, ** P <0.01, *** P <0.001; Tukey’s HSD test).

Article Snippet: The antibodies were used as follows; PE-conjugated anti-TNFRSF14 antibody (#130-101-593, Miltenyi Biotec), PE-conjugated REA Control human IgG1 (#130-104-612, Miltenyi Biotec), anti-TNFSF13/APRIL (#MAB5860, R&D SYSTEMS), mouse IgG1 isotype control (#MAB002, R&D SYSTEMS), HRP-conjugated anti-His tag antibody (#D291-7, MBL), and HRP-conjugated anti-HA tag antibody (#M180-7, MBL).

Techniques: Staining