mouse breast cancer cell line 4t1  (ATCC)

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 2.5 × 10 5 CT26 cells were subcutaneously injected into BALB/c mice for 11 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) five times at three-day intervals ( n = 5). The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 5). B The resected tumors from the CT26 tumor-bearing mice were analyzed by immunohistochemical analysis. ** p < 0.01. Unpaired t test ( n = 5). C A total of 2.5 × 10 5 CT26 cells were intravenously injected into BALB/c mice for 11 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 40 mg/kg) five times at three-day intervals ( n = 5). The tumor volume was recorded every three days. The resected lung tissues were analyzed by HE staining. *** p < 0.001. Unpaired t test ( n = 5). D 4T1 cells (5 × 105) were intravenously injected into BALB/c mice, which were then intraperitoneally administered anti-ENO1 antibodies (HuL001, 40 mg/kg) on the indicated days (n = 5). The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 5). E The resected tumors from 4T1-bearing mice were analyzed by HE staining. ** p < 0.01. Unpaired t test ( n = 5). F The resected lung tissues from 4T1-bearing mice were analyzed by HE staining, immunofluorescence staining and immunohistochemical analysis. ** p < 0.01 and *** p < 0.001. Unpaired t test ( n = 5). G The resected liver tissues from 4T1-bearing mice were analyzed by HE staining, immunofluorescence staining and immunohistochemical analysis. *** p < 0.001. Unpaired t test ( n = 5). H The density of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors from CT26- and 4T1-bearing mice was evaluated by immunofluorescence staining ( n = 3). I The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors from CT26-bearing mice. * p < 0.05 and *** p < 0.001. Unpaired t test ( n = 5). J The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors from 4T1-bearing mice. * p < 0.05 and *** p < 0.001. Unpaired t test ( n = 5). K The DEGs between HuL001- and Veh-treated resected CT26 tumors are shown in a volcano plot ( n = 2). L Compared with those in Veh.-treated resected tumors, the number of glycolysis-related gene sets significantly decreased in HuL001-treated resected tumors ( n = 2).

Article Snippet: The mouse colon cancer cell line CT26, mouse breast cancer cell line 4T1, human CRC cell lines (SW480, SW620, HCT116, LoVo and HT29) and human breast cancer cell lines (MDA-MB-468, BT-20, HS578T and SUM-159) were obtained from the ATCC.

Techniques: Injection, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 5 × 10 5 CT26 cells were subcutaneously injected into the left legs of BALB/c mice for 5 days and then intraperitoneally administered with anti-ENO1 antibodies (HuL001, 40 mg/kg) or clodronate liposomes (50 μL/mouse) on the indicated days ( n = 5). Local radiotherapy was given on Day 10. The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 4). B The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05 and *** p < 0.001. One-Way ANOVA test ( n = 3). C 4T1 cells (5 ×10 4 ) were subcutaneously injected into the left legs of BALB/c mice for 4 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) six times on the indicated days ( n = 5). Local radiotherapy was given on Days 10 and 12. The tumor volume was recorded every three days. * p < 0.05 and *** p < 0.001. Two-Way ANOVA test ( n = 4). D The resected tumors were weighed on Day 40. * p < 0.05. One-Way ANOVA test ( n = 4). E The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. F The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05. One-Way ANOVA test ( n = 3). G The frequencies of M1 (CD11c + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) and M2 (CD1206 + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) tumor-infiltrating macrophages were analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). H The quantification of the M1/M2 ratio is shown. * p < 0.05. One-Way ANOVA test ( n = 3). I A representative image of flow cytometric analysis of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells. J The frequency of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells was analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). K The density of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown. * p < 0.05. One-Way ANOVA test ( n = 3-4). L The density of IFNγ + (IFNγ hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown ( n = 3-4). * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3-4). M The proposed mechanism of TGFβ1/TGFβR/Smad3/PRMT5-mediated ENO1 translocation for lactate release via MCT4.

Article Snippet: The mouse colon cancer cell line CT26, mouse breast cancer cell line 4T1, human CRC cell lines (SW480, SW620, HCT116, LoVo and HT29) and human breast cancer cell lines (MDA-MB-468, BT-20, HS578T and SUM-159) were obtained from the ATCC.

Techniques: Injection, Liposomes, Immunofluorescence, Staining, Flow Cytometry, Translocation Assay

Journal: Frontiers in Immunology

Article Title: FACS-based genome-wide CRISPR screening platform identifies modulators of CD47

doi: 10.3389/fimmu.2025.1684539

Figure Lengend Snippet: Genome-wide CRISPR-Cas9 screen identifies genetic regulators of CD47 expression in murine cancer cell lines. (A) Schematic of the FACS-based CRISPR screening pipeline. Mouse cancer cells (B16F10A, MC38, and EMT6) were transduced with the mTKO genome-wide CRISPR-Cas9 library, followed by puromycin selection and passaging for 5 days to ensure stable sgRNA integration. Cells were stained with fluorophore-conjugated anti-CD47 antibodies and sorted by FACS into the top 30% (CD47 high ) and bottom 30% (CD47 low ) populations. Genomic DNA was extracted, and sgRNA abundance was determined by NGS and analyzed using DrugZ to calculate NormZ scores. (B) Ranked NormZ scores of sgRNAs in B16F10A, MC38, and EMT6 cells. Negative NormZ scores indicate positive regulators of CD47 (gene knockouts reduce CD47 expression), while positive NormZ scores indicate negative regulators (gene knockouts increase CD47 expression). CD47 itself ranked as the top positive regulator in all three cell lines, validating the screen’s robustness. (C) GO enrichment analysis of significant negative regulators of CD47 expression (|NormZ| > 3) in B16F10A and MC38 cells. Negative regulators were significantly enriched in pathways related to mitochondrial signaling, including oxidative phosphorylation, mitochondrial translation, and respiratory chain complex assembly.

Article Snippet: Mouse melanoma cell line B16F10A, mouse colon cancer cell line MC38, mouse breast cancer cell line EMT6, two mouse macrophage cells J774A-1, RAW264.7 are purchased from the American Type Culture Collection (ATCC).

Techniques: Genome Wide, CRISPR, Expressing, Transduction, Selection, Passaging, Staining, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: FACS-based genome-wide CRISPR screening platform identifies modulators of CD47

doi: 10.3389/fimmu.2025.1684539

Figure Lengend Snippet: DNAJC13 is a conserved positive regulator of CD47 expression identified across multiple cancer cell lines. (A) Venn diagram of significant positive regulators of CD47 expression identified in the genome-wide CRISPR screen. CD47 and DNAJC13 were the only two genes consistently identified as significant positive regulators (|NormZ| > 3) across all three cell lines (B16F10A, MC38, and EMT6). (B) Correlation of DNAJC13 and CD47 expression in human cancers. Scatter plots generated from TCGA datasets via GEPIA2 show a positive correlation between DNAJC13 and CD47 expression in breast cancer (BRCA), colon adenocarcinoma (COAD), and skin cutaneous melanoma (SKCM). Pearson correlation coefficients (r) and p-values are indicated. (C) Western blot validation of DNAJC13 regulation of CD47 expression. CRISPR-Cas9–mediated DNAJC13 knockout (three independent sgRNAs: DNAJC13#1, #2, #3) markedly reduced CD47 protein levels compared with vector control in B16F10A, MC38, and EMT6 cells. GAPDH was used as a loading control. (D) Flow cytometry analysis of surface CD47 expression. Representative FACS histograms show reduced surface CD47 expression in DNAJC13 KO cells (green, yellow, and orange peaks; three independent sgRNAs) compared with vector controls (red) in B16F10A, MC38, and EMT6 cells. Isotype control is shown in blue. Quantification of Mean fluorescence intensity (MFI) is shown on the right.

Article Snippet: Mouse melanoma cell line B16F10A, mouse colon cancer cell line MC38, mouse breast cancer cell line EMT6, two mouse macrophage cells J774A-1, RAW264.7 are purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Genome Wide, CRISPR, Generated, Western Blot, Biomarker Discovery, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: FACS-based genome-wide CRISPR screening platform identifies modulators of CD47

doi: 10.3389/fimmu.2025.1684539

Figure Lengend Snippet: DNAJC13 negatively correlates with macrophage infiltration and regulates macrophage-mediated phagocytosis. (A) Correlation between gene expression and macrophage infiltration in human cancers. Scatter plots generated using TIMER2 show negative correlations between CD47 expression (top row) or DNAJC13 expression (bottom row) and macrophage infiltration levels in breast cancer (BRCA), colon adenocarcinoma (COAD), and skin cutaneous melanoma (SKCM). Spearman’s correlation coefficients (Rho) and p-values are indicated. (B, C) DNAJC13 loss enhances macrophage phagocytosis in vitro . Flow cytometry analysis of macrophage-mediated phagocytosis in co-culture assays. DNAJC13 knockout (KO) or CD47 KO cancer cells (B16F10A, MC38, EMT6) were co-cultured with (B) RAW 264.7 or (C) J774 macrophages for the indicated time. Representative FACS plots show increased phagocytosis (higher engulfment rate) in DNAJC13-KO and CD47-KO groups compared with vector controls.

Article Snippet: Mouse melanoma cell line B16F10A, mouse colon cancer cell line MC38, mouse breast cancer cell line EMT6, two mouse macrophage cells J774A-1, RAW264.7 are purchased from the American Type Culture Collection (ATCC).

Techniques: Gene Expression, Generated, Expressing, In Vitro, Flow Cytometry, Co-Culture Assay, Knock-Out, Cell Culture, Plasmid Preparation

Journal: Exploration

Article Title: A Groundbreaking Electric Field‐Induced Cascade Gas Therapy Against Large Volume Solid Tumor Through Electro‐Stress Storm

doi: 10.1002/EXP.20240410

Figure Lengend Snippet: In vitro EGT performance and cytotoxicity of PR@L. (A) CLSM observation of RhB‐labeled PR internalized by 4T1 cells after different culture time. scale bar: 10 µm. (B) Schematic diagram of the experimental device for cytotoxicity evaluation of EGT system. (C) Cytotoxicity of 4T1 cells cultured with various formulations at diverse concentrations for 24 h. (D) Relative cytotoxicity of EGT‐treated 4T1 cells after incubation with different formulations (at 100 µg mL −1 Pt concentration) and (E) PR@L under various square‐wave AC parameters (10 mHz). (F) Intracellular RNS production after diverse treatments with or without electrical stimulation monitored by CLSM (scale bar: 50 µm) and (G) flow cytometer. (H) Intracellular ROS (DCF) and NO (DAF‐FM‐NO) CLSM images and corresponding fluorescence intensity profiles along the arrow (scale bar: 50 µm). n = 3 and * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: 4T1 mouse breast cancer cell line was provided by the American Type Culture Collection.

Techniques: In Vitro, Labeling, Cell Culture, Incubation, Concentration Assay, Flow Cytometry, Fluorescence

Journal: Exploration

Article Title: A Groundbreaking Electric Field‐Induced Cascade Gas Therapy Against Large Volume Solid Tumor Through Electro‐Stress Storm

doi: 10.1002/EXP.20240410

Figure Lengend Snippet: In vivo EGT treatment for solid tumor with large size by electro‐stimulating gas donor‐encapsulated PR. (A) Schematic illustration of in vivo EGT treatment operation. (B) Tumor growth curves of 4T1 tumor‐bearing mice after different treatments ( n = 5). (C) Average tumor weights and (D) mouse weight at the end of the experiment (at day 10, n = 5). (E) Tumor growth curves of individual mouse from each group. (F) Ki67, H&E, and TUNEL staining analyses of tumor tissue slices collected from different groups after treatment. Scale bar: 50 µm. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: 4T1 mouse breast cancer cell line was provided by the American Type Culture Collection.

Techniques: In Vivo, TUNEL Assay, Staining

Journal: Exploration

Article Title: A Groundbreaking Electric Field‐Induced Cascade Gas Therapy Against Large Volume Solid Tumor Through Electro‐Stress Storm

doi: 10.1002/EXP.20240410

Figure Lengend Snippet: EGT‐induced in vivo immunogenic effect boosted tumor immunotherapy. (A) Scheme illustration of immune activation mechanisms of the EGT platform through releasing immunogenic DAMPs and generating PR@L‐mediated electro‐stress storm. (B) Immunofluorescence analysis for in situ CRT exposure in 4T1 tumors from mice after various treatments. Scale bar: 100 µm. (C) Flow cytometric analysis and (D) corresponding quantitative analysis ( n = 3) of DCs maturation (gated on CD11c + CD80 + CD86 + ) in tumor draining lymph node cells collected from mice 10 days after treatments. (E) Immunofluorescence analysis (scale bar: 100 µm), (F) flow cytometric analysis, and (G) corresponding quantitative analysis ( n = 3) of infiltrating CD8 + T cells (gated on CD45 + CD3 + CD8 + ) in tumor tissues from mice after various treatments. (H) ELISA analysis of cytokines in serum from 4T1 tumor‐bearing mice, including TNF‐α, INF‐γ, and IL‐10 ( n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: 4T1 mouse breast cancer cell line was provided by the American Type Culture Collection.

Techniques: In Vivo, Activation Assay, Immunofluorescence, In Situ, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Mapping the Tissue‐of‐Origins of Mesenchymal Stromal Cells in Injury Repair

doi: 10.1002/advs.202509533

Figure Lengend Snippet: BM‐MSC‐derived myofibroblasts or CAFs were not detected in distal tissue fibrosis or tumors. A) Confocal imaging of kidney sections from normal and UUO‐induced renal fibrotic Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Myofibroblasts were indicated with anti‐Col1 antibody staining. B) Confocal imaging of lung sections from normal and bleomycin‐induced pulmonary fibrotic Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Myofibroblasts were indicated with anti‐Col1 antibody staining. C) Confocal imaging of liver sections from normal and CCl 4 ‐induced liver fibrotic Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Myofibroblasts were indicated with anti‐Col1 antibody staining. D) Confocal imaging of liver sections from normal and DDC‐induced liver fibrotic Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Myofibroblasts were indicated with anti‐Col1 antibody staining. E–H) Quantification of the percentages of DAPI + Col1 + myofibroblasts that were ZsGreen + and tdTomato + in renal fibrosis (E), pulmonary fibrosis (F), CCl 4 ‐induced liver fibrosis (G) and DDC‐induced liver fibrosis (H). n = 5 mice from 4 independent experiments. I) Confocal imaging of E0771‐induced subcutaneous tumors from Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Cancer associated fibroblasts (CAFs) were indicated with anti‐Col1 antibody staining. J) Confocal imaging of normal colons and AOM/DSS‐induced colorectal cancer from Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. CAFs were indicated with anti‐Col1 antibody staining. K,L) Quantification of the percentages of DAPI + Col1 + CAFs that were ZsGreen + and tdTomato + in subcutaneous tumors (K) and colorectal cancer (L). n = 3 mice from 3 independent experiments.

Article Snippet: The E0771 mouse breast cancer cell line (CRL‐3461, RRID:CVCL_GR23) was obtained from the American Tissue Type Collection (ATCC) in 2018.

Techniques: Derivative Assay, Imaging, Staining