Microfluidic devices for the interrogation of single circulating tumor cells

Yoon Sun Yang, Hoon Suk Rho, Joost F. Swennenhuis, Michiel Stevens, Arjan G.J. Tibbe, Severine le Gac, Han Gardeniers, Leon W.M.M. Terstappen

Research output: Contribution to conferencePoster

Abstract

Introduction: Genetic and phenotypic characterization of Circulating Tumor Cells (CTC) offer the opportunity for a “real time liquid biopsy”. However, heterogeneity and rarity of CTC command the need for individual cell characterization. Following an enrichment procedure of CTC from blood, the identification, isolation and manipulation of single CTC for further analysis without cell loss remains challenging. Here, we present microfluidic devices for parallel single cell whole genome amplification (psc WGA) and parallel probing of drug response of single cancer cells (psc probing).

Method: Microfluidic devices were designed using AUTOCAD software, and fabricated using PDMS multilayer soft-lithography. Cells from the SKBR-3 and MCF-7 breast cancer cell lines were used in the devices and identified using fluorescence microscopy after immunofluorescence staining. For pscWGA, the GE Single Cell GenomiPhi DNA Amplification kit was used under isothermal conditions.

Results: In the 1st scWGA device, single cancer cells were addressed in 16 individual reaction chambers, subsequently lyzed, and their DNA amplified on a chip. We successfully amplified DNA of single cancer cells in a ca. 23 nanoliter reaction volume. 1,000-fold amplified DNAs were validated using qPCR targeting a set of genes on different chromosomes. For WGA of CTC present in a large number of other cells, we developed a 2nd scWGA platform by combining a self-sorting microwell cell sorter and a microfluidic device. After filtration of a cell suspension using a microwell plate, cancer cells were identified using fluorescence microscopy at the bottom of the plate. Cells of interest were subsequently punched into the open-well structures of the microfluidic device for further analysis. The lysis and WGA reaction buffers were loaded using peristaltic pumping of integrated micro-valves. After cell lysis, DNA was amplified in the open-well reaction chamber. For validation ∼ 100 ng of DNA was pipetted out of the well. We also developed microfluidic devices to study drug-dose response of single cancer cells. This device is capable of capturing single cells, dosing various concentrations of drugs and exposing the cells to the drugs. We optimized the capturing efficiency using different sizes of beads (3 μm, 6 μm, and 15 μm) as well as MCF-7 cells. We demonstrated that single cancer cells could be exposed to different drug candidates in the reagent chambers and their response measured.

Conclusion: We successfully developed various microfluidic devices for individual cell characterization to be applied for CTC analysis. For genetic make-up, whole genome amplification of single cells either in suspension or in a self-sorting microwell plate was demonstrated. On-chip cell lysis and DNA amplification were performed and validated by qPCR targeting specific genes. In addition microfluidic devices were designed and tested to investigate single cell response to cancer drugs.
Original languageEnglish
DOIs
Publication statusPublished - 20 Apr 2015
Event106th AACR Annual Meeting 2015 - Philadelphia, United States
Duration: 17 Apr 201522 Apr 2015
Conference number: 106

Conference

Conference106th AACR Annual Meeting 2015
CountryUnited States
CityPhiladelphia
Period17/04/1522/04/15

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Lab-On-A-Chip Devices
Circulating Neoplastic Cells
DNA
Neoplasms
Pharmaceutical Preparations

Keywords

  • METIS-311902

Cite this

Yang, Y. S., Rho, H. S., Swennenhuis, J. F., Stevens, M., Tibbe, A. G. J., le Gac, S., ... Terstappen, L. W. M. M. (2015). Microfluidic devices for the interrogation of single circulating tumor cells. Poster session presented at 106th AACR Annual Meeting 2015, Philadelphia, United States. https://doi.org/10.1158/1538-7445.AM2015-367
Yang, Yoon Sun ; Rho, Hoon Suk ; Swennenhuis, Joost F. ; Stevens, Michiel ; Tibbe, Arjan G.J. ; le Gac, Severine ; Gardeniers, Han ; Terstappen, Leon W.M.M. / Microfluidic devices for the interrogation of single circulating tumor cells. Poster session presented at 106th AACR Annual Meeting 2015, Philadelphia, United States.
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abstract = "Introduction: Genetic and phenotypic characterization of Circulating Tumor Cells (CTC) offer the opportunity for a “real time liquid biopsy”. However, heterogeneity and rarity of CTC command the need for individual cell characterization. Following an enrichment procedure of CTC from blood, the identification, isolation and manipulation of single CTC for further analysis without cell loss remains challenging. Here, we present microfluidic devices for parallel single cell whole genome amplification (psc WGA) and parallel probing of drug response of single cancer cells (psc probing).Method: Microfluidic devices were designed using AUTOCAD software, and fabricated using PDMS multilayer soft-lithography. Cells from the SKBR-3 and MCF-7 breast cancer cell lines were used in the devices and identified using fluorescence microscopy after immunofluorescence staining. For pscWGA, the GE Single Cell GenomiPhi DNA Amplification kit was used under isothermal conditions.Results: In the 1st scWGA device, single cancer cells were addressed in 16 individual reaction chambers, subsequently lyzed, and their DNA amplified on a chip. We successfully amplified DNA of single cancer cells in a ca. 23 nanoliter reaction volume. 1,000-fold amplified DNAs were validated using qPCR targeting a set of genes on different chromosomes. For WGA of CTC present in a large number of other cells, we developed a 2nd scWGA platform by combining a self-sorting microwell cell sorter and a microfluidic device. After filtration of a cell suspension using a microwell plate, cancer cells were identified using fluorescence microscopy at the bottom of the plate. Cells of interest were subsequently punched into the open-well structures of the microfluidic device for further analysis. The lysis and WGA reaction buffers were loaded using peristaltic pumping of integrated micro-valves. After cell lysis, DNA was amplified in the open-well reaction chamber. For validation ∼ 100 ng of DNA was pipetted out of the well. We also developed microfluidic devices to study drug-dose response of single cancer cells. This device is capable of capturing single cells, dosing various concentrations of drugs and exposing the cells to the drugs. We optimized the capturing efficiency using different sizes of beads (3 μm, 6 μm, and 15 μm) as well as MCF-7 cells. We demonstrated that single cancer cells could be exposed to different drug candidates in the reagent chambers and their response measured.Conclusion: We successfully developed various microfluidic devices for individual cell characterization to be applied for CTC analysis. For genetic make-up, whole genome amplification of single cells either in suspension or in a self-sorting microwell plate was demonstrated. On-chip cell lysis and DNA amplification were performed and validated by qPCR targeting specific genes. In addition microfluidic devices were designed and tested to investigate single cell response to cancer drugs.",
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Yang, YS, Rho, HS, Swennenhuis, JF, Stevens, M, Tibbe, AGJ, le Gac, S, Gardeniers, H & Terstappen, LWMM 2015, 'Microfluidic devices for the interrogation of single circulating tumor cells' 106th AACR Annual Meeting 2015, Philadelphia, United States, 17/04/15 - 22/04/15, . https://doi.org/10.1158/1538-7445.AM2015-367

Microfluidic devices for the interrogation of single circulating tumor cells. / Yang, Yoon Sun; Rho, Hoon Suk; Swennenhuis, Joost F.; Stevens, Michiel ; Tibbe, Arjan G.J.; le Gac, Severine ; Gardeniers, Han; Terstappen, Leon W.M.M.

2015. Poster session presented at 106th AACR Annual Meeting 2015, Philadelphia, United States.

Research output: Contribution to conferencePoster

TY - CONF

T1 - Microfluidic devices for the interrogation of single circulating tumor cells

AU - Yang, Yoon Sun

AU - Rho, Hoon Suk

AU - Swennenhuis, Joost F.

AU - Stevens, Michiel

AU - Tibbe, Arjan G.J.

AU - le Gac, Severine

AU - Gardeniers, Han

AU - Terstappen, Leon W.M.M.

PY - 2015/4/20

Y1 - 2015/4/20

N2 - Introduction: Genetic and phenotypic characterization of Circulating Tumor Cells (CTC) offer the opportunity for a “real time liquid biopsy”. However, heterogeneity and rarity of CTC command the need for individual cell characterization. Following an enrichment procedure of CTC from blood, the identification, isolation and manipulation of single CTC for further analysis without cell loss remains challenging. Here, we present microfluidic devices for parallel single cell whole genome amplification (psc WGA) and parallel probing of drug response of single cancer cells (psc probing).Method: Microfluidic devices were designed using AUTOCAD software, and fabricated using PDMS multilayer soft-lithography. Cells from the SKBR-3 and MCF-7 breast cancer cell lines were used in the devices and identified using fluorescence microscopy after immunofluorescence staining. For pscWGA, the GE Single Cell GenomiPhi DNA Amplification kit was used under isothermal conditions.Results: In the 1st scWGA device, single cancer cells were addressed in 16 individual reaction chambers, subsequently lyzed, and their DNA amplified on a chip. We successfully amplified DNA of single cancer cells in a ca. 23 nanoliter reaction volume. 1,000-fold amplified DNAs were validated using qPCR targeting a set of genes on different chromosomes. For WGA of CTC present in a large number of other cells, we developed a 2nd scWGA platform by combining a self-sorting microwell cell sorter and a microfluidic device. After filtration of a cell suspension using a microwell plate, cancer cells were identified using fluorescence microscopy at the bottom of the plate. Cells of interest were subsequently punched into the open-well structures of the microfluidic device for further analysis. The lysis and WGA reaction buffers were loaded using peristaltic pumping of integrated micro-valves. After cell lysis, DNA was amplified in the open-well reaction chamber. For validation ∼ 100 ng of DNA was pipetted out of the well. We also developed microfluidic devices to study drug-dose response of single cancer cells. This device is capable of capturing single cells, dosing various concentrations of drugs and exposing the cells to the drugs. We optimized the capturing efficiency using different sizes of beads (3 μm, 6 μm, and 15 μm) as well as MCF-7 cells. We demonstrated that single cancer cells could be exposed to different drug candidates in the reagent chambers and their response measured.Conclusion: We successfully developed various microfluidic devices for individual cell characterization to be applied for CTC analysis. For genetic make-up, whole genome amplification of single cells either in suspension or in a self-sorting microwell plate was demonstrated. On-chip cell lysis and DNA amplification were performed and validated by qPCR targeting specific genes. In addition microfluidic devices were designed and tested to investigate single cell response to cancer drugs.

AB - Introduction: Genetic and phenotypic characterization of Circulating Tumor Cells (CTC) offer the opportunity for a “real time liquid biopsy”. However, heterogeneity and rarity of CTC command the need for individual cell characterization. Following an enrichment procedure of CTC from blood, the identification, isolation and manipulation of single CTC for further analysis without cell loss remains challenging. Here, we present microfluidic devices for parallel single cell whole genome amplification (psc WGA) and parallel probing of drug response of single cancer cells (psc probing).Method: Microfluidic devices were designed using AUTOCAD software, and fabricated using PDMS multilayer soft-lithography. Cells from the SKBR-3 and MCF-7 breast cancer cell lines were used in the devices and identified using fluorescence microscopy after immunofluorescence staining. For pscWGA, the GE Single Cell GenomiPhi DNA Amplification kit was used under isothermal conditions.Results: In the 1st scWGA device, single cancer cells were addressed in 16 individual reaction chambers, subsequently lyzed, and their DNA amplified on a chip. We successfully amplified DNA of single cancer cells in a ca. 23 nanoliter reaction volume. 1,000-fold amplified DNAs were validated using qPCR targeting a set of genes on different chromosomes. For WGA of CTC present in a large number of other cells, we developed a 2nd scWGA platform by combining a self-sorting microwell cell sorter and a microfluidic device. After filtration of a cell suspension using a microwell plate, cancer cells were identified using fluorescence microscopy at the bottom of the plate. Cells of interest were subsequently punched into the open-well structures of the microfluidic device for further analysis. The lysis and WGA reaction buffers were loaded using peristaltic pumping of integrated micro-valves. After cell lysis, DNA was amplified in the open-well reaction chamber. For validation ∼ 100 ng of DNA was pipetted out of the well. We also developed microfluidic devices to study drug-dose response of single cancer cells. This device is capable of capturing single cells, dosing various concentrations of drugs and exposing the cells to the drugs. We optimized the capturing efficiency using different sizes of beads (3 μm, 6 μm, and 15 μm) as well as MCF-7 cells. We demonstrated that single cancer cells could be exposed to different drug candidates in the reagent chambers and their response measured.Conclusion: We successfully developed various microfluidic devices for individual cell characterization to be applied for CTC analysis. For genetic make-up, whole genome amplification of single cells either in suspension or in a self-sorting microwell plate was demonstrated. On-chip cell lysis and DNA amplification were performed and validated by qPCR targeting specific genes. In addition microfluidic devices were designed and tested to investigate single cell response to cancer drugs.

KW - METIS-311902

U2 - 10.1158/1538-7445.AM2015-367

DO - 10.1158/1538-7445.AM2015-367

M3 - Poster

ER -

Yang YS, Rho HS, Swennenhuis JF, Stevens M, Tibbe AGJ, le Gac S et al. Microfluidic devices for the interrogation of single circulating tumor cells. 2015. Poster session presented at 106th AACR Annual Meeting 2015, Philadelphia, United States. https://doi.org/10.1158/1538-7445.AM2015-367