Investigating the transcriptional regulation of intratumoral hypoxia in CTCs
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE240127 | GEO | 2024/07/02
REPOSITORIES: GEO
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Project description:Investigating the transcriptional regulation of intratumoral hypoxia in CTCs
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Project description:Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To determine the relevance of ECM protein expression to human disease, CTCs were isolated from the blood of metastatic PDAC patients and subjected to single cell RNA-sequencing. Analysis of 7 pancreatic CTCs from 3 patients revealed that the majority expressed keratins defining their epithelial origin. A total of 13 of 60 extracellular protein genes enriched in mouse CTCs (see GEO GSE51372) were expressed at high levels (>100 rpm) in at least one human pancreatic CTC. Human SPARC was the only gene found at high levels in all human pancreatic CTCs. To achieve a deep RNA sequencing profile of CTCs at the single cell level, we applied a novel inertial focusing-enhanced device, the CTC-iChip, which allows high efficiency negative depletion of normal blood cells, leaving unattached CTCs in solution where they can be selected and analyzed as single cells (Pubmed ID 23552373). CTCs were then subjected to single cell RNA-sequencing (Pubmed ID 20203668).
2014-09-22 | E-GEOD-60407 | biostudies-arrayexpress
Project description:We designed a novel approach to fate-map hypoxic cells in order to determine their cellular response to physiological O2 gradients. Our system causes a change in the expressing fluorescent protein upon hypoxic exposure (DsRed -> GFP). We generated hypoxia fate-mapping tumors using MDA-MB-231 cells expressing our system. Tumors were resected 2 weeks post-implantation, mechanically disrupted and digested with collagenase to obtain a cell suspension. The cell suspension was enriched using magnetic-activated cell sorting (MACS) and DsRed+ cells were isolated from GFP+ cells by fluorescence-activated cell sorting (FACS) directly into Tris Reagent (Zymo Research). Total RNA was extracted from cells using TRIzol (Invitrogen) and purified using Direct-zol RNA mini kit (Zymo Research) with DNase I treatment. After RNA purification, samples were confirmed to have a RIN value > 9.0 when measured on an Agilent Bioanalyzer. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. The workflow consisted of mRNA enrichment, cDNA generation, end repair to generate blunt ends, A-tailing, adaptor ligation and 12 cycles of PCR amplification. Unique adaptors were used for each sample in order to multiplex samples into several lanes. Sequencing was performed on Illumina Hiseq 4000 with a 150bp pair-end run. A data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program.
2019-02-16 | GSE126609 | GEO
Project description:We designed a novel approach to fate-map hypoxic cells in order to determine their cellular response to physiological O2 gradients. Our system causes a change in the expressing fluorescent protein upon hypoxic exposure (DsRed -> GFP). We generated hypoxia fate-mapping tumors using MDA-MB-231 cells expressing our system. Metastatic lungs were resected 2 weeks after primary tumor removal, mechanically disrupted and digested with collagenase to obtain a cell suspension. The cell suspension was enriched using magnetic-activated cell sorting (MACS) and DsRed+ cells were isolated from GFP+ cells by fluorescence-activated cell sorting (FACS) directly into Tris Reagent (Zymo Research). Total RNA was extracted from cells using TRIzol (Invitrogen) and purified using Direct-zol RNA mini kit (Zymo Research) with DNase I treatment. After RNA purification, samples were confirmed to have a RIN value > 9.0 when measured on an Agilent Bioanalyzer. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. The workflow consisted of mRNA enrichment, cDNA generation, end repair to generate blunt ends, A-tailing, adaptor ligation and 12 cycles of PCR amplification. Unique adaptors were used for each sample in order to multiplex samples into several lanes. Sequencing was performed on Illumina Hiseq 3000/4000 with a 150bp pair-end run. A data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program.
2019-08-27 | GSE136372 | GEO
Project description:Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To determine the relevance of ECM protein expression to human disease, CTCs were isolated from the blood of metastatic PDAC patients and subjected to single cell RNA-sequencing. Analysis of 7 pancreatic CTCs from 3 patients revealed that the majority expressed keratins defining their epithelial origin. A total of 13 of 60 extracellular protein genes enriched in mouse CTCs (see GEO GSE51372) were expressed at high levels (>100 rpm) in at least one human pancreatic CTC. Human SPARC was the only gene found at high levels in all human pancreatic CTCs.
2014-09-22 | GSE60407 | GEO
Project description:For further validation that the circulating tumor cells (CTCs) from head and neck squamous cell carcinomas (HNSCC) patients were indeed cancer cells rather than non-specific contaminated cells such as white blood cells (WBCs), we examined the copy number alterations (CNAs) of the captured CTCs by aCGH (array comparative genomic hybridization).
2018-08-25 | GSE114122 | GEO
Project description:We developed a new method for the isolation of bronchial smooth muscle cells. This method is built upon a double fluorescent mouse SMA-GFP;NG2-DsRed and cell sorting. Using this method, we isolated bronchial smooth muscle cells from control and asthmatic C57BL/6J mice for gene profiling.
2014-03-13 | GSE45723 | GEO
Project description:Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging, hence sampling circulating tumor cells (CTCs) may reveal drug resistance mechanisms. We established single cell RNA-sequencing profiles of 77 intact CTCs isolated from 13 patients (mean 6 CTCs/patient) using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing on AR inhibitor, compared with untreated cases indicates activation of noncanonical Wnt signaling (P=0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, while its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure. A total of 221 single candidate prostate CTCs were isolated from 18 patients with metastatic prostate cancer and 4 patients with localized prostate cancer. Of these, 133 cells (60%) had RNA of sufficient quality for amplification and next generation RNA sequencing, and 122 (55%) had >100,000 uniquely aligned sequencing reads. In addition to candidate CTCs, we also obtained comprehensive transcriptomes for 12 bulk primary prostate cancers (macrodissected for >70% tumor content), 30 single cells derived from four different prostate cancer cell lines, and 5 patient-derived leukocyte controls. The leukocytes were readily distinguished by their expression of hematopoietic lineage markers and served to exclude any CTCs with potentially contaminating signals. Strict expression thresholds were used to define lineage-confirmed CTCs, scored by prostate lineage-specific genes (PSA, PSMA, AMACR, AR) and standard epithelial markers (KRT7, KRT8, KRT18, KRT19, EpCAM). 28 cells were excluded given the presence of leukocyte transcripts suggestive of cellular contamination or misidentification during selection, and 17 cells were excluded given low expression of both prostate lineage-specific genes and 5 standard epithelial markers. The remaining 77 cells, defined as lineage-confirmed CTCs, displayed expression of either prostate lineage-specific or epithelial genes, and low expression of leukocyte-specific genes, consistent with their tumor of origin.
2015-09-17 | E-GEOD-67980 | biostudies-arrayexpress
Project description:Blood-borne metastasis to the brain is a major complication of breast cancer, but cellular pathways that enable cancer cells to selectively grow in the brain microenvironment are poorly understood. We find that cultured circulating tumor cells (CTCs), derived from blood samples of women with advanced breast cancer and directly inoculated into the mouse frontal lobe, exhibit striking differences in proliferative potential in the brain. Derivative cell lines generated by serial intracranial injections acquire selectively increased proliferative competency in the brain, with reduced orthotopic tumor growth. Increased Hypoxia Inducible Factor 1A (HIF1A)-associated signaling correlates with enhanced proliferation in the brain, and shRNA-mediated suppression of HIF1A or drug inhibition of HIF-associated glycolytic pathways selectively impairs brain tumor growth while minimally impacting mammary tumor growth. In clinical specimens, brain metastases have elevated HIF1A protein expression, compared with matched primary breast tumors, and, in patients with brain metastases, hypoxic signaling in CTCs predicts decreased overall survival. The selective activation of hypoxic signaling by metastatic cancers in the brain may be an opportunity for therapeutic intervention.
2020-10-17 | GSE156943 | GEO