Journal: Frontiers in Oncology

Article Title: Radiolabeled Monoclonal Antibody Against Colony-Stimulating Factor 1 Receptor Specifically Distributes to the Spleen and Liver in Immunocompetent Mice

doi: 10.3389/fonc.2021.786191

Figure Lengend Snippet: In vitro characteristics of CSF1R mAb, DFO- N -suc-conjugated, and 89 Zr-labeled mAb. (A) Representative binding curve of CSF1R mAb and IgG2a binding to mouse CSF1R recombinant protein. (B) Representative binding curve of DFO- N -suc-CSF1R-mAb and CSF1R mAb binding to mouse CSF1R recombinant protein. (C) Representative SE HPLC of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb 280-nm signal (black) with the radiochemical signal overlay (green). mAb, monoclonal antibody; AU, absorbance unit; CSF1, colony-stimulating factor 1; CSF1R, colony-stimulating factor 1 receptor; SE HPLC, size-exclusion high-performance liquid chromatography.

Article Snippet: A Nunc 96-well polystyrene conical bottom microwell plate (Thermo Fisher Scientific) was coated overnight at 4°C with 1 µg/ml of recombinant mouse CSF1R protein (Sino Biological) in a 100 µl of 0.05 M Na 2 CO 3 solution, pH 9.6.

Techniques: In Vitro, Labeling, Binding Assay, Recombinant, High Performance Liquid Chromatography

Journal: Frontiers in Oncology

Article Title: Radiolabeled Monoclonal Antibody Against Colony-Stimulating Factor 1 Receptor Specifically Distributes to the Spleen and Liver in Immunocompetent Mice

doi: 10.3389/fonc.2021.786191

Figure Lengend Snippet: Biodistribution of 0.4 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb in non-tumor-bearing FVB/N mice. (A) Representative maximal intensity projection PET images of non-tumor-bearing FVB/N mice 24 and 72 h after intravenous administration of 0.4 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb. (B) PET quantification of spleen, liver, and blood pool at 24 ( n = 8) and 72 ( n = 4) h after [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb administration. Data are represented as mean SUV mean ± SD. (C) Ex vivo biodistribution at 24 ( n = 4) and 72 ( n = 4) h after administration of 0.4 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb intravenously. (D) Ex vivo autoradiography of spleen, mesenteric lymph node, and ileum tissue (upper panel) and matching H&E staining on the same tissue slide. Spleen, mesenteric lymph node, and ileum were exposed to different phosphor plates. Ileum magnification depicting autolysis. L, liver; S, spleen; BAT, brown adipose tissue; MLN, mesenteric lymph nodes; ALN, axillary lymph nodes; %ID/g, percentage injected dose per gram of tissue.

Article Snippet: A Nunc 96-well polystyrene conical bottom microwell plate (Thermo Fisher Scientific) was coated overnight at 4°C with 1 µg/ml of recombinant mouse CSF1R protein (Sino Biological) in a 100 µl of 0.05 M Na 2 CO 3 solution, pH 9.6.

Techniques: Ex Vivo, Autoradiography, Staining, Injection

Journal: Frontiers in Oncology

Article Title: Radiolabeled Monoclonal Antibody Against Colony-Stimulating Factor 1 Receptor Specifically Distributes to the Spleen and Liver in Immunocompetent Mice

doi: 10.3389/fonc.2021.786191

Figure Lengend Snippet: Biodistribution of 4 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb in non-tumor-bearing FVB/N mice. (A) Representative maximal intensity projection PET images of non-tumor-bearing FVB/N mice 24 and 72 h after administration of 4 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb intravenously. (B) PET quantification of spleen, liver, and blood pool at 24 ( n = 7) and 72 ( n = 4) h after [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb administration. Data are presented as mean + SD. (C) Ex vivo biodistribution at 24 ( n = 4) and 72 ( n = 4) h after administration of 4 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb intravenously. Data are expressed as mean + SD. B, blood pool; L, liver; S, spleen; SUV mean , mean standardized uptake value; BAT, brown adipose tissue; MLN, mesenteric lymph nodes; ALN, axillary lymph nodes; %ID/g, percentage injected dose per gram of tissue.

Article Snippet: A Nunc 96-well polystyrene conical bottom microwell plate (Thermo Fisher Scientific) was coated overnight at 4°C with 1 µg/ml of recombinant mouse CSF1R protein (Sino Biological) in a 100 µl of 0.05 M Na 2 CO 3 solution, pH 9.6.

Techniques: Ex Vivo, Injection

Journal: Frontiers in Oncology

Article Title: Radiolabeled Monoclonal Antibody Against Colony-Stimulating Factor 1 Receptor Specifically Distributes to the Spleen and Liver in Immunocompetent Mice

doi: 10.3389/fonc.2021.786191

Figure Lengend Snippet: Biodistribution of 10 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb in non-tumor-bearing FVB/N mice. (A) Representative maximal intensity projection PET images of non-tumor-bearing FVB/N mice 24 and 72 h after intravenous administration of 10 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb. B, blood pool; L, liver; S, spleen. (B) PET quantification of spleen, liver, and blood pool at 24 ( n = 6) and 72 ( n = 3) h after [ 89 Zr]Zr-DFO-N-suc-CSF1R-mAb administration. Data are presented as mean SUV mean ± SD. (C) Ex vivo biodistribution at 24 ( n = 3) and 72 ( n = 4) h after administration of 10 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb. Data are expressed as mean + SD. B, blood pool; L, liver; S, spleen; SUV mean , mean standardized uptake value; BAT, brown adipose tissue; MLN, mesenteric lymph nodes; ALN, axillary lymph nodes; %ID/g, percentage injected dose per gram of tissue.

Article Snippet: A Nunc 96-well polystyrene conical bottom microwell plate (Thermo Fisher Scientific) was coated overnight at 4°C with 1 µg/ml of recombinant mouse CSF1R protein (Sino Biological) in a 100 µl of 0.05 M Na 2 CO 3 solution, pH 9.6.

Techniques: Ex Vivo, Injection

Journal: Frontiers in Oncology

Article Title: Radiolabeled Monoclonal Antibody Against Colony-Stimulating Factor 1 Receptor Specifically Distributes to the Spleen and Liver in Immunocompetent Mice

doi: 10.3389/fonc.2021.786191

Figure Lengend Snippet: Biodistribution of 10 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb and [ 89 Zr]Zr-DFO- N -suc-IgG2a antibody in KEP tumor-bearing FVB/N mice. (A) Representative maximal intensity projection PET images of KEP tumor-bearing FVB/N mice 24 and 72 h after intravenous administration of 10 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb or [ 89 Zr]Zr-DFO- N -suc-IgG2a antibody. PET quantification of (B) tumor, (C) blood pool, (D) liver, and (E) spleen at 24 ( n = 3) and 72 ( n = 3) h after [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb or [ 89 Zr]Zr-DFO- N -suc-IgG2a antibody administration. Data are presented as mean + SD. (F) Ex vivo biodistribution at 72 h after administration of 10 mg/kg of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb ( n = 3) or [ 89 Zr]Zr-DFO- N -suc-IgG2a ( n = 3) antibody. Data are expressed as mean + SD. (G) Representative immunohistochemistry of F4/80 in KEP tumors of FVB/N mice at 72 h after administration of [ 89 Zr]Zr-DFO- N -suc-CSF1R-mAb or [ 89 Zr]Zr-DFO- N -suc-IgG2a intravenously. (H) Quantification of tumoral F4/80 immunohistochemistry. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 (unpaired t-test). B, blood pool; L, liver; S, spleen; T, tumor; SUV mean , mean standardized uptake value; BAT, brown adipose tissue; MLN, mesenteric lymph nodes; ALN, axillary lymph nodes; %ID/g, percentage injected dose per gram of tissue. n.s., not significant.

Article Snippet: A Nunc 96-well polystyrene conical bottom microwell plate (Thermo Fisher Scientific) was coated overnight at 4°C with 1 µg/ml of recombinant mouse CSF1R protein (Sino Biological) in a 100 µl of 0.05 M Na 2 CO 3 solution, pH 9.6.

Techniques: Ex Vivo, Immunohistochemistry, Injection

Journal: European Journal of Medical Research

Article Title: NDC80 promotes epithelial to mesenchymal transition of esophageal squamous cell carcinoma through macrophages polarization and PI3K/AKT pathway activation

doi: 10.1186/s40001-025-03397-3

Figure Lengend Snippet: NDC80 modulated tumor-associated macrophages polarization to promote ESCC progression. ( A ) Immunohistochemistry was used to detect the expression of NDC80 protein and macrophage markers in ESCC tumor tissue, n = 61. B, C Correlation between the expression of NDC80 protein and the number of macrophages infiltrations in ESCC tumor tissue was analyzed by simple linear regression, n = 61. Flow cytometry ( D ) and qPCR ( E ) were used to detect the regulation of macrophages polarization by ESCC cells overexpressing NDC80, n = 3, multiple unpaired t tests. F qPCR was used to detect the regulation of macrophages polarization by ESCC cells with silenced NDC80, n = 3, multiple unpaired t tests. G, H qPCR was used to assay the mRNA expression levels of inflammatory cytokines in tumor cells with different NDC80 expression levels, n = 3, multiple unpaired t tests. I, J ELISA detection of M-CSF and CXCL-2 levels in the supernatant of ESCC cells with overexpression or knockdown of NDC80 protein, n = 3, multiple unpaired t tests. M-CSF: Standard curve range = 31.25—2000 pg/mL, R 2 = 0.9997. CXCL-2: Standard curve range = 15.63—1000 pg/mL, R 2 = 0.9986. (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: The concentrations of cytokines M-CSF (E-EL-H0097) and CXCL-2 (E-EL-H1904) in the cell supernatants were detected using ELISA kits (Elabscience, China).

Techniques: Immunohistochemistry, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown

Journal: Advanced Science

Article Title: FAPα + Macrophages Orchestrate Immune Evasion in Multiple Myeloma by Dual Regulation of PD‐L1 and T Cell Senescence

doi: 10.1002/advs.202506239

Figure Lengend Snippet: FAPα + Macrophages Are Enriched in the Myeloma Microenvironment and Correlate with Disease Progression. (A) Heatmap of gene expression in macrophages from patients with MM (n = 3) or healthy donors (HDs, n = 3). (B,C) Cell percentages of FAPα + macrophages (CD11b + CD14 + ) and FAPα + BMSC (CD45 − CD38 − CD29 + ) in PBMCs or BMMCs from patients with MM (n = 9). (D) Reprehensive immunofluorescence staining (IF) images of CD138, CD68, and FAPα in the BM of a NDMM patient (Magnification ×400. Scale bar, 50 µm). (E–G) Flow cytometry analysis of the cell percentages of FAPα + macrophages and FAPα + BMSCs in BMMCs from patients with MM at different disease stages (n = 27) or HDs (n = 4). (H) Correlation analysis of the cell percentages between CD138 + MM cells and FAPα + macrophages or FAPα + BMSCs in patients with NDMM (n = 15). (I) Reprehensive immunohistochemical staining (IHC) images of CD138 and FAPα in patients with NDMM or MM‐CR (Magnification ×200. Scale bar, 50 µm). (J,K) Western blot (J) and flow cytometry (K) analysis of FAPα expression in macrophages co‐cultured with MM cells (n = 3). (L) TGFβ1 expression in different MM cell lines. (M‐N) TGFβ1‐induced FAPα protein expression in macrophages (Magnification ×400. Scale bar, 50 µm). (O) M‐CSF, TGFβ1, and FAPα levels in BM supernatants from patients with MM at different disease stages (n = 37) using ELISA assay. (P) Correlation analysis of FAPα and TGFβ1 or M‐CSF in BM supernatant from patients with NDMM (n = 17). (Q) Reprehensive IHC images of bone marrow samples from patients with MM (n = 18) (Magnification ×200. Scale bar, 50 µm). (R) IOD values of CD138, CD68, and FAPα in patients with NDMM or RRMM (n = 18). (S) Correlation analysis between CD138 and FAPα in patients with NDMM. (T) Kaplan–Meier curves of PFS and overall OS in the set of patients with NDMM based on FAP protein expression level detected in tumor tissues. The median value of FAP RNA expression in the was 88.26 (IOD). The expression value of the FAP high group (n = 9) was >88.26(IOD) and the FAP low group (n = 9) was <88.26(IOD). Data are presented as mean ± SD. Each dot means independent samples. ns, no significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Statistical analysis was performed using a 2‐tailed Student's t ‐test in C, E, F, G, K, O, and R, a Pearson correlation in H, P, and S, a log‐rank test in T. MM, multiple myeloma; HD, healthy donors; BMSCs, bone marrow mesenchymal stem cells; PBMCs, peripheral blood mononuclear cells; BMMCs, bone marrow mononuclear cells; BM, bone marrow; NDMM, newly diagnosed MM; RRMM, relapsed or refractory MM; CR, complete response; TGFβ1, Transforming growth factor beta 1; M‐CSF, macrophage colony stimulating factor; IHC, Immunohistochemistry; IOD, Integrated Optical Density; RRMM, Relapsed/Refractory MM; PFS, Progression‐free survival; OS, overall survival.

Article Snippet: The bone marrow supernatants of patients with different MM stages were collected, and different cytokines were detected as described by the respective manufacturers: Human M‐CSF AccuSignal ELISA Kit (Cat #KOA0253, Rockland, USA), Human Seprase/FAP AccuSignal ELISA Kit (Cat #KOA0627, Rockland, USA), Human TGFβ1 ELISA Kit (Cat #1117102) (All from Dakewe Bioengineering Co., Ltd, China).

Techniques: Biomarker Discovery, Gene Expression, Immunofluorescence, Staining, Flow Cytometry, Immunohistochemical staining, Western Blot, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, RNA Expression, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

doi: 10.1038/s41419-018-0956-4

Figure Lengend Snippet: a CSF1 expression with Hoechst 33342 and their merges of testicular sections from mice under four different treatments in the short term (W2, 2 weeks) and long term (W8, 8 weeks) experiments. Scale bar, 200 μm. b ELISA of CSF1 in mice serum of the four different groups in W2 and W8 experiments. c RT-qPCR detected Csf1 expression in mice testicular tissue under the four different treatments in W2 and W8 experiments. d CSF1 expression level detected by western blot analysis in the four groups in W2 and W8. Histograms are the statistical result of Image J (V1.48d) gradation analysis for the western blot experiments. (The results are shown as the mean ± S.E.M of at least three mice ( n = 3) and the statistical significance was expressed as follows: * p < 0.05; ** p < 0.01). C control group, M normal group treated with MLT, D diabetic group, DM MLT-treated diabetic group

Article Snippet: Mouse CSF1 level of mouse plasma was quantified using an ELISA kit (Boster, Wuhan, China) according to the instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

doi: 10.1038/s41419-018-0956-4

Figure Lengend Snippet: a CSF1 expression with Hoechst 33342 and their merges of MLTC-1 under three different ERS states (C control, Tm Tm treatment induced excessive ERS, Tm + 4PBA Tm treatment in combination with 4PBA for alleviated ERS). b CSF1 concentration in culture medium of MLTC-1 under the three different treatments was detected using ELISA. c RT-qPCR detected the mRNA expression level of Csf1 in MLTC-1 under the three treatments. d Expression of CSF1 in MLTC-1 under different treatments was measured by Western blot. Histograms are the statistical result of Image J (V1.48d) gradation analysis for the western blot experiments. e A graphical conclusion of this study is displayed: DM-induced high glucose activated ERS response factors (Grp78 and CHOP) in Leydig cells, resulting in apoptosis of Leydig cells and growth arrest of SSCs in testes. In response to these changes, melatonin treatment alleviated the apoptosis of Leydig cells via inhibition of ERS and recovered the CSF1 secretion in testes. CSF1 then resumed the self-renewal capacity of SSCs. (The results are expressed as the mean ± S.E.M of three separated wells of cells ( n = 3) in at least three different experiments and the statistical significance is expressed as follows: * p < 0.05; ** p < 0.01)

Article Snippet: Mouse CSF1 level of mouse plasma was quantified using an ELISA kit (Boster, Wuhan, China) according to the instructions.

Techniques: Expressing, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Inhibition

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet: Clinical relevance of the expression levels of IL-10 and CSF-1R in patients with cancer (A and B) The correlation of RNA level between IL-10/IL-10-RA axis and CSF-1R of head and neck squamous carcinoma (HNSCC; N = 566), breast cancer (BRCA; N = 1,218), and colon adenocarcinoma (COAD; N = 329) in The Cancer Genome Atlas (TCGA) database. The correlation was determined by Pearson correlation (r). (C) Kaplan-Meier survival analysis of HNSCC-, BRCA-, or COAD- patients with different expression profiles of IL - 10 , IL - 10RA , and CSF1R . High, upper quartile; low, lower quartile. p values were estimated by log-rank test. (D and E) The correlation of RNA level between IL-10/IL-10-RA axis and HPV positivity in TCGA-HNSCC cases. The correlation was determined by Pearson correlation (r). (F–I) 3D scatterplots for analyzing the expression of CD8 + T score and tumor-associated macrophage (TAM) score related to IL-10/IL-10RA axis and CSF1R in TCGA-HNSCC. CD8 + T or TAM scores were expressed from blue to red gradient color dots (low to high). The value expressed by RNA level (log2(norm_count+1)). (J) 3D scatterplots for the expression of different cytokine genes related to IL - 10 and CSF1R in TCGA-HNSCC. (K) Schematic outlines represent the approach of digital spatial profiling (DSP) analysis of human HNSCC (14 slides with 171 regions of interest [ROIs] analyzed from 6 patients). ROIs were categorized as peri-normal tissue (normal), primary tumor (inner tumor and outer tumor), and metastatic lymph node (LN). Scale bar, 100 or 200 μm as indicated. (L) DSP analysis of CSF1R , IL - 10 , and IFNG in ROIs of HNSCC samples. The ROIs (500 μm in diameter) were analyzed using a DSP CTA (Cancer Transcriptome Atlas) panel with 1825 RNA-binding oligonucleotide probes. (M) Correlation between expression levels of IL - 10 and IFNG from DSP data. The correlation was determined by Pearson correlation (r). (N) DSP analysis of the TAM score and CD8 + T score related to tumor spatial location in ROIs of HNSCC samples. (O) Left, a scatterplot of IL - 10 and CSF1R gene expression levels in TCGA HNSCC patients. Red dots indicate the patients with the top 50% of IL - 10 (IL-10 high ) and the bottom 50% of CSF1R (CSF1R low ). Right, survival analysis between groups of IL-10 high CSF1R low (red line) and others (blue line). p value was estimated by a log-rank test.

Article Snippet: Recombinant human CSF1R-His (Biolegend), human CSF1R-Fc, mouse CSF1R-Fc (Sino Biological), or mouse/human IL-10Ra fusion protein (R&D Systems) were immobilized on 96-well microtiter plates overnight at 4°C.

Techniques: Expressing, RNA Binding Assay

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet: BF10 repopulates infiltrated immune cells in tumor tissues and LNs (A) Experimental scheme. The HNSC/Q1-2 tumor cells (5 × 10 5 cells) were orthotopically inoculated into mice, followed by administrations of the indicated components (Ctrl-IgG, IL-10-Fc, αCSF1R, and BF10) with a 3 day interval for 3 doses. Tumor-infiltrated CD45 + immune cells were assessed by flow cytometry. (B–D) Population of CD8 + T cells (B), TAMs (C), and CD4 + T cells (D) in tumors from the indicated groups. (E) Representative plots (left) and population (right) of the granzyme B (GZMB)-producing tumor-infiltrating T cells from the indicated tumors. (F) Representative population of the TCF1 + Tim3 − T cells (Prog Tex) and the TCF1 − Tim3 + T cells (Term Tex) among total tumor-infiltrating CD44 + PD-1 + CD8 + T cells from the indicated mice. (G–H) Representative images of CD8 + T cell numbers from the indicated groups. (G) Multiplexed immunofluorescence (mIF) staining performed with Opal 7-Color IHC kit (PerkinElmer) for CD4 (green), CD8 (sky blue), GZMB (red), and nuclei (hyacinth). Representative composite images obtained and quantified by the Vectra Polaris Imaging System and Inform software. Scale bar, 100 μm. (H) Quantitative result of CD8 + T cells. (I–K) Representative image of Ki67 expression in tumor-draining LNs (tdLNs). (I) The isolated tdLN examined by immunohistochemistry (IHC). The brown color indicates Ki67 + cells. Scale bar, 300 μm. (J) Representative mIF images of tdLN. The isolated tdLNs were stained with DAPI (blue), CD8 (green), PD-1 (yellow), FoxP3, CD19 (pink), and Ki67 (red). Whole-tissue composite images were captured and analyzed with the Vectra Polaris Imaging System and Inform software. Scale bar, 400 μm. (K) Quantification of the cell number of CD8 + PD1 + , CD8 + PD1 + Ki67 + , and CD19 + cells in the BF10-treated versus control group. The data were presented as mean ± SD, and the statistics were calculated using unpaired Student’s t test. (two group comparison) and one-way ANOVA (more than 3 groups) with an appropriate test. ∗∗p < 0.01.

Article Snippet: Recombinant human CSF1R-His (Biolegend), human CSF1R-Fc, mouse CSF1R-Fc (Sino Biological), or mouse/human IL-10Ra fusion protein (R&D Systems) were immobilized on 96-well microtiter plates overnight at 4°C.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Imaging, Software, Expressing, Isolation, Immunohistochemistry, Control, Comparison

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet: Transcriptomic and TCR repertoire analysis of the samples from mice treated with BF10 versus its subcomponents (A) An illustration of mice receiving the indicated treatment followed by tumor isolation for bulk RNA-seq and gene set enrichment analysis (GSEA). (B–F) Enrichment plots of genes sets in BF10 versus control in mouse tumor samples. Significant pathways were identified by GSEA gene set of IFN-γ response (B), IFN-α response (C), inflammatory response (D), complement (E), and genes upregulated in CD8 T cells (GEO: GSE41867 ) (F). (G) The heatmap of GSEA enriched pathways in treatment groups. The normalized enrichment score (NES) and p value are shown. (H) Schema of bioinformatic strategy to study the effect of BF10 within tumor microenvironment. Tumors derived from two syngeneic tumor models (HNSC/Q1-2 and BRCA/4T1) treated with BF10, IL-10-Fc, αCSF-1R, or control were collected for bulk RNA-seq to investigate the differentially expressed genes (DEGs; >1.5-fold and p < 0.05 or <0.5-fold and p < 0.05). DEGs were analyzed using 2 modules: (1) Bioinformatics Database for Annotation, Visualization, and Integrated Discovery (DAVID; https://david.ncifcrf.gov/ ) and (2) Ingenuity Pathway Analysis (IPA). (I) DAVID functional Gene Ontology analysis. (J) IPA. (K) Immune repertoire TCR sequencing of CD8 + T cells. Tumor-bearing mice received treatments of Ctrl-IgG, IL-10, anti-CSF1R, or BF10 for 3 doses, followed by RNA extraction of isolated CD8 + T cells for TCR immune repertoire analysis. The data are shown as clonotype diversity and distribution of both TRAC and TRBC CDR3 sequencing from tumor and spleen. The number of the bracket indicates observed diversity in the enrichment metrics of QIAseq-RNA Immune Repertoire Application. (L) Examination of the TCR immune repertoire using Morisita-Horn index.

Article Snippet: Recombinant human CSF1R-His (Biolegend), human CSF1R-Fc, mouse CSF1R-Fc (Sino Biological), or mouse/human IL-10Ra fusion protein (R&D Systems) were immobilized on 96-well microtiter plates overnight at 4°C.

Techniques: Isolation, RNA Sequencing Assay, Control, Derivative Assay, Functional Assay, Sequencing, RNA Extraction

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet: Generation and characterization of the bifunctional anti-CSF-1R-IL-10 fusion proteins (A) Schematic representation of the bifunctional anti-CSF-1R-IL-10 fusion protein (BF10). BF10 was designed by genetically fusing human IL-10 to the C terminus of the CSF-1R fragment (mouse IgG2a) separated by a 14 amino acid linker. (B) SDS-PAGE of BF10 and the anti-CSF-1R antibody. N, non-reducing; R, reducing. (C) Dose-dependent binding affinity of anti-CSF-1R Ab, BF10, and IL-10-Fc to recombinant mouse CSF-1R protein by ELISA. Calculated curve of one representative data of three independent experiments. Each point (OD value) was expressed as the average of two repeats and was presented as mean ± SD. (D) The competition activity of anti-CSF-1R, BF10, and IL-10-Fc. Serial dilutions of the test proteins were preloaded into the well before mouse CSF-1 to CSF-1R binding reaction. The values were measured by absorbance and expressed as an OD value (450–650 nm). Calculated curve of one representative data of three independent experiments was shown. (E) STAT3 reporter assay. IL-10RA-overexpressed HeLa cells were seeded into a 96-well plate with complete DMEM medium (10% FBS) at 37°C for 4 h. Serial dilutions of IL-10-His tag, IL-10-Fc, anti-CSF1R, and BF10 were added into well for 18 h. Luciferase activity was measured using ONE-Glo Luciferase Assay (Promega). The calculated curve of one representative data of three independent experiments was presented as mean ± SD. (F) Effects of BF10 on T cell proliferation. CFSE-labeled CD8 + T cells were activated with anti-CD3 and anti-CD28 in absence or presence of IL-4-primed BMDMs. 50 ng/mL anti-CSF-1R, IL-10, or BF10 was added as indicated. Data are cumulative results of two independent experiments (n = 6 per group). The results were shown as mean ± SD, and the statistics were calculated using one-way ANOVA with Tukey test. (G) Oxygen consumption rate (OCR) of CD8 + T cells with BF10 treatment. Isolated splenic CD8 + T cells from tumor-bearing mice were pretreated with BF10 (0.05 mg/mL) for 30 min and then sequential addition of the Seahorse assay reagents of oligomycin (1 μM), FCCP (0.5 μM), and Rot/antimycin A (1 μM) to examine the effect of BF10 on T cell metabolism. Representative curve is one of three independent experiments and n = 3 repeats per each assay. p value was calculated using Student's t test. (H) The serum concentration of BF10. Mice received the equivalent dose treatments of BF10 (20 mg/kg), IL-10-Fc (10 mg/kg), or IL-10-tag (5 mg/kg) using intravenous injection. 10 μL blood was diluted and collected as the indicated times (0.25, 1, 2, 4, 8, 24, 48, and 72 h) and was subjected to ELISA to detect the IL-10 concentration. (I) The biodistribution of 111 In-BF10. Tumor-bearing mice with or without clodronate-liposome before BF10 injection. After 24 h, tissue samples were excised and weighted. The radioactivity of the sample was measured, and the value was expressed as a percentage of the injected dose per gram of sample (%ID/g). (J) The SPECT images of HNSCC/Q1-2 tumor-bearing mice receiving radiolabeled 111 In-BF10 injection. Mice were imaged at 24 h after injection. T, tumor; S, spleen; L, liver. (K) The blood clearance kinetic of 111 In-BF10. 111 In-BF10 were intravenously (i.v.) injected, and blood samples were collected at the indicated time points. Data were expressed as the percentage injected dose per milliliter (%ID/mL). The half-life (T 1/2 ) of the curve was estimated by Prism (v.9.2.0). p values were calculated using Student's t test. ∗p < 0.05 and ∗∗p < 0.01. (L and M) Representative images of the biodistribution of BF10. HNSC/Q1-2 Luciferase tumor-bearing mice were i.v. injected with Vivo680Tag-labeled BF10 (150μg) for 16 h before in vivo bioluminescence detection (L), and subsequently, tissues (tumor, spleen, or tdLNs) were isolated for fluorescence-Vivo680Tag detection (M). Scale bar, 100 or 800 μm as indicated.

Article Snippet: Recombinant human CSF1R-His (Biolegend), human CSF1R-Fc, mouse CSF1R-Fc (Sino Biological), or mouse/human IL-10Ra fusion protein (R&D Systems) were immobilized on 96-well microtiter plates overnight at 4°C.

Techniques: SDS Page, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Activity Assay, Reporter Assay, Luciferase, Labeling, Isolation, Concentration Assay, Injection, Radioactivity, Single Photon Emission Computed Tomography, In Vivo, Fluorescence

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet: BF10 suppresses tumor growth via increasing tumor-infiltrating CD8 + T cells and TAM depletion (A–C) Experimental scheme. The HNSCC/Q1-2 Luciferase tumor cells (5 × 10 5 cells) were subcutaneously inoculated into mice and received treatments of control (Ctrl)-IgG (30 mg/kg), IL-10-Fc (20 mg/kg), αCSF1R (30 mg/kg), or BF10 (36 mg/kg) for six doses as indicated. All mice received the equivalent dose treatment according to different molecular weights of antibody components as described in the . After 8 weeks, tumor growth curve (A), survival proportion (B), and spleen weight (C) were recorded and analyzed. The survival analysis was defined as death spontaneously within 1 month or a tumor burden size reaching 1,500 mm 3 or a length >1.5 cm. Statistical analyses were evaluated using one-way ANOVA with post-hoc Tukey test. The survival was analyzed by Kaplan-Meier method and a log rank test. ∗∗p < 0.01. (D) Experimental scheme for assessing liver toxicity. Mice received a three-dose treatment as indicated and were sacrificed at day 15. Mouse serum was collected and examined for the levels of ALT and AST. The quantified results were presented as mean ± SD, and these statistics were calculated using unpaired Student’s t test (two groups comparison) and one-way ANOVA (more than 3 groups) with Tukey test. ∗∗p < 0.01. (E and F) The experimental schema with BF10 treatment (E) and an illustration of different tumor regions (F). The orthotopic HNSCC tumor tissues were collected for staining. Tumor tissue was separated into 3 regions, which are defined as peri-tumor (PT; green), central-tumor (CT; blue), and peri-normal tissue (red). Scale bar, 250 μm. (G) Representative images of the distribution of tumor-infiltrated CD8 + T cells with BF10 treatment. (H) Quantitative results of tumor-infiltrated CD8 + T cells within tumor tissues. Images were captured and analyzed by the Vectra Polaris Imaging System and Inform software. The average numbers of the CD8 + T cells were counted and expressed per field (1,397 × 1,048 μm 2 ) using Inform software analysis. p values were calculated using Student's t test. ∗∗p < 0.01 and ∗∗∗p < 0.001. (I) Experimental scheme of CD8 + T cell depletion by administration with indicated treatments in orthotopic HNSCC tumor model. CD8 + T cell depletion was carried out using 200 μg anti-CD8 antibody (i.p. every 3 days) simultaneously with or without BF10. (J) Bioluminescence intensity measured and quantified using Xenogen IVIS 100 imaging system on days 6 and 22 for indicating the tumor growth. (K) The Kaplan-Meier survival curve of the tumor-bearing mice receiving control (n = 8), BF10 (n = 8), and anti-CD8 plus BF10 (n = 7). The death of mice was recorded when they died spontaneously, had a tumor diameter of 1.5 cm, or had their eating behavior affected at day 24. (L) Scheme of the macrophage depletion experiment. Tumor-bearing mice were treated with clodronate-liposome (200 μL per 20 g body weight), followed by cotreatment of IL-10-Fc or BF10 with a 3 day interval for following tumor growth and survival. (M and N) Tumor-bearing mice (N = 6–7 per group) received the treatments as indicated. Tumor size was measured by digital caliper, and tumor volume was calculated using the formula V = (length × width × width)/2. Values represent tumor growth curve in all groups (M). The survival was analyzed by Kaplan-Meier method and a log-rank test (N). ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: Recombinant human CSF1R-His (Biolegend), human CSF1R-Fc, mouse CSF1R-Fc (Sino Biological), or mouse/human IL-10Ra fusion protein (R&D Systems) were immobilized on 96-well microtiter plates overnight at 4°C.

Techniques: Luciferase, Control, Comparison, Staining, Imaging, Software

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet:

Article Snippet: Recombinant human CSF1R-His (Biolegend), human CSF1R-Fc, mouse CSF1R-Fc (Sino Biological), or mouse/human IL-10Ra fusion protein (R&D Systems) were immobilized on 96-well microtiter plates overnight at 4°C.

Techniques: Control, Flow Cytometry, RNA Sequencing Assay, Sequencing, Recombinant, Labeling, Enzyme-linked Immunosorbent Assay, Binding Assay, Polymer, Cell Isolation, Selection, Software