|
| Status |
Public on Feb 11, 2019 |
| Title |
PSCA3_post, biological replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
spleen of tumor-bearing mice, 30 days post transfer
|
| Organism |
Homo sapiens |
| Characteristics |
anti-psca car type: PSCA3
infusion timepoint: post-infusion
cell type: Cells expressing a third generation CAR
|
| Treatment protocol |
NSG mice bearing palpable subcutaneous tumors, derived from HPAC cells, were treated with human T cells. Treatment groups received CD8 T cells expressing either PSCA2 or PSCA3 CARs, plus untransduced CD4 cells for cytokine support. GFP-transduced CD8 T cells were used as a control, and 3 biological replicates were included per group. For the post-infusion samples, CD8+ T cells were isolated from the spleen (3 mice/group), using magnetic bead cell sorting.
|
| Growth protocol |
Human peripheral blood mononuclear cells were stimulated with anti-CD3 antibody (OKT3) in presence of IL-2. Two days post-stimulation, they were transduced with retroviral vectors encoding either a second generation CAR (PSCA2), a third generation CAR (PSCA3), or GFP as a control. Cells were cultured for 2 weeks in presence of IL-2, until collection of samples (pre-infusion samples) or transfer into mice (Post-infusion samples).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Isolation of total RNA was performed using the Qiagen RNeasy column (with QIAshredder) according to the manufacturer's instructions.
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA was prepared according to the Ambion Message Amp Premier protocol using 100 ng of total RNA (Ambion).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of cRNA was hybridized for 16 hr at 45C to the Affymetrix U133 Plus 2.0 arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GCS3000 scanner.
|
| Description |
A285
|
| Data processing |
The data were analyzed with the Affymetrix Expression Console v 1.4 software using the MAS 5.0 algorithm with default analysis settings and global scaling as the normalization method. The trimmed mean target intensity of each array was set to 500. Using the Affymetrix Transcriptome Analysis Console v3.0, a one-way unpaired ANOVA analysis was performed and differentially expressed probe sets with fold changes > 2.0 and p-values < .05 were retained.
|
| |
|
| Submission date |
Aug 18, 2017 |
| Last update date |
Feb 12, 2019 |
| Contact name |
Daniel Abate-Daga |
| E-mail(s) |
daniel.abatedaga@moffitt.org
|
| Phone |
813-745-6856
|
| Organization name |
H. Lee Moffitt Cancer Center and Research Institute
|
| Department |
Immunology
|
| Street address |
12902 Magnolia Dr.
|
| City |
Tampa |
| State/province |
Florida |
| ZIP/Postal code |
33612 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE102823 |
Gene expression profiling of CAR-T cells pre-and post-adoptive transfer |
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