Journal: NPJ Precision Oncology

Article Title: Multi-omics profiling reveals tumor microenvironment characteristics linked to immunotherapy response and prognosis in non-small cell lung cancer

doi: 10.1038/s41698-026-01474-2

Figure Lengend Snippet: A Identification of four celltypes in the LLC TME by scRNA-seq. B Anti-SPP1 therapy increases all immune subsets, decreases non-immune cells. C Subclustering of T/NK cells. D GO enrichment analysis of genes highly expressed in CD8 + T cells at different groups (control vs. anti-SPP1). E Anti-SPP1 therapy decreases M2-like macrophage proportion. F Immunofluorescence indicated increased CD8 + T infiltration with concurrent M2 reduction post-treatment.

Article Snippet: Five days post-inoculation, mice with palpable tumors were randomly assigned to four treatment groups ( n = 4 per group) using a random number table : Control (PBS) , Anti-PD-1 (clone RMP1-14, Bio X Cell, #BE0146) , Anti-SPP1 (anti-mouse osteopontin antibody, clone 103D6, MCE), and Anti-SPP1 combined with Anti-PD-1.

Techniques: Control, Immunofluorescence

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: After antibody stripping, the second round was performed using rabbit anti-human SPP1 monoclonal antibody (1:5000, Servicebio, Cat# GB11500) with HRP-conjugated goat anti-rabbit secondary antibody, followed by iF647-Tyramide (1:500, Servicebio, Cat# G1232).

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: After antibody stripping, the second round was performed using rabbit anti-human SPP1 monoclonal antibody (1:5000, Servicebio, Cat# GB11500) with HRP-conjugated goat anti-rabbit secondary antibody, followed by iF647-Tyramide (1:500, Servicebio, Cat# G1232).

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: After antibody stripping, the second round was performed using rabbit anti-human SPP1 monoclonal antibody (1:5000, Servicebio, Cat# GB11500) with HRP-conjugated goat anti-rabbit secondary antibody, followed by iF647-Tyramide (1:500, Servicebio, Cat# G1232).

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: After antibody stripping, the second round was performed using rabbit anti-human SPP1 monoclonal antibody (1:5000, Servicebio, Cat# GB11500) with HRP-conjugated goat anti-rabbit secondary antibody, followed by iF647-Tyramide (1:500, Servicebio, Cat# G1232).

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: After antibody stripping, the second round was performed using rabbit anti-human SPP1 monoclonal antibody (1:5000, Servicebio, Cat# GB11500) with HRP-conjugated goat anti-rabbit secondary antibody, followed by iF647-Tyramide (1:500, Servicebio, Cat# G1232).

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: After antibody stripping, the second round was performed using rabbit anti-human SPP1 monoclonal antibody (1:5000, Servicebio, Cat# GB11500) with HRP-conjugated goat anti-rabbit secondary antibody, followed by iF647-Tyramide (1:500, Servicebio, Cat# G1232).

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: After antibody stripping, the second round was performed using rabbit anti-human SPP1 monoclonal antibody (1:5000, Servicebio, Cat# GB11500) with HRP-conjugated goat anti-rabbit secondary antibody, followed by iF647-Tyramide (1:500, Servicebio, Cat# G1232).

Techniques: Activity Assay, Control