Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Spinal injection of TNF-α-activated astrocytes induces mechanical allodynia via Cx43-mediated CXCL1 release. (A) Intrathecal injection of TNF-α-activated astrocytes elicited persistent mechanical allodynia for >48 h. Note this allodynia is reduced by pretreatment of astrocytes with Cx43 small interfering RNA (1 µg/ml, 18 h). *P < 0.05, compared with TNF-α or TNF-α + non-targeting control small interfering RNA treated group; n = 6 mice/group. (B) ELISA analysis shows increased CXCL1 release in the CSF at 3 h after the intrathecal injection of TNF-α-activated astrocytes. *P < 0.05, compared with vehcile group; #P < 0.05, compared with non-activated astrocytes; n = 4 mice/group. (C) Intrathecal injection of a CXCL1 neutralizing antibody (4 µg) transiently and partially reversed mechanical allodynia, induced by TNF-α-treated astrocytes. *P < 0.05, compared with control IgG group; n = 6 mice/group. (D) Intrathecal injection of the CXCR2 antagonist SB225002 (20 µg = 57 nmol) transiently and partially reversed mechanical allodynia, induced by TNF-α-activated astrocytes. *P < 0.05, compared with vehicle (PBS); n = 5–6 mice/group. All data are mean ± SEM. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Injection, Small Interfering RNA, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: CXCL1, upregulated in spinal cord astrocytes after nerve injury, enhances excitatory synaptic transmission in spinal cord neurons and maintains neuropathic pain via CXCR2. (A) Western blotting shows CXCL1 upregulation in the spinal cord dorsal horn 21 days after CCI. Right, quantification of Cx43 levels in the dorsal horn. The western blot results are presented as a fold of sham control. *P < 0.05, compared to sham control, Student’s t-test, n = 4 mice/group. (B) Intrathecal injection of SB 225002 (20 µg), 21 days after CCI, reduced CCI-induced mechanical allodynia in the late phase. *P < 0.05, compared with vehicle (saline), Student’s t-test, n = 6 mice/group. (C) Double immunostaining of CXCL1 and GFAP in the dorsal horn 21 days after CCI. Note CXCL1 is primarily colocalized with GFAP. Arrows indicate doubled-labelled cells. Scale bar = 50 µm. (D and E) CXCL1 superfusion (100 ng/ml) increases spontaneous EPSC frequency (revealed by patch clamp recordings) in lamina IIo neurons of spinal cord slices. (E) Spontaneous EPSC frequency. *P < 0.05, Student’s t-test, n = 5 neurons/group. (F and G) CCI (21 d) increases spontaneous EPSC frequency, which is reversed by the CXCR2 antagonist SB225002 (1 µM). *P < 0.05, Student’s t-test, n = 5 neurons/group.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Transmission Assay, Western Blot, Control, Injection, Saline, Double Immunostaining, Patch Clamp

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Schematic of working hypothesis for astrocytic Cx43-mediated late-phase neuropathic pain. CCI induces a persistent upregulation of Cx43 in spinal cord astrocytes. Cx43 expression and activity is also upregulated by TNF-α, secreted from microglia. Upregulation of Cx43 hemichannel activities results in CXCL1 release. Astrocytic CXCL1 secretion activates CXCR2 on neurons (central terminals of primary sensory neurons and spinal cord neurons), leading to enhanced excitatory synaptic transmission in nociceptive neurons (e.g. lamina IIo excitatory interneurons) and sustained neuropathic pain in the late-phase. Additionally, CXCL1 can also be secreted from intact or injured primary afferents in the spinal cord especially in the early phase of CCI.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Expressing, Activity Assay, Transmission Assay

Journal: International Journal of Cancer

Article Title: Cancer‐associated fibroblasts promote PD‐L1 expression in mice cancer cells via secreting CXCL5

doi: 10.1002/ijc.32278

Figure Lengend Snippet: CAFs‐derived CXCL5 promotes PD‐L1 expression in cancer cells. ( a ) The expression of PD‐L1 in B16, CT26, A375 and HCT116 was determined by flow cytometry after treated with different concentrations of recombined CXCL5. ( b ) The mRNA expression of PD‐L1 in B16, CT26, A375 and HCT116 was detected by RT‐PCR. * p < 0.05. ( c ) Western blot showed the protein levels of CXCR2 in B16 and CT26 after transfected with siCXCR2. ( d ) Flow cytometry showed the expression of PD‐L1 in B16 and CT26 cocultured with CAFs after transfected with siCXCR2. ( e ) The protein levels of CXCR2 in A375 and HCT116 after transfected with siCXCR2 were analyzed by western blotting. ( f ) The expression of PD‐L1 in A375 and HCT116 treated with CXCL5 after transfected with siCXCR2 was determined by flow cytometry. ( g ) CXCL5 and α‐SMA expression in 3T3 cells were detected by western after transfected with CXCL5‐overexpression plasmid. ( h ) PD‐L1 protein expression in B16 and CT26 cocultured with/without 3T3‐CXCL5 cells was analyzed by western blot and the normalized protein expression was analyzed by Image J. * p < 0.05.

Article Snippet: The following antibodies were used for IHC, flow cytometry and western blotting: rabbit monoclonal anti‐PD‐L1 (1:200, ab205921, Abcam, Cambridge, MA), mouse monoclonal anti‐α‐smooth muscle actin (α‐SMA; 1:200, BM0002, BOSTER Biological Technology, Pleasanton, CA), rabbit polyclonal anti‐CXCR2 (1:200, BA0732‐2, BOSTER).

Techniques: Derivative Assay, Expressing, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Over Expression, Plasmid Preparation

Journal: International Journal of Cancer

Article Title: Cancer‐associated fibroblasts promote PD‐L1 expression in mice cancer cells via secreting CXCL5

doi: 10.1002/ijc.32278

Figure Lengend Snippet: The correlation of CXCR2, PD‐L1 and p‐AKT in tumor tissues in vivo . ( a ) The expression level of CXCR2 and PD‐L1 in human melanoma and CRC tissues were detected by IHC and evaluated. ( b ) The correlation of CXCR2 and PD‐L1 in xenograft tumor tissues was analyzed by IHC and evaluated. ( c ) The correlation of p‐AKT and PD‐L1 in xenograft tumor tissues was detected by IHC and quantized. ( d ) Representative immunostaining images of PD‐L1, CXCR2 and p‐AKT in B16 and CT26 xenograft tumor tissues. Scale bar, 200 μm.

Article Snippet: The following antibodies were used for IHC, flow cytometry and western blotting: rabbit monoclonal anti‐PD‐L1 (1:200, ab205921, Abcam, Cambridge, MA), mouse monoclonal anti‐α‐smooth muscle actin (α‐SMA; 1:200, BM0002, BOSTER Biological Technology, Pleasanton, CA), rabbit polyclonal anti‐CXCR2 (1:200, BA0732‐2, BOSTER).

Techniques: In Vivo, Expressing, Immunostaining

Journal: Advanced Science

Article Title: A Novel Self‐Regulated, Non‐Directional Magnetic Thermos‐Brachytherapy 125 I Seed Enhances Anticancer Efficacy by Rescuing Immune Escape

doi: 10.1002/advs.202508091

Figure Lengend Snippet: 125 I brachytherapy‐induced Cd274 + neutrophil recruitment promotes immune escape. A) Bar plot of the GSEA results showing functional differences in malignant epithelial cells between the radiotherapy/brachytherapy (R) and control (C) groups, with the bar length representing the normalized enrichment score. B) Dot plot showing differential expression levels and proportions of neutrophil chemokine‐related genes between the R and C groups. C) Violin plot of the differential expression of Cxcl2 between the R and C groups (Kruskal‐Wallis test). D) Violin plot showing the differential expression of Cxcr2 across general cell types. E) Stacked bar chart showing the proportions of neutrophil subtypes across different treatment groups. F) Dot plot illustrating the expression levels and proportions of functional markers in neutrophil subtypes. G) Flow cytometry analysis of subcutaneous tumors comparing the proportion of Cd274+ neutrophils between the C and R groups. Data are presented as mean ± s.d. (unpaired two‐sided t‐test, n = 5 per group). H) Representative images of multiplex immunofluorescence (mIF) staining showing colocalization of Cxcl2‐Cxcr2 interactions between malignant epithelial cells and neutrophils in subcutaneous tumors in the R and C groups (5 mice per group). I) Representative images of mIF staining comparing differential infiltration levels of CD274 + neutrophils in subcutaneous tumors between the R and C groups (5 mice per group). J) GSEA showing functional differences in malignant epithelial cells between the 125 I@MH (RM) and R groups. * : P < 0.05, ** : P < 0.01, *** : P < 0.001, **** : P < 0.0001.

Article Snippet: The primary antibodies included Epcam ( Cat : GB11274, Company : CiteAB, 1:2,000), Cxcl2 (16325‐1‐AP, Proteintech, 1:200), Csf3r (orb1676847, Biorbyt, 1:2,000), Cxcr2 (CSB‐ PA009567 , CUSABio, 1:200), Cd274/Pd‐l1 (28076‐1‐AP, Proteintech, 1:200), Cd3 (17‐0031‐82, Thermo Fisher, 1:200), Cd8 (11‐0081‐82, Thermo Fisher, 1:200), Ifn‐γ (12‐7311‐82, Thermo Fisher, 1:200), Pd‐1 (66220‐1‐lg, Proteintech, 1:200), F4/80 (123116, 1:100, BioLegend), Ccr2 (MAB55382, R&D Systems, 1:200), Apoe (66830‐1‐lg, Proteintech, 1:200).

Techniques: Functional Assay, Control, Quantitative Proteomics, Expressing, Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: A Novel Self‐Regulated, Non‐Directional Magnetic Thermos‐Brachytherapy 125 I Seed Enhances Anticancer Efficacy by Rescuing Immune Escape

doi: 10.1002/advs.202508091

Figure Lengend Snippet: MH rescues immune escape induced by 125 I brachytherapy. A) Stacked bar chart showing the proportions of lymphocyte subtypes across different treatment groups. B) Flow cytometry analysis of subcutaneous tumors comparing the proportions of exhausted T cells (upper) and effector T cells (lower) among the C, R, and RM groups. Data are presented as mean ± s.d. (unpaired two‐sided t ‐test, n = 5 per group). C) Representative image of multiplex immunofluorescence (mIF) staining comparing differential infiltration levels of exhausted T cells (upper) and effector T cells (lower) in subcutaneous tumors between the RM and R groups (5 mice per group). D) Comparison of subcutaneous tumor sizes in mice following no treatment, 125 I treatment, 125 I + anti‐PD‐1 treatment, and 125 I + anti‐PD‐1 + anti‐Cxcr2 treatment (6 mice per group). E) Stacked bar chart showing the proportions of phagocyte subtypes across different treatment groups. F) Bar plot of the GSEA results showing functional differences between Apoe + macrophages and Ccr2 + macrophages, with the bar length representing the normalized enrichment score. G) Flow cytometry analysis of subcutaneous tumors comparing the proportions of Ccr2 + macrophages (upper) and Apoe + macrophages (lower) among the C, R, and RM groups. Data are presented as mean ± s.d. (unpaired two‐sided t ‐test, n = 5 per group). H) Representative image of mIF staining comparing the differential infiltration levels of Ccr2 + macrophages (upper) and Apoe + macrophages (lower) in subcutaneous tumors between the RM and R groups. Data are presented as mean ± s.d. (unpaired two‐sided t ‐test, n = 5 per group). * : P < 0.05, ** : P < 0.01, *** : P < 0.001, **** : P < 0.0001.

Article Snippet: The primary antibodies included Epcam ( Cat : GB11274, Company : CiteAB, 1:2,000), Cxcl2 (16325‐1‐AP, Proteintech, 1:200), Csf3r (orb1676847, Biorbyt, 1:2,000), Cxcr2 (CSB‐ PA009567 , CUSABio, 1:200), Cd274/Pd‐l1 (28076‐1‐AP, Proteintech, 1:200), Cd3 (17‐0031‐82, Thermo Fisher, 1:200), Cd8 (11‐0081‐82, Thermo Fisher, 1:200), Ifn‐γ (12‐7311‐82, Thermo Fisher, 1:200), Pd‐1 (66220‐1‐lg, Proteintech, 1:200), F4/80 (123116, 1:100, BioLegend), Ccr2 (MAB55382, R&D Systems, 1:200), Apoe (66830‐1‐lg, Proteintech, 1:200).

Techniques: Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining, Comparison, Functional Assay