Defining a molecular signature of breast cancer circulating tumor cells using an in vitro model
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE224841 | GEO | 2023/03/23
REPOSITORIES: GEO
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Project description:Circulating tumor cells (CTCs) and disseminated tumour cells with mesenchymal traits are difficult to detect by epithelial marker proteins. Particularly, triple negative breast cancers (TNBC) that are prone to therapy failure release a subpopulation of circulating tumour cells (CTCs) with mesenchymal traits. To provide tools that support their detection and analysis, the cell line BC-M1 established from disseminated tumour cells in the bone marrow of a breast cancer patient and a bone metastasis subline of MDA-MB-231 were analysed. Mass spectrometry analysis revealed high levels of CUB domain-containing protein 1 (CDCP1) in BC-M1. CDCP1 was found in other carcinoma cell lines (MDA-MB-231, MDA-MB-468) and other DTC cell lines (LC-M1, PC-E1) as well. Peripheral blood mononuclear cells were virtually negative for CDCP1 by Western Blot and immunofluorescent staining. Presence of CDCP1 in CTCs was confirmed by CellSearch. Here, CDCP1 positive CTCs were detected in eight of 30 analysed breast cancer patients. For the isolation of CTCs from the blood of breast cancer patients, we established a sandwich magnetic-activated cell sorting (MACS). The extracellular domain of CDCP1 served for cell catching and the cytoplasmic domain of CDCP1 for immunofluorescent detection of CDCP1 in CTCs. We showed that the MACS approach is suitable for the isolation of EpCam/keratin negative breast cancer cells from the blood and isolated CDCP1 positive CTCs from breast cancer patients by MACS. Hence, our approach is particularly suited for the detection and isolation of CTCs from TNBC when low EpCam or keratin levels limit the application of conventional approaches.
2023-03-20 | PXD035203 | Pride
Project description:Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. The effect of Wnt2 is correlated with fibronectin upregulation, and it is mediated in part through non-canonical Wnt signaling and suppressed by inhibition of the Map3k7 (Tak1) kinase, an integrator of Wnt, BMP and TGF-beta signaling. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed significant enrichment for non-canonical Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify novel therapeutic targets to prevent the distal spread of cancer. Expression profiling of primary tumor, circulating tumor cells and ascites in a mouse model of pancreatic cancer suggested WNT signaling plays a role in pancreatic cancer metastasis. Induction of Wnt2 signaling in mouse pancreatic NB508 cells supported the hypothesis.
2012-08-17 | E-GEOD-40171 | biostudies-arrayexpress
Project description:Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. The effect of Wnt2 is correlated with fibronectin upregulation, and it is mediated in part through non-canonical Wnt signaling and suppressed by inhibition of the Map3k7 (Tak1) kinase, an integrator of Wnt, BMP and TGF-beta signaling. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed significant enrichment for non-canonical Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify novel therapeutic targets to prevent the distal spread of cancer. Expression profiling of circulating tumor cells in human pancreatic cancer patients support a hypothesis that WNT signaling plays a role in pancreatic cancer metastasis.
2012-08-17 | E-GEOD-40174 | biostudies-arrayexpress
Project description:Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. The effect of Wnt2 is correlated with fibronectin upregulation, and it is mediated in part through non-canonical Wnt signaling and suppressed by inhibition of the Map3k7 (Tak1) kinase, an integrator of Wnt, BMP and TGF-beta signaling. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed significant enrichment for non-canonical Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify novel therapeutic targets to prevent the distal spread of cancer.
2012-08-17 | GSE40174 | GEO
Project description:Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. The effect of Wnt2 is correlated with fibronectin upregulation, and it is mediated in part through non-canonical Wnt signaling and suppressed by inhibition of the Map3k7 (Tak1) kinase, an integrator of Wnt, BMP and TGF-beta signaling. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed significant enrichment for non-canonical Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify novel therapeutic targets to prevent the distal spread of cancer.
2012-08-17 | GSE40171 | GEO
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:BACKGROUND: We characterized the whole transcriptome of circulating tumor cells (CTCs) in Stage II-III breast cancer to evaluate correlations with primary tumor biology. METHODS: CTCs were isolated from peripheral blood (PB) via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE-FACS). CTCs, PB and fresh tumors were profiled with RNA Seq. Formalin-fixed, paraffin-embedded (FFPE) tumors were subjected to RNA Seq and NanoString PAM50 assays with Risk of Recurrence (ROR) scores. RESULTS: CTCs were detected in 29/33 (88%) of patients. We selected 21 cases to attempt RNA Seq (median number of CTCs=9). 16 CTC samples yielded results that passed quality control metrics. These samples had a median of 4,311,255 uniquely mapped reads, less than PB or tumors. Intrinsic subtype predicted by comparing estrogen receptor (ER), progesterone receptor (PR) and HER2 versus PAM50 for FFPE tumors was 85% concordant. However, CTC RNA Seq subtype assessed by the PAM50 classification genes was highly discordant both with the subtype predicted by ER/PR/HER2 as well as by tumor PAM50. Two patients died of metastatic disease - both had high ROR scores and high CTC counts. We identified significant genes, canonical pathways, upstream regulators and molecular interaction networks comparing CTCs by various clinical factors. We identified a 75-gene signature with highest expression in CTCs and tumors taken together that was prognostic in The Cancer Genome Atlas and METABRIC datasets. CONCLUSION: It is feasible to use RNA Seq of CTCs in non-metastatic patients to discover novel tumor biology characteristics.
2019-02-04 | GSE111842 | GEO
Project description:CTCs in cancer patients are thought to be responsible for metastasis. Currently, there is no ex vivo model that can isolate this group of cells. We have developed an ex vivo 4D lung cancer model that forms perfusable tumor nodules and form CTCs. Gene array analyses show 2504 differentially expressed genes when comparing CTCs from the 4D model seeded with A549 cells to the same cells grown on a petri dish (2D). We compared the gene expression profile (Human OneArray v5 chip) of A549 cells, human lung cancer cell line, grown on petri dish (2D) and same cells circulating as tumor cells in our ex vivo model (4D/CTC).
2015-03-12 | E-GEOD-50991 | biostudies-arrayexpress
Project description:By applying RNA-ISH and RNAseq to circulating tumor cells (CTCs), the study provides definitive evidence of epithelial to mesenchymal transition (EMT) across all histological types of breast cancer, identifying mediators such as FOXC1 and TGF-β signaling, and demonstrating dynamic treatment-associated changes in EMT within clusters of CTCs. Epithelial to mesenchymal transition (EMT) has been postulated to contribute to the migration and dissemination of cancer cells, but supporting histopathological evidence is limited. We used a microfluidic device to isolate circulating tumor cells (CTCs), combined with multiplex fluorescent RNA-in-situ hybridization (ISH) and RNA sequencing, to quantify and characterize EMT in breast cancer cells within the bloodstream. Whereas only rare (0.1-10%) cells in the primary tumor expressed both mesenchymal and epithelial markers, such biphenotypic as well as purely mesenchymal cells were enriched among CTCs, across all histological subtypes of breast cancer. In an index patient followed longitudinally, fluctuation in epithelial and mesenchymal states was observed as a function of initial response and subsequent resistance to therapy. Mesenchymal markers were predominant in clusters of tumor cells, many of which had adherent platelets. Finally, RNA sequencing of CTC clusters identified TGF-β and other EMT-related signatures, which were absent from more epithelial CTCs. FOXC1, a known regulator of EMT, was abundantly expressed in mesenchymal CTCs and was detectable within localized regions of the primary breast tumor. Together, these data support a role for EMT in the blood-borne dissemination of breast cancer and point to the dynamic nature of this cell fate change.
2013-02-01 | E-GEOD-41245 | biostudies-arrayexpress
Project description:CTCs in cancer patients are thought to be responsible for metastasis. Currently, there is no ex vivo model that can isolate this group of cells. We have developed an ex vivo 4D lung cancer model that forms perfusable tumor nodules and form CTCs. Gene array analyses show 2504 differentially expressed genes when comparing CTCs from the 4D model seeded with A549 cells to the same cells grown on a petri dish (2D).
2015-03-12 | GSE50991 | GEO