Journal: STAR Protocols

Article Title: Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes

doi: 10.1016/j.xpro.2021.100537

Figure Lengend Snippet:

Article Snippet: E0771 , CH3 BioSystems , SKU:940001-Vial RRID: N/A.

Techniques: Recombinant, Staining, Blocking Assay, Saline, Multiplex Assay, Software

Journal: Current Issues in Molecular Biology

Article Title: Marine Bromophenol Derivatives as a Novel Class of Potent Small-Molecule STING Agonists

doi: 10.3390/cimb48010061

Figure Lengend Snippet: Tumor growth in mice treated with OSBP63. C57BL/6J mice bearing a single subcutaneous E0771 breast tumor (~100 mm 3 ) received intratumoral OSBP63 (5 mg/kg). Tumor volumes were measured over time and plotted as a line graph. ( n = 3; **** p < 0.0001 vs. control).

Article Snippet: The 4T1 and E0771 cells were purchased from Ubigene Biosciences Co., Ltd. (Guangzhou, China).

Techniques: Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Dysregulation of metabolic‐associated pathways in muscle of breast cancer patients: preclinical evaluation of interleukin‐15 targeting fatigue

doi: 10.1002/jcsm.12294

Figure Lengend Snippet: Mammary tumour‐induced skeletal muscle dysfunction in wild type mice. (A) Representative live in vivo bioluminescent images of luciferase‐containing E0771 tumour cells through 4 weeks of tumour growth in the mammary fat pads of wild type mice. (B) Bioluminescent quantification of luciferase‐containing E0771 tumour cells through 4 weeks of tumour growth in the mammary fat pads of wild type mice. (C) Changes in body weight in wild type after 4 weeks of tumour growth in the mammary fat pads of wild type mice. These weights do not include the weight of the tumour, which was resected. (D) Differential expression of the Il15 gene in skeletal muscles of wild type mice with no tumour, and following 2 and 4 weeks of tumour growth in the mammary fat pads. (E) Ex vivo skeletal muscle fatigue curves from wild type mice with no tumour, and following 2 and 4 weeks of tumour growth in the mammary fat pads. The leftward shift of the fatigue curve in the 4WK mice is indicative of a greater rate of muscle fatigue. (F) Quantification of the area under the fatigue curve in muscles from wild type mice with no tumour, and following 2 and 4 weeks of tumour growth in the mammary fat pads. (G) Isometric force output at increasing stimulation frequencies in muscles from wild type mice with no tumour, and following 2 and 4 weeks of tumour growth in the mammary fat pads. * P < 0.05; ** P < 0.001; *** P < 0.0001. CON, control mice; EDL, extensor digitorum longus; IL‐15, interleukin‐15.

Article Snippet: E0771 cells were screened for the presence of contaminants prior to use (IDEXX Bioresearch).

Techniques: In Vivo, Luciferase, Quantitative Proteomics, Muscles, Ex Vivo, Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Dysregulation of metabolic‐associated pathways in muscle of breast cancer patients: preclinical evaluation of interleukin‐15 targeting fatigue

doi: 10.1002/jcsm.12294

Figure Lengend Snippet: Muscle weights following E0771 tumour growth

Article Snippet: E0771 cells were screened for the presence of contaminants prior to use (IDEXX Bioresearch).

Techniques: Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Dysregulation of metabolic‐associated pathways in muscle of breast cancer patients: preclinical evaluation of interleukin‐15 targeting fatigue

doi: 10.1002/jcsm.12294

Figure Lengend Snippet: Skeletal muscle function in IL15TG mice following mammary tumour growth. (A) Representative live in vivo bioluminescent images of luciferase‐containing E0771 tumour cells after 4 weeks of tumour growth in the mammary fat pads of B6 littermate control and IL15TG mice. (B) Bioluminescent quantification of luciferase‐containing E0771 tumour cells through 4 weeks of tumour growth in the mammary fat pads of B6 littermate control and IL15TG mice. (C) Changes in body weight in wild type after 4 weeks of tumour growth in the mammary fat pads of wild type mice. These weights do not include the weight of the tumour, which was resected. (D) Ex vivo skeletal muscle fatigue curves from B6 littermate control mice with no tumour, and following 4 weeks of tumour growth in the mammary fat pads. The leftward shift of the fatigue curve in the CON tumour mice is indicative of a greater rate of muscle fatigue. (E) Quantification of the area under the fatigue curve in muscles from B6 littermate control mice with no tumour, and following 4 weeks of tumour growth in the mammary fat pads. (F) Isometric force output at increasing stimulation frequencies in muscles from B6 littermate control mice with no tumour, and following 4 weeks of tumour growth in the mammary fat pads. (G) Ex vivo skeletal muscle fatigue curves from IL15TG mice with no tumour, and following 4 weeks of tumour growth in the mammary fat pads. (H) Quantification of the area under the fatigue curve in muscles from IL15TG mice with no tumour, and following 4 weeks of tumour growth in the mammary fat pads. (F) Isometric force output at increasing stimulation frequencies in muscles from IL15TG mice with no tumour, and following 4 weeks of tumour growth in the mammary fat pads. In Figures C, E, and F, solid lines represent the average value from wild type non‐tumour‐bearing mice, and dotted lines represent the average value from wild type mice following 4 weeks of tumour growth. * P < 0.05; ** P < 0.001; *** P < 0.0001. EDL, extensor digitorum longus.

Article Snippet: E0771 cells were screened for the presence of contaminants prior to use (IDEXX Bioresearch).

Techniques: In Vivo, Luciferase, Control, Ex Vivo, Muscles

Journal: Nature

Article Title: Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming

doi: 10.1038/nature21724

Figure Lengend Snippet: a) . Flow cytometry quantification of E0771 tumour-infiltrating endothelial cells (CD31 + ) and pericytes (NG2 + ) (WT, n=11; CD4KO, n=9; CD8KO, n=12; TCRKO, n=8). b) . Staining and quantification of endothelial cells (green) attached by pericytes (red) (n=4/group; Scale bar, 50μm) c) . Normalized absorbance of Evans blue in tumours (BC (blank control), n=5; WT, n=11; CD4KO, n=11; CD8KO, n=12; TCRKO, n=12). d) . Blood CTC frequencies from tumour-bearing mice (WT, n=11; CD4KO, n=11; CD8KO, n=12; TCRKO, n=12). e) . Lung metastatic area from H&E images (WT, n=14; CD4KO, n=14; CD8KO, n=12; TCRKO, n=8; Scale bar, 1mm). Mean ± s.e.m shown. Smaller dots are values from individual fields ( b ) and whole lung sections ( e ). Outlined circles are mean values taken over multiple fields/sections from the same mouse. P values were calculated by comparing individual animals using two-tailed unpaired Student’s t-test (a–c,e) or two-tailed unpaired Mann–Whitney U test (d) . n.s., not significant.

Article Snippet: E0771 cells were harvested from culture with 2 mg·ml −1 EDTA (Versene, Lonza).

Techniques: Flow Cytometry, Staining, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming

doi: 10.1038/nature21724

Figure Lengend Snippet: a) . Flow cytometric plots of CD3 + cells of 4T1 tumour harvested from WT and CD4KO mice. Five animals were examined in each group. Representative plots are shown. b–g) . Quantification of tumour vascular normalization markers including pericyte coverage (b) , vessel density and vessel length (c) , VE-cadherin expression (white arrow heads show vessels without VE-cadherin expression) (d) , hypoxia measured by pimonidazole staining (e) , lectin perfusion efficiency (f) , and dextran leakage (g) (n=5/group; Scale bars, 50μm (d,f,g) , 1mm (e) ). h). (Top) Schematic of the experimental design. (Bottom) The two doses of antibody deplete CD4 + -TLs for 2 weeks. One point represents one mouse. Whole blood was collected for the flow cytometric analysis. i) . Dot plots showing that tumours were resected at similar size/weight j) . Representative whole animal bioluminescence images showing spontaneous 4T1 metastasis in αIgG or αCD4 treated mice. Dots representing mice with detected metastases are labeled with a red boundary. k) . Kaplan-Meier curves showing the metastasis-free frequency of 4T1 tumour-bearing mice treated with αIgG or αCD4. n=10 and 9 for αIgG and αCD4 groups, respectively (i–k) . l). (Top) Flow cytometry quantification of tumour-infiltrating CD4 + -TLs across three murine tumour models (4T1, n=4; E0771, n=5; AT3, n=4). The tumours were resected at similar size/weight (around 1 gram) from the same batch of experiments. (Bottom) A table summarizing the results from vessel normalization assays. The number indicates the fold change increased (red) or decreased (green) in WT mice compared to CD4KO mice. In E0771 model, RNA-seq reveals an increase in extracellular matrix and adhesion molecule gene expression in WT mice over CD4KO mice. m–p) . Quantification of tumour vascular normalization markers (n=5/group; Scale bars, 50μm (m,o,p) ; n=10/group; Scale bars,1mm (n) ). q) . Scatter plot showing the Cd4 and Ifng gene expression levels of different p53 −/− murine breast tumour models. The Cd8 gene expression and ER status are denoted by the colors of the dots and dot outlines, respectively. Two models chosen for hypoxia measurement are highlighted. r) . Hypoxia quantification for T1 and T11 tumours in WT and CD4KO background as measured by pimonidazole staining (T1: n=5/group; T11: WT, n=5; CD4KO, n=8; Scale bars,1mm). Data are presented as means ± s.e.m. Animal numbers used in (i–k) are denoted in (k) . P values are determined by two-tailed unpaired Student’s t-test ( b–g,i,m–p,r ), Fisher’s exact test ( j ) and Log-Rank test ( k ).

Article Snippet: E0771 cells were harvested from culture with 2 mg·ml −1 EDTA (Versene, Lonza).

Techniques: Expressing, Staining, Labeling, Flow Cytometry, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: Nature

Article Title: Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming

doi: 10.1038/nature21724

Figure Lengend Snippet: a,b) . Flow cytometry quantification validating that M-II KO mice have decreased MHC-II expression in tumour-infiltrating immune cells (CD45 + ), including macrophages (Mϕ, CD45 + CD11b + Ly6G F4/80 + ), dendritic cells (DC, CD45 + CD11b + Ly6G − F4/80 − CD11c + ), and B cells (CD45 + B220 + ) (CTRL: n=10; M-II KO : n=11). c) . Flow cytometry gating of suspension cells dissociated from thymus, characterized as CD45 + EpCAM − immune cells and CD45 − EpCAM + epithelial cells. d,e) . Quantification of MHC-II expression of thymus showing that MHC-II expression is inhibited in immune cells but preserved in epithelial cells in M-II KO mice (CTRL: n=7; M-II KO : n=6). f) . Quantification of different types of tumour-infiltrating stroma cells (n=10/group). g) . Quantification of MHC-II expression in different cell types (CTRL: n=10; Tie2Cre;H2Ab +/floxP : n=5; M-II KO : n=11). h) . Quantification of T cells in spleens from 5 – 6 week-old female mice showing the number of T cells is independent of MHC-II expression on Tie2Cre + cells (CTRL: n=7; M-II KO : n=6). i) . Quantification of activated CD4 + -TLs and effector CD4 + -TLs from tumours of similar sizes. (activated CD4 + -TL: CD45 + CD3 + CD4 + CD25 + FoxP3 − Treg: CD45 + CD3 + CD4 + CD25 + FoxP3 + ; Effector memory cell: CD44 + CD62L − Naïve CD4 + -TL: CD44 − CD62L + ) (n=11/group). j) . The percentages of CD4 + -TL activation markers in spleen showing a similar pattern as in tumour (i) (CTRL: n=7; M-II KO : n=6). k) . Quantification of different E0771 tumour-infiltrating T helper cells (IFNγ + Th1, IL4 + Th2 and IL17A + Th17) (n=11/group). l) . Quantification of E0771 tumour-infiltrating CD4 + -TL cells, macrophage, dendritic cells, B cells and neutrophils (CD45 + CD11b + Ly6G high ) (n=11/group). Data are presented as means ± s.e.m. The genetic backgrounds of mice are denoted with different colors shown on the right of (l) . WT, Tie2Cre and H2Ab floxP/floxP were combined as CTRL group. P values were calculated using two-tailed unpaired Student’s t-test (a–b, d–f, h–k) or two-tailed unpaired Mann–Whitney U test ( l ). n.s., not significant.

Article Snippet: E0771 cells were harvested from culture with 2 mg·ml −1 EDTA (Versene, Lonza).

Techniques: Flow Cytometry, Expressing, Suspension, Activation Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming

doi: 10.1038/nature21724

Figure Lengend Snippet: a) . Schematic of the experimental design. b) . ICB leads to CD4 + -TL dependent tumour growth inhibition, measured by tumour weight at Day 15 post E0771 injection. c,d) .Total number of immune cells (c) and T cell (d) in tumours from different groups. Although the number of pan tumour-infiltrating immune cells (CD45 + ) is not changed, the number of CD4 + -TLs increased after immune checkpoint blockade therapy. e) . A heatmap summarizing changes to tumour-infiltrating immune components after ICB therapy. The number of different immune cells (rows) is shown for each tumour (columns) after control or checkpoint blockade treatment. The weight of each tumour is shown (top panel). Row-side annotations show p values comparing between CD8KO (αIgG) and CD8KO (αPD1αCTLA4) groups (far left column), and between CD8KO (αPD1αCTLA4) and TCRKO (αPD1αCTLA4) (far right column) (EM T: effector memory T cells). f) . Quantification of different subsets among CD45 + CD11b + cells showing the effect of ICB on innate immune microenvironment (Eosinophil: CD45 + CD11b + SiglecF + ). g,h) . Quantification of the percentage of Tregs among total CD4 + -TLs, and the ratio of effector memory CD4 + -TLs to naïve CD4 + -TLs after ICB in CD8 KO mice. i) . Quantification of the percentage of different CD4 + T helper cells. j) . Percentage of IFNγ + cells in CD4 + or CD4 − cells among all the CD45 + tumour-associated immune cells, indicating CD4 + -TLs make up the majority of IFNγ + cells. Data are presented as means ± s.e.m. Animal numbers used in (b–j) are denoted in (a) . P values were calculated using two-tailed unpaired (b–i) or paired (j) Student’s t-test.

Article Snippet: E0771 cells were harvested from culture with 2 mg·ml −1 EDTA (Versene, Lonza).

Techniques: Inhibition, Injection, Control, Two Tailed Test

Journal: Nature

Article Title: Mutual Regulation of Tumour Vessel Normalization and Immunostimulatory Reprogramming

doi: 10.1038/nature21724

Figure Lengend Snippet: a,b) . Quantitative RT-PCR analysis showing the effect of IFNγ and sCD40L on the mRNA levels of adhesion molecules, VEGFA (a) , and T cell attractant chemokines (b) . The experiments were repeated independently for three times (batches) with technical duplicates in each time. c) . Schematic of the experimental design and hypothetical model. d) . Tumours resected at Day12 – 13 post E0771 injection have similar size/weight, and the effect of Th1 adoptive transfer on vessel normalization as measured by the CD31 + endothelial cells to NG2 + pericytes ratio. e) . Flow cytometry quantification CD45.1 + adoptive transferred Th1 cells, and CD45.2 + host immune cells. f) . Characterization and quantification of CD45.2 + host immune cells showing that Th1-mediated immune infiltration is partially dependent on pericyte coverage. g) . Effect of Th1 adoptive transfer and pericyte depletion on CD11b + Ly6G + immune cells demonstrating different pattern with other tumour-infiltrating immune cells as from (f) . h) . Schematic summary of CD4 + -TL-mediated vessel normalization, and subsequent formation of positive feedback loop through cell-cell interaction, cytokine production and increased pericyte coverage. Checkpoint blockade therapy and antigen presentation enhance Th1-skewed CD4 + -TL activation and promote the vessel normalization/immunostimulatory reprogramming positive feedback loop. Data are presented as means ± s.e.m. Animal numbers used in (d–g) are denoted in (c) . P values were calculated using two-tailed unpaired Student’s t-test based on biological replicates ( a,b,d–g ). Technical replicates are averaged within each biological replicate ( a,b ).

Article Snippet: E0771 cells were harvested from culture with 2 mg·ml −1 EDTA (Versene, Lonza).

Techniques: Quantitative RT-PCR, Injection, Adoptive Transfer Assay, Flow Cytometry, Immunopeptidomics, Activation Assay, Two Tailed Test

Journal: Research Square

Article Title: The acid-sensing nociceptor TRPV1 controls breast cancer progression in bone via regulating HGF secretion from sensory neurons

doi: 10.21203/rs.3.rs-3105966/v1

Figure Lengend Snippet: A. Radiograph of osteolytic lesions associated with mouse E0771 BC colonization in tibiae in WT (left) and TRPV1 −/− (right) mice taken at day 21 prior to sacrifice under general anesthesia. B. Quantitative analysis of . Ratio represents osteolytic area of TRPV1 −/− mice /osteolytic area of WT mice x 1. Data are shown as mean ± SD (N=7). *p<0.05 vs WT mice. C. Macroscopic view of lung metastasis of E0771 BC cells from tibiae in the same experiment as . D. Quantitative analysis of . The number of 4T1 BC metastatic foci in lung was macroscopically counted as described in . Data are shown as mean ± SD (N=7). *p<0.05 vs WT mice. E. Hind-paw mechanical allodynia assessed at day 21 in WT and TRPV1 −/− mice that were intratibially injected with E0771 BC cells. See legend for experimental details. Data are shown as mean ± SD (N=7). *p<0.05 vs sham WT mice. # p<0.05 vs WT E0771 mice. F. Expression of molecular marker for SN excitation, pERK1/2 and pCREB, in DRGs harvested from WT and TRPV1 −/− mice that were intratibially injected with E0771 BC cells at day 21 by Western analysis. G. Co-expression of pERK1/2 (left) and CGRP (center) in SNs in DRGs harvested from WT andTRPV1 −/− mice intratibially injected with or without E0771 at day 21 by immunofluorescence. Scale bar=100μm.

Article Snippet: Mouse 4T1 or E0771 BC cells (2×10 5 /10μl) or PBS (sham) were inoculated into the bone marrow cavity of the right tibiae in 4- to 6-week-old female BALB/c (Harlan Laboratories, Indianapolis, IN), or C57BL/6J (E0771 BC) (Jackson Laboratory, Bar Harbor, ME), respectively, under general anesthesia with ketamine (Ketaset; 90–150 mg/kg, ip) and xylazine (AnaSed; 5–10 mg/kg, ip).

Techniques: Injection, Expressing, Marker, Western Blot, Immunofluorescence

Journal: Research Square

Article Title: The acid-sensing nociceptor TRPV1 controls breast cancer progression in bone via regulating HGF secretion from sensory neurons

doi: 10.21203/rs.3.rs-3105966/v1

Figure Lengend Snippet: A. Co-expression of HGF (left) and CGRP (center) on SNs in DRGs in WT and TRPV1 −/− mice intratibially injected with or without E0771 BC cells. DRGs were harvested from mice at day 21 and sections were immunofluorostained with anti-CGRP and anti-HGF antibody. Scale bar=50μm. B. Quantitative analysis of . Ratio represents TRPV1 −/− sham, E0771 BC-injected WT or E0771 BC-injected TRPV1 −/− mice /WT sham mice x 1. Data are shown as mean ± SD (N=7). *p<0.05 vs WT sham mice. # p<0.05 vs WT E0771 BC mice. C. Western analysis of HGF expression in DRG SNs harvested at day 21 from WT and TRPV1 −/− mice that were intratibially injected with or without E0771 BC cells.

Article Snippet: Mouse 4T1 or E0771 BC cells (2×10 5 /10μl) or PBS (sham) were inoculated into the bone marrow cavity of the right tibiae in 4- to 6-week-old female BALB/c (Harlan Laboratories, Indianapolis, IN), or C57BL/6J (E0771 BC) (Jackson Laboratory, Bar Harbor, ME), respectively, under general anesthesia with ketamine (Ketaset; 90–150 mg/kg, ip) and xylazine (AnaSed; 5–10 mg/kg, ip).

Techniques: Expressing, Injection, Western Blot