RNA seq analysis of T cells，EpCAM CAR-T cells and rapamycin-pretreated EpCAM CAR-T cells
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ABSTRACT:
PROVIDER: PRJNA683033 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13208500_1.fastq.gz | Fastqsanger.gz | |||
| SRR13208500_2.fastq.gz | Fastqsanger.gz | |||
| SRR13208501_1.fastq.gz | Fastqsanger.gz | |||
| SRR13208501_2.fastq.gz | Fastqsanger.gz | |||
| SRR13208502_1.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: Compared the differences in the transcriptome of T cells versus EpCAM CAR-T cells and EpCAM CAR-T cells versus rapamycin-pretreated EpCAM CAR-T cells.Methods: Next-generation sequencing (NGS) has revolutionized the systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) between T cells and EpCAM CAR-T cells，and between EpCAM CAR-T cells and rapamycin-pretreated EpCAM CAR-T cells. Results: RNA-seq showed that CAR-T cells significantly upregulated the expression of exhaustion markers compared to T cells. Gene Set Enrichment Analysis (GSEA) analysis showed that CAR-T cells cells were enriched for signatures of apoptosis. Compared with T cells, Kyoto Encyclopedia of Genes and Genomessignaling(KEGG) pathway analysis showed that the PI3K/AKT/mTOR signaling pathway was significantly enriched. Likewise, GSEA showed that the mTOR signaling pathway was significantly upregulated in CAR-T cells. Next, we analyzed the RNA-seq data between CAR-T cells and rapamycin-pretreated CAR-T cells. RNA-seq showed that rapamycin-treated CAR T down-regulated the expression of exhaustion markers compared with untreated CAR-T cells. GSEA analysis showed that the apoptosis-related gene set was enriched in untreated CAR-T cells.Conclusions:In vitro culture promotes the terminal differentiation of CAR-T cells.CAR-T cells present a highly activated mTOR phenotype. Rapamycin pretreatment prevents CAR-T cells terminal differentiation.
2021-12-05 | GSE162796 | GEO
Project description:Chimeric antigen receptor designs that incorporate pharmacologic control are desirable, however designs suitable for clinical translation are needed. We designed a fully human, rapamycin regulated, drug product for targeting CD33+ tumors called dimerization agent regulated immunoreceptor complex (DARIC33). T cell products demonstrated target specific and rapamycin dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1nM rapamycin. Rapamycin withdrawal paused DARIC33-stimulated T cell effector functions, which were restored following re-exposure to rapamycin, demonstrating reversible effector function control. While rapamycin-regulated DARIC33 T cells were highly sensitive to target antigen, CD34+ stem cell colony forming capacity was not impacted. We benchmarked DARIC33 potency relative to CD19 CAR T cells to estimate a T cell dose for clinical testing. In addition, we integrated in vitro and preclinical in vivo drug concentration thresholds for OFF-ON state transitions, as well as murine and human rapamycin pharmacokinetics, to estimate a clinically applicable rapamycin dosing schedule. A phase 1 DARIC33 trial has been initiated (PLAT-08, NCT05105152), with initial evidence of rapamycin-regulated T cell activation and anti-tumor impact. Our findings provide evidence that the DARIC platform exhibits sensitive regulation and potency needed for clinical application to other important immunotherapy targets.
2024-04-18 | GSE255002 | GEO
Project description:Lentiviral vectors (LV) have become the dominant tool for stable gene transfer into lymphocytes including chimeric antigen receptor (CAR) gene delivery to T cells, a major breakthrough in cancer therapy. Yet, room for improvement remains, especially for the latest LV generations delivering genes selectively into T cell subtypes, a key requirement for in vivo CAR T cell generation. Towards improving gene transfer rates with these vectors, we conducted whole transcriptome analyses on human T lymphocytes after exposure to CAR-encoding conventional vector VSV-LV, and vectors targeted to CD8+ (CD8-LV) or CD4+ T cells (CD4-LV). Genes related to quiescence and antiviral restriction were found to be upregulated in CAR-negative cells exposed to all types of LVs. Down-modulation of various antiviral restriction factors including the interferon-induced transmembrane proteins (IFITMs) was achieved with rapamycin as verified by mass spectrometry (LC-MS). Strikingly, rapamycin enhanced transduction by up to 7-fold for CD8-LV and CD4-LV without compromising CAR T cell activities, but did not improve VSV-LV. When administered to humanized mice, CD8-LV resulted in higher rates of GFP gene delivery as well as faster in vivo CAR T cell generation and tumor control. The data favors multi-omics approaches for improvements in gene delivery.
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Project description:Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing
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Project description:EpCAM is frequently overexpressed in human invasive breast cancer. We reported EpCAM overexpression to be an independent prognostic marker for poor overall survival in node-positive breast cancer. We used microarrays in order to investigate changes of the transcriptome on EpCAM gene overexpression in human breast cancer cells
2010-12-02 | GSE25743 | GEO
Project description:EpCAM is frequently overexpressed in human invasive breast cancer. We reported EpCAM overexpression to be an independent prognostic marker for poor overall survival in node-positive breast cancer. We used microarrays in order to investigate changes of the transcriptome on EpCAM gene overexpression in human breast cancer cells Hs578T cells were found to express only very little EpCAM mRNA and protein in comparison to established and well characterized breast cancer cell lines such as MCF-7 or SK-BR-3. Hs578T cells were stably transfected with the EpCAM cDNA containing construct pIRESpuro3_EpCAM and the respective empty vector control.
2010-12-02 | E-GEOD-25743 | biostudies-arrayexpress
Project description:Chimeric antigen receptor (CAR) T cells are known to be potent in recognizing and eliminating tumor elimination, and effector functions. They induce marked responses in patients with refractory leukemia and lymphoma, but dysfunction of T cells is an inevitable obstacle for persistent curative effect. Extending the persistence of these cells without compromising their potency is supposed to improve treatment outcomes. We demonstrated that the SOCE (Store-Operated Calcium Entry) inhibitor BTP-2, rather than calcium chelator BAPTA-AM, diminished exhaustion and terminal differentiation of CAR-T cells both in tonic signaling and tumor antigen exposure models. And BTP-2 pretreated CAR-T cells showed improved antitumor potency and prolonged survival in NSG mice. To further investigate the biological impact of calcium signaling at the molecular level, we performed RNA-seq on CAR-T cells and CAR-T cells exposed to Nalm6 stimulation followed by treatment with DMSO, BAPTA and BTP-2.The high-throughput RNA sequencing (RNA-Seq) revealed that treatment with BTP-2 downregulates glycolysis pathway.
2021-11-19 | GSE178998 | GEO
Project description:To characterize transfer of molecules from target cells into CAR T cells via trogocytosis we cultured NALM-6 leukemia cell line expressing a CD19-mCherry fusion protein with CAR T cells. NALM6-CD19-mCherry were loaded with heavy amino acid and cocultured with CAR T cells for 1 hour. CAR T cells were next sorted into two fractions, mCherry-positive (TrogPos), and -negative (TrogNeg). Proteomics analysis revealed the presence of targeted antigen (CD19) in the TrogPos only.
2019-03-29 | PXD012833 | Pride
Project description:Adoptive cell therapy, a subset of cancer immunotherapy, is collection of therapeutic approaches which aim to redirect the immune system by reprogramming patient T-cells to target antigenic molecules differentially and specifically expressed in certain cancers. One promising immunotherapy technique is CAR T-cell therapy, where cancer cells are targeted through the expression a chimeric antigen receptor (CAR), a synthetic trans- membrane receptor that functionally compensates for the T-cell receptor (TCR) but targets a tumor associated antigen on the cancer cell surface. While CAR T-cell therapy is promising with two clinically approved second-generation CARs (Kymriah and Yescarta), few studies have investigated the mechanism of signal propagation in T-cells and no studies have investigated the potential signaling response in the target cells. To gain further insight to CAR-based signaling, we stimulated third generation CD19 CAR-expressing Jurkat T-cells by co-culture with SILAC labeled CD19HI Raji B-cells and used two phosphoenrichment strategies coupled with liquid chromatography-tandem mass spec- trometry (LC-MS/MS) to detect and analyze global phosphorylation changes in both cell populations. Analysis of the phosphopeptides originating from the CD19-CAR T cells revealed an increase in many phosphorylation events necessary for canonical TCR signaling. We also observed for the first time a significant decrease in B-cell receptor- related phosphopeptide abundance in CD19HI Raji B-cells after co-culture with CD19-targetted CAR T-cells.
2022-01-28 | PXD028109 | Pride
Project description:Epithelial cell adhesion molecule EpCAM is a single transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration, and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP-7) as a target gene of EpCAM signalling via EpICD nuclear translocation. EpCAM and MMP-7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP-7 is a novel target of EpCAM signalling. FaDu hypopharynx carcinoma cells were transiently transfected with a control siRNA or an EpCAM-specific siRNA. After two days, expression of EpCAM was assessed upon flow cytometry with antibodies, which specifically bind within the EGF-repeat in the extracellular domain of EpCAM (Balzar, et al., 1999). Compared to control siRNA, EpCAM siRNA induced a mean 43% reduction of EpCAM expression at the cell surface. Reduction of EpCAM expression upon siRNA treatment was confirmed by RT-PCR and qPCR. EpCAM down-regulation resulted in diminished cell proliferation assessed upon cell counting. The observed 43% decrease in cell surface expression of EpCAM translated in cell numbers diminished by 35%. This is a confirmation of earlier findings on the reduced proliferation of breast and colon cancer cell lines following siRNA-mediated knock-down of EpCAM (Maetzel, et al., 2009, Munz, et al., 2004, Osta, et al., 2004). Equal amounts of total RNA (RIN score = 10) from control- and EpCAM-siRNA-treated FaDu cells were next subjected to a single, explorative cDNA microarray analysis with the aim to identify potential target genes of the signal transduction cascade of EpCAM. All genes, which were up- or down-regulated by at least two-fold (>2-fold; 716 genes up-regulated, 552 genes down-regulated), were then subjected to a Panther classification software analysis (http://www.pantherdb.org/). Special emphasis was put on the regulation of genes associated with signalling pathways. Angiogenesis, Alzheimer's disease-presenilin pathway, Wnt signalling pathway, inflammation and cadherin signalling pathways were most strongly affected by EpCAM knock-down. mRNA levels of selected genes differently regulated following partial knock-down of EpCAM included the co-regulated genes for secreted frizzled-related protein 5, protocadherin 10, dickkopf homolog 2, MMP24, SPARC, Myst3, ErbB4, MMP12, WISP2, MMP7, Nanos1, WNT9A, and the counter-regulated genes for MMP11, fibronectin 1, DACT3, WNT2B, WNT5A, APC2, Spondin 1, and cateninα2. All genes except for protocadherin 10, Myst3, ErbB4, and WNT2B were validated in RT-PCR assays. Since several matrix metalloproteases were regulated after EpCAM knock-down and EpCAM is known to correlate with invasion, we chose this protein family for in-depth analysis. Matrix metalloprotease 7 (MMP-7) is a soluble factor, which, unlike other family members, is preferentially expressed in epithelial cells (Wilson, et al., 1995). Since EpCAM also displays highest expression in epithelia, we chose to proceed with the analysis of MMP7. SiRNA-mediated knock-down of EpCAM resulted in a comparable 50% down-regulation of MMP7 mRNA levels in RT-PCR and qPCR.
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