Journal: Nature immunology

Article Title: Unique pattern of neutrophil migration and function during tumor progression

doi: 10.1038/s41590-018-0229-5

Figure Lengend Snippet: a . Transwell assays evaluating the ability of BM neutrophils from mice bearing s.c. 4662 tumors derived from KPC mice to spontaneously migrate (left) and chemotaxis in response to CXCL1 (middle) or to fMPL (right). Mean, and SD are shown (n=4). P values are calculated in two-sided Student’s t-test (not shown due to lack of significance). b . Transwell assays evaluating the ability of neutrophils to spontaneously migrate. In one week LL2 experiments, n=5 for control group and n=6 for LL2 mice. In three week LL2 experiments, n=3 for control mice and n=4 for LL2 mice. Mean, and SD are shown. c . Transwell assays of neutrophil chemotaxis in response to CXCL1. Mean and SD are shown. (d, e) Flow cytometry analysis of CD45.2 + BM neutrophils from control mice and CD45.1 + neutrophils from one-week or three-week LL2 TB mice to migrate into the spleen and lung 1 hour after injection to naive CD45.1 + CD45.2 + recipients. d . Flow cytometry gating strategy with representative result from 6 performed experiments, e . Ratio between CD45.1 + and CD45.2 + CD11b + Ly6G + Ly6C lo neutrophils. In each experiment, the ratio of CD45.1 + /CD45.2 + cells injected into recipient mice was set as the baseline= 1. For one week LL2 experiments n=8, for three-week LL2 experiments n=6. Mean, and SD are shown. P values are calculated in two-sided Student’s t-test from baseline within each group of LL2 mice and between the two groups of LL2 mice are shown. f . Transwell migration assay of BM neutrophils from control and KPC mice with PanIn and invasive PDA. N=5 (control), n=5 (PanIn), n=3 (PDA). Mean and SD are shown. g . The mean-squared displacements of neutrophil migration. Each curve represents cumulative results of traces 45–60 individual cells. In a–c, e,f p values were calculated in two-sided Student’s t-test. * − p <0.05, **−p<0.01. In g . p values were calculated in two-way ANOVA test with Bonferroni correction for multiple comparisons. ***−p<0.0001 Two experiments with similar results were performed.

Article Snippet: For BM Ly6G + cells isolation, cells were labeled with biotinylated anti-Ly6G antibody (Miltenyi Biotec), incubated with streptavidin-coated microbeads (Miltenyi Biotec) and separated on MACS columns (Miltenyi Biotec).

Techniques: Derivative Assay, Chemotaxis Assay, Control, Flow Cytometry, Injection, Transwell Migration Assay, Migration

Journal: Nature immunology

Article Title: Unique pattern of neutrophil migration and function during tumor progression

doi: 10.1038/s41590-018-0229-5

Figure Lengend Snippet: a . Antigen-specific proliferation of CD8 + T cells in the presence of BM Ly6G + cells isolated from control mice and one or three weeks LL2 mice. Transgenic, Pmel splenocytes stimulated with specific peptide were used as responders. Proliferation was measured in triplicate by 3 H thymidine uptake. Baseline of T cell proliferation in the absence of added neutrophils was set as 100%. Control -n=7, week 1 LL2 - n=10, week 3 LL2 - n=11. Mean, and SD are shown. b . Antigen-specific proliferation of CD8 + T cells in the presence of BM Ly6G + cells from naive mice, RET melanoma mice (left panel), LLC, or EL4 (right panel) mice. Pmel splenocytes stimulated with specific peptide were used as responders. Proliferation was measured in triplicate by 3 H thymidine uptake. One experiment is shown. Three experiments with the same results were performed. Each experiment included 3 mice. Mean and SD are shown. c . Suppressive activity of PMN from BM of KPC and TRAMP transgenic mice. OT-1 splenocytes stimulated with specific peptide were used as responders. Proliferation was measured in triplicate by 3 H thymidine uptake. For KPC mice 5 experiments with similar results were performed. In TRAMP mice two experiments with the same results were performed. Mean and SD are shown. d . Suppressive activity of neutrophils from KPC mice with PanIN and PDA. Proliferation was evaluated by 3 H-thymidine uptake in triplicates. Means and SD of cumulative result from two mice are shown. e . Cytokine protein expression array in sera of one- and three-week orthotopic LL2 TB mice. Experiments were performed in duplicates and individual data are shown. On the left cytokines with more than 2SD increase in sera of three-week LL2 mice over one-week LL2 mice. On the right cytokines with less than 2SD increase or decreased in three-week LL2 mice. * − p <0.05; **−p<0.01 in two-sided Student’s t-test.

Article Snippet: For BM Ly6G + cells isolation, cells were labeled with biotinylated anti-Ly6G antibody (Miltenyi Biotec), incubated with streptavidin-coated microbeads (Miltenyi Biotec) and separated on MACS columns (Miltenyi Biotec).

Techniques: Isolation, Control, Transgenic Assay, Activity Assay, Expressing

Journal: Nature immunology

Article Title: Unique pattern of neutrophil migration and function during tumor progression

doi: 10.1038/s41590-018-0229-5

Figure Lengend Snippet: Spontaneous (n=24 for both healthy donors and cancer patients groups) ( a ) or chemokine induced (for healthy donor n=24 for media, n=10 for CXCL8 stimulation, n=6 for fMLP stimulation; for cancer patients n= 14 for media, n=3 for CXCL8, and n=3 for fMLP); ( b) migration of neutrophils from healthy individuals and cancer subjects. Individual results for each subject, mean and SD are shown. c . CXCR1 and CXCR2 expression in neutrophils from healthy individual (n=7) and cancer patients (n=4 for CXCR1 and n=6 for CXCR2) assessed by flow cytometry. Individual results for each subject, mean and SD are shown. d, e . Spontaneous (n=12) (d) and CXCL1 stimulated (n=11 for PMN and n=13 for PMN-MDSC) ( e ) migration of neutrophils and PMN-MDSC from the same cancer patients. f . Expression of chemokine receptors in human neutrophils. Indicated chemokine receptors were measured by flow cytometry on PMN-MDSC and neutrophils from cancer patients. N=4 for CXCR1 and n=6 for CXCR2. g . Percentage of CD45 − CFSE + LLC cells in lungs from naive mice 12 hours after intravenous injection. BM Ly6G + neutrophils (PMN) from naïve (n=7), one week (n=5), or three-week (n=5) LL2 mice were injected 6 hours before tumor cells. h . IVIS-based analysis of ex-vivo luciferase activity in excised lungs from naive mice, which were injected i.v. with indicated BM Ly6G + cells 5 hours before the i.v. injection of LL2 tumor cells. N=3. Detection was performed two weeks after the injection. In all panels mean and SD are shown. P values are calculated in two-sided Student’s t-test. * − p <0.05; **−p<0.01; ***−p<0.001; ****−p<0.0001.

Article Snippet: For BM Ly6G + cells isolation, cells were labeled with biotinylated anti-Ly6G antibody (Miltenyi Biotec), incubated with streptavidin-coated microbeads (Miltenyi Biotec) and separated on MACS columns (Miltenyi Biotec).

Techniques: Migration, Expressing, Flow Cytometry, Injection, Ex Vivo, Luciferase, Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Polymorphonuclear myeloid-derived suppressor cells and phosphatidylinositol-3 kinase gamma are critical to tobacco-mimicking oral carcinogenesis in mice

doi: 10.1136/jitc-2023-007110

Figure Lengend Snippet: Inhibition of p110γ reduces tobacco-mimicking carcinogenesis and reduces granulocyte infiltration. (A) 4NQO carcinogenesis and treatment regimen: mice received 50 µg/mL 4NQO in the drinking water for 18 weeks and returned to regular drinking water thereafter. Mice received IPI549 or vehicle treatment for the final 4 weeks. (B) Representative gross images of tongues from mice treated with vehicle or IPI549 (left). Quantification of gross, exophytic lesions per tongue (right; **p<0.01 determined by unpaired Student’s t-test). (C) The carcinogenesis and treatment protocol described in A was carried out three times and pathology scored by two treatment-blinded pathologists. Mild, moderate, or severe dysplasia was assigned a score of 1, 2 or 3, respectively. The average dysplasia score of three to six animals per group is presented across all three experiments (*p<0.05 determined by paired Student’s t-test). (D) The rate of SCC malignancy at week 22 across all three experiments (analyzed by χ 2 analysis). (E) Representative Ly6G immunostaining and quantification across all three experiments. *p<0.05 determined by unpaired Student’s t-test. SCC, squamous cell carcinoma; wk, week; 4NQO, 4-nitroquinoline 1-oxide.

Article Snippet: Leukocyte populations were isolated using magnetic beads: CD8a T cells were isolated using Miltenyi Biotec CD8a+ T cell isolation kit (#130-104-075) and Ly6G+ cells were isolated using Miltenyi Biotec Myeloid-Derived Suppressor Cell Isolation kit (#130-094-538; using only the anti-Ly6G+ magnetic beads steps) following manufacturer’s instructions.

Techniques: Inhibition, Immunostaining

Journal: Journal for Immunotherapy of Cancer

Article Title: Polymorphonuclear myeloid-derived suppressor cells and phosphatidylinositol-3 kinase gamma are critical to tobacco-mimicking oral carcinogenesis in mice

doi: 10.1136/jitc-2023-007110

Figure Lengend Snippet: Tumorigenic tongue SCC cell lines have increased systemic PMN-MDSCs. (A) Transplanted A223, A1206 and A1207 tumors (~1000 mm 3 ) were harvested from C57BL/6 mice, dissociated and stained for CyTOF analysis using antibody panel 1. tSNE analysis was performed to identify immune cell clusters in A223, A1206 and A1207 tumors. (B) Representative image of spleens from a wild-type C57BL/6 mouse and a C57BL/6 mouse bearing an A1206 tumor (left). The percentage of Ly6G+ cells in spleens from C57BL/6 mice without tumors or with A1206 tumors (right). (C) Ly6G+ cells isolated from the spleen of a C57BL/6 mouse without a tumor or the spleen of a mouse bearing an A1206 tumor were co-cultured with CD8+ T cells isolated from a naïve mouse at the indicated cell number to determine whether Ly6G+ cells suppress T-cell proliferation (induced by IL-2 and anti-CD3, anti-CD28 beads). Representative data of three independent repeats. **p<0.01, ***p<0.001 as determined by Student’s t-test. IL, interleukin; PMN-MDSCs; polymorphonuclear MDSCs; SCC, squamous cell carcinoma; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: Leukocyte populations were isolated using magnetic beads: CD8a T cells were isolated using Miltenyi Biotec CD8a+ T cell isolation kit (#130-104-075) and Ly6G+ cells were isolated using Miltenyi Biotec Myeloid-Derived Suppressor Cell Isolation kit (#130-094-538; using only the anti-Ly6G+ magnetic beads steps) following manufacturer’s instructions.

Techniques: Staining, Isolation, Cell Culture

Journal: Journal for Immunotherapy of Cancer

Article Title: Polymorphonuclear myeloid-derived suppressor cells and phosphatidylinositol-3 kinase gamma are critical to tobacco-mimicking oral carcinogenesis in mice

doi: 10.1136/jitc-2023-007110

Figure Lengend Snippet: Carcinogenic microenvironment enables A1207 p110γ-dependent tumor growth. (A–E) 4NQO carcinogenesis and treatment regimen: mice received 50 µg/mL 4NQO in the drinking water (or regular drinking water for control mice) for 16 weeks and returned to regular drinking water thereafter. Mice received daily IPI549 or vehicle treatments beginning week 15 for the duration of the experiment and all mice were transplanted with A1207 cells to the dorsal tongue on week 16. (B) Representative images of whole tongue sections from mice treated with water versus 4NQO+IPI549 versus 4NQO+Vehicle all transplanted with A1207 cells on week 16. Black dotted line outlines the tumor border. (C) Tumor volume in each treatment group was determined. *p<0.05, **p<0.01, **p<0.001 determined by one-way ANOVA with Tukey’s multiple comparison test (right). (D–E) Ly6G immunostaining and quantification of tongue tumors. *p<0.05 determined by unpaired Student’s t-test. (F–H) C57BL/6 mice were transplanted with A1206 tumor cells to the right flank or not transplanted. When A1206 tumors reached 300 mm 3 , all mice (naïve and A1206 tumor-bearing) were transplanted with A1207 cells to the left flank. (G) Size of left flank A1207 tumor was monitored over time. Due to the size of A1206 tumors, all mice required euthanasia 34 days after A1207 transplant. **p<0.01 determined by unpaired Student’s t-test at the indicated time points. (H) Rate of complete A1207 tumor clearance was monitored over time. *p<0.05 determined by log-rank test. ANOVA, analysis of variance; wk, week; 4NQO, 4-nitroquinoline 1-oxide.

Article Snippet: Leukocyte populations were isolated using magnetic beads: CD8a T cells were isolated using Miltenyi Biotec CD8a+ T cell isolation kit (#130-104-075) and Ly6G+ cells were isolated using Miltenyi Biotec Myeloid-Derived Suppressor Cell Isolation kit (#130-094-538; using only the anti-Ly6G+ magnetic beads steps) following manufacturer’s instructions.

Techniques: Control, Comparison, Immunostaining

Journal: Journal for Immunotherapy of Cancer

Article Title: Polymorphonuclear myeloid-derived suppressor cells and phosphatidylinositol-3 kinase gamma are critical to tobacco-mimicking oral carcinogenesis in mice

doi: 10.1136/jitc-2023-007110

Figure Lengend Snippet: PMN-MDSCs from tumor bearing mice enable A1207 tumor growth. Ly6G+ cells were harvested from spleens of C57BL/6 mice without tumors or bearing A1206 tumors on day 0, 5, 11 and 16. 0.5 million A1207+1.5 million Ly6G+ cells were transplanted to the flanks of recipient C57BL/6 mice on day 0. Initiated tumors were transplanted with another 1.5 million Ly6G+ cells on days 5, 11 and 16. Rate of complete A1207 tumor clearance was monitored over time. *p<0.05 determined by log-rank test. PMN-MDSCs; polymorphonuclear myeloid-derived suppressor cells; WT, wild type.

Article Snippet: Leukocyte populations were isolated using magnetic beads: CD8a T cells were isolated using Miltenyi Biotec CD8a+ T cell isolation kit (#130-104-075) and Ly6G+ cells were isolated using Miltenyi Biotec Myeloid-Derived Suppressor Cell Isolation kit (#130-094-538; using only the anti-Ly6G+ magnetic beads steps) following manufacturer’s instructions.

Techniques: Derivative Assay

Journal: Scientific Reports

Article Title: Essential roles of C-type lectin Mincle in induction of neuropathic pain in mice

doi: 10.1038/s41598-018-37318-8

Figure Lengend Snippet: Elimination of Mincle mRNA by depletion of neutrophils and macrophages/monocytes from injured SN cells. ( a ) Representative flow cytometry dot plots of Ly6G + and/or F4/80 + cells and ( b ) the proportions of these cells in samples of contralateral total, ipsilateral total, ipsilateral depleted fraction, and ipsilateral trapped fraction cells 12 h after PNI. ( a ) Lower-right (red frame) and upper-left (blue frame) quadrant of the panels representing neutrophils (Ly6G + ) and macrophages/monocytes (F4/80 + ), respectively. Note that neutrophils and macrophages/monocytes were adequately removed from the ipsilateral depleted fraction (lower-left panel in a and lower panels in b). ( c ) The Mincle mRNA expression in each sample. The expression level of Mincle gene was normalized to that of Gapdh gene. PCR amplifications were done in triplicate. Data are representative of two independent experiments. Note that Mincle mRNA expression in the ipsilateral depleted fraction was virtually eliminated.

Article Snippet: Two-thirds of the cells obtained from injured SNs were incubated with rat anti mouse Ly6G-FITC (2 μg/mL, clone: 1A8, Tonbo Biosciences, RRID_AB_2621704) and rat anti mouse F4/80-PE (2 μg/mL, clone: BM8.1, Tonbo Biosciences) for 30 min on ice, washed with MACS Running buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), and then incubated with anti-FITC and anti-PE antibody-coated immunomagnetic microbeads (Miltenyi Biotec, RRID: AB_244371, AB_244373, respectively) for 15 min on ice.

Techniques: Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Essential roles of C-type lectin Mincle in induction of neuropathic pain in mice

doi: 10.1038/s41598-018-37318-8

Figure Lengend Snippet: PNI up-regulates Mincle mRNA at the transection site. ( a – c ) Dark field and bright field images of the same area of ISHH revealed mRNA distribution of Mincle mRNA after PNI in the L4 DRG and SN, 12 h after PNI, contralateral ( a ), ipsilateral ( b ), and 3 days after PNI, ipsilateral site ( c ). Arrowheads indicate positive cells. Scale bar: Dark field and bright field images, 400 μm, transection site, 80 μm. Counterstained with haematoxylin. ( d,e ) Bright-field photomicrographs of combined ISHH for Mincle mRNA with immunostaining with Ly6G or F4/80 in the ipsilateral SN. Arrowheads indicate cells single-labelled with ISHH (aggregation of grains). Arrows indicate cells double-labelled with ISHH (aggregation of grains) and IHC (brown staining). Counterstained with haematoxylin. Scale bar: 20 μm.

Article Snippet: Two-thirds of the cells obtained from injured SNs were incubated with rat anti mouse Ly6G-FITC (2 μg/mL, clone: 1A8, Tonbo Biosciences, RRID_AB_2621704) and rat anti mouse F4/80-PE (2 μg/mL, clone: BM8.1, Tonbo Biosciences) for 30 min on ice, washed with MACS Running buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), and then incubated with anti-FITC and anti-PE antibody-coated immunomagnetic microbeads (Miltenyi Biotec, RRID: AB_244371, AB_244373, respectively) for 15 min on ice.

Techniques: Immunostaining, Staining

Journal: Scientific Reports

Article Title: Essential roles of C-type lectin Mincle in induction of neuropathic pain in mice

doi: 10.1038/s41598-018-37318-8

Figure Lengend Snippet: Normal leucocyte infiltration at the injured site of Mincle −/− mice. Representative flow cytometry dot plot of CD11b + Ly6G + cells in the SN ( a ) and DRG ( b ) 1 day after PNI. Upper-right quadrant (red frame) of the left panels representing infiltrating neutrophils (CD11b + Ly6G + ). The bar graph (right) summarises the proportions of neutrophils in the ipsilateral or contralateral tissues of WT or Mincle −/− mice ( n = 5 in each group, one-way ANOVA followed by Tukey’s post hoc test, *** P < 0.001 vs. contralateral side). Representative flow cytometry dot plot of CD11b + F4/80 + cells in the SN ( c ) and DRG ( d ) 3 days after PNI. Upper-right quadrant (red frame) of the left panels representing infiltrating monocytes/macrophages (CD11b + F4/80 + ). The bar graph (right) summarises the proportions of monocytes/macrophages in the ipsilateral or contralateral tissues of WT or Mincle −/− mice ( n = 4 in each group, one-way ANOVA followed by Tukey’s post hoc test, ** P < 0.01 vs. contralateral side). Values are mean ± SEM.

Article Snippet: Two-thirds of the cells obtained from injured SNs were incubated with rat anti mouse Ly6G-FITC (2 μg/mL, clone: 1A8, Tonbo Biosciences, RRID_AB_2621704) and rat anti mouse F4/80-PE (2 μg/mL, clone: BM8.1, Tonbo Biosciences) for 30 min on ice, washed with MACS Running buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), and then incubated with anti-FITC and anti-PE antibody-coated immunomagnetic microbeads (Miltenyi Biotec, RRID: AB_244371, AB_244373, respectively) for 15 min on ice.

Techniques: Flow Cytometry

Journal: Scientific Reports

Article Title: Essential roles of C-type lectin Mincle in induction of neuropathic pain in mice

doi: 10.1038/s41598-018-37318-8

Figure Lengend Snippet: Increase of Mincle mRNA in injured SN depends on MyD88 in neuropathic pain model. ( a ) Real-time PCR analysis of Mincle mRNA in the injured SN and DRG 12 h after PNI in WT and MyD88 −/− mice. Bar graphs show fold-change compared to naïve WT mice ( n = 3 in each group, Student’s t test. * P < 0.05 vs. WT mice). ( b ) Representative flow cytometry dot plot of CD11b + Ly6G + cells (left) in the SN (upper) and DRG (lower) 1 day after PNI. Upper-right quadrant (red frame) representing infiltrating neutrophils (CD11b + Ly6G + ). The bar graph (right) summarises the proportions of neutrophils in the ipsilateral or contralateral tissues of WT or MyD88 −/− mice ( n = 3 in each group, one-way ANOVA followed by Tukey’s post hoc test, *** P < 0.001 vs. WT mice, ### P < 0.001 vs. contralateral side). Values are mean ± SEM.

Article Snippet: Two-thirds of the cells obtained from injured SNs were incubated with rat anti mouse Ly6G-FITC (2 μg/mL, clone: 1A8, Tonbo Biosciences, RRID_AB_2621704) and rat anti mouse F4/80-PE (2 μg/mL, clone: BM8.1, Tonbo Biosciences) for 30 min on ice, washed with MACS Running buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), and then incubated with anti-FITC and anti-PE antibody-coated immunomagnetic microbeads (Miltenyi Biotec, RRID: AB_244371, AB_244373, respectively) for 15 min on ice.

Techniques: Real-time Polymerase Chain Reaction, Flow Cytometry