Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: The frequency of ILC1 is decreased, whereas ILC3 is increased, in IDH1-mutant human glioma tissues compared with the IDH1-wild-type group. A Representative flow-cytometry gating strategies for ILCs in human tonsil (control) tissue, IDH1-wildtype, and IDH1-mutant glioma tissue (FSC-A = forward-scatter area; SSC-A = side-scatter area). B Comparison of total ILC percentages in tonsil controls (n = 11), IDH1-wild-type (n = 9), and IDH1-mutant (n = 3) glioma tissues (top center); comparison of ILC1 percentages (bottom left), ILC2 percentages (bottom center), and ILC3 percentages (bottom right) across the same groups. C Comparison of mean fluorescence intensity (MFI) of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) expression on ILC1s in tonsil control, IDH1-wild-type, and IDH1-mutant groups. D Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC2s. E Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC3s. Error bars represent ± SD. Statistical significance was determined by one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Flow Cytometry, Control, Comparison, Fluorescence, Expressing

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: The frequency of ILC3 is increased in the peripheral blood of IDH1-mutant glioma patients compared with the IDH1-wild-type group. A Representative flow-cytometry gating strategies for ILCs in peripheral blood of healthy controls, IDH1-wild-type glioma patients, and IDH1-mutant glioma patients (FSC-A = forward-scatter area; SSC-A = side-scatter area). B Comparison of total ILC percentages in blood samples from healthy controls (HC, n = 20), IDH1-wild-type (n = 12), and IDH1-mutant (n = 7) groups (top center); comparison of ILC1 (bottom left), ILC2 (bottom center), and ILC3 (bottom right) frequencies across the same groups. C Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC1s in HC, IDH1-wild-type, and IDH1-mutant groups. D Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC2s. E Comparison of MFI of KLRG1 (top left), PD-1 (top right), and CTLA-4 (bottom center) on ILC3s. Error bars represent ± SD. Statistical significance was determined by one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Mutagenesis, Flow Cytometry, Comparison

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: Co-culture of tonsil-derived ILCs with U87-MG significantly increased surface PD-1, KLRG1, and CTLA-4 compared with IDH1-mutant U87-MG. A – D Human tonsil-derived ILCs were cultured alone or co-cultured with U87-MG or IDH1-mutant U87-MG glioma cell lines, either with cytokine supplementation (recombinant human IL-2 [5 ng/mL], IL-7 [50 ng/mL], IL-12 [50 ng/mL], IL-1β [50 ng/mL], IL-23 [50 ng/mL]) or without cytokines for four days. Data points represent two independent experiments (ILC n = 3; U87-MG + ILC n = 6; IDH1-mutant U87-MG + ILC n = 6; technical replicates) A Representative flow-cytometry gating strategy for human tonsil-derived ILCs. B Flow-cytometry contour plots showing CTLA-4, KLRG1, and PD-1 surface expression percentages on ILCs. C Quantification of CTLA-4, KLRG1, and PD-1 expression percentages on ILCs. D Mean fluorescence intensity (MFI) of CTLA-4, KLRG1, and PD-1 expression on ILCs. E – F ILCs were cultured alone or exposed to glioma-conditioned medium (GCM) from U87-MG or IDH1-mutant U87-MG cell lines under the same cytokine conditions for four days. Data points represent two independent experiments (each with three technical replicates). E Percentages of CTLA-4, KLRG1, and PD-1–expressing ILCs following GCM exposure. F Corresponding MFI of CTLA-4, KLRG1, and PD-1 expression on ILCs. G – H Proliferation of CFSE-labeled tonsil ILCs co-cultured with U87-MG or IDH1-mutant U87-MG cells was analyzed after four days. G Representative flow-cytometry plots of CFSE dilution. H Quantification of proliferating CFSE-labeled ILCs from three independent experiments (each with four technical replicates). Error bars represent ± SD. Statistical analyses were performed using one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Co-Culture Assay, Derivative Assay, Mutagenesis, Cell Culture, Recombinant, Flow Cytometry, Expressing, Fluorescence, Labeling

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: IL-17 and IFN-γ production is increased in tonsil-derived ILCs exposed to glioma-conditioned medium (GCM) from U87-MG and IDH1-mutant U87-MG cell lines. A – E Tonsil-derived ILCs were cultured alone or with glioma-conditioned medium (GCM) obtained from U87-MG or IDH1-mutant U87-MG cell lines for four days, with or without cytokine supplementation (recombinant human IL-2 [5 ng/mL], IL-7 [50 ng/mL], IL-12 [50 ng/mL], IL-1β [50 ng/mL], IL-23 [50 ng/mL]). Golgi Stop was added during the final nine hours of incubation. A Percentages (left) and mean fluorescence intensity (MFI; right) of TNF-α–producing ILCs. B Percentages (left) and MFI (right) of IL-2–producing ILCs. C Percentages (left) and MFI (right) of IL-17–producing ILCs. D Percentages (left) and MFI (right) of IFN-γ–producing ILCs. E Percentages (left) and MFI (right) of GM-CSF–producing ILCs. Data represents two independent experiments (each with three technical replicates). Error bars indicate ± SEM. Statistical significance was determined by one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Mutagenesis, Cell Culture, Recombinant, Incubation, Fluorescence

Journal: Inflammation Research

Article Title: IDH1 Mutant Glioma Favors Group 3 Innate Lymphoid Cells and Is Resistant to Immune Checkpoint Expression

doi: 10.1007/s00011-026-02223-8

Figure Lengend Snippet: D-2-HG levels are comparable in-patient plasma but elevated in glioma-conditioned medium (GCM) from IDH1-mutant U87-MG + ILC co-cultures compared with U87-MG + ILC. A Quantification of D-2-HG (OD₄₅₀) in plasma samples from healthy controls (HC, n = 12), IDH1-wild-type glioma patients (n = 12), and IDH1-mutant glioma patients (n = 7). B Quantification of D-2-HG (OD₄₅₀) in glioma-conditioned medium (GCM) collected from cultures of ILCs alone, U87-MG, IDH1-mutant U87-MG cell lines, and tonsil ILCs co-cultured with U87-MG or IDH1-mutant U87-MG cells under cytokine-supplemented (recombinant human IL-2 [5 ng/mL], IL-7 [50 ng/mL], IL-12 [50 ng/mL], IL-1β [50 ng/mL], IL-23 [50 ng/mL]) and cytokine-free conditions for four days. Data points represent two independent experiments (ILC n = 4, U87-MG + ILC n = 2, IDH1-mutant U87-MG + ILC n = 2 technical replicates). Error bars indicate ± SEM. (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

Article Snippet: Human glioma U87-MG and its isogenic IDH1 (R132H)-mutant U87-MG (#HTB14IG) cell lines were obtained from the American Type Culture Collection (ATCC).

Techniques: Clinical Proteomics, Mutagenesis, Cell Culture, Recombinant

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Lis1 and CD133 gene expression in neurosphere-like U87 cells. (A) Exposure of U87 glioma cells to stem-conditioned medium (SM) induces phenotypic modifications resulting in neurospheres after five days, in both regular U87 cells (left) and shLis1-U87 cells (right). The expression of Lis1 (B) and CD133 (D) is induced in U87 cells exposed to SM, with the highest level at day 5 of incubation. (C) As expected, silencing Lis1 gene in U87 cells (shLis-U87) inhibits its induction in cells incubated with SM. CD133 induction is almost abrogated in shLis-U87 cells (E) compared with U87 cells (D). Lis1 is highly expressed in CD133+ cells isolated from U87 cell line and primary glioblastoma (HTC1 and HTC2) cell cultures in normal culture medium (CM) or SM (F) . Data show enrichment up to 60 fold in Lis1 expression in CD133 + fractions for cells grown in CM and up to 32 fold in cells incubated in SM. Lis1 expression in CD133+ fraction isolated from U87 cells grown in CM is 32 times higher than that of CD133- fraction (U87 columns); in CD133+ fraction isolated from U87 incubated in SM Lis1 expression is 35 times higher than in CD133 negative cells from the same culture. The negative control is represented by CD133+ cells isolated from shLis-U87 incubated in CM or SM for which Lis1 was not increased as compared with CD133- cells (shLis-U87/CM and SM columns).

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Gene Expression, Expressing, Incubation, Isolation, Negative Control

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Proliferation of irradiated U87 and shLis-U87 cells. Cells having Lis1 silenced or not were irradiated with X-ray doses from 5 to 50 Gy. Cells seeded at a density of 1x10 4 cells/well, in quadruplicates or triplicates in E-plates and placed in xCelligence RTCA instrument, were followed-up for 100 hours (A) . Alternatively, irradiated or not irradiated cells were seeded in 24-well plates and the DNA amount per well was determined using Hoechst 33342 (B) . Both methods showed that irradiated U87 cells recovered better their proliferative capacity than shLis-U87 cells.

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Irradiation

Journal: Journal of Cancer

Article Title: Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells

doi: 10.7150/jca.17635

Figure Lengend Snippet: Cell adhesion, migration and proliferation of CD133 + cells isolated from U87 and shLis-U87 cells . CD133+ cells were isolated from control U87 and shLis-U87 cells. The purity of the fraction is revealed by higher CD133 expression (assessed by RT-PCR) in CD133+ fraction as compared with CD133- fraction (A) . CD133+ cells isolated from control U87 (blue circles) or from shLis-U87 (red squares) cultures were subjected to functional assays using xCELLigence Real-Time Cell Analysis instruments. The data representing the recorded cell index at different times show that CD133+ cells isolated from shLis-U87 culture present two times lower (B) adherence to the surface, (C) migratory potential and (D) proliferative rate, as compared with the those isolated from control U87 culture.

Article Snippet: U87 cells were transfected with a mix of three plasmids, each containing specific shRNA for Lis1 using Plasmid Transfection Reagent in Plasmid Transfection Medium (Santa Cruz, CA).

Techniques: Migration, Isolation, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Functional Assay, Cell Analysis

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: Hypoxia regulated miRNAs. Hierarchical clustering of hypoxia-induced and down-regulated miRNAs (>1.5-fold) in response to hypoxia (0.2% O 2 ) in cell line U87MG (a) . List of hypoxia-regulated miRNA clusters in U87MG cells (b) . A table showing correlation of microRNAs altered in hypoxia or in GBM tumor tissues (c) .

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques:

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: Quantitative RT-PCR data showing miRNA levels in response to hypoxia or HIF1A. Graph showing miRNAs that are upregulated (a) or downregulated (b) in response to hypoxia. (c) U87MG cells were transfected with pCDNA3.1or a HIF1A over-expressing plasmid (pCDNA3.1-HIF1A), and miRNA levels were determined. U87MG cells were transfected with pLK0.1-shGFP or a shHIF1A over-expressing plasmid (pLK0.1-shHIF1A) and levels of HIF1A (d) and miRNAs (e) in response to hypoxia were determined. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: MiR-210-3p induces HIF transcriptional activity. U87MG cells were transfected with a HRE luciferase vector, along with either miR-210-3p overexpression - [(pBABE-miR210) or control (pBABE)] or inhibition - [(miR-210 inhibitor) or (control)] vectors, and HRE transcriptional activity was assayed (a) . U87MG cells were transiently transfected with either a miR-210-3p over-expression vector (pBABE-miR-210) or the empty pBABE-puro parent vector (b) or with either a miR-210-3p inhibitor or control oligos (c) , and VEGF/CA9 levels were determined by qRT-PCR. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Over Expression, Control, Inhibition, Quantitative RT-PCR

Journal: BMC Genomics

Article Title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

doi: 10.1186/1471-2164-15-686

Figure Lengend Snippet: MiR-210-3p functions. Graphs showing MTT assay results of cell survival on the 3 rd day in U87MG, U251MG and A172 cells in response to either miR-210 overexpression - [miR-210 polyclonals (pBABE-miR-210) or control (pBABE)] or inhibition - [(miR-210 inhibitor) or (control)] under (a) 0.2% hypoxia (b) or serum starvation or (c) chemo drug temozolomide treatment. The graphical data points represent mean ± S.D. of at least three independent experiments. (*P > 0.01 and < 0.05; **P < 0.01). Error bars denote ± S. D.

Article Snippet: Cell line U87MG was obtained from the National Centre for Cell Sciences, Pune.

Techniques: MTT Assay, Over Expression, Control, Inhibition

Journal: iScience

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

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet: TIM-3 is one of the most highly elevated immune checkpoints in GBM and is expressed by both non-tumor and tumor cells (A) The expression analyses of immune checkpoints in GBM, in comparison with non-tumor tissue and LGG, identified 7 overlapping elevated immune checkpoints, including 2 co-inhibitory immune checkpoints ( TIM- 3 and LAIR1 ) and 5 co-stimulatory immune checkpoints ( SLAMF8 , CD300A , TYPOBP , CD58 , and BTN3A2 ) (TCGA-seq, GBM, n = 155; LGG, n = 515; non-tumor, n = 4; GSE16011 , GBM, n = 155; LGG, n = 116; non-tumor, n = 8). (B) RT-qPCR analyses of CTLA4 , TIM3 , LAIR1 , PD1 , PDL1 , PDL2 and ID O 1 in clinical GBM samples (n = 10, means ± SEM, one-way ANOVA). (C) Immunohistochemical staining (Left, scale bar, 50 μm) and analyses (Right, non-tumor, n = 13; Grade II, n = 6; Grade III, n = 17; Grade IV, n = 77; means ± SEM, one-way ANOVA) of TIM-3 in clinical samples. (D) The Kaplan-Meier analyses of clinical glioma samples reveal the correlation of TIM-3 with poor prognosis in GBM (TIM-3 high vs. low, p = 0.0003; log rank test). (E) CIBERSORT analysis of non-tumor cell populations associated with high TIM-3 expression in TCGA RNA-seq GBM dataset (Pearson correlation analysis). (F) The tSNE plot of TIM-3 expression profile in GBM and immune cells with single-cell RNA-seq data ( GSE131928 ). (G) Representative immunofluorescence images of TIM-3 and GFAP staining in clinical GBM samples (scale bar, 50 μm). (H) FACS analysis shows that clinical GBM samples contained low CD45, and high TIM-3 cell populations (n = 7). (I) qPCR analysis of TIM-3 mRNA expression in NHA, indicated glioma cell lines (U87, U251, and LN229), primary glioma cells (PGC1228, PGC21, PGC24, and PGC40), and PBMC from patients with glioma (G-PBMC1 and G-PBMC2) and healthy donor (PBMC1 and PBMC2) (n = 3, means ± SEM, one-way ANOVA). (J) Western blot analyses of TIM-3 in NHA, indicated glioma cell lines, and primary glioma adherent and neurosphere cells (n = 3, means ± SEM, one-way ANOVA). (K) The schematic diagram describing the co-culture system of THP-1-derived anti-inflammatory/pro-tumorigenic (M2) macrophages and glioma cells (left panel), and representative western blot images of TIM-3 in indicated glioma cells cocultured with THP-1-derived anti-inflammatory/pro-tumorigenic TAMs (right panel). (ns p ≥ 0.05, ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001).

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Immunohistochemical staining, Staining, RNA Sequencing, Immunofluorescence, Western Blot, Co-Culture Assay, Derivative Assay

Journal: iScience

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

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet: TIM-3 increases IL6 expression via activating NF-κB signaling in glioma cells (A) Representative western blotting images of Gal-9 in conditioned medium (CM) from indicated glioma cells separately transfected with TIM-3 overexpression, control, knockdown, or siNC vector. (B) The detection (left) and quantification (right) of cytokines in culture supernatants from GSC40 transduced with TIM-3 overexpression or control vector by proteome profiler cytokine array. (C-E) Representative western blotting images of IL6 in U87 cells transduced with control (U87-NC) or TIM-3 overexpression vectors (U87-TIM3 OE), respectively, and then with indicated treatment at indicated incubation time (C, with CHX; D, with CHX after MG132 pretreatment; E, with CHX after CQ pretreatment). (F) Representative western blot images of indicated glioma cells transduced with TIM-3 overexpression vector with or without blockade of Gal-9 (10 μg/mL). (G) IL6 concentration in CM from indicated cells measured by ELISA (n = 3, means ± SEM, one-way ANOVA). (H) Representative western blot images of indicated cells incubated with indicated concentrations (ng/mL) of IL6. (I-K) The decreased growth (I, n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm), migration (J, n = 3, means ± SEM, one-way ANOVA; scale bar, 50 μm), and neurosphere formation abilities (K, upper: n = 10, extreme limiting dilution assay; lower: stem cell frequency, n = 3, means ± SEM, one-way ANOVA) of GSC40 induced by TIM-3 knockdown was partially rescued by IL6 supplement (40 ng/mL). (ns p ≥ 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

Techniques: Expressing, Western Blot, Transfection, Over Expression, Control, Knockdown, Plasmid Preparation, Transduction, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Migration, Limiting Dilution Assay

Journal: iScience

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

doi: 10.1016/j.isci.2022.105329

Figure Lengend Snippet:

Article Snippet: Human: U87 cells , GeneChem , RRID: CVCL_0022.

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