4t1 2 crl 3406 mouse mammary tumor cells  (ATCC)

Journal: The Journal of Clinical Investigation

Article Title: Inflammation- and resolution-programmed myeloid circuits govern therapeutic resistance in epithelial and mesenchymal triple-negative breast cancer

doi: 10.1172/JCI198815

Figure Lengend Snippet: ( A ) Flow cytometry analysis of peripheral blood (PB) neutrophils from BALB/c mice (pink) with sham (surgery without tumor implantation), T11, T12, and 2208L tumors and C57BL/6 mice (blue) with sham, AT3, E0771, and PyMT-M tumors. ( B and C ) Neutrophil ( B ) and macrophage ( C ) infiltration in the indicated tumors at day 20 after implantation. ( D ) Growth of AT3, E0771, PyMT-M, and T11 tumors under vehicle or paclitaxel (PTX) plus anti–PD-1 antibody. Numbers indicate cured/total mice per group. Dark lines represent group averages; light lines represent individual tumor size. ( E ) Schematic of the experimental design: following combined treatment, mice with tumor eradication were monitored for recurrence. Recurrence-free mice were rechallenged to assess immune memory. Relapsed tumors were excised to generate resistant cell lines. ( F ) Growth of E0771 tumors under vehicle or combined treatment. ( G ) Analysis of PB monocytes from mice with E0771, E0771-Res1, E0771-Res2 tumors and from mice with AT3 and AT3-Res tumors. ( H ) Growth of E0771, E0771-Res1, and E0771-Res2 tumors under vehicle or PTX plus anti–PD-1 antibody. ( I ) Average myeloid cell infiltrates in 2208L and E0771 tumors and their resistant derivatives ( n = 3) determined by single-cell RNA-seq. ( J and K ) Relative expression of selected Hallmark gene sets in 2208L and E0771 parental tumors and resistant derivatives by bulk RNA-seq. Points represent individual samples, which are color-coded by group. ( J ) 2208L parental (P, n = 3), 2208L-resistant tumors (R, n = 5). ( K ) E0771 parental (P, n = 4), E0771-Res1 (purple, n = 4), and E0771-Res2 (pink, n = 4). ( L and M ) Tumor growth of 2208L-Res2 ( L ) and E0771-Res1 ( M ) tumors receiving vehicle or 100 mg/kg celecoxib daily and treated with vehicle or combined treatment. ( A – C , G , L , and M ) Statistical significance was determined using 1-way ANOVA followed by Tukey’s test. ( D , G , and H ) Significance was calculated using unpaired 2-tailed Student’s t test. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: Single cells were then subjected to red blood cell lysis, and tumor cells were isolated using the Mouse Tumor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Tumor Implantation, Single Cell, RNA Sequencing, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Inflammation- and resolution-programmed myeloid circuits govern therapeutic resistance in epithelial and mesenchymal triple-negative breast cancer

doi: 10.1172/JCI198815

Figure Lengend Snippet: ( A ) Schematic of the experimental design: Combined treatments started on day 3 after tumor implantation. Anti–M-CSF plus clodrosome or control IgG1 plus liposome were administered as indicated. ( B ) Flow cytometry of PB monocytes in E0771-Res1 tumor-bearing mice under control or combined therapy, with or without macrophage depletion ( n = 5). ( C ) Representative immunohistochemistry of F4/80 in E0771-Res1 tumors treated with control IgG plus liposome or anti–M-CSF plus clodrosome. Scale bar: 100 μm. ( D ) Tumor growth of E0771-Res1 tumors under vehicle or combined therapy, with or without macrophage depletion ( n = 5). The values 5.04 × 10 3 and 4.31 × 10 –4 represent P values for the comparison of tumor volumes between combined paclitaxel and anti–PD-1 treatment and vehicle control at day 24 in control and macrophage-depleted mice, respectively. ( E ) Flow cytometry of immune infiltrates in E0771-Res1 tumors at endpoint. (Left) Log 2 fold change of major immune cells relative to the vehicle-treated tumor group. (Right) Average immune cell number per 1,000 CD45 – cells. ( F ) Quantification of major immune cells per 1,000 CD45 – cells in E0771-Res1 tumors under indicated treatments. ( G ) PB monocytes in E0771-Res1 tumor-bearing WT or CCR2-KO mice treated with control or combined treatment. Samples collected on day 1 and 17 after implantation. Significance was calculated using paired 2-tailed Student’s t test. ( H and I ) Growth of E0771-Res1 ( H ) and T12 tumors ( I ) in WT or CCR2-KO mice treated with vehicle or PTX combined with anti–PD-1 ( n = 5 mice per group). ( J ) Flow cytometry analysis of the immune infiltrates in E0771-Res1 tumors. (Left) Log 2 fold change of major immune cells relative to the vehicle-treated tumor group. (Right) Average immune cell number per 1,000 CD45 – cells. ( K ) Quantification of major immune cells per 1,000 CD45 – cells in E0771-Res1 tumors in WT versus CCR2-KO mice. ( B , D , F , H , I , and K ) Statistical significance was determined using 1-way ANOVA followed by Tukey’s test. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: Single cells were then subjected to red blood cell lysis, and tumor cells were isolated using the Mouse Tumor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Tumor Implantation, Control, Flow Cytometry, Immunohistochemistry, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Inflammation- and resolution-programmed myeloid circuits govern therapeutic resistance in epithelial and mesenchymal triple-negative breast cancer

doi: 10.1172/JCI198815

Figure Lengend Snippet: ( A ) Growth of E0771-Res1 tumor in CCR2-KO mice receiving WT or TREM2-KO monocytes, under combined therapy ( n = 5). Arrowheads indicate monocyte transfers. ( B ) Representative flow plots showing CFSE dilution and CD25 expression in CD8 + T cell treated with activation beads and indicated concentrations of murine C1q. ( C – E ) Quantification of total CD8 + T cells ( C ), CD25 + /total CD8 + T cell ratios ( D ), and IFN-γ production by CD8 + T cells ( E ). ( F ) Quantification of surviving E0771-OVA cells after coculture with pretreated OT-1 CD8 + T cells. ( G ) ELISA quantification of C1q in tumor lysates from E0771, E0771-Res1, and E0771-Res2 tumors ( n = 4). ( H ) Immunofluorescence of MitoTracker Deep Red, C1q, C1QBP, and DAPI in CD8 + T cells. Scale bar: 1 μm. ( I ) Immunoblot of OPA1, DRP1, and phosphor-DRP1 (Ser616) in CD8 + T cells. (Bottom) Quantification of DRP1 levels normalized to β-ACTIN (3 independent experiments). ( J ) TEM images of activated CD8 + T cells treated with vehicle or C1q for 48 hours. Scale bar: 2 μm; 0.5 μm (zoomed-in). ( K ) Representative flow plots of MitoTracker Deep Red/Green (MDR/MG) populations. ( L and M ) CD25 expression ( L ) and IFN-γ production ( M ) in MDR/MG lo versus MDR/MG hi CD8 + T cells. ( N ) Representative plots and quantification of MDR/MG hi CD8 + T cells from E0771-Res1 tumors in WT ( n = 5) or C1qa -KO mice ( n = 4). ( O ) Growth of E0771-Res2 tumors in WT or C1qa -KO mice under vehicle or combined therapy. The values 0.2266 and 1.5 × 10 4 indicate represent P values for the comparison of tumor volumes between the combined paclitaxel and anti–PD-1 treatment group and the vehicle control at day 18, in wild-type and C1qa knockout mice, respectively. ( P ) Tumor growth of E0771-Res1 tumors in CCR2-KO mice receiving WT or C1qa -KO monocytes under combined therapy ( n = 5). ( Q ) Flow cytometry of CD8 + T cells from tumors in P . Significance was calculated using 1-way ANOVA followed by Tukey’s test ( C – G , and I ); unpaired 2-tailed Student’s t test ( A and L – Q ). * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: Single cells were then subjected to red blood cell lysis, and tumor cells were isolated using the Mouse Tumor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Western Blot, Comparison, Control, Knock-Out, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: Inflammation- and resolution-programmed myeloid circuits govern therapeutic resistance in epithelial and mesenchymal triple-negative breast cancer

doi: 10.1172/JCI198815

Figure Lengend Snippet: ( A ) Immunoblot of ELOVL5 protein expression in E0771-Res1 and E0771-Res2 cells with Elvol5 shRNA transduction. GAPDH serves as loading control. ( B ) ELISA quantification of RvD1 in BMDMs cocultured with PTX-treated E0771-Res1 or E0771-Res2 cells transduced with control, Elovl5 shRNA#1, or shRNA#2. ( C ) ELISA analysis of RvD1 in TAMs isolated from E0771-Res1 cells transduced with control, Elovl5 shRNA#1, or shRNA#2 tumors. Each data point represents TAMs isolated from an individual tumor ( n = 3). ( D and E ) Growth of E0771-Res1 tumor ( D ), E0771-Res2 tumor ( E ), and their derivatives with Elovl5 shRNA under vehicle or combined treatment. Significance was calculated using unpaired 2-tailed Student’s t test. ( F ) Flow cytometry of immune infiltrates in E0771-Res1 tumors transduced with control, Elovl5 shRNA#1, or shRNA#2 at endpoint. Log 2 fold change relative to the average cell number of the vehicle-treated tumor group is displayed. Each square represents a cell type within an individual tumor. ( G ) Quantification of CD8 + T cells from tumors in D . ( H ) Immunoblot of ZEB1 expression in E0771, E0771-Res1, and E0771-Res1 cells with induced microRNA200c expression. β-ACTIN served as loading control. ( I ) Relative murine Elovl5 expression in E0771-Res1 cells without or with microRNA200c induction ( n = 3). Statistical significance was calculated using unpaired 2-tailed Student’s t test. ( J ) ELISA quantification of DHA in E0771, E0771-Res1, and E0771-Res1 cells with induced microRNA200c expression ( n = 3). ( K ) Tumor growth of E0771-Res1 without or with microRNA200c induction under control or PTX plus anti–PD-1 antibody treatment. Numbers indicate cured/total mice in each group. Statistical significance of tumor volume at day 18 was assessed using 1-way ANOVA followed by Tukey’s test. ( B , C , G , and J ) Statistical significance was determined using 1-way ANOVA followed by Tukey’s test.

Article Snippet: Single cells were then subjected to red blood cell lysis, and tumor cells were isolated using the Mouse Tumor Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Western Blot, Expressing, shRNA, Transduction, Control, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry