Specific Binding of Cancer Cells Using a MicroChamber Array Functionalized with Antibodies X. Zheng1, S.L. Cheung1, L. Wang2, J. Schroeder3, R. Heimark4, J.C. Baygents2, R.Guzman2 & Y. Zohar1 November 19, 2009 1Aerospace

& Mechanical Engineering, 2Chemical & Environmental Engineering 3Molecular and Cellular Biology, 4Department of Surgery

University of Arizona, Tucson, AZ, USA THE UNIVERSITY OF ARIZONA

OUTLINE • Introduction – Motivation, Background & Objective

• Experimental Arrangements – Device fabrication – Surface coating

• Experimental Results – – – – –

Concentration effect Incubation time effect Ambient temperature effect Surface coating effect Cell type effect

• Conclusions THE UNIVERSITY OF ARIZONA

MOTIVATION Microchip for sorting CTCs

• Challenge: Sort 1 CTC in 1-10 billion blood cells

• Approach: Single step from whole blood

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BACKGROUND Cadherins can be used as a highly selective tool to specifically capture target cells in tissue or blood.

Cell

Find the proper lock to a given key ! N-cad E-cad

9 THE UNIVERSITY OF ARIZONA

X

Cell

BACKGROUND Antibody Structure

MD A -M B -2 31

PC 3N

BT -2 0

Western Blotting

Binding site Heavy chain Fab region

N-Cad Light chain

E-Cad

Cad-11

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Hinge

HOOC

COOH

Fc region

OBJECTIVE ¾ Conceptual Design

Advantages: 9 9 9 9 9

Liquid/gas interface (surface tension & liquid evaporation) is eliminated Number & type of cells loaded into each chamber can be determined Total sample volume in each chamber is accurately known Number & type of cells captured in each chamber can be determined Chamber array (5x5) provides multiple data points for statistical analysis THE UNIVERSITY OF ARIZONA

EXPERIMENTAL ARRANGEMENTS ¾ Device fabrication

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EXPERIMENTAL ARRANGEMENTS ¾ Surface functionalization 100ug/ml antibody in PBS, 1 hr 2mg/ml BSA in PBS, 1 hr

O=CH N

O=CH N

O=CH N

CH

CH

CH

=

P

=

P

=

P

NH2

NH2

NH2

Si

Si

Si

OH OH OH OH OH OH OH OH OH

50ug/ml protein G in PBS, overnight, 40C 2% glutaraldehyde in PBS, 2 hrs

P

H 2N

O HC

1% APTES in acetone, 30 mins

C H2

H2 C

O C H2

CH

OCH 2CH 3 CH 3CH 2O

Si OCH 2CH 3

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NH 2

EXPERIMENTAL ARRANGEMENTS ¾ Device testing Anti-Mouse IgG (CY3) 15ug/ml, 1hr

P

P

N=CH

N=CH

N=CH

=

CH

CH

CH

=

=

O

Anti-N-Cadherin

P

N

N

N

Si

Si

Si

O

O

O

O

O

O

O

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(IgG1 from mouse)

• O

Uniform intensity → Uniform antibody activity

EXPERIMENTAL RESULTS

‰ Cell concentration • 104/ml - 2.5x105/ml ‰ Incubation time • 15 min - 2 hrs

2

‰ PC3N on anti-N-cad

Attached Cells [cells/mm ]

¾ Cell concentration effect on capture rate 800

600

400 120 min 60 min 30 min 15 min

200

0 0.0

0.5

1.0

1.5

5

2.0

3

2.5

Concentration [10 cells/cm ]

•Density increases linearly with concentration (within the test concentration range; i.e. every cell contact the functionalized surface) THE UNIVERSITY OF ARIZONA

3.0

EXPERIMENTAL RESULTS

‰ Cell concentration • 104/ml - 2.5x105/ml ‰ Incubation time • 15 min - 2 hrs

2

‰ PC3N on anti-N-cad

Attached Cells [cells/mm ]

¾ Incubation time effect on capture rate 1200

5 2.5105cells/ml 2.5x10 cells/ml

5 105cells/ml 10 cells/ml

1000

4 5104cells/ml 5x10 cells/ml

4 104cells/ml 10 cells/ml

800 600

Asymptotic density level

400 200 0 0

20

Time constant

40 60 80 100 Incubation time [min]

120

• Captured cell density increased exponentially with incubation time • Asymptotic density level increased almost linearly with concentration • Time constant increases with cell concentration THE UNIVERSITY OF ARIZONA

EXPERIMENTAL RESULTS ¾ Ambient temperature effect on capture rate ‰ PC3N on anti-N-cad

‰ Temperature • 4oC - 37oC ‰ Utilize NaN3 treatment to prevent possible receptor internalization at 37oC

Attached Cells [%]

‰ Cell concentration • 105/ml

100% 37 °C NaN3 treated 37 °C 25 °C 4 °C

80% 60% 40% 20% 0% 0

• 4oC, capture no cells up to 60 minutes • 25oC & 37oC, 90% after 15 minutes THE UNIVERSITY OF ARIZONA

10

20 30 40 Incubation time [min]

50

60

EXPERIMENTAL RESULTS ¾ Surface coating effect on PC3N capture rate

anti-N-cad

Mouse-IgG

SiO2

Captured PC3N cells, from 5x105 cells/ml suspensions, on various coated surfaces.

Attached Cells [%]

100% 80% 60% 40% 20% 0%

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Anti-N-Cadherin Mouse IgG SiO2

EXPERIMENTAL RESULTS ¾ Cell type effect on cell capture rate

PC3N

BT20

MDA-MB-231 PC3N BT20 MDA-MB-231

Different cell types (5x105/ml) captured on anti-N-cad surfaces

Attached Cells [%]

100% 80% 60% 40% 20% 0%

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1

EXPERIMENTAL RESULTS ¾ Cell type effects N-cad-expressing MDA-MB-231 cells; Green color marks N-cad molecules.

MDA-MB-231

Ncdh+MDA-MB-231

Ncdh+MDA-MB-231 MDA-MB-231

Capture of MDA-MB-231 and Ncdh+MDA-MB-231 cells (105/ml) on anti-Ncadherin surfaces.

Attached Cells [%]

80%

60%

40%

20%

0%

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1

CONCLUSIONS ¾ Microchambers have been fabricated in Si wafers and functionalized with bio-active layers. ¾ Capture of target cells from suspensions increases: • linearly with concentration (dilute suspension). • exponentially with incubation time.

¾ Cell receptor – surface ligand binding affinity is highest at a temperature range of 25-37°C. ¾ The receptor – antibody binding is highly specific; changing cell-type or immobilized-antibody results in very low binding affinity.

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ACKNOWLEDGEMENTS ¾ This work is supported by a BCRP grant, BC061859, and an Arizona Biomedical Research Commission grant 06-080. ¾ The coding of human N-cadherin cDNA was a gift from Dr. Brian McCray (The University of Arizona). ¾ The Si-DRIE was carried out at the Arizona State University, courtesy of Prof. Jun Chae.

Thank you THE UNIVERSITY OF ARIZONA

EXPERIMENTAL RESULTS ¾ Surface coating effect on BT20 capture rate

anti-E-cad

anti-N-cad

Mse-IgG

Captured BT20 cells, from 5x105c cells/ml suspensions, on various coated surfaces.

Attached Cells [%]

100% 80% 60% 40% 20% 0%

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SiO2 Anti-E-Cadherin Anti-N-Cadherin Mouse IgG SiO2

Specific Binding of Cancer Cells Using a ...

Jan 16, 2010 - X. Zheng1, S.L. Cheung1, L. Wang2, J. Schroeder3, R. Heimark4,. J.C. Baygents2, R.Guzman2 & Y. Zohar1. November 19, 2009. 1Aerospace & Mechanical Engineering, 2Chemical & Environmental Engineering. 3Molecular and Cellular Biology, 4Department of Surgery. University of Arizona, Tucson, AZ, ...

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