cxcl5 neutralizing antibody  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: Robust transcriptomic hallmarks targeting intratumor heterogeneity in intrahepatic cholangiocarcinoma

doi: 10.1016/j.xcrm.2026.102708

Figure Lengend Snippet: Distinct immune microenvironment across LIHV-defined subgroups (A) Immune cell abundance comparison across subgroups using the indicated immune analysis tools across four independent iCCA cohorts. Circle color and size represent log 2 fold change and p value, respectively. (B) Boxplots of CD66b + neutrophils, CD68 + macrophages, CD3 + T cells, CD20 + B cells, and αSMA + fibroblasts across subgroups (Mann-Whitney U test). (C) Heatmap of immune signatures and checkpoint expression summarized as mean Z scores per subgroup across four cohorts. ∗FDR < 0.05; ∗∗FDR < 0.01; ∗∗∗FDR < 0.001. (D) Heatmap of Spearman correlations between chemokine expression and neutrophil infiltration. IHC, immunohistochemistry. (E) Boxplot comparing CXCL5 expression across subgroups in the Fu-iCCA cohort (Mann-Whitney U test). (F) Dot heatmap of chemokine gene expression across major cell types in Xue’s scRNA-seq dataset. (G) Boxplots of average CXCL5 expression in tumor cells and macrophages across subgroups (Mann-Whitney U test). (H) Western blot validating CXCL5 knockdown and overexpression efficiency in RBE and HuCCT1 cells. (I) Quantification of migrated neutrophils in transwell assays co-cultured with modified RBE and HuCCT1 cells ( n = 4 replicates per group; mean ± SD; Student’s t test). (J) Western blot analysis of CXCL5 overexpression in KTP cells. (K) Tumor growth curves of mice injected with control or CXCL5-overexpressing KTP cells ( n = 6 per group; mean ± SEM; Student’s t test). (L) Proportion and number of infiltrated neutrophils in control and CXCL5-overexpressing KTP tumors ( n = 6 per group; Student’s t test). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also and and .

Article Snippet: A CXCL5 neutralizing antibody (R&D Systems, AF254) was added at 5 μg/mL.

Techniques: Comparison, MANN-WHITNEY, Expressing, Immunohistochemistry, Gene Expression, Western Blot, Knockdown, Over Expression, Cell Culture, Modification, Injection, Control

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Schematic of the protocol to compare the effects of conditioned media (CM) from CCD-18Co human normal colon tissue fibroblasts (NF CM), primary human colon cancer-associated fibroblasts (CAF CM), and human HCT 116 colon cancer cell-stimulated CAF CM (CAF CCM) on C2C12 myotube wasting. B Myosin heavy chain 2 (MYH2) immunostaining of C2C12 myotubes cultured in normal myotube differentiation media (DM) and treated with NF CM, CAF CM, and CAF CCM for 72 h (scale bar = 150 μm). C Calculation of mean myotube diameter. D Western blot analysis of MYH2 and atrogin-1 expression in the treated myotubes. E Densitometry of MYH2 and atrogin-1 expression relative to GAPDH. F Cytokine array analysis CAF CM, CAF CCM, and HCT 116 cancer cell CM. Red boxes and numbers indicate upregulated cytokines in the CAF CCM compared to CAF CM. G Quantification of the fold-change for the upregulated cytokines. H ELISA-based detection of CXCL5 in NF CM, CAF CM, CAF CCM, and HCT 116 cancer cell CM. The CAF CM and CCM values are the mean obtained from three sources of CAF: two derived from patients and one provided commercially. All experiments were conducted 3 times independently and the values are indicated as the mean ± SD. For C and E : * = p < 0.05 and *** = p < 0.001 compared to DM. For H : ** = p < 0.01 compared to CAF CM

Article Snippet: CM was treated to the differentiated myotubes for 72 h. CXCL5 neutralizing antibody MAB-254 was purchased from R&D system (MN, USA) and CXCR2 antagonist SSB225005 was obtained from MedChemExpress (NJ, USA).

Techniques: Immunostaining, Cell Culture, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Myosin heavy chain 2 (MYH2) immunostaining of C2C12 myotubes cultured in normal differentiation media (DM), DM plus 10 ng/mL CXCL5, or DM plus 20 ng/mL CXCL5 for 72 h. (scale bar = 150 μm) B Calculation of mean myotube diameter. C Western blot analysis of atrogin-1 and MuRF-1 expression in the treated myotubes. D Densitometry of atrogin-1 and MuRF-1 expression relative to GAPDH. E Western blot analysis of total extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 in myotubes treated with CAF CCM or 20 ng/mL CXCL5 for 0.5 h and 2 h. F Densitometry of phosphorylated ERK1/2 relative to ERK1/2. G MHY2 immunostaining of C2C12 myotubes cultured for 72 h as follows: (1) Normal differentiation media (DM), (2) DM:CAF CCM (1:1) plus vehicle (0.1% DMSO), (3) DM:CAF CCM (1:1) plus 0.5 μg/mL CXCL5 neutralizing antibody, and (4) DM:CAF CCM (1:1) plus 22 nM SB225005 for 96 h (scale bar = 150 μm). H Calculation of mean myotube diameter. All experiments were conducted 3 times independently and the values are indicated as the mean ± SD. For B , D , F and H : * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 indicate significantly increased compared to untreated. For H : # = p < 0.05 and ## = p < 0.01 indicates significantly increased compared to the DM:CCM (1:1) group

Article Snippet: CM was treated to the differentiated myotubes for 72 h. CXCL5 neutralizing antibody MAB-254 was purchased from R&D system (MN, USA) and CXCR2 antagonist SSB225005 was obtained from MedChemExpress (NJ, USA).

Techniques: Immunostaining, Cell Culture, Western Blot, Expressing

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A IVIS imaging of NOD-SCID mice 3 weeks post-xenograft with 1 × 10 6 human HCT 116 luc2 colon cancer cells, or 1 × 10 6 HCT 116 luc2 cancer cells plus 2 × 10 6 human colon CAF. B Mean total flux detected from the tumor at the end point (ns = not significant). C Dissected tumors and tumor mass at the 3 week endpoint. D Tumor free body weight (B.W) at the 3 week end point. E Representative images of H&E stained gastrocnemius muscle (scale bar = 50 μm). F Calculation of the myofiber cross sectional area. G qPCR analysis of CXCL1, 2, 3, 5, 6, 7, and 8 (known CXCR2 ligands) and IL-6 in the dissected tumor. H IVIS imaging of NOD-SCID mice at 3 weeks post-xenograft with human HCT 116 luc2 cancer cells plus human colon CAF, with or without CXCL5 neutralization (CXCL5 Neu Ab). I Mean total flux detected from the tumor at the 3 week end point. J Dissected tumors and tumor mass. K Tumor free body weight (B.W) at the 3 week end point. L Hanging tolerance in the treated mice. M Tibialis anterior (TA) muscle mass. N Representative H&E staining of the TA muscle (scale bar = 100 µm). O Calculation of the myofiber cross sectional area. P CXCR2 immunostaining of the TA muscle (scale bar = 100 µm). Q Proportion of CXCR2 positive myofibers. 5 mice per group were used for the experiments and the values are indicated as the mean ± SEM. For D and F * = p < 0.05 and ** = p < 0.01 indicate significantly decreased compared to PBS treated mice. For G , * = p < 0.05 indicates significantly increased compared to HCT 116 injected mice. For I – Q 5–7 mice per group were used for the experiments and the values are indicated as the mean ± SEM. * = p < 0.05 and ** = p < 0.01 indicate significantly decreased compared to vehicle alone. # = p < 0.05 and ## = p < 0.01 indicate significantly increased compared to HCT 116 plus CAF

Article Snippet: CM was treated to the differentiated myotubes for 72 h. CXCL5 neutralizing antibody MAB-254 was purchased from R&D system (MN, USA) and CXCR2 antagonist SSB225005 was obtained from MedChemExpress (NJ, USA).

Techniques: Imaging, Staining, Neutralization, Immunostaining, Injection

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Heat map of genes showing differential expression between the tibialis anterior (TA) muscles of NOD-SCID mice treated as follows: (1) No xenograft (Normal); (2) Xenograft with HCT 116 human cancer cells plus CAF (HCT 116 + CAF); (3) Xenograft with HCT 116 human cancer cells plus CAF, followed by treatment with a CXCL5 neutralizing antibody (Neu Ab). B KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis for the HCT-116 + CAF compared with Neu Ab treatment groups. C Gene ontology (GO) functional analysis for the HCT-116 + CAF group compared with Neu Ab treatment group. D Western blot analysis of PI3K-AKT and ERK1/2 phosphorylation in the dissected TA muscle from vehicle alone, HCT 116 + CAF, and Neu Ab-treated groups. E Densitometry of PI3K-AKT and ERK1/2 phosphorylation relative to PI3K-AKT and ERK1/2. F Heat map for genes showing differential expression between (1) HCT 116 + CAF xenograft compared to vehicle alone (HCT-116 + CAF/Normal), and (2) HCT 116 + CAF xenograft treated with a CXCL5 neutralizing antibody compared to HCT 116 + CAF xenograft alone (Neu Ab/HCT 116 + CAF). G qPCR analysis of the differentially expressed genes, in addition to atrogin-1 and MuRF-1 in the TA muscles. H Western blot analysis of PI3K-AKT phosphorylation in CAF CCM treated myotubes. I Densitometry of PI3K-AKT phosphorylation relative to PI3K-AKT. J Protein synthesis rate as determined by western blot analysis of puromycin incorporation (SUnSET assay). K Densitometry of puromycin incorporation normalized by GAPDH expression. For E and G : 4–5 mice per group were used for the experiments and the values are indicated as the mean ± SEM. * = p < 0.05, ** = p < 0.01 and *** = p < 0.001 indicate significantly increased or decreased compared to vehicle alone-injected mice (Normal). # = p < 0.05, ## = p < 0.01 and ### = p < 0.001 indicate significantly decreased compared to HCT 116 + CAF. For I and K : All values are indicated as the mean ± SD. * = p < 0.05 and *** = p < 0.001 indicate significantly decreased compared to vehicle alone. # = p < 0.05 indicates significantly increased compared to CAF CCM treatment

Article Snippet: CM was treated to the differentiated myotubes for 72 h. CXCL5 neutralizing antibody MAB-254 was purchased from R&D system (MN, USA) and CXCR2 antagonist SSB225005 was obtained from MedChemExpress (NJ, USA).

Techniques: Quantitative Proteomics, Muscles, Functional Assay, Western Blot, Phospho-proteomics, Expressing, Injection

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Schematic of the protocol to investigate CXCL5 neutralization in a model of cytokine-induced skeletal muscle wasting. C57BL6/J mice were treated with 40 ng/kg CXCL5 and 80 ng/kg IL-6 for 4 weeks with or without 120 μg/kg CXCL5 neutralizing antibody. 120 μg/kg IgG1 was used as control. B Body weight at the end point of experiment. C Quadriceps muscle mass. D Gastrocnemius muscle mass. E Tibialis anterior (TA) muscle mass. F Laminin staining of the TA muscle (scale bar = 150 µm). G TA myofiber cross sectional area. H CXCR2 staining of the TA muscle (scale bar = 150 µm). I The proportion of CXCR2 positive fibers in TA muscle. J Western blot analysis of atrogin-1, MuRF-1 and CXCR2 expression in the TA muscle. K Densitometry of atrogin-1, MuRF-1 and CXCR2 expression normalized by the expression of GAPDH. For B – I, 7 mice per group were used for the experiments and for J – K, 4 mice per group were used for the experiments and the analysis was carried out two times. The values were indicated as the mean ± SEM. * = p < 0.05 and ** = p < 0.01 indicate significantly increased or decreased compared to IgG1 control. # = p < 0.05 and ## = p < 0.01 indicate significantly decreased compared to CXCL5 + IL-6 + IgG1

Article Snippet: CM was treated to the differentiated myotubes for 72 h. CXCL5 neutralizing antibody MAB-254 was purchased from R&D system (MN, USA) and CXCR2 antagonist SSB225005 was obtained from MedChemExpress (NJ, USA).

Techniques: Neutralization, Control, Staining, Western Blot, Expressing

Journal: Journal of Biomedical Science

Article Title: CXCL5 neutralization mitigates cancer cachexia by disrupting CAF-cancer cell crosstalk

doi: 10.1186/s12929-025-01192-0

Figure Lengend Snippet: A Representative MYH2-stained images of human donor myotubes cultured as follows: (1) Differentiation media (DM) for 72 h; (2) Treatment with human HCT 116 colon cancer cell-stimulated CAF CM (CAF CCM) for 72 h; (3) Treatment with CAF CCM and CXCL5 neutralizing antibody (CAF CCM + Neu Ab) for 72 h (scale bar = 100 μm). B Calculation of mean myotube diameter. C Western blot analysis of MYH2 and atrogin-1 expression. D Densitometry of MYH2 and atrogin-1 expression relative to α–tubulin. E Immunohistochemical analysis of CXCL5 and vimentin expression in tumor-stromal and normal tissues obtained from a colon carcinoma patient. White arrows indicate overlapping CXCL5 and vimentin immunostaining. Quantification of CXCL5 fluorescence in the tumor-stromal tissues is also shown (** = p < 0.01 compared to normal). All experiments were conducted 3 times independently and the values were indicated as the mean ± SD. For B and D : * = p < 0.05 indicate significantly increased or decreased compared to DM alone. # = p < 0.05 and ## = p < 0.01 indicate significantly increased or decreased compared to CAF CCM. G Working model of the role of CAF in cancer cachexia progression. Molecular crosstalk between cancer cells and CAF in the tumor microenvironment induces the secretion of chemokine CXCL5 by CAF. CXCL5 activates muscle atrophy signaling, as shown by decreased PI3K-AKT and ERK phosphorylation and upregulation of the key atrogenes, atrogin-1 and MuRF-1, causing the skeletal muscle loss observed in cancer cachexia

Article Snippet: CM was treated to the differentiated myotubes for 72 h. CXCL5 neutralizing antibody MAB-254 was purchased from R&D system (MN, USA) and CXCR2 antagonist SSB225005 was obtained from MedChemExpress (NJ, USA).

Techniques: Staining, Cell Culture, Western Blot, Expressing, Immunohistochemical staining, Immunostaining, Fluorescence, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Absence of Langerhans cells resulted in over-influx of neutrophils and increased bacterial burden in skin wounds

doi: 10.1038/s41419-024-07143-1

Figure Lengend Snippet: A The expression of a range of neutrophil chemokines at mRNA level in normal skin, normal wounds and LC-deficient wounds on hour 48 post wounding. B Cxcl5 transcripts in normal wounds and LC-deleted wounds on hour 24 post wounding. C Immunohistostaining of CXCL5 expression in the wounds at hour 48 post wounding. D At hour 48 after wounding, LC-depleted mice (DT) were treated with or without brefeldin A (BFA). Four hours later, the wound tissues were harvested and were subject to CXCL5 and keratin immunohistostaining. Representative control wound tissue (Saline) without BFA treatment were also provided. Scale bar: 50 μm. E – G Langerin DTR mice were pre-treated with saline or DT. Right after wounding, they were both separated to two groups receiving either an anti-CXCL5 antibody or an isotype antibody. E Immunohistostaining of Ly6G in the wounds were performed at hour 48 after wounding. F Kinetic statistics of wound area changes over time and curve fitting rate are shown. G. Frequencies of healed and unhealed wounds on day 9 after wounding. n = 6. H – J . Langerin DTR mice were pre-treated with saline or DT. Right after wounding, they were both separated to two groups receiving either reparxin or vehicle. H Immunostaining of Ly6G in the wounds were performed at hour 48 after wounding. I Kinetic statistics of wound area changes over time and curve fitting rate are shown. J Frequencies of healed and unhealed wounds on day 9 after wounding. n = 6. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns not significant by ordinary one-way ANOVA test ( A , E , F , H , I ) and two-tailed unpaired t test ( B , C ). Data are depicted as mean ± SEM. Data are derived from at least 2 independent experiments.

Article Snippet: A CXCL5 neutralizing antibody (MAB433, RD) was i.p. injected at the dose of 50 μg/mouse right after wounding, and the control group was given a same dose of isotype antibody (E-AB-F098430, Elabscience).

Techniques: Expressing, Control, Saline, Immunostaining, Two Tailed Test, Derivative Assay

Journal: Cell Death & Disease

Article Title: Absence of Langerhans cells resulted in over-influx of neutrophils and increased bacterial burden in skin wounds

doi: 10.1038/s41419-024-07143-1

Figure Lengend Snippet: A The number of bacteria in the wounds of LC-deficient mice (DT) and LC-intact mice (saline) at the indicated time after wounding. CFU: clone forming unit. B Mass spectrum results of wound bacteria indicate that the majority of the bacteria in the LC-deficient wounds were Staphylococcus xylosus and Staphylococcus sciuri . C Primary keratinocytes were stimulated with the indicated PAMPs or DAMPs molecules and their production of Cxcl5 was measured. D Morphology analyses of LCs in Langerin-GFP reporter mice on hour 6 after wounding. Each dot indicates the quantification from one LC cell, 8 cells were selected randomly per mouse ( n = 4) for statistical analysis. Scale bar: 20 μm. E Quantification of LC branch ramification complexity (illustration of Sholl analysis). F Two-photon microscopy analysis of LC dendrite changes in a 1-h observation period. The skin samples were derived from naïve Langerin EGFP mice or the mice at hour 6 post wounding. Each dot represents the average frequency of all dendrites from one LC cell. 8 cells were selected randomly per mouse ( n = 4) for statistical analysis. G – I SYTO 64-labeled bacteria were applied on top of the fresh wounds made on the backskins of Langerin EGFP mice. G The interaction between bacteria (red) and LCs (green) was monitored in the indicated time points after wounding. Dotted line, border of wounds. Arrows, bacteria-phagocytosed LCs. Scale bar: 50 μm. H Time lapse of an LC and its associated bacteria at wound periphery 5 h post wounding. Arrows: free bacteria. I A representative two-photon image of wound epidermal sections show the bacteria (arrows, red) phagocytosed by LCs (green). Z-projections of 30 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns. not significant by ordinary one-way ANOVA test ( A ) and two-tailed unpaired t test ( C , D and F ). Data are depicted as mean ± SEM. Data are derived from at least 2 independent experiments.

Article Snippet: A CXCL5 neutralizing antibody (MAB433, RD) was i.p. injected at the dose of 50 μg/mouse right after wounding, and the control group was given a same dose of isotype antibody (E-AB-F098430, Elabscience).

Techniques: Bacteria, Saline, Microscopy, Derivative Assay, Labeling, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Absence of Langerhans cells resulted in over-influx of neutrophils and increased bacterial burden in skin wounds

doi: 10.1038/s41419-024-07143-1

Figure Lengend Snippet: Langerin DTR mice were pre-treated with saline or DT. Right after wounding, they received daily topical application of benzalkonium chloride or saline. For A – C , Scale bar: 50 μm. A CXCL5 expression in the epidermis on day 2 after wounding. Scale bar: 50 μm. B Neutrophil (Ly6G) infiltration in the wounds on day 2 after wounding. Scale bar: 50 μm. C Ki67 + proliferating cells in the epidermis on day 2 after wounding. The white dotted area represents the epidermis at the edge of the wound. Epi: epidermis; Der: dermis. D Migration of epidermal progenitor cells (K14 + ) was measured in the indicated time points after wounding. The dotted lines indicate the initial wound edge, and arrows mark wound tongues. w.b., wound bed. Scale bar: 1 mm. E Representative wound images, kinetic statistics of wound area changes over time and curve fitting rate are shown. * P < 0.05, ** P < 0.01, ns not significant by ordinary one-way ANOVA test ( A , B , C and E ) or two-way ANOVA with multiple comparisons ( D ). Data are depicted as mean ± SEM. Data are derived from at least 2 independent experiments.

Article Snippet: A CXCL5 neutralizing antibody (MAB433, RD) was i.p. injected at the dose of 50 μg/mouse right after wounding, and the control group was given a same dose of isotype antibody (E-AB-F098430, Elabscience).

Techniques: Saline, Expressing, Migration, Derivative Assay

Journal: Cell Death & Disease

Article Title: Absence of Langerhans cells resulted in over-influx of neutrophils and increased bacterial burden in skin wounds

doi: 10.1038/s41419-024-07143-1

Figure Lengend Snippet: Primers’ details.

Article Snippet: A CXCL5 neutralizing antibody (MAB433, RD) was i.p. injected at the dose of 50 μg/mouse right after wounding, and the control group was given a same dose of isotype antibody (E-AB-F098430, Elabscience).

Techniques: