Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A Double staining of CD8 and αSMA in mouse various subcutaneous tumors. Scale bar, 200 μm. The quantitative evaluation of sections from each tumor stained for B αSMA ( n = 12 fields from four mice per group) and C CD8 ( n = 12 fields from four mice per group). D The correlation between the number of CD8 + T cells and αSMA + area in each mouse tumor tissue. E Double staining of collagen 1 and CD45 to detect fibrocytes in mouse various subcutaneous tumors. Scale bar, 100 μm. F The quantitative evaluation of sections from each tumor stained for fibrocytes ( n = 12 fields from four mice per group). G The correlation between the number of fibrocytes and αSMA + area in each mouse tumor tissue. The correlation was estimated by Spearman’s correlation and a linear regression analysis (the best-fit line is indicated). Representative images of tumor sections resected from non-small cell lung carcinoma (NSCLC) patients stained to detect αSMA + cancer-associated fibroblasts ( H , αSMA + FAP + ) and fibrocytes ( I , CD45 + FSP-1 + ). Scale bar, 100 μm. J Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in NSCLC tumor tissue stained in Fig. 1H and I ( n = 50 patients). K Representative images of resected NSCLC tissue stained to detect CD8 + T cells and EpCAM + tumor cells. Scale bar, 100 μm. L Quantitative evaluation of tumor-infiltrating fibrocytes (CD45 + FSP-1 + ) in each NSCLC group divided by the immune phenotypes of tumor-infiltrating CD8 + T cells (inflamed, desert, and exclusion). * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: The FAP and CD45 cell surface expression in tumor-infiltrating fibrocytes isolated from AB1-HA tumor tissue were detected by PE-Cy7 conjugated anti-mouse CD45 antibody (1:100 dilution, 30-F11, eBioscience) and PE conjugated anti-mouse FAP antibody (1:100 dilution, Novus Biologicals), respectively.

Techniques: Double Staining, Staining

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A A clustermap of relative transcription factor-activities in tumor-infiltrating fibrocytes and macrophages was estimated by wPGSA from a single cell RNA-seq (scRNA-seq) dataset of mouse tumor-infiltrating CD45 + immune cells in subcutaneous AB1-HA tissue. B UMAP plots of clock genes in scRNA-seq data generated from CD45 + cells in AB1-HA tumor tissue. C The gene expression levels of clock genes in tumor-infiltrating fibrocytes (CD34 + CD45 + cells) and macrophages (CD34 - CD45 + F4/80 + cells) isolated from AB1-HA tumor tissue ( n = 3 per group). * P < 0.05 by Student’s t- test. Data are shown as the mean ± s.e.m. D Monocle-generated plots presenting pseudotime ordering and differentiation trajectory of tumor-infiltrating fibrocyte cluster from AB1-HA tumor tissue. E Monocle-generated plots showing the pseudotime-ordered expression of selected marker genes and clock genes. Lines show the relative expression of each marker in pseudotime.

Article Snippet: The FAP and CD45 cell surface expression in tumor-infiltrating fibrocytes isolated from AB1-HA tumor tissue were detected by PE-Cy7 conjugated anti-mouse CD45 antibody (1:100 dilution, 30-F11, eBioscience) and PE conjugated anti-mouse FAP antibody (1:100 dilution, Novus Biologicals), respectively.

Techniques: RNA Sequencing, Generated, Gene Expression, Isolation, Expressing, Marker

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A A diagram showing the strategy used to isolate fibrocytes from AB1-HA tumor-bearing mice. B The isolation of fibrocytes (CD34 + CD45 + F4/8 mid cells) and macrophages (CD34 - CD45 + F4/80 high cells) from AB1-HA tumor tissue C The gene expression levels of COL1A1 and ACTA2 in AB1-HA tumor-infiltrating fibrocytes treated with KL001 and/or TGF-β ( n = 3 per group). * P < 0.05 by Student’s t- test. D A diagram showing the strategy used to isolate fibrocytes from LLC tumor-bearing mice. E The gene expression level of ACTA2 in LLC tumor-infiltrating fibrocytes treated with KL001 ( n = 3 per group). * P < 0.05 by Student’s t- test. F A diagram showing the strategy used to isolate fibrocytes and fibroblasts from LLC tumor-bearing COL1A2-GFP reporter mice. G The isolation of fibrocytes (CD45 + GFP + cells), fibroblasts (CD45 - GFP + cells), and other immune cells (CD45 + GFP - cells) from LLC tumor - bearing COL1A2-GFP reporter mice. H The gene expression levels of COL1A1, ACTA2, and CD45 in each population isolated in Fig. ( n = 3 per group). * P < 0.05 by Student’s t- test. I The gene expression levels of ACTA2 in LLC tumor-infiltrating fibrocytes and fibroblasts treated with KL001 and/or TGF-β ( n = 3 per group). J A diagram showing the strategy used to isolate fibrocytes from mouse lung. K The gene expression levels of COL1A1 and ACTA2 in lung fibrocytes treated with KL001 and/or TGF-β ( n = 3 per group). * P < 0.05 by Student’s t- test. * P < 0.05 by Student’s t- test. All data are shown as the mean ± s.e.m.

Article Snippet: The FAP and CD45 cell surface expression in tumor-infiltrating fibrocytes isolated from AB1-HA tumor tissue were detected by PE-Cy7 conjugated anti-mouse CD45 antibody (1:100 dilution, 30-F11, eBioscience) and PE conjugated anti-mouse FAP antibody (1:100 dilution, Novus Biologicals), respectively.

Techniques: Isolation, Gene Expression

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume of AB1-HA-bearing mice treated with KL001 from 5 days after tumor cell injection ( n = 6 per group). * P < 0.05 by the Mann-Whitney U test. B The evaluation of the tumor volume of LLC-bearing mice treated with KL001 from 5 days after tumor cell injection ( n = 6 per group). * P < 0.05 by the Mann-Whitney U test. C Representative images and the D quantitative evaluation of sections from AB1-HA tumors stained for αSMA ( n = 15 fields from five mice per group). Scale bar, 200 μm. E Representative images and F the quantitative evaluation of sections from AB1-HA tumors stained for collagen 1a1 ( n = 15 fields from five mice per group). Scale bar, 200 μm. G Representative images and H the quantitative evaluation of sections from AB1-HA tumors stained for collagen 1a1 and CD45 to detect fibrocytes ( n = 16 fields from four mice per group). Scale bar, 100 μm. The tumors were harvested at day 16 from each group studied in Fig. 4A. * P < 0.05 by the Mann-Whitney U test. I The evaluation of the tumor volume of AB1-HA-bearing mice treated with CLK8 from 5 days after tumor cell injection ( n = 5 per group). * P < 0.05 by the Mann-Whitney U test. The quantitative evaluation of sections from AB1-HA tumors stained for αSMA ( J , n = 15 fields from five mice per group), collagen 1a1 ( K , n = 15 from five mice fields per group), and fibrocytes ( L , n = 15 fields from five mice per group). The tumors were harvested at day 16 from each group studied in Fig. 4G. * P < 0.05 by the Mann-Whitney U test. All data are shown as the mean ± s.e.m.

Article Snippet: The FAP and CD45 cell surface expression in tumor-infiltrating fibrocytes isolated from AB1-HA tumor tissue were detected by PE-Cy7 conjugated anti-mouse CD45 antibody (1:100 dilution, 30-F11, eBioscience) and PE conjugated anti-mouse FAP antibody (1:100 dilution, Novus Biologicals), respectively.

Techniques: Injection, MANN-WHITNEY, Staining

Journal: NPJ Precision Oncology

Article Title: Clock pathway inhibitor overcomes tumor immune-exclusion via regulation of fibrocyte differentiation

doi: 10.1038/s41698-025-01066-6

Figure Lengend Snippet: A The evaluation of the tumor volume and B the representative image of tumor tissue of LLC-bearing mice treated with KL001 and/or αPD-L1 Ab (n = 7 per group). * P < 0.05 by a one-way ANOVA. C Representative images and D the quantitative evaluation of sections from LLC tumors stained for αSMA ( n = 15 fields from five mice per group). The tumors were harvested at day 21 from each group studied in Fig. 6A. Scale bar, 200 μm. Representative images and the quantitative evaluation of sections from LLC tumors stained for collagen 1 + CD45 + fibrocytes ( E, F ), CD8 + T cells ( G, H ) and CD4 + T cells ( I, J ). Scale bar, 100 μm. * P < 0.05 by a one-way ANOVA. K The evaluation of the tumor volume of AB1-HA-bearing mice treated with KL001 and/or αCTLA-4 Ab ( n = 6 per group). * P < 0.05 by a one-way ANOVA. The quantitative evaluation of sections from AB1-HA tumors ( n = 15 fields from five mice per group) stained for CD8 ( L ), CD4 ( M ), and Foxp3 ( N ). The tumors were harvested at day 18 from each group studied in Fig. 6K. * P < 0.05 by a one-way ANOVA. All data are shown as the mean ± s.e.m.

Article Snippet: The FAP and CD45 cell surface expression in tumor-infiltrating fibrocytes isolated from AB1-HA tumor tissue were detected by PE-Cy7 conjugated anti-mouse CD45 antibody (1:100 dilution, 30-F11, eBioscience) and PE conjugated anti-mouse FAP antibody (1:100 dilution, Novus Biologicals), respectively.

Techniques: Staining

Journal: International Journal of Oncology

Article Title: ADAM17-regulated CX3CL1 expression produced by bone marrow endothelial cells promotes spinal metastasis from hepatocellular carcinoma

doi: 10.3892/ijo.2020.5045

Figure Lengend Snippet: Interaction between BMECs and HCC cells enhances tumor growth in the spine. (A) si-CX3CL1 was transfected into BMECs to construct CX3CL1 low BMECs. Transwell assays were used to examine recruitment of BMECs co-cultured with or without HMCC97H cells. The number of the recruited cells was quantified. n=3. Cell recruitment were analyzed using ANOVA test. (B) Cell vitality of BMECs was assessed using CCK-8 assay; n=3. Data were analyzed using ANOVA followed by Tukey's test. (C) Tumor size was measured based on the volume. n=5. Scale bar, 5 mm. Data were analyzed using an unpaired t-test. (D) Samples from 2 groups were examined using immunofluorescence. Magnification, ×20. The result was analyzed using an unpaired t-test. Gray bars represent the CX3CL1 nor BMECs group and black bars represent the CX3CL1 low BMECs group (E) Flow cytometry revealed the percentage and number of F4/80 + CD11b + cells; n=5. (F) Flow cytometry was used for detecting expression of M2 macrophage surface markers, CX3CR1, MRC1 and CD163; n=5. Cells were selected based on higher expression of CD45 for macrophages. Subsequently, CD11b and F4/80 expression were used for further characterization of macrophages. FACS (fluorescent-activated cell sorting) data were analyzed using a Kruskal Wallis test followed by Dunn's non-parametric post hoc test. HCC, hepatocellular carcinoma; BMECs, bone marrow endothelial cells; si, small interfering; CCK-8, Cell Counting kit-8; CX3CL1, C-X3-C motif chemokine ligand 1.

Article Snippet: To stain for the cell surface markers, cells were incubated with following fluorescence-labeled monoclonal antibodies for 30 min on ice respectively: PE-conjugated anti-CD11b (cat. no. 333142; BD Biosciences; 1:300), FITC-conjugated anti-F4/80 (cat. no. orb223773; Biorbyt; 1:300), PE-Cy7-conjugated-CD45 (cat. no. 147703; BioLegend; 1:300), APC-conjugated CX3CR1 (cat. no. 341609; BioLegend; 1:300), PE-Cy3-conjugated MRC1 (cat. no. sc-376232; Santa Cruz Biotechnology, Inc.; 1:300) and PE-Cy5-CD163 (cat. no. sc-33715; Santa Cruz Biotechnology, Inc.; 1:300) antibodies.

Techniques: Transfection, Construct, Cell Culture, CCK-8 Assay, Immunofluorescence, Flow Cytometry, Expressing, FACS, Cell Counting