Download Integrated microfluidic device for drug accumulation measurement

Integrated microfluidic device for drug accumulation
measurement on a single MDR transporter-expressing
prostate cancer cell isolated among blood cells
Paul Li
Department of Chemistry,
Department of Molecular Biology and Biochemistry
Simon Fraser University (SFU)
Outline
• Why measuring single cells? We can measure rare cells
even though there are only a few of them
• Are the rare cells representative of the whole? High
statistical power of the same single cell analysis
(SASCA)
• Results confirmed by flow cytometry which analyze
thousand of different cells
• SASCA on prostate, lung cancer cells
• SASCA on AML patient cells
Hard to do bulk analysis with a
small amount of rare cells
Richard Cote, U Miami
CTC microfluidic chip
20 mm
1
1
2
E
30 mm
D
3
2
D
D
Chamber 1
E
Chamber 2
30 mm
B
A
B
E
A
2
B
A
Chamber 1
Label-free cell isolation
based on size difference
C
C
3
Chamber 2
Cell retention structure
for capturing a single cancer cell
1
2
3
2
C
1
2
3
100 μm
Preparation of the mixture of PCa and blood cells
RBC
RBC
WBC
1st PBS washing
PCa
RBC
WBC
RBC
WBC
2nd PBS washing
50 μm
Separation of the prostate cancer cell among
other blood cells
200 μm
Blood cells only
(WBCs +RBCs)
Prostate cancer cells
only
Prostate cancer cell
mixed with blood cells
Isolation of tumor cells from blood cells
Drug accumulation measured in a single PCa cell
Cell signal
Fluorescence Intensity
4000
Fi
3000
Fe
2000
Ft
Background signal
1000
0
0
200
400
600
Time (second)
Ft: total drug fluorescent intensity
Fi: intracellular drug fluorescent intensity
Fe: extracellular drug fluorescent intensity
800
1000
Drug accumulation in a single 22Rv1cell in the presence
of Fumitremorgin C (FTC) as a MDR inhibitor
Trypan blue treatment conducted in Chamber 2
of the CTC chip
0s
1.5 s
3s
5s
7s
8s
4s
200 μm
Comparison of drug accumulation in captured single
prostate cancer cells and in normal white blood cells
DNR: daunorubicin
FTC: fumitremorgin C
CsA: cyclosporine A
PTX: paclitaxel
Pgp: P-glycoprotein
HBSS: Hank’s balanced
salt buffer
Enhancement of OG-PTX accumulation in single
22Rv1 cells due to ABCG2 and ABCB1 inhibitors
Lung cancer cell study
Drug accumulation on a single NCI-H1650 cell in the presence of gefitinib.
Further in vivo tests are required
to confirm utility of treatment of
paclitaxel and gefitinib
Drug accumulation on a single NCI-HCC827 cell in the presence of gefitinib, followed by CsA.
Even though other issues of
bioavailabity, tumor penetration, etc,
are resolved, there is no drug
accumulation in the cell
Beyond cell lines
AML patient cells from British Columbia Cancer Agency:
10 CR samples and 10 NR samples,
AML: acute myeloid leukemia
CR: complete remission
NR: non-responsive
Assay of AML
patient cell samples
15
Three types of cells (M:MDR, N:non-B1C1-MDR and W: white blood cells)
Daunorubicin treatment on NR samples
16
AML patient data (CR samples: diagnosis and relapse)
Relapse samples
Diagnosis samples
DNR: daunorubicin
MK571: ABCC1 inhibitor
FTC: fumitremorgin C (ABCG2 inhibitor)
CsA: cyclosporine A
H: Hank’s balanced salt buffer
Detection of CD34 expression after
drug accumulation measurement
17
Single-Cell Bioanalysis (SCB)
Paul C.H. Li, PhD, Professor, Bioanalytical Chemistry, Microfluidic Biochips, [email protected]
Measure drug uptake in rare multidrug resistant (MDR) cancer cell
Anal. Chem. 2008, 80, 4095, Can. J Pure & Appl. Sci, 2009, 3, 765
LabChip, 2011, 11, 1378, Integrat. Biol. 2009, 1, 90
Prostate cancer cell study
(Australian Prostate Centre)
Discovery of MDR inhibitors
Lung cancer cell study
Drug accumulation on a single NCI-H1650 cell in
the presence of gefitinib.
Drug accumulation on a single NCI-HCC827 cell in the
presence of gefitinib, followed by CsA.
AML patient data (3 types of cells)
How many samples?
One sample, one single cell
One sample, 23 single cells
Acknowledgements
 Dr. James Li for initiating the SASCA drug
accumulation experiments
 Dr. Donna Hogge (BCCA) for providing AML patient
cell samples (CR and NR)
 Dr. Maher (Australian blood bank) for AML patient cell
CR samples (diagnosis and relapse)
 Dr. Ricky Wong and Dr. Patrick Yue (HK Baptist U) for
providing ginsenosides, and Yuchun Chen and Iryna
Kolesnyk for help with experiments
 Avid Khamenehfar for prostate and lung cancer cell
measurement
Supporting slides
Isolation of white blood cells from blood through a
Ficoll gradient
Plasma + Platelets
Buffy coat including
White blood cells (WBCs)
Ficoll solution
Red blood cells (RBCs)
After 1st
PBS washing
After 2nd
PBS washing
Optimization of DNR and OG-PTX concentrations for
drug accumulation measurements
Effective concentrations of MDR inhibitors for
experiments on the 22Rv1 cells
3000
2500
2000
FTC
Same cell treated with DNR
Series1
1500
Same cell treated with OG-PTX
Series2
1000
500
0
1
+FTC
102 μM
+20
3
+40
+80
4
5
CsA
Fluorescence Intensity
3000
2500
2000
Same cell treated with DNR
Series1
1500
Same cell treated with OG-PTX
Series2
1000
500
0
+CsA
0.5 μM
+2.5
+5
+10
+20
Enhancement of DNR accumulation in single
22Rv1 cells due to ABCG2 and ABCB1 inhibitors