A microfluidic device for label-free, physical capture of circulating tumor cell-clusters
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE67939 | GEO | 2015/05/17
SECONDARY ACCESSION(S): PRJNA281253
REPOSITORIES: GEO
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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
Project description:Circulating tumor cell clusters/micro-emboli (CTM) possess greater metastatic capacity and survival advantage compared to individual circulating tumor cell (CTCs). However, the formation of CTM subtypes and their role in tumor metastasis remain unclear. In this study, we used a microfluidic Cluster-chip with easy operation and high efficiency to isolate CTM from peripheral blood, which confirmed their correlation with clinicopathological features and identified the critical role of CTC-platelet clusters in BC metastasis. The correlation between platelets and CTM function was further confirmed in a mouse model and RNA-seq of CTM identified high-expressed genes related to hypoxia stimulation and platelet activation which possibly suggested the correlation of hypoxia and CTC-platelet cluster formation. In conclusion, we successfully developed the Cluster-chip platform to realize the clinical capture of CTMs and analyze the biological properties of CTC-platelet clusters, which could benefit the design of potential treatment regimens to prevent CTM-mediated metastasis and tumor malignant progression.
2024-02-28 | GSE256445 | GEO
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:Extremely rare circulating tumor cell (CTC) clusters are both increasingly appreciated as highly metastatic precursors and virtually unexplored. Technologies are primarily designed to detect single CTCs and often fail to account for the fragility of clusters or to leverage cluster-specific markers for higher sensitivity. Meanwhile, the few technologies targeting CTC clusters lack scalability. Here, we introduce the Cluster-Wells, which combines the speed and practicality of membrane filtration with the sensitive and deterministic screening afforded by microfluidic chips. The >100,000 microwells in the Cluster-Wells physically arrest CTC clusters in unprocessed whole blood, gently isolating virtually all clusters at a throughput of >25 mL/h, and allow viable clusters to be retrieved from the device. Using the Cluster-Wells, we isolated CTC clusters ranging from 2–100+ cells from prostate and ovarian cancer patients and analyzed a subset using RNA sequencing. Routine isolation of CTC clusters will democratize research on their utility in managing cancer.
2022-05-24 | GSE202650 | GEO
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.
2014-07-11 | GSE51827 | GEO
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: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:Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tu-mor cells, CTCs) either alone or Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tu-mor cells, CTCs) either alone or as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell–cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tis-sue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on com-panion cells in clusters.as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell–cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils. Our results suggest that the EV proteome of CTCs contain signaling proteins that can modulate degranulation and granule mobilization in neutrophils and, also, contain tis-sue plasminogen activator and other proteins that can regulate cluster formation. By exposing naïve neutrophils to EVs isolated from CTCs, we further show how these changes are modulated in a dynamic fashion indicating evidence for a deeper EV based remodulatory effect on com-panion cells in clusters.
2021-06-07 | PXD026340 | Pride
Project description:Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, with the latter displaying an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the intravasation of CTC clusters are largely unknown. Here, we compare gene expression of hypoxic and normoxic CTC clusters to identify genes that are not only upregulated as a consequence to hypoxia, but that may also play a pivotal role in promoting CTC clusters generation and metastasis.
2020-09-08 | GSE126669 | GEO