py8119 mouse breast cancer cells  (ATCC)

Journal: bioRxiv

Article Title: Single-cell profiling of synchronous multi-organ metastasis reveals a systemic CD74 + lipid-associated macrophage niche driving polymetastatic breast cancer

doi: 10.64898/2026.01.31.701004

Figure Lengend Snippet: a. qPCR analysis of Mif expression in VO-PyMT cells transduced with control or two independent Mif-targeting short hairpin RNAs (shRNA). Bar heights show means and error bars depict standard deviation of technical triplicates. Data was reproduced in 3 independent experiments. b. Immunoblot of Mif protein in shControl- or shMif-transduced VO-PyMT cells. B-Actin is shown as loading control. Data is representative of two independent experiments. c. MIF protein concentration in cell culture supernatants of shControl and shMif-transduced VO-PyMT cells as determined by enzyme linked immunosorbent assay (ELISA). Bar heights depict means and error bars show standard deviation of two technical replicates. d. Relative Mif expression levels in Py8119 cells harboring an shControl or shMif hairpin, as determined by qPCR. Error bars show standard deviations. Data is representative of 3 separate experiments. e. Western blot analysis of MIF protein in shControl or shMif Py8119 cells. B-Actin is shown as loading control. Data is representative of two independent experiments. f. Number of spheres per well quantified in in vitro spheroid cultures of VO-PyMT or Py8119 cells upon Mif knockdown. Sphere count was performed 7 days post seeding. Ns, not significant. Two-tailed Student’s t tests were used to estimate significance. g. Representative images of spheroids quantified in (f). Scale bars: 1 mm.

Article Snippet: Py8119 mouse breast cancer cells were purchased from ATCC (cat. no. CRL-3278), and cultured in F-12K medium supplemented with 2 mM L-Glutamine, 1,500 mg/L sodium bicarbonate, 10% vol/vol FBS, and P/S.

Techniques: Expressing, Transduction, Control, shRNA, Standard Deviation, Western Blot, Protein Concentration, Cell Culture, Enzyme-linked Immunosorbent Assay, In Vitro, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Single-cell profiling of synchronous multi-organ metastasis reveals a systemic CD74 + lipid-associated macrophage niche driving polymetastatic breast cancer

doi: 10.64898/2026.01.31.701004

Figure Lengend Snippet: a. Quantification of metastatic burden using ex vivo BLI of FVB mice injected VO-PyMT cells transduced with control vectors (shControl) and Mif-knockdown vectors (shMif 1; shMif 2). Data was acquired at day 12 post i.c. injections. shControl, n = 11; shMif (1), n = 11; shMif (2), n = 5. Data represents results from 2 independent experiments. In each experiment, raw photon flux values (p/s) from each organ were normalized to the average photon flux (p/s) values of the shControl group. Boxes depict 25th and 75th percentiles. Median is shown as horizontal lines. Whiskers depict data range. All data points are shown as dots. P values were determined using two-tailed Mann-Whitney tests by comparing shControl ( n = 11) versus the two shMif groups combined ( n = 16). Images show representative ex vivo BLI in brain, lung, liver or lower limb bones from each experimental group. b. Quantification of multi-organ metastatic burden ex vivo using BLI in albino C57/BL6 mice injected intracardially with Py8119 breast cancer cells transduced with control (shControl) and Mif-knockdown vector (shMif 2). Representative images are shown for each organ analyzed. Ex vivo metastatic burden was quantified 10 days after cancer cell injection. shControl, n = 14; shMif (2), n = 14. Data is representative of 3 independent experiments. Data in each organ was normalized to the average photon flux (p/s) of the shControl group in each experiment. Boxes boundaries define the interquartile ranges. Horizontal lines depict median values in each group. The whiskers show data range. The dots depict data points. P values were calculated by one-tailed Mann-Whitney t tests. c. Schematic showing pre-clinical model of MIF-CD74 targeting using 4-IPP, a small molecule inhibitor of MIF binding capacity. Multi-metastatic VO-PyMT cells were injected intracardially at day 0. Seeding and micrometastatic growth was allowed for 3 days, followed by i.p. administration of either a vehicle solution or 4-IPP. At day 12 post i.c. injection of VO-PyMT cells, metastatic colonization was analyzed ex vivo in brain, lung, liver and lower limb bones. d. Box plots showing quantification of metastatic burden of experiment as determined by ex vivo BLI in brain, lung, liver and bones of FVB mice injected intracardially with VO-PyMT cells and treated with the MIF inhibitor 4-IPP as described in (c). Vehicle group, n = 14; 4-IPP group, n = 13. For bone metastatic burden, right and left lower limb bones were analyzed separately. Data represents results from 2 independent experiments. In each experiment, photon flux (p/s) in each organ was normalized to the average photon flux (p/s) in the Vehicle control group. Boxes boundaries indicate 25 th and 75 th percentiles. Median is indicated by a horizontal line in boxes. Whiskers show data range. Data points are indicated as dots. P values were calculated using one-tailed Mann-Whitney t tests.

Article Snippet: Py8119 mouse breast cancer cells were purchased from ATCC (cat. no. CRL-3278), and cultured in F-12K medium supplemented with 2 mM L-Glutamine, 1,500 mg/L sodium bicarbonate, 10% vol/vol FBS, and P/S.

Techniques: Ex Vivo, Injection, Transduction, Control, Knockdown, Two Tailed Test, MANN-WHITNEY, Plasmid Preparation, One-tailed Test, Binding Assay

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: ACS Omega

Article Title: Feasibility of a Protease Activity-Based Nanosensor for Breast Cancer Screening

doi: 10.1021/acsomega.5c09454

Figure Lengend Snippet: Catalytic efficiency of MMP-2 on fluorogenic probes and assessment of the ABN toxicity. (a) Schematic showing the catalytic efficiency of MMP-2 on TIAH–LH, immobilized on the surface of microplate wells and incubated with the 50 nM catalytic domain for 2 h at 37 °C. Initial cleavage velocity of the substrate conjugated onto the nanocarrier surface at (b) 32.5 Å and (c) 53.4 Å from the core, highlighting the catalytic efficiency. The lines represent the Michaelis–Menten model fit to the data. mean ± s.e.m. is denoted by a horizontal line with error bars; n = 2 wells per probe concentration; N = 3; R 2 = 0.87; R 2 = 0.99. K cat , catalytic constant; K M , Michaelis–Menten constant. (d–g) Blood levels of hepatotoxicity markers of the mice injected with the ABN were equal to the levels found in the controls. The mean body weight remained stable for 5 d after ABN injection. Each square represents one mouse, and the mean value is denoted by the red bar. The Shapiro–Wilk test is followed by the unpaired two-tailed t -test; * P > 0.05. ALT, alanine aminotransferase; AST, aspartate aminotransferase. (h, i) Viability of 4T1 and ID8 cells after incubation with the ABN at different concentrations for 24 h. mean ± s.e.m. is denoted by a horizontal line with error bars; n = 2–3 wells per condition; N = 3; one-way ANOVA with Bonferroni multiple comparisons; ** P < 0.05. Ctrl, positive control: acetaminophen at 25 mM in cell culture media.

Article Snippet: Mouse breast cancer cell 4T1 was cultured in ATCC-formulated RPMI medium supplemented with 10% fetal bovine serum and mouse ovarian surface epithelial cell ID8 in high glucose DMEM supplemented with 4% fetal bovine serum, 5 μg mL –1 insulin and transferrin, and 5 ng mL –1 sodium selenite.

Techniques: Incubation, Concentration Assay, Injection, Two Tailed Test, Positive Control, Cell Culture

Journal: ACS Omega

Article Title: Feasibility of a Protease Activity-Based Nanosensor for Breast Cancer Screening

doi: 10.1021/acsomega.5c09454

Figure Lengend Snippet: ABN performance in the 4T1 mammary tumor model. (a) Kinetics of the ABN signal in the plasma of mice bearing a four-week-old tumor. The lines denote the behavior predicted by a one-phase exponential decay model. Values are represented as mean ± s.e.m; n = 2–4 mice; R 2 = 0.99, tumor; R 2 = 0.98, nontumor. (b) Kinetics of the biomarker signal in the plasma of tumor-bearing mice versus normal counterparts, indicating the exacerbation of protease activity in the blood. Lines denote the kinetic behavior by a one-phase exponential decay model. Values are represented as mean ± s.e.m.; n = 2–4 mice; R 2 = 0.78, tumor; R 2 = 0.74, nontumor.

Article Snippet: Mouse breast cancer cell 4T1 was cultured in ATCC-formulated RPMI medium supplemented with 10% fetal bovine serum and mouse ovarian surface epithelial cell ID8 in high glucose DMEM supplemented with 4% fetal bovine serum, 5 μg mL –1 insulin and transferrin, and 5 ng mL –1 sodium selenite.

Techniques: Clinical Proteomics, Biomarker Discovery, Activity 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