Journal: Cancer Biology & Medicine

Article Title: Nuclear PHGDH regulates macrophage polarization through transcriptional repression of GLUD1 and GLS2 in breast cancer

doi: 10.20892/j.issn.2095-3941.2024.0398

Figure Lengend Snippet: Inhibition of PHGDH enhances the M2-like macrophage polarization phenotype and immunosuppressive function. (A, B) Overexpression and knockdown of PHGDH validated at the mRNA level by qRT-PCR and the protein level by Western blot in THP1 cells. (C, D) Validation of overexpression and knockdown of PHGDH at the mRNA level by qRT-PCR and the protein level by Western blot in BMDM cells. (E, F) Effects of PHGDH overexpression or knockdown in tumor-associated macrophages on polarization function and (G, H) immunosuppressive function ( t -test, * P < 0.05, ** P < 0.01, *** P < 0.001) . (I) Correlation analyses of the transcriptional levels of macrophage-associated markers ( TGF-β and TNF-α ) with PHGDH in human breast cancer, according to data from the TCGA database. (J–L) PHGDH overexpressing BMDMs (OE-PHGDH) and control BMDMs (OE-NC) were mixed with EO771 cells and grafted into C57BL/6 mice ( n = 7). (J) Schematic diagram of the cell transfer experiment and tumor image. (K) Growth curve and tumor weight ( t -test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant). (L) Representative immunohistochemistry images of paraffin-embedded xenograft sections. Scale bar, 20 μm.

Article Snippet: The mouse breast cancer cell line EO771 (ATCC, Cat. CRL-3461) was obtained from the ATCC and grown according to the standard protocols on the ATCC website.

Techniques: Inhibition, Over Expression, Knockdown, Quantitative RT-PCR, Western Blot, Biomarker Discovery, Control, Immunohistochemistry

Journal: Cancer Cell International

Article Title: EO771, the first luminal B mammary cancer cell line from C57BL/6 mice

doi: 10.1186/s12935-020-01418-1

Figure Lengend Snippet: EO771 cells display a luminal B phenotype. a – d The relative expression of mRNA coding for ERα ( a ), ERβ ( b ), PR ( c ) and ERBB2 ( d ) was evaluated on MCF-7, MDA-MB-231 and EO771 cells. The values are normalized to the GAPDH gene expression. The data from MCF-7 were set to 1 and the relative quantity of mRNA is shown. P values of < 0.05 (*) using a Wilcoxon-Mann Whitney test indicate a significant difference. E–H: ERα ( e ), ERβ ( f ), PR ( g ) and ERBB2 ( h ) protein levels was assayed by western blot on MCF-7, MDA-MB-231 and EO771 cells (representative of 3 experiments) and normalized to the GAPDH or actin protein levels

Article Snippet: Mouse mammary cancer cell line EO771 (CH3 BioSystems, Amherst, NY), human triple negative breast cancer cell line MDA-MB-231 (American Type Culture Collection (ATCC), Molsheim, France) and human luminal A breast cancer cell line MCF-7 (ATCC) were grown in DMEM supplemented with foetal calf serum (10%), L-Glutamine (1%) and penicillin / streptomycin (1%) at 37 °C in 5% CO 2 .

Techniques: Expressing, Gene Expression, MANN-WHITNEY, Western Blot

Journal: Cancer Cell International

Article Title: EO771, the first luminal B mammary cancer cell line from C57BL/6 mice

doi: 10.1186/s12935-020-01418-1

Figure Lengend Snippet: Anti-estrogenic treatments reduce EO771 cell viability. MCF-7, MDA-MB-231 and EO771 cells were treated with increasing tamoxifen ( a , e and i ), 4-OH-tamoxifen ( b , f and j ), endoxifen ( c , g and k ) and fulvestrant ( d , h and l ) concentrations for 48 h ( a – d ), 72 h (E–H) and 96 h ( i – l ). Cell viability was measured by fluorescence using resazurin solution. The untreated condition corresponds to a viability of 100%

Article Snippet: Mouse mammary cancer cell line EO771 (CH3 BioSystems, Amherst, NY), human triple negative breast cancer cell line MDA-MB-231 (American Type Culture Collection (ATCC), Molsheim, France) and human luminal A breast cancer cell line MCF-7 (ATCC) were grown in DMEM supplemented with foetal calf serum (10%), L-Glutamine (1%) and penicillin / streptomycin (1%) at 37 °C in 5% CO 2 .

Techniques: Fluorescence

Journal: Cancer Cell International

Article Title: EO771, the first luminal B mammary cancer cell line from C57BL/6 mice

doi: 10.1186/s12935-020-01418-1

Figure Lengend Snippet: Estradiol and leptin do not influence EO771 cell phenotype and viability. a , b ERα, ERβ and PR protein levels was assayed by western blot on EO771 cells (representative of 3 experiments) and normalized to the actin protein levels in presence of estradiol (Low: 1.5 ng/mL; High: 225 ng/mL) or leptin (Low: 10 ng/mL; High: 100 ng/mL) in untreated conditions (DMSO) ( a ) or with tamoxifen ( b ). c , d Cell viability was measured by fluorescence using resazurin solution. EO771 cells were cultured in the presence of estradiol, leptin or vehicle ( c ) and EO771 cells were treated with increasing tamoxifen concentration ( d ). The untreated conditions corresponded to a viability of 100%. The tamoxifen IC50 corresponded to the tamoxifen concentration inducing 50% of EO771 viability ( e )

Article Snippet: Mouse mammary cancer cell line EO771 (CH3 BioSystems, Amherst, NY), human triple negative breast cancer cell line MDA-MB-231 (American Type Culture Collection (ATCC), Molsheim, France) and human luminal A breast cancer cell line MCF-7 (ATCC) were grown in DMEM supplemented with foetal calf serum (10%), L-Glutamine (1%) and penicillin / streptomycin (1%) at 37 °C in 5% CO 2 .

Techniques: Western Blot, Fluorescence, Cell Culture, Concentration Assay

Journal: Cancer Cell International

Article Title: EO771, the first luminal B mammary cancer cell line from C57BL/6 mice

doi: 10.1186/s12935-020-01418-1

Figure Lengend Snippet: Tamoxifen modifies intracellular signalling pathways in EO771 cells. EO771 cells were treated by the tamoxifen IC50 (14 µM) or vehicle (DMSO) for 48 h. The expression of both total and phosphorylated forms of signalling pathway proteins (CREB, JNK, NFκB, p38, ERK1/2, AKT, p70S6K, STAT3 and STAT5) and MMP3 was analysed by measure of mean of fluorescence intensity (MFI) using a Luminex system. P values of < 0.05 (*) using a Wilcoxon-Mann Whitney test indicate a significant difference

Article Snippet: Mouse mammary cancer cell line EO771 (CH3 BioSystems, Amherst, NY), human triple negative breast cancer cell line MDA-MB-231 (American Type Culture Collection (ATCC), Molsheim, France) and human luminal A breast cancer cell line MCF-7 (ATCC) were grown in DMEM supplemented with foetal calf serum (10%), L-Glutamine (1%) and penicillin / streptomycin (1%) at 37 °C in 5% CO 2 .

Techniques: Expressing, Fluorescence, Luminex, MANN-WHITNEY

Journal: Cancer Cell International

Article Title: EO771, the first luminal B mammary cancer cell line from C57BL/6 mice

doi: 10.1186/s12935-020-01418-1

Figure Lengend Snippet: The EO771 phenotype and their sensitivity to tamoxifen. EO771 cells express ERβ, PR and HER2. In presence of estrogens, ERβ are translocated to the nucleus where they bind to the estrogen response elements (ERE) inducing transduction of target genes. Anti-estrogen treatments, such as tamoxifen, block this effect. On the contrary, tamoxifen induces the activation of p38 and JNK pathway, leading to apoptosis, but also increases the MMP3 level

Article Snippet: Mouse mammary cancer cell line EO771 (CH3 BioSystems, Amherst, NY), human triple negative breast cancer cell line MDA-MB-231 (American Type Culture Collection (ATCC), Molsheim, France) and human luminal A breast cancer cell line MCF-7 (ATCC) were grown in DMEM supplemented with foetal calf serum (10%), L-Glutamine (1%) and penicillin / streptomycin (1%) at 37 °C in 5% CO 2 .

Techniques: Transduction, Blocking Assay, Activation Assay

Journal: Scientific Reports

Article Title: Photon versus carbon ion irradiation: immunomodulatory effects exerted on murine tumor cell lines

doi: 10.1038/s41598-020-78577-8

Figure Lengend Snippet: Radiation survival curves and RBE calculation for EO771 and PDA30364/OVA cells in response to photon or carbon ion irradiation. Clonogenic survival assays applying graded doses of photon or carbon ion irradiation to EO771 cells ( a ) and PDA30364/OVA cells ( b ). Mean values ± SD of triplicates from one (EO771) or two (PDA30364/OVA) independent experiments are shown. Physical doses of carbon ions biologically defined as equivalent to 1, 3, 5 and 10 Gy photon radiation applying dose-dependent RBEs are shown in ( c ).

Article Snippet: The breast cancer cell line EO771 was cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, USA) containing 10% heat-inactivated FCS, 10 mmol/l HEPES (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific) and 100 μg/ml streptomycin (Thermo Fisher Scientific).

Techniques: Irradiation

Journal: Scientific Reports

Article Title: Photon versus carbon ion irradiation: immunomodulatory effects exerted on murine tumor cell lines

doi: 10.1038/s41598-020-78577-8

Figure Lengend Snippet: Cell cycle analysis of EO771 and PDA30364/OVA cells after photon or carbon ion irradiation. Quantification of cell cycle stages within EO771 cells ( a ) and PDA30364/OVA cells ( b ) 12, 36 and 60 h after irradiation with photons ( a , left and Ref. ) or carbon ions ( a , right and b ) biologically equivalent to the indicated photon doses. DNA content was determined by propidium iodide staining followed by flow cytometric analysis. Representative results of one out of two independent experiments performed are presented.

Article Snippet: The breast cancer cell line EO771 was cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, USA) containing 10% heat-inactivated FCS, 10 mmol/l HEPES (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific) and 100 μg/ml streptomycin (Thermo Fisher Scientific).

Techniques: Cell Cycle Assay, Irradiation, Staining

Journal: Scientific Reports

Article Title: Photon versus carbon ion irradiation: immunomodulatory effects exerted on murine tumor cell lines

doi: 10.1038/s41598-020-78577-8

Figure Lengend Snippet: Cell surface expression of immunomodulatory molecules on EO771 cells irradiated with photons or carbon ions. Flow cytometric analysis of PD-L1, CD73 and MHC-I cell surface expression on EO771 cells 12 and 36 h after irradiation with graded doses of photons ( a ) or biologically equivalent doses of carbon ions ( b ). Depicted are fold changes of MFI (mean fluorescence intensity) normalized to MFI of non-irradiated cells. At least 40,000 viable cells were acquired per sample. Results of two experiments performed are shown (squares: experiment 1; dots: experiment 2).

Article Snippet: The breast cancer cell line EO771 was cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, USA) containing 10% heat-inactivated FCS, 10 mmol/l HEPES (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific) and 100 μg/ml streptomycin (Thermo Fisher Scientific).

Techniques: Expressing, Irradiation, Fluorescence

Journal: Scientific Reports

Article Title: Photon versus carbon ion irradiation: immunomodulatory effects exerted on murine tumor cell lines

doi: 10.1038/s41598-020-78577-8

Figure Lengend Snippet: Irradiation enhances susceptibility of EO771/Luci/OVA cells to CTL recognition. Irradiation of EO771/Luci/OVA cells with increasing doses of photons ( a ) or carbon ions ( c ) enhanced cytolysis of target cells as measured by luciferase-based cytotoxicity assay, but did not affect IFNγ secretion of OVA-specific CTLs in IFNγ ELISpot assays ( b , d ). Representative results of one out of three (photons) and two (carbon ions) independent experiments performed are shown.

Article Snippet: The breast cancer cell line EO771 was cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, USA) containing 10% heat-inactivated FCS, 10 mmol/l HEPES (Thermo Fisher Scientific), 100 U/ml penicillin (Thermo Fisher Scientific) and 100 μg/ml streptomycin (Thermo Fisher Scientific).

Techniques: Irradiation, Luciferase, Cytotoxicity Assay, Enzyme-linked Immunospot

Journal: Journal of Translational Medicine

Article Title: Pharmaceutical targeting Th2-mediated immunity enhances immunotherapy response in breast cancer

doi: 10.1186/s12967-022-03807-8

Figure Lengend Snippet: IPD enhances the antitumor activity of cytotoxic CD8 + T cells in breast cancer. A Representative contour plots and quantification of CD8 + T cells ratios in EO771 and 4T1 tumors (n = 5, t test). B , C Flow cytometry analysis of IFN-γ + and TNF-α + CD8 + T cells from EO771 tumor (B) and 4T1 tumor (C) (n = 5, t test). D Representative immunofluorescence images of CD8 + T cells (green) and granzyme B (red) in EO771 tumors. Scale bar, 25 μm. E , F Representative histogram of Ki-67 expression and percentages of Ki-67 + CD8 + cells in EO771 (E) and 4T1 (F) tumors (n = 5, t test). (G and H) Representative contour plots of PD-1 + Tim-3 + CD8 + T cells of EO771 (G) and 4T1 (H) tumors. Mean ± SEM; ** p < 0.01; *** p < 0.001; ns, not significant

Article Snippet: MDA-MB-231 cell (human breast cancer cell line), EO771 cells (a C57BL/6 murine breast cancer cell line), EMT6 cells (a BALB/c murine breast cancer cell line), and 4T1 cells (a BALB/c murine breast cancer cell line) were obtained from the American Type Culture Collection.

Techniques: Activity Assay, Flow Cytometry, Immunofluorescence, Expressing

Journal: Journal of Translational Medicine

Article Title: Pharmaceutical targeting Th2-mediated immunity enhances immunotherapy response in breast cancer

doi: 10.1186/s12967-022-03807-8

Figure Lengend Snippet: Inhibiting Th2-mediated immunity reshapes the immune landscape in the TME. A , B Representative quantification of myeloid-derived suppressor cell (MDSC), tumor-associated macrophage (TAM), and regulatory T cell (Treg) of CD45 + live cells in EO771 (A) and 4T1 (B) tumors (n = 5, two-way ANOVA). C T-SNE plot of TILs overlaid with color-coded clusters in EO771 tumors. D T-SNE plot of tumor-infiltrating leukocytes overlaid with color-coded clusters from vehicle-treated or IPD-treated mice. E Frequency of clusters of indicated immune cell subsets from vehicle-treated and IPD-treated mice (n = 5, t-test). F Macrophage-associated density t-SNE plots and quantification of an equal number of CD45 + tumor-infiltrating leukocytes in vehicle-treated versus IPD-treated mice. G , H Relative MHC II:CD206 ratio of TAMs in EO771 and 4T1 tumors from vehicle-treated versus IPD-treated mice (n = 5, t test). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant

Article Snippet: MDA-MB-231 cell (human breast cancer cell line), EO771 cells (a C57BL/6 murine breast cancer cell line), EMT6 cells (a BALB/c murine breast cancer cell line), and 4T1 cells (a BALB/c murine breast cancer cell line) were obtained from the American Type Culture Collection.

Techniques: Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Pharmaceutical targeting Th2-mediated immunity enhances immunotherapy response in breast cancer

doi: 10.1186/s12967-022-03807-8

Figure Lengend Snippet: IPD synergizes with anti-PD1 treatment in breast cancer. A EO771 (left) and 4T1 (right) tumor growth treated with vehicle, IPD, anti-PD1, or IPD + anti-PD1 (n = 5, two-way ANOVA). B Kaplan-Meir survival analysis of 4T1 tumor-bearing mice with indicated treatments (n = 10, log-rank test). C Lung metastatic nodules from 4T1 tumor-bearing mice treated with indicated treatments (n = 5, one-way ANOVA). D Quantification of CD8 + T cells ratios in EO771 and 4T1 tumors treated with indicated treatments (n = 5, one-way ANOVA). E , F Representative flow cytometry images and percentages of IFN-γ + CD8 + T cells from EO771 (E) and 4T1 (F) tumors (n = 5, one-way ANOVA). Mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: MDA-MB-231 cell (human breast cancer cell line), EO771 cells (a C57BL/6 murine breast cancer cell line), EMT6 cells (a BALB/c murine breast cancer cell line), and 4T1 cells (a BALB/c murine breast cancer cell line) were obtained from the American Type Culture Collection.

Techniques: Flow Cytometry