Journal: Signal Transduction and Targeted Therapy

Article Title: Low-dose radiotherapy synergizes with PD-1 blockade to achieve durable survival in advanced NSCLC through antitumor neutrophil programming

doi: 10.1038/s41392-026-02712-6

Figure Lengend Snippet: TRIDENT induces chemotactic and polarizing signals that recruit and program neutrophils toward an antitumor phenotype. a Schematic of the in vitro Transwell assay evaluating neutrophil migration toward tumor-conditioned medium derived from the indicated treatments. b Quantification of neutrophil migration across Transwell membranes ( n = 4 per group). c Experimental design of the in vivo neutrophil-trafficking assay in a dual-tumor model after indicated therapy. d Representative flow cytometry plots and quantification of transferred CD45.1⁺ neutrophils in tumors ( n = 7 per group). e Single-cell RNA-seq showing Cxcr2 upregulation in neutrophils from TRIDENT-treated tumors. Genes were classified as upregulated if increased in both comparisons, downregulated if decreased in both, oppositely regulated if significant in both but with opposite direction, and unchanged if non-significant in ≥1 comparison or showed minimal change. f Flow cytometric quantification of CXCR2⁺ neutrophils in second tumors in the LLC model across treatment groups ( n = 7 per group). g Heatmap comparing relative mRNA expression of Cxcl1, Cxcl2, Cxcl3 , and Cxcl5 from averaged qPCR values in tumor tissues after the indicated treatment ( n = 4 per group). Tumor growth curves ( h , i ) and survival curve ( j ) of LLC-bearing mice treated with TRIDENT alone or with CXCR2 blockade ( n = 9 per group). k, l qPCR analysis showing increased expression of Csf2 and Ifng in tumor tissues after indicated treatment ( n = 4 per group). m – p Flow cytometric analysis of antigen presentation and co-stimulatory molecules (CD14, ICAM-1, MHC-I, and CD86) on neutrophils cultured with tumor-conditioned medium derived from control, LDRT-, or HDRT-treated tumors ( n = 10 per group). Tumor growth curves ( q , r ) and survival curve ( s ) of LLC-bearing mice treated with TRIDENT alone or with GM-CSF/IFN-γ blockade ( n = 6 per group). Data are mean ± SEM. Statistical significance was assessed by one-way ANOVA ( b, f, g, k – p ), Student’s t test ( d ), two-way ANOVA ( h , i, q , r ) or log-rank test for survival ( j , s ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: For CXCR2 blockade, a CXCR2 antagonist (HY-101022, MedChemExpress; 2 mg/kg, i.p.) was administered every 3 days from randomization.

Techniques: In Vitro, Transwell Assay, Migration, Derivative Assay, In Vivo, Flow Cytometry, Single Cell, RNA Sequencing, Comparison, Expressing, Immunopeptidomics, Cell Culture, Control

Journal: bioRxiv

Article Title: Mechanistic insights of radiation-induced endothelial senescence impelling glioblastoma genomic instability at relapse

doi: 10.1101/2021.12.13.472364

Figure Lengend Snippet: a. CXCR2 expression in U251 and human primary GBM cells by FACS analysis. Left panel : Representative histograms of CXCR2 staining in U251 cells. Right panel : Geomean of fluorescence compared to isotype. Geomean of CXCR2 expression in U251 cells and GBM primary cells is indicated respectively as black and white circles. b. Radio-induced MN production per cell after 5Gy in U251 cells in CM or SASP after CXCR2 blocking using monoclonal antibody MAb331(250ng/ml) or SB332235 antagonist (1nM). (mean±SD, n=3, one-way ANOVA; * p<0.05, *** p<0.001). c. Polynucleated cell frequency after 5Gy-radiation in U251 cells in CM or SASP after CXCR2 blocking using monoclonal antibody MAb331(250ng/ml) or SB332235 antagonist (1nM). (mean±SD; n=3; two-way ANOVA, * p<0.05, ** p<0.01). d. Elongation rate of U251 cells in CM (black) or SASP (blue) supplemented or not with CXCR2 monoclonal antibody (MAb331, 250ng/ml). When indicated, cells were irradiated with 5Gy-radiation. (mean±SD, n=3, two-way ANOVA; ** p<0.01, *** p<0.001).

Article Snippet: Anti-CXCR2 blocking antibody (Mab331) and antagonist drug (SB332235) were purchased from R&D and TOCRIS (Bioscience) respectively.

Techniques: Expressing, Staining, Fluorescence, Blocking Assay, Irradiation

Journal: bioRxiv

Article Title: Mechanistic insights of radiation-induced endothelial senescence impelling glioblastoma genomic instability at relapse

doi: 10.1101/2021.12.13.472364

Figure Lengend Snippet: a. Median survival (days) of tumor-bearing mice after orthotopic injections of radiation-surviving U251 cells obtained in presence of CM (R15CM), SASP (R15SASP), CM supplemented with CXCL8 and CXCL5 (R15CM -CXCL5/8 ), SASP supplemented with either CXCR2 monoclonal antibody Mab331 (R15SASP- Mab ) or CXCR2 antagonist SB3322235 (R15SASP- SB ). Log-rank p-values are indicated as compare to mice survival injected with either R15CM radioresistant cells or R15SASP radioresistant cells. b. Tumor-bearing mice survival after orthotopic injections of R15CM-CXCL (left panel), R15SASP-mAbR2 (middle panel) and R15SASP-SBR2 (right panel) (n=8/group) as compare to R15CM and R15SASP tumor-bearing mice. c. Immunofluorescence of CXCL5 and CXCL8 in primary and recurrent human GBM. d. Kaplan-Meier plots overall survival of GBM patients depending on CXCL5 and CXCL8 mRNA expression using the TCGA-glioblastoma database. (Log-rank tests analysis).

Article Snippet: Anti-CXCR2 blocking antibody (Mab331) and antagonist drug (SB332235) were purchased from R&D and TOCRIS (Bioscience) respectively.

Techniques: Injection, Immunofluorescence, Expressing

Journal: Journal of translational medicine

Article Title: RBM15 recruits myeloid-derived suppressor cells via the m6A-IGF2BP3/CBR3-AS1/miR-409-3p/CXCL1 axis, facilitating radioresistance in non-small-cell lung cancer.

doi: 10.1186/s12967-025-06205-y

Figure Lengend Snippet: Fig. 4 CBR3-AS1 recruits MDSCs through the miR-409-3p/CXCL1/CXCR2 axis. A Relative mRNA expression levels of CXCL1 in H520 cells transfected with a control empty vector (vecCtrl), miR-409-3p mimic plasmid (miR-409-3p), control shRNA (shCtrl), or specific interfering hairpin RNAs targeting miR-409-3p (shmiR-409-3p). The cells were exposed to radiation after 24 h of transfection. B Relative protein expression levels of CXCL1 in H520 cells. β-actin was used as the reference for normalization. C Relative luciferase activity of CXCL1 under miR-409-3p overexpression in the wild-type and mutant CXCL1 groups treated with radiation. D, E Relative mRNA and protein expression levels of CXCL1 in H520 cells transfected with shCtrl, shCBR3-AS1, or shmiR-409-3p. F Migration ability of MDSCs toward the conditioned medium of H520 cells transfected with CXCL1, shCXCL1, or treated with a CXCR2 inhibitor (SB265610, 10 mM) and treated with radiation. G, H Levels of interferon (IFN)-γ secreted by CD8+ and CD4.+ T cells co-cultured with H520 cells transfected with CXCL1, shCXCL1 or treated with a CXCR2 inhibitor (SB265610, 10 mM). IFN-γ expression was reported as the mean fluorescence intensity. The significance between two groups was analyzed using Student’s t-test. Data are presented as mean ± SD from three independent experiments. Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Membranes were probed with primary antibodies: RBM15 (1:500, Abclonal, USA, A4936), IGF2BP3 (1:1000, Abclonal, A4444), CXCL1 (1:500, Abclonal, USA, A5802), CXCR2 (1:1000, Proteintech, Wuhan, China, 20634-1-AP), or β-actin (1:500, Abclonal, USA, WL01372).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, shRNA, Luciferase, Activity Assay, Over Expression, Mutagenesis, Migration, Cell Culture, Fluorescence

Journal: Mucosal Immunology

Article Title: Butyrate regulates neutrophil homeostasis and impairs early antimicrobial activity in the lung

doi: 10.1016/j.mucimm.2023.05.005

Figure Lengend Snippet: Effect of butyrate on acute Pseudomonas aeruginosa immunity. (A) Quantification of the total number of cells in BALF 18 hours post-inoculation with 1 × 10 6 CFU of P. aeruginosa strain PAO1 in Ctrl and But mice. (B) Frequency and quantification of cell types in cytospins of BALF from control and butyrate-treated mice 18 hours after PAO1 infection; (C) Quantification of P. aeruginosa CFU in BALF 18 hours post-challenge. (D, E) Cytokine and chemokine production in BALF 18 hours following infection. (F) MFI of CXCR2 on lung AM and IM from Ctrl and But mice. (G) MFI of CXCR2 on Ctrl and But lung macrophages US or stimulated for 2 hours with LPS in vitro . (H) transmigration of Ctrl and But neutrophils toward a CXCL2 or CCL2 chemokine gradient after 3 hours. Results are a mean of two independent experiments. Values are expressed as mean ± standard error of mean; n = 6–14. Statistical significance was determined with one-way analysis of variance in (A–E, G, H) and Student’s t test (unpaired, two-tailed) in (F). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. AM = alveolar macrophages; BALF = bronchoalveolar lavage fluid; But = butyrate-treated; CFU = colony-forming units; Ctrl = control; IFN = interferon; IL = interleukin; IM = interstitial macrophages; LPS = lipopolysaccharide; MFI = mean fluorescence intensity; PAO1 = P. aeruginosa strain 1; TNF = tumor necrosis factor; US = unstimulated.

Article Snippet: To differentiate progenitor and neutrophil populations in the BM, cells were stained with antibodies to lineage cocktail, CD115 (BD Biosciences, clone 2B8), SiglecF (BD Biosciences, clone 2B8), CD117 BUV117 (BD Biosciences, clone 2B8), Sca1 APCFire (Biolegend, clone D7), FcgR biot (Miltenyi, clone 93), CD34 BV421 (Biolegend, clone SA376A4), CD11b BV510 (BD Biosciences, clone M1/70), Ly6G Pecy5 (eBioscience, clone 1A8-Ly6g), CXCR2 Pecy7 (Miltenyi, clone REA942) and streptavidin BUV395 (BD Biosciences).

Techniques: Control, Infection, In Vitro, Transmigration Assay, Two Tailed Test, Fluorescence

Journal: Nature immunology

Article Title: Locally instructed CXCR4 hi neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps

doi: 10.1038/s41590-019-0496-9

Figure Lengend Snippet: a , Representative dot plots of lung CD45 + CD11b + Ly-6G + neutrophils from vehicle or LPS lo mice treated or not with Sch527123, a CXCR2 antagonist (anti-CXCR2), 2 h before and 4, 8 and 20 h after LPS, and analyzed 24 h after LPS lo . Insets indicate % of cells within the gate. b , Absolute numbers of lung CD45 + CD11b + Ly-6G + neutrophils as in a. c , Eosinophil cell counts in the BALF, assessed 3 days after the second HDM administration in LPS lo -HDM mice treated or not with anti-CXCR2, 2 h before and 4 and 8 h after LPS lo , and 2 h before and 4 h after i.n.sensitization with HDM. d , ELISA measurement of cytokine production by HDM-restimulated BLN cells of mice as in c. e , Inflammatory score estimating perivascular and peribronchial inflammation, quantified from H&E-stained lung sections of mice as in c. f , Representative H&E staining of lung sections of mice as in c. g , Quantification of PAS-stained epithelial cells per bronchi showing airway mucus production in mice as in c. h , Representative PAS staining of lung sections of mice as in c. i , Eosinophil cell counts in the BALF, assessed 3 days after the second HDM administration in mice transferred i.t. with vehicle, LPS lo and LPS hi neutrophils and exposed to HDM. j , ELISA measurement of cytokine production by HDM-restimulated BLN cells of mice as in i. k , Inflammatory score estimating perivascular and peribronchial inflammation, quantified from H&E-stained lung sections of mice as in i. l , Representative H&E staining of lung sections of mice as in i. m , Quantification of PAS-stained epithelial cells per bronchi showing airway mucus production in mice as in i. n , Representative PAS staining of lung sections of mice as in i. (b-e,g,i-k,m) Data show mean + s.e.m. and are pooled from ( b-e,g ) 2 independent experiments, each symbol representing individual mice (n=6/group) or ( i-k,m ) ≥3 independent experiments, each symbol representing ( i,k,m ) individual mice (n=9/group) or ( j ) independent experiments in which cells from 2-3 mice were pooled by group. P values were calculated using using a ( b,i-k,m ) one-way ANOVA with Tukey's post hoc test, ( c ) a two-sided unpaired Student's t test or ( d-e,g ) a two-sided Mann-Whitney test. * P <0.05; ** P <0.01; *** P <0.001. ns, not significant. BALF, bronchoalveolar lavage fluid. i.t., intra-tracheal ns, not significant. Scale bar = 32 μm.

Article Snippet: To inhibit LPS-induced lung neutrophil recruitment , an antagonist of the chemokine receptor CXCR2 (i.e., Sch527123 , MedChem Express) was given orally, by gavage, at the dose of 3mg/kg, 2 h before, 4 h after and 8 h after LPS treatment, as well as 2 h before and 4 h after i.n. administration of 40 μg HDM.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY