Journal: Molecular Therapy Oncology

Article Title: Potency-enhancing mutations in E3-19K and i-leader increase the cytolytic activity of the PH20/ SPAM1 -armed oncolytic adenovirus Ad5Δ24RGD

doi: 10.1016/j.omton.2026.201137

Figure Lengend Snippet: The 19K SS and iL Q125Ter modifications mutually increase the potency of OAds regardless of the type of fiber modification (A) Sizes of MTT-stained plaques in cancer cell monolayers under an agarose overlay. Two to three independent experiments were conducted. The total number ( n ) of analyzed plaques is indicated for each group. Data are shown as means (SD) and analyzed using Brown-Forsythe and Welch ANOVA tests with Games-Howell post hoc multiple comparisons. (B) IC 50 values derived from viability curves after infection of cells (2.5 × 10 4 per well) in suspension with serial 3-fold dilutions of the indicated OAds. Data were collected on day 6 post-infection for all cell cultures except LN18 and LN229 cells infected with Ad5Δ24RGD-derived OAds (on day 7). Two to three independent experiments with technical triplicates were performed. Data are shown as means (SD). Only ≥3-fold differences from the parental virus Ad5Δ24RGD are indicated. (C) Sizes of fluorescent plaques and MTT-stained plaques in cancer cell monolayers under an agarose overlay. Three to four independent experiments were performed. The total number ( n ) of analyzed plaques is indicated for each group. Data are shown as means (SD) and analyzed using Brown-Forsythe and Welch ANOVA tests and Games-Howell post hoc multiple comparisons. ∗∗∗∗ p ≤ 0.0001; ∗∗∗ p ≤ 0.001; ∗∗ p ≤ 0.01; ∗ p ≤ 0.05; NS, not significant. See also .

Article Snippet: Human embryonic kidney HEK293, lung adenocarcinoma A549 and H441, prostate adenocarcinoma LNCap and PC3, hepatocellular carcinoma HepG2 and Hep3B, glioblastoma LN18, LN229, DBTRG, T98G, and U87 (American Type Culture Collection, ATCC), murine glioma CT-2A (#SCC194, Sigma) and GL261 cells (#ACC802, DSMZ Cell Culture Collection), and human short-term glioma (III–IV grade) primary cell cultures (GliSav, GliShat, GliBah, GliDu, AG-AASH, GBM100622, and GBM160323) were grown in Dulbecco’s modified Eagle medium (DMEM) with L-alanyl-glutamine, 4.5 g/L glucose, and Na pyruvate (#C415, Paneco), supplemented with 10% fetal bovine serum (FBS; HyClone/Cytiva, neoFroxx, or Capricorn Scientific) and penicillin-streptomycin solution at a final concentration of 50 U/mL and 50 μg/mL (#А063, Paneco).

Techniques: Modification, Staining, Derivative Assay, Infection, Suspension, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA675-5p inhibitor’s dual role as novel therapeutic alternative or sensitizing treatment in resistant glioma models

doi: 10.1016/j.omtn.2025.102647

Figure Lengend Snippet: miRNA657-5p inhibitor has an anti-proliferative effect mediated by HIF-1α down-regulation (A) Viability of TMZ-resistant glioma cell lines (T98, LN18, and U118) cells, assessed by means of Trypan blue exclusion test, and expressed as the percentage of viable cells compared with untreated cells after treatment with miRNA675-5p inhibitor for 18 h. ∗∗ p < 0.01 vs. scramble-treated cells. (B) ATP level in T98, LN18, and U118 cells analyzed by Cell Tox Green kit and expressed as relative fluorescence units compared with scramble-treated cells. ∗∗∗ p < 0.001 vs. control cells. (C) Gene expression analysis for BAX , BAD , and BCL-2 analyzed by real-time PCR in T98, LN18, and U118 cells after treatment with miRNA675-5p inhibitor. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as fold of induction (FOI) of the ratio between treated and scramble-treated cells. ∗ p < 0.05; ∗∗ p < 0.01 inhibitor- vs. scramble-treated cells. (D) Analysis of caspase 3/7 activity evaluated by Caspase-Glo 3/7 Assay and expressed as relative luminescent units. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 inhibitor- vs. scramble-treated cells. (E) In silico analysis of an integrative map reconstructed after the public database (The Cancer Genome Atlas Program) interrogation based on protein-protein interaction (called interactome). (F) Gene expression analysis for HIF-1α analyzed with real-time PCR in T98, LN18, and U118 cells after treatment with miRNA675-5p inhibitor. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as the FOI of the ratio between treated and scramble-treated cells. ∗∗ p < 0.01 inhibitor- vs. scramble-treated cells. (G) ELISA-based HIF-1α nuclear (black columns) and cytoplasm (gray columns) quantification after miRNA675-5p inhibitor treatment. The data are expressed as absorbance at 450 nm. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 vs. control cells in nuclear vs. cytoplasmatic localization. Graphs represent mean values ± SD of three independent experiments.

Article Snippet: U251 (ICLC, Ospedale San Martino, Genova, Italy), T98, LN18 and U118 ATCC (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI1640 (U251 and T98) or DMEM High Glucose (LN18 and U118), both supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, and 2 mM glutamine (all Euroclone, Milan, Italy) in a humidified atmosphere of 5% of CO 2 at 37°C.

Techniques: CellTox Assay, Fluorescence, Control, Gene Expression, Real-time Polymerase Chain Reaction, Activity Assay, Caspase-Glo Assay, In Silico, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA675-5p inhibitor’s dual role as novel therapeutic alternative or sensitizing treatment in resistant glioma models

doi: 10.1016/j.omtn.2025.102647

Figure Lengend Snippet: miRNA675-5p inhibitor effect is due to oxidative stress (A) Luminescent assay (ROS-Glo H2O2 Assay kit) was applied to measure H 2 O 2 levels in cell culture medium of different cell lines after treatment with scramble (as control)- or miRNA675-5p inhibitor. Data were expressed as relative luminescence units (RLUs) obtained by luciferase counts normalized for the amount of total proteins quantified by Bradford assay. ∗∗∗ p < 0.001 vs. control cells; # p < 0.05 U251 vs. average ROS level in T98, LN18, and U118 cells. (B) Gene expression analysis for detox-related genes ( SOD-1 and CAT-1 ) and (C) for HIF-1α-related genes ( NRF-2 , Nf-kb , and KEAP1 ) analyzed by means of Real-time PCR in all cells. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as the FOI of the ratio between treated and scramble-treated cells. ∗ p < 0.05, ∗∗ p < 0.01; ∗∗∗ p < 0.001 vs. scramble-treated cells. (D) ATP levels in U251, T98, LN18, and U118 cells after treatment with MitoTEMPO, a ROS scavenger, and inhibitor analyzed by Cell Tox Green expressed as relative fluorescence units compared with inhibitor-treated cells alone. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. inhibitor-treated cells; ### p < 0.001 inhibitor + MitoTEMPO vs. inhibitor alone. (E) Gene expression analysis of apoptosis-related genes ( BAX , BAD , and BCL-2 ) by real-time PCR in all cells after inhibitor of inhibitor + MitoTEMPO treatment. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as the FOI of the ratio between MitoTEMPO plus inhibitor and inhibitor-treated cells. (F–H) Relative GSH (F) and GSSG (G) abundance and GSH/GSSG ratio (H) in U251 and T98 cell line in control condition vs. inhibitor-treated cells obtained by LC-MS analysis ( n = 9). ∗∗∗ p < 0.001 vs. scramble-treated cells; # p < 0.05 and ### p < 0.001 U251 scramble-treated (control) cells vs. T98 scramble-treated (control) cells. Graphs represent mean values ± SD of three independent experiments.

Article Snippet: U251 (ICLC, Ospedale San Martino, Genova, Italy), T98, LN18 and U118 ATCC (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI1640 (U251 and T98) or DMEM High Glucose (LN18 and U118), both supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, and 2 mM glutamine (all Euroclone, Milan, Italy) in a humidified atmosphere of 5% of CO 2 at 37°C.

Techniques: Luminescence Assay, H2O2 Assay, Cell Culture, Control, Luciferase, Bradford Assay, Gene Expression, Real-time Polymerase Chain Reaction, CellTox Assay, Fluorescence, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA675-5p inhibitor’s dual role as novel therapeutic alternative or sensitizing treatment in resistant glioma models

doi: 10.1016/j.omtn.2025.102647

Figure Lengend Snippet: miRNA675-5p sensitizes resistant cells to subsequent TMZ treatment (A and B) Representative images of immunoblots (A) and quantification histograms (B) of regulatory associated protein of mtor complex 1 (RPTOR) expression. RPTOR level was assessed in U251 (left) and T98 (right) scramble (black bars) cell lines, treated with TMZ (light gray bars) miRNA675-5p inhibitor (gray bars), or inhibitor followed by TMZ treatment (dark gray bars). Mean ± SD. ∗Significant difference, ANOVA and Tukey’s test, n = 3, p < 0.05. (C) Viability of resistant glioma cell lines (T98) cells, assessed by means of Trypan blue exclusion test, and expressed as live or dead cells after miRNA675-5p inhibition and subsequent TMZ challenge, in absence or presence of the refresh period. ∗∗ p < 0.01 dead vs. live cells; ## p < 0.01 dead cells in control vs. TMZ-treated cells. (D) Cytotoxicity evaluation of the average of T98, LN18, and U118 cells analyzed by Cell Tox Green expressed as relative fluorescence units (RFUs) compared with scramble-treated cells, in absence or presence of the refresh period. (E) Gene expression analysis of apoptosis related genes ( BAX , BAD , and BCL-2 ) by real-time PCR in all cells after TMZ treatment subsequent to miRNA675-5p inhibition. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as fold of induction (FOI) of the ratio between inhibitor + TMZ- and scramble + TMZ-treated cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 of inhibitor + TMZ- vs. scramble + TMZ-treated cells. (F) Comparison of HIF-1α expression level between U251 basal level and T98 basal and treated cells. Data were normalized to β-ACTIN , and the ΔCt values were depicted in the graph. ∗∗ p < 0.01 inhibitor alone vs. inhibitor + TMZ treatment. (G) Gene expression analysis of HIF-1α and miRNA675-5p levels by real-time PCR in all cells after TMZ treatment subsequent to miRNA675-5p inhibition. Data were normalized to β-ACTIN or U6 , respectively housekeeping reference for gene and miRNA expression, and the ΔΔCt values were expressed as FOI of the ratio between inhibitor + TMZ- and scramble + TMZ-treated cells. ∗ p < 0.05, ∗∗∗ p < 0.001 inhibitor + TMZ-vs. scramble + TMZ-treated cells.

Article Snippet: U251 (ICLC, Ospedale San Martino, Genova, Italy), T98, LN18 and U118 ATCC (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI1640 (U251 and T98) or DMEM High Glucose (LN18 and U118), both supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, and 2 mM glutamine (all Euroclone, Milan, Italy) in a humidified atmosphere of 5% of CO 2 at 37°C.

Techniques: Western Blot, Expressing, Inhibition, Control, CellTox Assay, Fluorescence, Gene Expression, Real-time Polymerase Chain Reaction, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dasatinib boosts γδ T cell expansion and memory phenotypes with enhanced antitumor immunity

doi: 10.1007/s00262-026-04335-w

Figure Lengend Snippet: Phenotypic characterization of ex vivo expanded γδ2 T cells. A Overview of Zol-based γδ2 T cell expansion from human PBMCs. B – C Time-course analysis of γδ2 cell population dynamics from five healthy donors (HD-1 to HD-5), showing initial decline followed by robust expansion. The blue line indicates the poor expander (HD-3 and HD-5), whose γδ2 T cells constitute less than 60% of all CD3 + cells. D t-SNE and FlowSOM clustering identified nine phenotypically distinct CD3 + VD2 + -γδ2 T cell subsets (P0–P8) during cell expansion based on the expression of surface markers from the pool sample of five healthy donors. E Marker expression heatmap defines γδ2 T cell subsets (P0–P8). F Histogram analysis of key memory and cytotoxic markers over culture duration, data from the pool sample of five healthy donors. G Cytotoxicity of γδ2 T cells from HD-1 against Ln18 cells decreases with extended culture, as measured by co-culture apoptosis assays

Article Snippet: Human glioblastoma cell lines Ln18 were purchased from the American Type Culture Collection (Manassas, VA); human glioblastoma cell lines GBM8901 and human breast adenocarcinoma cell line MDA-MB231 were purchased from Bioresources Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: Ex Vivo, Expressing, Marker, Co-Culture Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Dasatinib boosts γδ T cell expansion and memory phenotypes with enhanced antitumor immunity

doi: 10.1007/s00262-026-04335-w

Figure Lengend Snippet: Dasatinib-cultured γδ2 T cells exhibit superior in vitro antitumor activity and persistence. A Cytotoxicity of γδ2 T cells expanded with dasatinib (γδ2 T-Da) or vehicle (γδ2 T-Ve) against MDA-MB-231 (TNBC), Ln18, and GBM8901 (GBM) cancer cells in a 5-h co-culture assay. Tumor cell apoptosis was measured by Annexin V positivity. While γδ2 T-Ve cells showed donor-variable cytotoxicity, γδ2 T-Da cells consistently exhibited enhanced tumor cell killing in a majority of tested donors. B Polyfunctional cytokine profiling of γδ2 T cells post co-culture, assessed by intracellular staining for TNF-α, IFN-γ, IL2, IL4, and IL17A. γδ2 T-Da cells displayed higher proportions of TNF-α⁺ and IFN-γ⁺ cells, indicating a more robust effector response. C Long-term cytotoxicity against GBM8901 cells using γδ2 T-Da and γδ2 T-Ve cells harvested at day 7, 10, and 13 of ex vivo expansion. γδ2 T-Da cells retained greater cytotoxicity over time, whereas γδ2 T-Ve cells showed a progressive loss of killing ability. D Serial killing assay demonstrating that γδ2 T-Da cells maintain cytotoxic function over repeated tumor cell challenges, suggesting enhanced persistence and durability of antitumor activity. Student t -test is used, * p < 0.05, ** p < 0.01 and *** p < 0.001. The effector-to-target (E:T) ratios used in A , C , and D were 1:1

Article Snippet: Human glioblastoma cell lines Ln18 were purchased from the American Type Culture Collection (Manassas, VA); human glioblastoma cell lines GBM8901 and human breast adenocarcinoma cell line MDA-MB231 were purchased from Bioresources Collection and Research Center (Food Industry Research and Development Institute, Hsinchu, Taiwan).

Techniques: Cell Culture, In Vitro, Activity Assay, Co-culture Assay, Co-Culture Assay, Staining, Ex Vivo