Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells
Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enume...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2014-07-01
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| Series: | Frontiers in Oncology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fonc.2014.00201/full |
| _version_ | 1818162975860588544 |
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| author | Erica L Carpenter JulieAnn eRader Jacob eRuden Eric F Rappaport Kristen N Hunter Paul L Hallberg Kate eKrytska Peter J O'Dwyer Yael P Mosse Yael P Mosse |
| author_facet | Erica L Carpenter JulieAnn eRader Jacob eRuden Eric F Rappaport Kristen N Hunter Paul L Hallberg Kate eKrytska Peter J O'Dwyer Yael P Mosse Yael P Mosse |
| author_sort | Erica L Carpenter |
| collection | DOAJ |
| description | Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells. Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control white blood cells. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples from patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients. |
| first_indexed | 2024-12-11T16:42:13Z |
| format | Article |
| id | doaj.art-1c3ad05571ea4da58a7971470f1713e3 |
| institution | Directory Open Access Journal |
| issn | 2234-943X |
| language | English |
| last_indexed | 2024-12-11T16:42:13Z |
| publishDate | 2014-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Oncology |
| spelling | doaj.art-1c3ad05571ea4da58a7971470f1713e32022-12-22T00:58:17ZengFrontiers Media S.A.Frontiers in Oncology2234-943X2014-07-01410.3389/fonc.2014.0020199305Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cellsErica L Carpenter0JulieAnn eRader1Jacob eRuden2Eric F Rappaport3Kristen N Hunter4Paul L Hallberg5Kate eKrytska6Peter J O'Dwyer7Yael P Mosse8Yael P Mosse9Perelman School of Medicine at the University of PennsylvaniaChildren's Hospital of PhiladelphiaChildren's Hospital of PhiladelphiaChildren's Hospital of PhiladelphiaChildren's Hospital of PhiladelphiaPerelman School of Medicine at the University of PennsylvaniaChildren's Hospital of PhiladelphiaPerelman School of Medicine at the University of PennsylvaniaChildren's Hospital of PhiladelphiaPerelman School of Medicine at the University of PennsylvaniaOur understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells. Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control white blood cells. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples from patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.http://journal.frontiersin.org/Journal/10.3389/fonc.2014.00201/fullNeuroblastomacirculating tumor cellwhole genome amplificationtargeted sequencingdisseminated tumor cellsingle cell capture |
| spellingShingle | Erica L Carpenter JulieAnn eRader Jacob eRuden Eric F Rappaport Kristen N Hunter Paul L Hallberg Kate eKrytska Peter J O'Dwyer Yael P Mosse Yael P Mosse Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells Frontiers in Oncology Neuroblastoma circulating tumor cell whole genome amplification targeted sequencing disseminated tumor cell single cell capture |
| title | Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells |
| title_full | Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells |
| title_fullStr | Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells |
| title_full_unstemmed | Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells |
| title_short | Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells |
| title_sort | dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells |
| topic | Neuroblastoma circulating tumor cell whole genome amplification targeted sequencing disseminated tumor cell single cell capture |
| url | http://journal.frontiersin.org/Journal/10.3389/fonc.2014.00201/full |
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