Journal: BMC Cancer

Article Title: Aberrant methylation of the M-type phospholipase A 2 receptor gene in leukemic cells

doi: 10.1186/1471-2407-12-576

Figure Lengend Snippet: PLA2R1 gene methylations in different human cell lines

Article Snippet: In addition, genomic DNA from Raji (human B-cell leukemia), MCF7 (human mammary adenocarcinoma), and A431 (human melanoma) cell lines was purchased from BioCat GmbH (Heidelberg, Germany).

Techniques: Methylation

Journal: Scientific Reports

Article Title: Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis

doi: 10.1038/srep24730

Figure Lengend Snippet: ( a ) Single MCF-7 cells were extracted with different time, and the vertical axis represents the MS intensity of ADP (m/z 426). ( b ) The mass spectrum of unicellular metabolites.

Article Snippet: MCF-7 (human breast cancer cell) cell lines were purchased from ABGENT.

Techniques:

Journal: Scientific Reports

Article Title: Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis

doi: 10.1038/srep24730

Figure Lengend Snippet: ( A – C ) ATP, ADP and AMP in MCF-7 cells. ( a – c ) Standard solutions of ATP, ADP and AMP with the concentration of 10 μM. ( D – F ) GSH, GSSG and UDP-Glc-NAc in MCF-7 cells. ( d , e ) Standard solutions of GSH and GSSG with the concentration of 10 μM. ( f ) MS/MS spectrum of UDP-Glc-NAc from MassBank database ( http://www.massbank.jp ).

Article Snippet: MCF-7 (human breast cancer cell) cell lines were purchased from ABGENT.

Techniques: Concentration Assay, Tandem Mass Spectroscopy

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) GDC-0810 structure. ( B ) MCF7 ERα In-Cell Western assay comparing GDC-0810 potency to fulvestrant and 4-hydroxytamoxifen. ERα levels are quantified by immunofluorescence assay, in triplicate, 4-hr post compound treatment. Error bars are SEM. ( C ) Western blot analysis assessing the effect of GDC-0810 (100 nM), and fulvestrant (100 nM) ( D ) on ERα levels at 2, 4 or 6 hr of treatment, in the presence or absence of the 26S proteasome inhibitor MG132 (10 μM). ( E ) MCF7 cell viability assay comparing GDC-0810 activity to fulvestrant and 4OH-tamoxifen, in the absence of exogenous estradiol. Viable cells are presented as percent CellTiter-Glo luciferase activity relative to the vehicle control after 5 day compound incubation. Error bars represent standard deviation from the mean, from biological quadruplicates. ( F ) MCF7 cell viability assay comparing GDC-0810 activity to fulvestrant and 4OH-tamoxifen, in the presence of 0.1 nM estradiol. Viable cells are presented as percent CellTiter-Glo luciferase activity relative to the vehicle control after 5 day compound incubation.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: In-Cell ELISA, Immunofluorescence, Western Blot, Viability Assay, Activity Assay, Luciferase, Control, Incubation, Standard Deviation

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) ERα conformational profiling. A mammalian 2-hybrid assay was performed to monitor interaction of ERα with 14 conformation selective peptide probes. Luciferase signal was measured after 24 hr of compound treatment (1 μM for all compounds). Interaction profiles of ERα therapies, GDC-0810 and vehicle control from biological triplicates were analyzed by hierarchical clustering using the Ward algorithm and standardized data. ( B ) Transcriptional activity of benchmark ERα ligands in MCF7 cells, in the absence of exogenous estrogen. Transcriptional activity was monitored using a SERM discriminatory target gene set following 24 hr 1 μM ligand treatment. Data was log2 normalized followed by standardization and hierarchical clustering. ( C ) Doxycycline-inducible MCF7 cells were pre-cultured in estrogen-depleted medium with 100 ng/ml doxycycline for 2 weeks. Cells were then cultured in medium containing 10, 100 and 500 nM fulvestrant or GDC-0810 for 5 days, and the effect on levels of ER, PR and Cyclin D1 were assessed by Western Blot analysis. ( D , E ) Doxycycline-inducible MCF7 cells were pre-cultured in estrogen-depleted medium with 10 ng/ml doxycycline for 2 weeks before treatment. Cells were treated with a range of doses of fulvestrant or GDC-0810 for 7 days in estrogen-depleted medium with or without 0.1 nM E2. Cell viability was determined by CellTiter-Glo assay.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Hybrid Assay, Luciferase, Control, Activity Assay, Cell Culture, Western Blot, Glo Assay

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ERα ChIP was performed on MCF7 cells following 45 min or 4 hr compound incubation. qPCR quantification was performed using oligonsucleotides directed to 10 distinct ERα binding sites. Data for ERα and non-specific IgG control is plotted as a percent of total input.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Incubation, Binding Assay, Control

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) Tamoxifen-sensitive MCF7 tumor bearing animals were dosed with vehicle, fulvestrant (50 mg/kg on days 1, 3, 8; then 25 mg/kg 2x/week, s.c.) or GDC-0810 (1, 10, 100 mg/kg/day, p.o.) for 28 days in the presence of 60-day release 0.36 mg 17β-estradiol pellets. ( B , C ) Gene expression analysis of tumors treated with 100 mg/kg GDC-0810, compared to tumors in the presence or absence of estrogen pellets. Tumors were harvested on day 28 of the study; this was a separate study from that shown in ( A ). GDC-0810 plasma concentration is also shown. *p<0.05, n = 3. See for an extended panel of genes. ( D ) Representative FES-PET images of MCF7 tumors in the right dorsum (arrow) of mice treated with vehicle or GDC-0810 (100 mg/kg). Images were taken 1–2 hr after the dosing on the seventh day of treatment. ( E ) Percent change in FES SUVR after 6 days of treatment. Each bar represents the mean percent change in 18 F-labeled estradiol SUVR. Vehicle-treated mice exhibited an average increase of SUVR of 26.1% whereas mice treated with 10 mg/kg and 100 mg/kg exhibited a 45.2% and 63.3% reduction in SUVR, respectively, compared to baseline (*p<0.0001 vs. vehicle). ( F ) HCI-003 patient derived xenograft tumors were implanted in mice containing a 1 mg 17β-estradiol beeswax pellet. Tumor bearing animals were dosed with vehicle, fulvestrant (200 mg/kg, 3x/week, s.c.), GDC-0810 (10 or 100 mg/kg/day, p.o.) for 43 days. One vehicle treated group had the 17β-estradiol pellets removed at treatment start to assure growth dependence on estradiol.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Gene Expression, Clinical Proteomics, Concentration Assay, Labeling, Derivative Assay

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) Tamoxifen and fulvestrant anti-tumor activity in MCF7 xenograft. Crl:NU-Foxn1 nu mice implanted with 17-β Estradiol pellets (0.72 mg/pellet/60 days, Innovative Research of America) were injected with 1 × 10 7 MCF7 cells. 8 days later, animals bearing viable tumors were dosed orally with Vehicle (9% Peg-400:0.5% Tween–80:0.5% Povidone:90% 0.5% Carboxymethylcellulose) or tamoxifen (60 mg/kg/day), or subcutaneously with fulvestrant (200 mg/kg, 3x/week, 10% ETOH:Castor Oil) for 28 days. * Denotes significance (p<0.05) compared to Vehicle (+E2) in 1-Way ANOVA and Dunnett's Multiple Comparison Test. ( B ) Gene expression analysis, assessed using Fluidigm, of MCF7 tumors treated with GDC-0810, 100 mg/kg/day, p.o., on day 28, compared to tumors grown in the presence and absence of estrogen pellets at the same timepoint, n = 3. ( C ) Tamoxifen and fulvestrant display anti-tumor activity in the patient derived tumor, HCI-003. NOD.CB17-Prkdc scid /NcrCrl mice were implanted with HCI-003 tumor fragments and estradiol impregnated beeswax pellets. Animals were ovariectomized 19 days after fragment implant. Animals with viable tumors were dosed orally with Vehicle (9% Peg-400:0.5% Tween–80:0.5% Povidone:90% 0.5% Carboxymethylcellulose), tamoxifen (60 mg/kg/day), or fulvestrant (200 mg/kg 3x/week) for 38 days. Estradiol pellets were removed from one group of animals on the first day of dosing to serve as a control for estradiol dependence for cell growth (Vehicle (-E2)). For all graphs * denotes significance (p<0.05) compared to Vehicle (+E2) in 1-Way ANOVA and Dunnett's Multiple Comparison Test. ( D ) GDC-0810 displayed anti-tumor activity in ZR75-1 ER+ breast cancer tumors. Crl:NU-Foxn1 nu mice implanted with 17-β Estradiol pellets (0.72 mg/pellet/60 days, Innovative Research of America) were injected with 5 × 10 6 ZR-75-1 cells. 21 days later, animals bearing viable tumors were dosed orally with Vehicle (9% Peg-400:0.5% Tween–80:0.5% Povidone:90% 0.5% Carboxymethylcellulose), tamoxifen (60 mg/kg/day), or GDC-0810 (100 mg/kg/day) for 28 days. Estradiol pellets were removed from one group of animals on the first day of dosing to serve as a control for estradiol dependence for cell growth [Vehicle (-E2)]. * Denotes significance (p<0.05) compared to Vehicle (+E2) in 1-Way ANOVA and Dunnett's Multiple Comparison Test.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Activity Assay, Injection, Comparison, Gene Expression, Derivative Assay, Control

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) MCF7 cells were injected s.c. into athymic ovariectomized mice supplemented with a 14-day release, 0.17-mg 17β-estradiol pellets. Four weeks later, mice bearing tumors ≥250 mm 3 were randomized to treatment with vehicle, GDC-0810 10 mg/kg/day or 100 mg/kg/day (p.o.) for 9 days (*p<0.01 vs. vehicle, **p<0.005 vs. vehicle). ( B ) Left panel: Representative images of IHC for ERα and PR in mice treated with vehicle or GDC-0810. Scale bars: 200 µm. Right panel: Quantitative histoscores from tumors in left panel (H-score; *p<0.05, **p<0.005). Each bar: mean ± SEM.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Injection

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) Tamoxifen-resistant MCF7 tumors were implanted in animals supplemented with 60-day release 0.18 mg 17β-estradiol pellets. Tumor bearing animals were dosed with vehicle, tamoxifen (120 mg/kg/day p.o.), fulvestrant (200 mg/kg, 3x/week, s.c.) or GDC-0810 (10, 30 or 100 mg/kg/day, p.o.) for 27 days ( B ) Representative images of IHC for ERα and Ki67 in tamoxifen-resistant MCF7 xenograft tumors from ( A ) treated with vehicle, tamoxifen or GDC-0810. ( C ) Quantitative PCR analysis of ER-regulated genes in tamoxifen-resistant MCF7 xenograft tumors from ( A ), treated with vehicle, fulvestrant or GDC-0810. S.c. is sub-cutaneous dosing, and p.o. is per os (by mouth) oral gavage dosing. * Denotes significance (p<0.05) compared to Vehicle in 1-Way ANOVA and Dunnett's Multiple Comparison Test.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Real-time Polymerase Chain Reaction, Comparison

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: TamR1 tumor fragments were implanted into Crl:NU-Foxn1 nu mice implanted with either 0.72 or 0.18 mg/60 day 17-β estradiol pellets. Once tumors were established pellets were removed from 1 group. 28 days after pellet removal ERα protein levels were determined for 4 tumors from each group. Data is presented as average compared to Vehicle (-E2). Error is standard deviation. * Denotes significance (p<0.05) compared to Vehicle (-E2) by T-test.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Standard Deviation

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) A cell free, FRET-based E2 competitive binding assay (E2 present at EC80) was used to determine the binding of GDC-0810 to ER.WT, ER.Y537S and ER.D538G ligand binding domains. Shown are the IC50 values calculated from multiple independent experiments, with mean and standard deviation indicated ( B ) A cell free, FRET-based PGC1α recruitment assay was used to evaluate the effect of GDC-0810 on ER:PGC1α interaction in the presence of agonist (EC80), using either purified wild-type or mutant ERα ligand binding domains (LBD). Shown are the IC50 values calculated from multiple independent experiments, with mean and standard deviation indicated. ( C ) Quantitative RT-PCR analysis of ER-regulated genes from CRISPR-Cas9 engineered ER.Y537S cells, in the absence and presence of estrogen, highlighting the E2 independent pathway activity of MCF7 cells expressing ER.Y537S. ( D ) Cell viability assays, measuring the effect of GDC-0810, were performed on MCF7 ER.WT (black lines) and ER.Y537S (blue lines) cells, in the absence (solid lines) and presence (dotted lines) of estrogen. ( E ) Western blot analysis evaluating levels of ERα, as well as PR and cyclin D1 as ER targets, in ER.WT and ER.Y537S cells. Cells were treated with GDC-0810 for 24 hr.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Competitive Binding Assay, Binding Assay, Ligand Binding Assay, Standard Deviation, Purification, Mutagenesis, Quantitative RT-PCR, CRISPR, Activity Assay, Expressing, Western Blot

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) Schematic detailing the strategy to engineer the ER.Y537S mutation into the endogenous ESR1 locus in MCF7 cells. ( B ) Y537S-specific ddPCR on genomic DNA was used to evaluate the mutant allele frequency in 24 independent knock-in clones. ( C ) Expression of the mutant allele was evaluated in 7 clones, as well as in the WHIM20 PDX model by performing Y537S-specific ddPCR on cDNA. ( D ) The effect of GDC-0810 on the viability of MCF7 ER.WT and multiple MCF7 ER.Y537S clones in the absence of estrogen; all mutant clones exhibit a similar right shift relative to ER.WT cells. ( E ) The effect of fulvestrant on cell viability was compared across MCF7 ER.WT (black lines) and ER.Y537S (blue lines) cells in the absence (solid lines) and presence (dotted lines) of estrogen. ( F ) Western blot analyses were performed on ER.WT and ER.Y537S cells in the presence and absence of fulvestrant and estrogen. Cells were harvested 24 hr after drug administration. ( G ) The effect of 4OH-tamoxifen on cell viability was compared across MCF7 ER.WT (black lines) and ER.Y537S (blue lines) cells in the absence (solid lines) and presence (dotted lines) of estrogen. ( H ) Western blot analyses were performed on ER.WT and ER.Y537S cells in the presence and absence of 4OH-tamoxifen and estrogen. Cells were harvested 24 hr after drug administration.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Mutagenesis, Knock-In, Clone Assay, Expressing, Western Blot

Journal: eLife

Article Title: The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer

doi: 10.7554/eLife.15828

Figure Lengend Snippet: ( A ) MCF7 HA-ER.Y537S overexpressing tumors were implanted in animals without supplemental 17β-estradiol pellets. Tumor bearing animals were dosed with vehicle, tamoxifen (60 mg/kg/day p.o.), fulvestrant (200 mg/kg, 3x/week, s.c.) or GDC-0810 (100 mg/kg/day, p.o.) for 28 days. ( B ) Gene expression analysis of WHIM20 tumors after 4 days of dosing, harvested 8 hr after the final dose. ( C ) Western blot and quantification of ER levels by RPPA, assessing the effect of GDC-0810 and fulvestrant on ER and PR levels in the WHIM20 PDX model. ( D ) Representative IHC images of WHIM20 tumors treated with either vehicle, fulvestrant or GDC-0810 and probed using anti-ER, anti-PR, or anti-Ki67 antibodies. ( E ) WHIM20 tumors were implanted in mice without supplemental 17β-estradiol pellets. Upon reaching ~200 mm 3 ,tumor bearing animals were dosed with vehicle, tamoxifen (60 mg/kg/day p.o.), fulvestrant (50 mg/kg, days 1, 3 and 8, 25 mg/kg 1x/week thereafter, s.c.) or GDC-0810 (100 mg/kg/day, p.o.) for 35 days. * Denotes significance (p<0.05) compared to Vehicle in 1-Way ANOVA and Dunnett's Multiple Comparison Test.

Article Snippet: The tamoxifen-resistant derivative of the ER+ human tumor cell line, MCF7 (TamR1) was developed by Seragon Pharmaceuticals, Inc ( ).

Techniques: Gene Expression, Western Blot, Comparison