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: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

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: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

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: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

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: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

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: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

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: To inhibit CXCL5, 120 μg/kg of neutralizing antibody (R&D Systems, MAB-254, clone 33160, MN, USA) and IgG1 monoclonal antibody (R&D Systems, MAB-002, clone 11711, MN, USA) were treated for 3 d prior to transplantation via intraperitoneal (IP) injection.

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