ApoStream™, a New Dielectrophoretic Device for Antibody Independent Isolation and Recovery of

Viable Cancer Cells from Blood

May 26th, 2013

Presenter: Anoop Menachery, Ph.D.

Special DEP Session at Advances in Microfluidics & Nanofluidics (AMN 2013) May 24-26, 2013, University of Notre Dame, Notre Dame, Indiana, USA

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Introduction • Isolation and enumeration of circulating tumor cells (CTCs) are

used to monitor metastatic disease progression and guide cancer therapy.

• Currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone.

• ApoCell developed a device, ApoStream™, that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to separate CTCs from blood using dielectrophoretic technology in a microfluidic flow chamber.

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ApoStream™ Technology Theory of Operation

Dielectric properties (polarizability) of cells are dependant upon cell diameter, membrane morphology and conductivity. Inherent differences in morphology of CTCs and normal cells result from differences in dielectric polarization when exposed to an AC electric current.

Normal cell

Tumor cell

For the separation of cancer cells from healthy blood cells, the ApoStream™ device operates in a modified form to conventional DEP-FFF, in that the cancer cells are attracted by positive DEP forces towards the electrode plane, and thus away from the bulk of the blood cells that are levitated by negative DEP into the higher velocity region. Gupta et al., Biomicrofluidics 6, 024133 (2012)

flow

DEP levitation +

hydrodynamic lift forces

sedimentation force

Electrode array

Tumor Cells

PBMCs

(A) Dielectrophoretic, hydrodynamic and sedimentation forces are utilized to attract CTCs and repel normal cells from the chamber floor.

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DEP Crossover Frequency Forms the Basis for Separation of CTCs from Blood Cells

• DEP crossover frequency differs between cancer cells and normal blood cells.

• This difference enables ApoStream™ to separate cancer cells from normal blood cells.

Cancer Cells

Blood Cells

Attracted

Repelled

Sangjo Shim et al. Biomicrofluidics, 7, 011808, 2013. P. R. C. Gascoyne et al. Electrophoresis 30, 1388, 2009.

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ApoStream™ Flow Schematic

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Separation Flow Chamber Design

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Video Footage of Spiked Cell Separation through ApoStream™

(Left) Video demonstrating the flow and collection of fluorescently labeled SKOV3 cancer cells through the collection port in the ApoStream™ flow chamber. Cancer cells are collected into the collection port when the DEP field is activated. (Right) Video demonstrating the flow of fluorescently labeled PBMCs through ApoStream™ flow chamber. The first half of the video (10 s) demonstrates that most PBMCs fall into the collection port when the DEP field is not active. The second half of the video (from 11 to 21 s) demonstrates that upon the activation of DEP field the PBMCs are repelled from the electrode causing them to move into the high velocity flow region and are no longer being pulled into the collection port .

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Design of Experiment (DOE) for Parameter Optimization

• The performance of the device was optimized using model cell lines in a design of experiment approach for key operating parameters.

– Frequency – Voltage – Flow rates – Buffer Formulation

• Optimized parameters were then used in cell spiking studies with SKOV3 and MDA-MB-231 cell lines (high and low EpCAM expression, respectively) to demonstrate linearity and precision of recovery.

• Frequency was selected to effect positive DEP forces in the model cancer cell lines while sufficiently below crossover frequency of normal blood cells.

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Inter-day and Intra-day Precision Day of Run Replicate % Cancer Cell Recovery

Average % Cancer Cell Recovery

Standard Deviation

Coefficient of Variation (%CV)

Sample 1 75.0Sample 2 73.8Sample 3 77.6Sample 4 73.1Sample 1 77.4Sample 2 71.0Sample 3 78.7Sample 4 77.5Sample 5 75.0Sample 6 73.8Sample 7 77.6Sample 8 73.1Sample 9 81.4

Sample 10 75.2Sample 11 72.7Sample 12 71.6Sample 1 69.1Sample 2 70.8Sample 3 70.0

Day 1 Sample 1 73.4Day 2 Sample 2 72.6Day 3 Sample 3 71.2

2.7

1.2

Day 1

Intra-day Precision (MDA-MB-231 cells) Day 1 70.0 0.9

Intra-day Precision (SKOV3 cells) 74.9 2.0

Inter-day Precision (MDA-MB-231 cells)

76.2

0.7 0.9Day 2 74.9

Day 3 75.2

Inter-day Precision (SKOV3 cells)

Day 1

72.4 1.1 1.5

• The average recovery of SKOV3 and MDA-MB-231 cancer cells was 75.4 ± 3.1% (n=12) and 71.2 ± 1.6% (n=6), respectively.

• The intra-day and inter-day precision coefficients of variation (CVs) of the device were both less than 3%.

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Linearity of Cancer Cell Recovery

• Linear regression analysis yielded a correlation coefficient (R2) of more than 0.99 for a spiking range of 4-2,600 cells.

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Images of cultured MDA-MB-231 cancer cells at day 2 and day 7: (a,b) control cells (no ApoStream™ separation); (c,d) cells captured with ApoStream™.

ApoStream™ recovered MDA-MB-231 cancer cells show exponential growth and no difference compared to control cells.

ApoStream™ Captured Cells Retain Viability MDA-MB-231 Breast Cancer Cells

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Conclusions

• ApoStream™ separation exploits the differences in dielectrophoretic properties between cancer cells and normal blood cells.

• The ApoStream™ device demonstrated high precision and linearity of recovery of viable cancer cells independent of their EpCAM expression level.

• Isolation and enrichment of viable cancer cells from the ApoStream™ has the potential to enable molecular characterization of CTCs from a wide range of cancer types.

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Acknowledgements

Funded in part by NCI Contract No. HHSN261200800001E Robert Kinders, PhD Priya Balasubramanian, PhD Lihua Wang, PhD

Anoop Menachery, PhD Vishal Gupta, PhD Vlada O. Melnikova, PhD David K. Hasegawa, MS Darren W. Davis, PhD

James H. Doroshow, MD Joseph E. Tomaszewski, PhD

Institute for Integrated Micro and Nano Systems Ronald Pethig, PhD

http://dctd.cancer.gov/�

ApoStream™, a New Dielectrophoretic Device for Antibody Independent Isolation and Recovery of Viable Cancer Cells from BloodIntroductionApoStream™ Technology �Theory of OperationDEP Crossover Frequency Forms the Basis for Separation of CTCs from Blood CellsApoStream™ Flow SchematicSeparation Flow Chamber Design Video Footage of Spiked Cell Separation through ApoStream™Design of Experiment (DOE) for Parameter OptimizationInter-day and Intra-day PrecisionLinearity of Cancer Cell RecoverySlide Number 11ConclusionsAcknowledgements