Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet: Glioma cell TIM-3/IL6 signaling promotes the migration and activation of anti-inflammatory/pro-tumorigenic TAMs (A) A venn diagram revealing TIM-3 positively correlated genes in TCGA RNA-seq and GSE16011 GBM datasets. (B) GO analysis of TIM-3 positively correlated genes showed an enrichment in myeloid leukocyte-related terms. (C) Clustering of GSVA scores and analyses of signaling pathways in TCGA RNA-seq dataset showing the enrichment of immune-related phenotype associated with high TIM-3 expression in GBM. (D) GSEA revealed the significant enrichment of macrophage chemotaxis (right) and differentiation (left) regulation phenotype in GBM with high TIM-3 expression. (E) Transwell assays demonstrated that the incubation of CM from indicated glioma cells transfected with siTIM3 vector significantly inhibited PBMC migration, which could be rescued by IL6 incubation (40 ng/mL) (n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm). (F and G) qPCR analyses of indicated pro-inflammatory/anti-tumorigenic and anti-inflammatory/pro-tumorigenic markers in THP-1 cells incubated with conditioned medium (CM) from indicated glioma cells (n = 3, means ± SEM, one-way ANOVA; anti-Gal-9: 10 μg/mL). (H and I) FACS analyses of MHC II and CD206 in PBMC incubated with CM from GSC40 transduced with indicated vectors. (J) Representative immunohistochemical staining images of Tim-3, Ki-67, Iba1, Ccr2, Cd3, Cd4, and Cd8 in the brain sections obtained from mice transplanted with indicated GL261 glioma cells (Scale bar, 20 μm; n = 3, means ± SEM, t -test). (ns p ≥ 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Article Snippet: Mouse: GL261 , iCell , RRID: CVCL_Y003.

Techniques: Migration, Activation Assay, RNA Sequencing, Protein-Protein interactions, Expressing, Chemotaxis Assay, Incubation, Transfection, Plasmid Preparation, Transduction, Immunohistochemical staining, Staining

Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet: Blocking IL6 signaling by Tocilizumab attenuates the enhanced proliferation, migration, and invasion of glioma cells, and their crosstalk with macrophages induced by TIM-3 in vitro (A) The elevated proliferation of indicated glioma cells induced by TIM-3 was efficiently decreased by Tocilizumab (500 ng/mL) and anti-TIM-3 antibody (10 μg/mL) (n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm). (B) Representative bioluminescence (at day 7, 14, 21) images, H&E staining images, and survival plot of mouse brains transplanted with indicated GL261 cells and treated with control, anti-Tim3 antibody or anti-Il6r antibody (n = 6). (C) In vitro proliferation assay showing the influence of indicated Tocilizumab treatment on the growth of GSC40 (n = 3, one-way ANOVA). (D and E) The increased proliferation (D) and invasion (E) of indicated glioma cells induced by TIM-3 was efficiently decreased by Tocilizumab (500 ng/mL) (n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm). (F) EdU assay showing the increased proliferation of PGC1228 cells induced by TIM-3 was efficiently decreased by Tocilizumab (500 ng/mL) (n = 3, means ± SEM, one-way ANOVA). (G and H) The increased migration (G), and invasion (H) of indicated PGC1228 cells induced by TIM-3 was efficiently decreased by Tocilizumab (500 ng/mL) (n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm). (I) FACS analyses showed that Tocilizumab (500 ng/mL) inhibited the phenotype transition of PBMC to anti-inflammatory/pro-tumorigenic TAMs induced by IL6 (40 ng/mL) (n = 3, means ± SEM, one-way ANOVA). (J) Representative western blot images of indicated proteins in THP-1-derived macrophages incubated with indicated glioma cell CM (Tocilizumab, 500 ng/mL). (K) Western blot analyses of indicated proteins in THP-1-derived M0 and pro-inflammatory/anti-tumorigenic (M1) macrophages transfected with lentiviral TIM-3 overexpression or control vector, followed with or without Tocilizumab treatment (500 ng/mL) (n = 3, means ± SEM, one-way ANOVA). ( ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: Mouse: GL261 , iCell , RRID: CVCL_Y003.

Techniques: Blocking Assay, Migration, In Vitro, Staining, Control, Proliferation Assay, EdU Assay, Western Blot, Derivative Assay, Incubation, Transfection, Over Expression, Plasmid Preparation

Journal: iScience

Article Title: Cancer cell intrinsic TIM-3 induces glioblastoma progression

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet:

Article Snippet: Mouse: GL261 , iCell , RRID: CVCL_Y003.

Techniques: Control, Purification, Recombinant, Modification, Sterility, Cell Culture, Lysis, Transfection, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Proliferation Assay, Imaging, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Software, Fluorescence, Microscopy

Journal: Materials Today Bio

Article Title: Delivery of extracellular vesicles loaded with immune checkpoint inhibitors for immunotherapeutic management of glioma

doi: 10.1016/j.mtbio.2024.101244

Figure Lengend Snippet: Barrier permeation enhancement effect of EVs and efficient delivery of aPD-L1/EV into brain. ( A ) Scheme of the in vitro barrier permeation test with C2BBe1 trans-well system (created from Biorender.com ). ( B ) Amounts of permeated aPD-L1 with free aPD-L1 or aPD-L1/EV administration through the C2BBe1 barrier. ( C ) Comparison of drug delivery efficiency with RO or IN administration of FITC-conjugated IgG or IgG/EV. The upper column displays the fluorescence of IgG within brain sections, and the lower column shows the cumulative fluorescence intensity of brain sections. ( D ) Distribution of aPD-L1 in GL261 tumor-bearing mice. Green fluorescence indicates aPD-L1, and nuclei were stained with DAPI. Statistics in ( B, C ) were conducted by one-way ANOVA with Bonferroni post hoc test. Asterisks represent a significant difference between indicated groups, with significance defined as p < 0.05. Data were presented as mean ± SD with n = 3.

Article Snippet: The GL261-luc murine glioblastoma cell line was purchased from Creative Biolabs (Shirley, NY, USA).

Techniques: In Vitro, Comparison, Fluorescence, Staining

Journal: Materials Today Bio

Article Title: Delivery of extracellular vesicles loaded with immune checkpoint inhibitors for immunotherapeutic management of glioma

doi: 10.1016/j.mtbio.2024.101244

Figure Lengend Snippet: Therapeutic effect of ICI/EVs in managing orthotopic GL261 tumors. ( A ) Schematic of experimental procedures for GL261 tumor management (created from Biorender.com ). ( B, C ) Tumor volumes were evaluated with IVIS, and representative images are shown in ( B ). Changes in tumor volume were calculated based on the total flux observed on day 0 ( C ). ( D ) The survival of orthotopic GL261 tumor-bearing mice post-treatment was analyzed. The aPD-L1/EV + aCTLA-4/EV group demonstrated an extended survival time compared to other treatment groups. ( E ) Ki67 staining of GL261 tumors. Scale bar: 50 μm. Statistics for ( C ) were obtained from GLM with Bonferroni post hoc test, and for ( D ) from the Kaplan-Meier survival analysis with log rank test. The asterisk in ( C ) indicates a significant difference from the PBS and aPD-L1 treatments. In ( D ), the asterisk and hash signs represent significant difference from all treatment groups and from the PBS group, respectively. Significance was defined as p < 0.05. Data are presented as mean ± SEM with n = 4 in ( C ) and in ( D ) with n = 8∼18.

Article Snippet: The GL261-luc murine glioblastoma cell line was purchased from Creative Biolabs (Shirley, NY, USA).

Techniques: Staining

Journal: Materials Today Bio

Article Title: Delivery of extracellular vesicles loaded with immune checkpoint inhibitors for immunotherapeutic management of glioma

doi: 10.1016/j.mtbio.2024.101244

Figure Lengend Snippet: Immune cells in GL261 tumors post treatment. ( A ) IHC images of cytotoxic T cells within the tumor tissue. Green fluorescence indicates CD8 expression on T cells, while nuclei are counterstained with DAPI. Scale bar: 50 μm. ( B ) IHC images of M1 macrophages within tumor tissue. Green fluorescence indicates F4/80-stained macrophages, and red fluorescence indicates iNOS expression. Nuclei are counterstained with DAPI. Scale bar: 20 μm. ( C-E ) Flow cytometry analysis results for total T cells ( C ), cytotoxic T cells ( D ), and M1 macrophages ( E ). Statistics for ( C, D, E ) were obtained from one-way ANOVA with Bonferroni post hoc test, with significance defined as p < 0.05. Data are expressed as mean ± SEM with n = 5.

Article Snippet: The GL261-luc murine glioblastoma cell line was purchased from Creative Biolabs (Shirley, NY, USA).

Techniques: Fluorescence, Expressing, Staining, Flow Cytometry