|
| Status |
Public on Oct 10, 2018 |
| Title |
ENT + CTLA4 2 |
| Sample type |
RNA |
| |
|
| Source name |
G-MDSCs, 3 week treated, breast tumor
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: FVB-Neu-N
tissue: G-MDSCs isolated from syngeneically transplanted breast tumor
|
| Treatment protocol |
The ENT dosing solution (5 mg/kg) was made by suspending solid ENT (generously provided by Syndax Pharmaceuticals) in 0.5% methylcellulose via sonication and given by oral gavage daily for three weeks. To imitate oral gavage in control groups, 0.5% methylcellulose was given to vehicle mice as well as mice receiving only antibody therapy during the three weeks of ENT dosing. Mice received anti-PD-1 and/or anti-CTLA-4 twice a week for three weeks. All studies in neu-N model were performed with and without an anti-HER2 antibody to mimic treatment with and without trastuzumab. Anti-HER2 antibody was given once a week for three weeks. Dosage of antibodies was as follows: anti–PD-1, 100 µg/mouse via intraperitoneal injection (i.p.); anti–CTLA-4, 100 µg/mouse i.p.; anti-HER2, 100 µg/mouse i.p. Monoclonal antibodies were obtained from BioXcell [anti–PD-1 (clone RPM1-14), anti–CTLA-4 (clone 9H-10), and anti-HER2 (clone 7.16.4)] and all were diluted to 0.5 mg/mL in PBS. Isotypes were used to treat vehicle mice and were also obtained from BioXcell [anti–PD-1 isotype: rat IgG2a (clone 2A3); anti–CTLA-4 isotype: polyclonal Syrian Hamster IgG; anti-HER2 isotype: mouse IgG2a (clone C1.18.4)]. All isotype antibodies were also diluted to 0.5 mg/mL in PBS.
|
| Growth protocol |
The neu-N model is a syngeneic model whereby NT2.5 cells that were derived from a spontaneous mammary tumor are implanted via injection of 5 x 10^4 cells into the mammary fat pad of 7-8 week old female neu-N mice . NT2.5 cells were cultured from frozen stocks and passaged twice before injection. Tumors were allowed to seed for 3 days prior to initiating treatment with various drug combinations as described below . Untreated mice developed palpable tumors within 1 week.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
G-MDSCs were isolated from single-cell suspensions from tumors following tumor dissociation using Miltenyi Biotec’s Myeloid-Derived Suppressor Cell Isolation Kit (cat. 130-094-538) according to the manufacturer’s protocol. RNA was extracted from whole tumors or G-MDSCs isolated from tumors using the Qiagen RNeasy extraction kit (cat. 74104). Using a NanoDrop ND-1000, RNA quality and quantity were determined (samples with A260/280 < 1.8 were excluded). Gene expression was measured on the NanoString nCounter Analysis System (NanoString Technologies). The NanoString PanCancer Immune CodeSet (cat. XT-CSO-MIP1-12) was used to perform the nCounter Gene Expression Assay for whole tumor samples, whereas the Mouse Myeloid CodeSet (cat. XT-CSO-MII2-12) was used to perform nCounter Gene Expression Assay for isolated G-MDSCs. Quantification of target mRNA in each sample was performed by detection within the nCounter Digital Analyzer. Ly6G+ cells were positively selected to isolate G-MDSCs from Ly6G- M-MDSCs and were passed through LS columns (Miltenyi Biotec, cat. 130-042-401) twice to increase purity. Eluted G-MDSCs were then used for downstream assays described below.
|
| Label |
Capture probe-3'biotin, Reporter probe-5' barcode signal
|
| Label protocol |
Followed NanoString nCounter XT gene expression assay protocol. Briefly, hybridization buffer was mixed with the nCounter Myeloid Innate Immunity Panel reporter codeset to prepare a master mix. Master mix, sample and Rnase-fee water was combined with nCounter Myeloid Innate Immunity Panel Capture codeset. Samples were then incubated for 18 h at 65 deg C.
|
| |
|
| Hybridization protocol |
Followed NanoString nCounter XT gene expression assay protocol. Briefly, hybridization buffer was mixed with the nCounter Myeloid Innate Immunity Panel reporter codeset to prepare a master mix. Master mix, sample and Rnase-fee water was combined with nCounter Myeloid Innate Immunity Panel Capture codeset. Samples were then incubated for 18 h at 65 deg C.
|
| Scan protocol |
Raw counts of mRNA hybridized to oligos were counted using the NanoString nCounter instrument.
|
| Description |
Please note, title describes what each tumor was treated with prior to cell isolation entinotat-ENT, anti-PD1-PD1, anti-CTLA4-CTLA4, vehicle-PBS+ methylcellulose
G-MDSCs were extracted from tumors following 3 full weeks of treatment with drug
|
| Data processing |
Data from the NanoString nCounter system was normalized to the internal positive controls and housekeeping genes using the recommended settings in the nSolver 4.0 software normalization module (NanoString Technologies). Normalized data was exported and differential expression analysis was performed using a linear model method with the limma package (40) for the R programming language. To identify pathways which were differentially expressed in each treatment, the gage package was used to test for differentially expressed KEGG pathways. Pathways of interest were visualized using the pathview package.
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| |
|
| Submission date |
Oct 09, 2018 |
| Last update date |
Oct 10, 2018 |
| Contact name |
Evanthia Theodosiou Roussos Torres |
| Organization name |
Johns Hopkins University
|
| Department |
Oncology
|
| Lab |
Jaffee Lab
|
| Street address |
1650 Orleans Street, CRB1 4M
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21287 |
| Country |
USA |
| |
|
| Platform ID |
GPL25266 |
| Series (1) |
| GSE121030 |
Mouse Myeloid panel on G-MDSCs from treated tumors |
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