Journal: bioRxiv

Article Title: Translational toolkit for reproducible, cross-study profiling of human ageing hallmarks in human blood and tissue

doi: 10.64898/2026.04.20.719545

Figure Lengend Snippet: A. Schematic overview of a novel 32-color spectral flow cytometry panel for profiling circulating immune populations and intracellular senescence markers p16 and p21. B. PCA analysis of young and aged peripheral blood mononuclear cells (PBMCs), integrating all identified immune cell subsets. C. IMMAge score calculated based on PBMC subsets for young and aged participants in young (n=8) and aged (n=12) participants. D. CD4+ CD57+CD28- and E. CD8+ CD57+CD28-senescent cells expressed as a percentage of total CD4+ or CD8 T cells in young (n=8) and aged (n=12) participants. Intracellular p21 expression in F. Naïve and G. EMRA CD8+ T cells from (n=9) young and aged (n=12) participants. H. Intracellular p16 expression assessed as median fluorescent intensity in Memory B cells (CD19+ CD38− CD24+) from (n=9) young and aged (n=12) participants. Expression of CD95 in I. CD4+ and J. CD8+ T cells between in young (n=8) and aged (n=12) participants. K. Schematic overview of cryosection and whole-tissue approaches for detecting cellular senescence using β-galactosidase staining (SA-beta gal) in human adipose tissue. Representative images of positive β-galactosidase staining (blue) of senescent cells in L. 20 µM human adipose tissue cryosections and M. whole human adipose tissue following 24h incubation. Expression of SASP markers N. GDF15, O. CXCL-9, P. TNF alpha, and Q. IL-6 in plasma samples from young (n=5) and aged (n=12) participants, determined by ELISA. Data are mean ± S.E.M. Statistical significance was assessed (C-F, H-J) unpaired t-tests or (G, N-Q) Mann Whitney U.

Article Snippet: Serum and EDTA plasma concentration of CXCL-9, GDF-15, IL-6 and TNFα, were measured using commercially available ELISA kits (CXCL9; DY392-05, GDF15; DY957, IL-6; DY206, TNF; DY210, R&D Systems, MN, USA) following the manufacturer’s protocol.

Techniques: Flow Cytometry, Expressing, Staining, Incubation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: The CXCL9/SPP1 polarity axis in tumor-associated macrophages: immunoregulatory and prognostic significance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1763652

Figure Lengend Snippet: Identification of key cells and annotation of macrophage subsets. (A, B) t-SNE plots showed that CXCL9 and SPP1 were distributed mainly in macrophages among the differential cells. (C) The localization of CXCL9 and SPP1 genes in different cell types was determined, and macrophages accounted for a high proportion. (D) The expression of CXCL9 and SPP1 in differential cells was analyzed, and both genes were differentially expressed in macrophages. (E, F) The identification of 30 PCs was performed. (G) Macrophages were clustered into 15 subclusters after dimensionality reduction. (H) A bubble plot was used to show the expression of CXCL9 and SPP1 in different cell types; both genes were highly expressed in subcluster 3. (I) Macrophage subsets were annotated into 4 subpopulations. (J) A bubble plot was generated to display the high expression of CXCL9 and SPP1 in the annotated cells.

Article Snippet: In the first round, sections were incubated with rabbit anti-human CXCL9 monoclonal antibody (1:500, Servicebio, Cat# 22355-1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (ready-to-use, Servicebio, Cat# G1302), and signal was developed using iF488-Tyramide (1:500, Servicebio, Cat# G1231).

Techniques: Expressing, Generated

Journal: Frontiers in Immunology

Article Title: The CXCL9/SPP1 polarity axis in tumor-associated macrophages: immunoregulatory and prognostic significance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1763652

Figure Lengend Snippet: RCTD cell type deconvolution. (A) Visualization of spatial distribution of cell types in spatial transcriptomics between disease and healthy groups; (B) Cell type composition of spatial transcriptomics between disease and healthy groups; (C) Comparative analysis of M1/M2 module scores in four macrophage subtypes (Significance levels were indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns for not statistically significant); Spatial distribution characteristics of macrophage subtypes in adjacent normal tissue (P24_B2, (D) ] and tumor tissue [P24_T2, (E) ] of non-small cell lung cancer (NSCLC) patient P24; (F) UMAP distribution of macrophage subtypes in NSCLC tumor and paratumor tissues; (G) Average proportion of the 8 nearest neighbor cells around the two macrophage subtypes; (H) Differences in neighborhood composition between the two macrophage subtypes; (I) Spatial lag Moran scatter plot analysis of CXCL9 gene expression; (J) Spatial lag Moran scatter plot analysis of SPP1 gene expression; (K) CXCL9/SPP1 spatial hotspot distribution map; (L) Spatial lag distribution map of SPP1; (M) Spatial lag distribution map of CXCL9.

Article Snippet: In the first round, sections were incubated with rabbit anti-human CXCL9 monoclonal antibody (1:500, Servicebio, Cat# 22355-1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (ready-to-use, Servicebio, Cat# G1302), and signal was developed using iF488-Tyramide (1:500, Servicebio, Cat# G1231).

Techniques: Spatial Transcriptomics, Gene Expression

Journal: Frontiers in Immunology

Article Title: The CXCL9/SPP1 polarity axis in tumor-associated macrophages: immunoregulatory and prognostic significance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1763652

Figure Lengend Snippet: Validation of CXCL9 + and SPP1 + macrophage expression and polarization characteristics in lung cancer tissues. (A) Representative mIF staining for CXCL9+ TAMs and SPP1+ TAMs in lung tissue sections from the normal and LUAD tumor groups. DAPI (blue), CD68 (red), CXCL9 (yellow), SPP1 (green) are shown, along with individual and merged channels. (n = 3 per group). Scale bar, 20 μm. (B) Representative mIF staining of CXCL9+ TAMs (yellow) and SPP1+ TAMs (green) in lung tissue sections from the normal and LUAD tumor groups (n = 3 per group). Scale bar, 20 μm. (C) qRT-PCR validation of macrophage polarization. The M1 group showed high expression of Cxcl9 and iNOS, while the M2 group showed high expression of Spp1 and Arg1. Asterisks indicate statistically significant differences (*p < 0.05).

Article Snippet: In the first round, sections were incubated with rabbit anti-human CXCL9 monoclonal antibody (1:500, Servicebio, Cat# 22355-1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (ready-to-use, Servicebio, Cat# G1302), and signal was developed using iF488-Tyramide (1:500, Servicebio, Cat# G1231).

Techniques: Biomarker Discovery, Expressing, Staining, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: The CXCL9/SPP1 polarity axis in tumor-associated macrophages: immunoregulatory and prognostic significance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1763652

Figure Lengend Snippet: Acquisition of DEGs. (A) The distribution of 485 DEGs1 between CXCL9 + SPP1 − and CXCL9 − SPP1 + macrophages was analyzed. (B, C) A total of 10,589 DEGs2 in the TCGA training cohort were identified. (D) Differential expression of CXCL9 between the high and low expression groups was detected. (E) Significant differences were observed in the K-M survival curves between the CXCL9 high and low expression groups. (F, G) A total of 1,838 DEGs between the CXCL9 high and low expression groups were identified. (H) Differential expression of SPP1 between the high and low expression groups was detected. (I) Significant differences were found in the KM survival curves between the SPP1 high and low expression groups. (J, K) A total of 2,100 DEGs between the SPP1 high and low expression groups were identified. (L) A Venn diagram was constructed to show the overlap of 91 intersecting genes.

Article Snippet: In the first round, sections were incubated with rabbit anti-human CXCL9 monoclonal antibody (1:500, Servicebio, Cat# 22355-1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (ready-to-use, Servicebio, Cat# G1302), and signal was developed using iF488-Tyramide (1:500, Servicebio, Cat# G1231).

Techniques: Quantitative Proteomics, Expressing, Construct

Journal: Frontiers in Immunology

Article Title: The CXCL9/SPP1 polarity axis in tumor-associated macrophages: immunoregulatory and prognostic significance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1763652

Figure Lengend Snippet: Gene set enrichment analysis (GSEA) of CXCL9 and SPP1 high/low expression groups. (A) GSEA was performed between the CXCL9 high- and low-expression groups, and 41 KEGG pathways were significantly enriched. (B) GSEA was conducted between the SPP1 high- and low-expression groups, and 14 KEGG pathways were significantly enriched. (C, D) Single−gene GSEA was performed for CXCL9 and SPP1, and 54 and 51 KEGG pathways were significantly enriched, respectively.

Article Snippet: In the first round, sections were incubated with rabbit anti-human CXCL9 monoclonal antibody (1:500, Servicebio, Cat# 22355-1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (ready-to-use, Servicebio, Cat# G1302), and signal was developed using iF488-Tyramide (1:500, Servicebio, Cat# G1231).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: The CXCL9/SPP1 polarity axis in tumor-associated macrophages: immunoregulatory and prognostic significance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1763652

Figure Lengend Snippet: Pseudotime analysis and cell-cell communication of macrophages. (A) Pseudotime analysis of macrophages. (B) Analysis of pseudotime differentiation trajectories of macrophage subsets with different CXCL9-SPP1 phenotypes. (C) Spatial distribution characteristics of core functional gene expression in macrophage pseudotime trajectories. (D) Heatmap of expression profiles of key macrophage marker genes across different cell clusters. (E) Network diagram of the number of connections between different cell types in the control group. (F) Network diagram of the number of connections between different cell types in the tumor group. (G) Network diagram of the number of connections between macrophages and other cell types in the control group. (H) Network diagram of the number of connections between macrophages and other cell types in the tumor group. (I) Network diagram of connection weights between different cell types in the control group. (J) Network diagram of connection weights between different cell types in the tumor group. (K) Network diagram of connection weights between macrophages and other cell types in the control group. (L) Network diagram of connection weights between macrophages and other cell types in the tumor group.

Article Snippet: In the first round, sections were incubated with rabbit anti-human CXCL9 monoclonal antibody (1:500, Servicebio, Cat# 22355-1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (ready-to-use, Servicebio, Cat# G1302), and signal was developed using iF488-Tyramide (1:500, Servicebio, Cat# G1231).

Techniques: Functional Assay, Gene Expression, Expressing, Marker, Control

Journal: Frontiers in Immunology

Article Title: The CXCL9/SPP1 polarity axis in tumor-associated macrophages: immunoregulatory and prognostic significance in non-small cell lung cancer

doi: 10.3389/fimmu.2026.1763652

Figure Lengend Snippet: Ligand-receptor interactions and metabolic activity in cell-cell communication. (A) Bubble plot of ligand-receptor interactions in cell-cell communication across different cell types in the control group. (B) Bubble plot of ligand-receptor interactions in cell-cell communication across different cell types in the tumor group. (C) Normalized activity of the top five transcription factors (TFs) in macrophage subsets. (D) Bubble plot of metabolic activity in macrophage subsets. (E, F) Correlation between prognostic genes and CXCL9/SPP1.

Article Snippet: In the first round, sections were incubated with rabbit anti-human CXCL9 monoclonal antibody (1:500, Servicebio, Cat# 22355-1-AP), followed by HRP-conjugated goat anti-rabbit secondary antibody (ready-to-use, Servicebio, Cat# G1302), and signal was developed using iF488-Tyramide (1:500, Servicebio, Cat# G1231).

Techniques: Activity Assay, Control