Single-Cell Identification of Melanoma Biomarkers in Circulating Tumor Cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE255299 | GEO | 2024/02/07
REPOSITORIES: GEO
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Project description:Circulating tumor cells (CTCs) are critical in the development of distant organ tumor metastasis, and are associated with advanced cancer stage and poor patient outcome. Here, we present the first genome-wide nucleotide-level characterization of CTCs. Our single-nucleotide polymorphism (SNP) analysis in patients with melanoma involved: 1) global comparative genomic analysis of CTCs and matched regional metastases, 2) identification of key genomic aberrations in CTCs, 3) verification of these target genes in aggressive distant tumor metastases, and 4) evidence of selective expression and functional consequence of CTC-associated genes in melanomas. We report 131 aberrant loci in CTCs that are potentially pro-metastatic, and show that such expression of a 5-marker gene panel (CSMD2, CNTNAP5, FLJ14051, ADAM6, TRPM2) in melanomas confers prognostic utility. Successful treatment of melanoma requires understanding of the metastatic process and identification of patients with tumors most likely to develop aggressive metastatic disease. Melanomas are heterogeneous, and CTCs have long been recognized as vehicles for cancer spread, representing particularly aggressive tumor clones that can evolve into successful clinical metastases. Elucidation of genomic aberrations in CTCs will aid in the development of prognostic biomarkers and therapeutic strategies to target CTCs to prevent or control distant cancer spread. This study provides the first detailed genomic confirmation of the close relation between CTCs and tumor metastases, and illustrates how CTCs can be utilized as a novel approach and rational source for identification of pro-metastatic genes in cancer research. Three individual patient cohorts were utilized in the study. CNV and LOH loci were evaluated initially in metastatic melanoma patients (n=13) in a discovery cohort. SNP loci that harbored CNV/LOH in CTCs were then separately verified for: (a) presence in distant organ metastasis (AJCC Stage IV melanoma) (n=27), and (b) relevance to prognosis in regional melanoma metastasis (AJCC Stage III melanoma) (n=35). The first discovery patient cohort group was utilized for capture of CTCs, and consisted of peripheral blood mononuclear cell (PBMC) and tumor specimens from metastatic melanoma patients (n=13). CTC-related loci were verified in a second cohort of patients with Stage IV distant organ metastases (n=27), including 15 brain, 4 lung, and 8 gastrointestinal (bowel, liver) metastases. The third patient cohort consisted of early passage (<12) established melanoma cell lines derived from 35 regional melanoma metastases (Stage III) for evaluation of the prognostic utility of the CTC-associated aberrant loci.
2014-07-01 | E-GEOD-43934 | biostudies-arrayexpress
Project description:Clusters of circulating tumor cells (CTC-clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Here, we first use mouse models to demonstrate that breast cancer cells injected intravascularly as clusters are more prone to survive and colonize the lungs than single cells. Primary mammary tumors comprised of tagged cells give rise to oligoclonal CTC-clusters, with 50-fold increased metastatic potential, compared with single CTCs. Using intravital imaging and in vivo flow cytometry, CTC-clusters are visualized in the tumor circulation, and they demonstrate rapid clearance in peripheral vessels. In patients with breast cancer, presence of CTC-clusters is correlated with decreased progression-free survival. RNA sequencing identifies the cell junction protein plakoglobin as most differentially expressed between clusters and single human breast CTCs. Expression of plakoglobin is required for efficient CTC-cluster formation and breast cancer metastasis in mice, while its expression is associated with diminished metastasis-free survival in breast cancer patients. Together, these observations suggest that plakoglobin-enriched primary tumor cells break off into the vasculature as CTC-clusters, with greatly enhanced metastasis propensity. RNA-seq from 29 samples (15 pools of single CTCs and 14 CTC-clusters) isolated from 10 breast cancer patients
2014-07-11 | E-GEOD-51827 | biostudies-arrayexpress
Project description:<p>Circulating tumor cells (CTCs) are recognized as direct seeds of metastasis. However, CTC count may not be the 'best' indicator of metastatic risk because their heterogeneity is generally neglected. In this study, we develop a molecular typing system to predict colorectal cancer metastasis potential based on the metabolic fingerprints of single CTCs. After identification of the metabolites potentially related to metastasis using mass spectrometry-based untargeted metabolomics, setup of a home-built single-cell quantitative mass spectrometric platform for target metabolite analysis in individual CTCs and use of a machine learning method composed of non-negative matrix factorization and logistic regression, CTCs are divided into two subgroups, C1 and C2, based on a 4-metabolite fingerprint. Both <em>in vitro</em> and <em>in vivo</em> experiments demonstrate that CTC count in C2 subgroup is closely associated with metastasis incidence. This is an interesting report on the presence of a specific population of CTCs with distinct metastatic potential at the single-cell metabolite level. </p>
2023-08-21 | MTBLS5028 | MetaboLights
Project description:Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice, rapidly declining after B-RAF inhibitor treatment. CTCs were shed early from localized tumors and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled comparison of RNA sequencing profiles with the matched primary tumor. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. In patients with metastatic melanoma, CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF targeted therapy. Together, the capture of CTCs and their molecular characterization provide insight into the hematogenous spread of melanoma. We adapted a microfluidic platform, the HbCTC-Chip (Stott et al., 2010, Pubmed ID: 20930119), to capture melanoma CTCs derived from mouse tumors, using panels of antibodies against melanoma-specific cell surface markers, followed by staining for melanoma antigens and optimized on-chip fluorescent imaging. We used a tamoxifen inducible BRAF(CA/+)/PTEN(flox/flox) melanoma mouse model (Dankort et al., 2009, Pubmed ID: 19282848) derived from a C57BL/6 background. Such mice received focal subcutaneous injection of tamoxifen (Sigma) (50ul at 5mg/ml in 50% EtOH suspension) at the left flank at 6-7 weeks after birth. Blood samples were collected from five mice with high tumor burden following tamoxifen injection. Blood from each mouse was split and processed through the CTC-chips functionalized with anti-CSPG4/MCAM antibody and control IgGs, respectively. Matched primary (from the tamoxifen injection site) and metastatic (from upper or lower back) tumors were harvested from the same mouse and immediately flash-frozen in liquid nitrogen. RNA extraction, single molecular sequencing and determination of Digital Gene Expression was as in Yu et al., 2012 (Pubmed ID: 22763454). One of the five mice yielded no CTC or IgG data. In addition, skin was taken from a sixth mouse, which was a complete wild type C57BL/6 mouse (without the BRAF/PTEN transgenes).The skin was taken after euthanasia of the animal and was processed as the tissue from the matched primary and metastatic tumors.
2014-03-31 | E-GEOD-52031 | biostudies-arrayexpress
Project description:Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice, rapidly declining after B-RAF inhibitor treatment. CTCs were shed early from localized tumors and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled comparison of RNA sequencing profiles with the matched primary tumor. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. In patients with metastatic melanoma, CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF targeted therapy. Together, the capture of CTCs and their molecular characterization provide insight into the hematogenous spread of melanoma.
2014-03-31 | GSE52031 | GEO
Project description:Circulating Tumour Cells (CTCs) act as tumour seeds for haematogeneous melanoma spread of melanoma. Since CTCs have been reported to be heterogeneous in melanoma, the characterisation of the different melanoma CTC subpopulations harbours biological and clinical significance. For detection of melanoma CTCs, melanoma-associated markers (i.e. MCSP, melanoma-associated chondroitin sulphate proteoglycan) have been used; however, recent studies have found that melanoma CTCs commonly express ABCB5 (ATP-binding cassette sub-family B member 5) a melanoma-initiating marker. Here, we used a genome-wide expression microarray to interrogate the transcriptome of MCSP-enriched and ABCB5-enriched CTC fraction in order to provide a better understanding of their biology and role in melanoma metastasis. Findings from this study highlited the distinct transcriptional programming of both CTC subpopulations.
2018-05-11 | GSE113166 | GEO
Project description:About 230 clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but no assays enable comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for CTC enrichment and isolation with 100% purity with a non-random whole genome amplifiation method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 breast cancer patients. We defined a genome integrity index (GII) to identify cells suited for molecular chracterization by different molecular assay in more than 90% of single cells, such as diagnostic profiling for point mutations, gene amplifications and whole genomes of single cells. The high reliability on clinical samples enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified therapeutically relevant genomic disparity between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered preexisting cells reistsant to ERBB2 targeted therapies suggesting ongoing microevolution at late stage disease whose exploration may provide essential information for personalized treatment decisions. The analysis aimed to indentify profiles of copy number changes in genomic DNA of single circulating tumor cells (CTCs). For this, CTCs were enrched using the FDA approved CellSearch System and single-cell were isolated using the DEPArray System. Subsequently, single-cell DNA was amplified using the Ampli1 WGA Kits and subjected to single-cell aCGH analysis according to previously published protocol (Czyz ZT et al., PLoS One. 2014 Jan 21;9(1):e85907). The analysis included 38 single CTCs and 10 white blood cells (WBCs) obtained from 16 breast cancer patient. WBCs were used as controls for the analysis. In addition, four CTC cell pools were included in the analysis. This was done to show the discrepancies between the profiles of individual cells and corresponing average genomic profile of CTCs in a patient material (cell pools), thereby demonstrating the importance of the analysis on the cell-by-cell basis. The reference sample used for all aCGH experiments consisted of a pool of four single-cell WGA products generated from WBCs of a healthy female donor.
2014-11-05 | E-GEOD-58192 | biostudies-arrayexpress
Project description:Circulating tumor cells (CTCs) represent the molecular characteristics of tumor sites and travel in the blood for seeding distant metastases. "EpCAM+/pan-cytokeratin (CK)+/CD45-/DAPI+" has been widely accepted as a CTC definition, especially in breast cancer, prostate cancer and colorectal cancer. However, reports on CTC detection in non-small cell lung cancer are limited due to a lack of efficient CTC marker. We describe hexokinase 2 (HK2) that assays elevated glycolysis of cancer cells, called Warburg effect, as a new marker for CTC detection in lung adenocarcinoma (LUAD), especially the CK negative CTCs. Single-cell sequencing was used to confirm the malignancy of putative CTCs by detecting genome-wide copy number alternations characteristic of malignant cells. We employed this marker in a variety of liquid biopsies from LUAD patients, including peripheral blood, pleural effusion and cerebrospinal fluid.
2021-02-28 | E-MTAB-8767 | biostudies-arrayexpress
Project description:The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Our DNA methylation analysis led us to hypothesize that CTC clusters are characterized by active TF networks that support both stemness and proliferation. To identify whether the stemness- and proliferation-related TF networks are also transcriptionally active in CTC clusters compared to single CTCs, we performed single-cell resolution RNA sequencing analysis of single CTCs and CTC clusters, matched within individual liquid biopsies and isolated from six breast cancer patients with progressive metastatic disease, and of single CTCs and CTC clusters isolated from three xenograft models. In addition, among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. We performed RNA sequencing analysis of BR16 and Brx50 cells upon treatment to assess the molecular consequences of clusters dissociation.
2019-01-11 | GSE111065 | GEO
Project description:Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Highly parallel architecture of the chip allows deterministic screening of clinically relevant volumes of blood samples at slow, and hence, non-damaging flow rates. Using the Cluster-Chip, we identify CTC-clusters in 30-40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as being tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. We used the Cluster-Chip to capture CTC-clusters from the blood of a breast cancer patient with high CTC counts, released CTC-clusters in solution, stained them with TexasRed-conjugated antibodies against the leukocyte cell surface markers CD45, CD14 and CD16, and then isolated intact CTC-clusters individually using a micromanipulator. From a single time point, we retrieved 15 CTC-clusters, and each of those clusters was individually subjected to RNA-sequencing analysis using a next generation platform (SOLiD 5500). In addition, two leukocytes were isolated from the blood of a healthy donor were individually subjected to RNA-sequencing analysis using the same platform.
2015-05-17 | E-GEOD-67939 | biostudies-arrayexpress