Selection and Enumeration of Low-Abundance Biological Cells from Complex Matrices

Udara Dharmasiri

Research Seminar April 19, 2010

1

“Highly Efficient Capture and Enumeration of Low Abundance Prostate Cancer Cells Using Prostate-Specific Membrane Antigen Aptamers Immobilized to a Polymeric Microfluidic Device”

Electrophoresis, 2009, 30: 1-12

“Microsystems for the Capture of Low-Abundance Cells”

Annual Reviews of Analytical Chemistry, 2010, Vol. 3

“Enrichment and Detection of Escherichia coli 0157:H7 Using An Antibody Modified Microfluidic Chip”

Analytical Chemistry, 2010, 82 (7), 2844–2849

“High-Throughput Isolation and Electrokinetic

Manipulation of Circulating Tumor Cells Using a Polymeric Microfluidic Device”

2

Manuscript in Preparation

Highly Efficient Capture and Enumeration of Low Abundance Prostate Cancer Cells

Using Prostate-Specific Membrane Antigen Aptamers Immobilized to a Polymeric

Microfluidic Device

Electrophoresis, 2009, 30: 1-12

3

Loeb, S. The oncologist 2008, 13, 299-305

www.metastasis.cauchoiscar.com

Clinical Utility of Circulating Tumor Cells (CTCs)

• Cancer metastasis by circulating tumor cells (CTCs)

• Elucidating the presence and number of CTCs is emerging as an effective method for

- diagnosis- prognosis- prediction of therapeutic benefits

4

Analyzing Low Abundance Material from Peripheral Blood

• Red Blood Cells– 109/mL– 3 – 5 µm– Biconcave discs

Allard, W. J. et al. 2004, Clinical Cancer Res., 10, 6897-904

• White Blood Cells– 106/mL– ~15 µm– Spherical

• Circulating Tumor Cells– 1 – 10/mL– 15 – 30 µm– Spherical

1 = CTC2 = Membrane Pore3 = Leukocyte

Hayes, D. F. J. et al. 2008, Clinical Cancer Res., 14, 3646-3650 5

Existing Tools for Analyzing CTCs in Peripheral Blood

• Nuclear tracked polycarbonate membranes– Separation based on size– Requires whole blood density gradient centrifugation – 1 CTC in 1 mL of peripheral blood– Enumeration of cells by fluorescence visualization

• Magnetic capture using microbeads coated with recognition elements– 5 log enrichment– Only mononucleated

cell fraction– 1 CTC in 106 MNC– Enumeration of cells by fluorescence visualization

Dynal

Vona et al., 2000, Am. J. Pathology 256: 576

CTC-chip

● Target CTCs interact with antibody (EpCAM) coated microposts

● Purity - ~50%

● CTCs in the peripheral blood are captured and isolated

● Made from silica using DRIE and contains microposts

● Cell enumeration - by cell staining Nagrath, S. Nature 2007, 450, 1235-1239

● Capture efficiency - ~65%

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● Selectively and specifically isolate breast cancer cells through a monoclonal antibody mediated process ● Sampling large input (1 ml) of whole blood in short time (<37 min)

● CTC capture efficiency >97% and purity ~100%● The released CTCs enumerated on-device using conductivity detector with 100% detection efficiency

High Throughput Microsampling Unit (HTMSU)

Adams, A. A. JACS 2008, 130, 8633-86418

● Prostate cancer developsin the prostate gland

● The most common type of cancer in men in the USA

● LNCaP (Prostate cancer cell line) - Metastasize into the lymph nodes

● LNCaP cell membrane contains Prostate Specific Membrane Antigen (PSMA)

www.prostatecancerfoundation.org

● Diagnosed by Prostate Specific Antigen (PSA) test - high false positive and negative errors (30%)

Prostate Cancer

PSMA - 750 amino acids - Mw = 110 kDa - 1 x 106 molecules/cell

Liu, T. Prostate, 2008, 68, 955-964

LNCaP Cells9

NH2-(CH2)6-(CH2-CH2-O)6- CCAAGACCUGACUUCUAACUAAGUCUACGUUCC

Aptamers for LNCaP Cell Capture

● Generated by in vitro

selection process- SELEX

● Oligonucleotides Advantages● Chemically robust● Greater surface density ● Properties can be changed on demand• End-point attachment to surfaces

PSMA Aptamer

Expression level/Molecules cell-1

Molecular weight / kDa

Recognition Element

PSMA 1 x 106 100 PSMA aptamer

EpCAM 5 x 105 33 EpCAM antibody

● Mw = ~ 10 kDa and Kd = ~ 50 nM-1

Lupold, S. E. Cancer Res. 2002, 62, 4029-4033

Parrott, A.M Nuc. 2003, Acids Res. 28, 489-497 10

Polymer-based High-Throughput Sampling Unit for Capturing CTCs (PMMA)

1. 150 µm (depth) x 30 µm (width) x 3 cm (length)2. Total volume = 180 nL3. Number of parallel channels = 514. Processing time for 1 mL input (4 mm/s) = 9.1 min5. 4-levels of specificity (capture – 30 µm; detection – 50 µm;

immunoaffinity; shear) 6. Integrated reader for enumerating cells

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Production of Plastic Fluidic Components from Metal Molding Tools

(1) CNC controller(2) 40,000 rpm spindle(3) Automated tool changer(4) Laser-based measurement

system(5) Real-time imaging system(6) X-Y translational stage

(1)

3)

• KERN MMP 2252– Precision of ±1 µm– Microstructure aspect

ratios ≤ 20:1– Milling in metals,

ceramics, polymers

(2)

(3)(5)

(6)

(4)

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Producing Parts from Metal Molding Tools

(1)

(3)

(5)

(4)

(2)

►Jenoptik Microtechnik HEX 02– Attach mould insert– Evacuate chamber– Heat substrate to Tg

(105oC - PMMA)– Thermal fusion bonding for

channel enclosure

(1) CNC controller(2) Telescopic upper stage(3) Lower heating platen(4) Fixed stage(5) Vacuum system51-channel curvilinear

51-channel linear

50 μm linear

20 μm linear

35 μm linear(1)

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Immobilization of Aptamers to Polymer Surfaces

8.4 x 1012 molecules/cm2

Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300

McCarley et al., J. Am. Chem. Soc. 2005, 127, 842-84314

Monitoring Fluidic Optimization Measurements

Carl Zeiss Axiovert

● Computer controlled● Programmable motorized stage● Inverted optical microscope● Video CCD inspection ● Fluorescence imaging

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Cell Capture Efficiency● 0.5 ml of 1,000 cells/ml suspension introduced at linear velocities between 0.1 to 10 mm/s ● Post capture rinse with 150 mM PBS at 50 mm/s linear velocity ● The number of selected cells onto the PSMA aptamer and EpCAM antibody HTMSU counted using fluorescence microscopy

0 2 4 6 8 10

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Cap

ture

Effi

cien

cy (%

)

Linear Velocity (mm s-1)

Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300Chang and Hammer, Biophys. J.,1999, 76, 1280

Capture determined by

Encounter Rate (ko)

DNuko

PeNu 2

DVaPe 47.0

■ Aptamer

■ Antibody

Capture determined by

reaction Probability (P)

)1(

in

in

k

kP

kTEsin eFk /

Va

38

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Non-Specific Cell Adsorption● PSMA aptamer immobilized onto the HTMSU i) MCF-7 - Human breast cancer cell line, does not express PSMA ii) WBC - White blood cells iii) RBC - Red blood cells introduced at 2.5 mm/s linear flow velocity

● A post capture rinse performed with PBS buffer at 50 mm/s linear velocity

● The number of cells adsorbed onto the HTMSU counted using fluorescence microscopy

Trial MCF-7 WBC RBC

I 0 0 0

II 0 0 0

III 0 0 0

● PSMA aptamer does not interact with MCF-7, WBC and RBC

Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300

● 0.25% (w/v) trypsin infused into the HTMSU ● Trypsin (23.8 KDa) – glycoprotein that proteolytically cleaves at arginine and lysine AA residues. pI = 10.5; Optimal activity at pH = 8.0● The process of typsination was evaluated by microscopy

Cell Release from the Capture Surface

A. Before trypsin infused B. Exposed to trypsin for 2 minC. Exposed to trypsin for 6.5 min - cell was

releasedD. Exposed to trypsin for 7.5 min - cell was

removed

180 1 2 3 4 5 6 7 8

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Ce

ll R

em

ov

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Time (min)Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300

Whole blood

Whole blood + LNCaP

SN ≥ 3

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Automated Cell Enumeration• One ml of blood containing 20 LNCaP cells seeded into the HTMSU

● The captured cells released using the trypsin prior to on-chip

conductivity enumeration

● Total of 18 cells were counted at a volume flow rate of 5 µl/min

● The conductance response from whole blood without LNCaP obtained

0 50 100 150 200 2500

50

100

150

200

250

Number of spiked LNCaP cells per mL

Num

ber of

con

duct

ance

sig

nal r

egis

tere

d

R2 = 0.9997

Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300

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Conclusions ● LNCaP cell capture efficiency for PSMA aptamer tethered

microfluidic device was 95 +1%

● MCF-7 cells, WBCs and RBCs do not interfere with LNCaP

cell capture and therefore cell separation purity is ~100%

● 0.25% (w/v) trypsin was an effective reagent for releasing

LNCaP cells from the capture surface

● Conductivity enumeration efficiency was ~100% for the

released LNCaP cells

Enrichment and Detection of E. coli O157:H7 from Water Samples Using an

Antibody Modified Microfluidic Chip

Analytical Chemistry, 2010, 82 (7), 2844–2849

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• E. coli O157:H7 - gram negative bacterium rod-shaped 2 μm long and 0.5 μm diameter

• The pathogenicity of E. coli O157:H7 is associated with the production of Shiga-like toxins

-bloody diarrhea -colitis

• In 2008, 2,000 Americans were hospitalized and ~60 died

E. coli O157:H7

Alocilja, E.C. 2003. Biosens. Bioelectron. 18: 841-84

2 m

Coliform Standards (colonies /100 ml)Drinking Water 1TC*

Swimming Water 200FC*Boating Water 1000FC

Treated Sewage Effluent < 200FC

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(TC*- Total Coliform , FC*- Fecal Coliform)

EPA Approved Method for E. coli Detection

Bennett, A.R. 1996. Letters in Applied Microbiology 22: 237-243

• ß-D glucuronidase production is not present in O157:H7 serotype• E. coli O157:H7 viable but not culturable• Presence of interfering agents alter the accuracy of chromogenic

media

Cell culturing using EPA Method 1603. (LSU-BR University Lake)

15 cfu/100mL

x100volume processed# colonies

CFU/100 mL24 cfu/10mL 4 cfu/1mL

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Anti E.Coli O157:H7 Antibody

Microchip Enrichment

• Mw = ~150 kDa

• Polyclonal (pAb)

• Kd = ~50 pM-1

• E. coli O157:H7 are identified by combination

of O and H antigens

• 9×106 molecules of O antigen/bacteria

www.kpl.com

(i) pAb can recognize O157 types for intact and non-culturable cells

(ii) Selective cell capture allows cell enrichment and enumeration from potentially contaminated samples

Fitzmaurice, J. Mol. Cell. Probes 2004, 18, 123-13224

Polymer-Based (PMMA) Sampling Unit for E. coli O157:H7 Selection

11 mm

5.5 mm

8 sub devices-16 curvilinear channels-9.5 mm long, 15 µm width/80 µm depth. Surface area (cell selection bed) = 40 mm2 , volume = 250 nL

25Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849

E. coli O157:H7 Selection and Enumeration

26150 mM PBS solution infused at 50 mm/s linear velocity to remove non-specifically absorbed cells Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849

System Operation

Syringe pump15 m

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• Inverted optical microscope• Fluorescence imaging with

high sensitivity CCD• Syringe pump • E. coli cells were stained

- FITC (PKH67)

- Lipophilic membrane linker

Carl Zeiss Axiovert

0 1 2 3 4 520

25

30

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200

500252

348300

Slt!

Uid

AD

Thr

esho

ld c

ycle

(C

t)

Log (cell density) [cfu]

10 cfu

6 cfu

• PCR directed to the conserved regions within the genes encoding for SLT-I (shiga-like toxins) and the uidA gene, encodes for ß-glucuronidase in E. coli O157:H7

• Mismatch in G residue, as opposed to the T residue found in other E. coli strains)

Recognition of Escherichia coli O157:H7 by mismatch amplification assay-multiplex PCR (Cebula et al.,1995, 33, 248)

E. Coli O157:H7 Enumeration via RT-qPCR

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RT-qPCR Experiment Amplification and Dissociation Curves

Created with PSI-Plot, Sun Jun 14 21:57:04 2009

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may27ap.txt

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Fluo

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ence

(dRn

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Cycle # Temperature (oC)Fl

uore

scen

ce (-

Rn(T

))

specific DNA product (80 oC)

< 75°C correspond to non-specific DNA

Ct

slt1

Single blinded sample(cfu in the sample)

Real-time qPCR results for uidA gene

Ct ±SDcfu detected in the sample

(RSD %)

30 39.3 ±0.2 34 ±4 (12%)90 37.6 ±0.1 94 ±2 (2.1%)

150 37.1 ±0.1 130 ±8 (6.2%)400 35.2 ±0.3 405 ±5 (1.2%)800 33.9 ±0.1 799 ±15 (1.8%)

Real-time qPCR results for the uidA gene

29Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849

30

Cell Capture Efficiency• 3 x 103 cells/mL introduced at different volumetric flow rates

• Total input volume analyzed = 500 µL

10 20 30 40

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80

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Capt

ure

Effici

ency

(%)

Linear Flow Velocity (mm/s)Chang/Hammer model for mobile cell interactions

(1) Encounter rate(2) Probability of the reaction

Data for curvilinear channel, width- 15 m and depth- 80 m

Chang, K. C.; Hammer, D. A. Biophys. J. 1999, 76, 1280–1292

Data for linear channel, depth- 80 m

Channel width (m)Ca

ptur

e Effi

cien

cy (%

)

Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849

Specificity of Polyclonal Anti-E. coli O157:H7 Antibody

15 µm

A micrograph of capture surface E. coli O157:H7

A micrograph of capture surface E. coli K12

• 3x103 cells/mL were introduced at 5 mm/s linear velocity

31Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849

Cell Release from the Capture Surface• Mixture of chelators infused into channels at 10 mm/s

• Captured cells were observed microscopically until removed by release solution and Stoke’s force

A. Brightfield micrograph of the captured cell before being infused the releasing solutionB. Fluorescent micrograph of the captured cell before being infused the releasing solutionC. Brightfield micrograph of the cell released surface (4 mins) D. Fluorescent field micrograph of the cell released surface (4 mins)

0 min 4 min 4 min0 min

Avg. stripping time: 3.4 min +0.35 (n=25)

Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–284932

Water Sample Evaluation

Water Sample cfu in the sample Real-time qPCR results for slt1 gene

Standard Sample 200 cfu/200 mL (Spiked) 198 11 cfu

Standard Sample 1,000 cfu/100 mL (Spiked) 1045 100 cfu

Baton Rouge University Lake, LA

15 cfu/100 mL(EPA Method 1603) 122 cfu

Lake Granbury, TX 8 cfu/100 mL(EPA Method 1603) 51 cfu

Water Treatment Plant , LA 2.6 x 106 cfu/100 mL(EPA Method 1603) 9.6 x 105 2000 cfu *

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* Max Capacity of the bed: 260 x 106 cells

LSU LakeDharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849

Conclusions

• Recovery of E. coli O157:H7 was ~72%

• E. coli O157:H7 was selected and enumerated without other serotype interferences

• The strategy developed offered the ability to monitor recreational water quality without the need for a cell

culture step

• The entire processing steps were implemented in under 5 h

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High-Throughput Isolation and Electrokinetic Manipulation of

Circulating Tumor Cells Using a Polymeric Microfluidic Device

35

Future Work

Objectives

No. ofPatients

No. ofspecimens

Average No. of CTCs in 7.5 ml

Prostate cancer 123 188 47 (+13)

Breast cancer 422 1316 80 (+14)

Lung cancer 99 168 92 (+12)

Allard, W.J. Clin. Cancer Res. 2004 10: 6897-904

• Design a microfluidic device for processing 7.5 mL of blood to select CTCs in a short time period (~30 min)

• Electrokinetic collection of selected CTCs for molecular profiling

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Cell Selection and Manipulation

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High-Throughput Microsampling Unit (HTMSU)

Adams, A. A. JACS 2008, 130, 8633-8641Dharmasiri, U. Electrophoresis 2009, 30, 3289–3300

• Selectively and specifically isolate breast and prostate cancer cells through an affinity agent mediated process

• Sampling 1 ml of whole blood in short time (<37 min)

• CTC capture efficiency >97% and purity ~100%

• The released CTCs enumerated on-device using conductivity detector with ~100% detection efficiency

38

460 curvilinear channels Volume -100 L Process 7.5 mL of sample in 30 min

Cell Capture Section of µHTMSU

Out put In put

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Electrophoresis

F - Coulomb force q- Net charge on the object E- The applied electric field

F = qE

Annu. Rev. Biomed. Eng. 2006. 8:425–54

• Electrophoresis (EP): The force on a charged particle exerted by an electric field

• Most mammalian cells are covered with negatively charged functional groups at neutral pH

• In water, the cells move at a velocity given by the balance of the Coulomb and viscous drag forces, a process known as EP

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Electrokinetics

CTC Type Electrophoretic Mobility , (m.u.)

Breast cancer 1.19

Blast cell leukemia (large cells) 1.62

Colon cancer 1.47

Lung cancer 1.32

Vassar, P.S. Nature 1963, 4873, 1215-26

• Utilizes the electroosmotic flow (eof) of the solution and the electrophoretic mobility (ep) of the material being transported

• The linear velocity (app) at which the material moves is governed by;

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Cell Manipulation Section of the Microfluidic Unit

In put

Future Directions

• CTCs in large volume of patients’ blood (>7.5 mL) will be selected in short time (<30 min)

• Molecular profiling of CTCs

• Determine biology of CTCs and cells at primary tumor

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Detection of point mutations Gene expression profiling

Genetic Make up Adhesion properties Metastatic potential

AcknowledgmentsSoper Research Group

National Science FoundationGrant NIH # - 1 R33 CA099246-01State of Louisiana Board of RegentsTexas Sea Grant (NA06OAR4170076)

Dr. Maggie WitekDr. Robert TruaxMs. Karen

Prof. Steven A. SoperProf. Robin McCarley

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THANK YOU

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