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Towards a fast, efficientassay for isolating circulating tumor cells
PI: Professor David Eddington
Grad Student: Cari Launiere
Me: Joey Labuz
July 30, 2009
Introduction
Breast, colon, prostate, and lung cancers accounted for nearly half of cancer deaths (American Cancer Society, 2008)
All 4 can be metastatic diseases Circulating tumor cells (CTCs)
Rare in blood (as low as 1 in 1,000,000,000) Alternative to biopsy screenings High expression of epithelial cell adhesion
molecule (EpCAM) (Went et al., 2004)
CTC-chip assay
Posts fabricated from Si wafer 100 µm diameter 100 µm tall
Posts coated with anti-EpCAM
Whole blood flowed through device by pressure source
mL-scale volumes
SEM of Si posts with captured cancer cell (colored red for visibility)
(S Nagrath, et al. 2007)
CTC-chip assay (cont.)
Pros Simpler than other
methods (immunomagnetic beads)
No pre-processing of blood necessary
High sensitivity (99.1%) Improved purity (over
two times better)
Cons Complex fabrication
process (DRIE) Max flow of ~1 mL/hr
1-2 hours to run sample We can do ~6x faster
High cost Low efficiency (~60%) Low purity (~50%)
Photo/SoftLithography Rapid prototyping of
polydimethylsiloxane channels
Benefits of PDMS Good optical clarity Good scalability
PDMS channel placed on glass slide with proteins Rapid prototyping of PDMS channels
(JS Mohammed, et al. 2008)
Caveolin-1 Capture
Cav-1 expression generally inversely proportional to EpCAM expression
Explore as way to isolate CTCs with low EpCAM expression (i.e. MDA-MB-231)
(Sieuwerts, et al, 2009) Computer generated images of various Cav-1 conformations (Cai, et al)
E-Selectin Binding
Present in physiological flow situations (e.g. blood vessels)
Binds to cancer as well as blood cells (e.g. leukocytes)
Catch bond mechanism pulls cells out of flow
Chinese finger trap of proteins
Catch bonds’ strength increases as tensile force, until a maximum, where the force begins to overcome the bond strength (Thomas W, 2009).
Mixer Optimization
Force cells down to proteins on slide Channel height: 100 µm Groove height: 160 µm Grooves lead to transverse flow
Flow Slide with protein coat
Transverse Flow
(NS Lynn and DS Dandy, 2007)
GrooveChannel
Imaging Problem – Clumped Cells Clumped cells are often
counted as one, instead of several
Watersheding methods inadequate for separating cells and maintaining image quality
Imaging Solution – Clumped Cells Use ImageJ
Macro executes series of commands
Output text file to MatLab Use MatLab
Find clumped cells based on average area and standard deviation
Using average, separate clumps into individual cells
Cell area histogram: All cells with areas greater than the mean + standard deviation are considered clumps
Imaging Solution – Clumped Cells Validate method by
using hand counts Image 1
By hand: 97 Using program: 98 Error: 1 %
Image 2 By hand: 841 Using program: 831 Error: 1.2 %
Image 1
Image 2
Imaging Solution – Mixer
Use subtract function in ImageJ Subtracts grayscale values pixel by pixel Subtract image from control
Control image Image with cells
_
Preliminary results
Run trials with HL-60 and MDA-MB-231 cells, respectively
Cells roll on E-selectin as expected Observed under the
microscope at 0.1 mL/min
Anti-EpCAM helped maintain new capture
Anti-CAV1 helped facilitate stationary capture
Cells detach upon entering mixer Could be due to overly
turbulent flow Or due to poor protein
coating – adjust method for future experiments
Summary
CTCs attractive option for cancer screening Less invasive than biopsy Broader, earlier detection
Channel optimized to increase cell contact with protein-functionalized surface
Use protein cocktail to optimize capture E-selectin to pull cells out of flow Anti-EpCAM and anti-CAV1 to bind CTCs
Wrote programs for rapid image analysis
Acknowledgements
Financial support NSF DoD
Cari Launiere Prof. David Eddington REU advisors The BML lab My roommate
ReferencesCai, Q. C. et al. Putative caveolin-binding sites in SARS-CoV proteins. Acta
Pharmacologica Sinica 24, 1051-1059 (2003).Cancer Facts & Figures 2008. American Cancer Society (2008).Lynn NS and DS Dandy. “Geometrical optimization of helical flow in grooved
micromixers” Lab on a Chip. 7: 580-587. 2007.Mohammed, JS, HH Caicedo, et al. “Microfluidic add-on for standard electrophysiology
chambers.” Lab on a Chip. 8: 1048-1055. 2008Monahan, J., Gewirth, A. A. & Nuzzo, R. G. A method for filling complex polymeric
microfluidic devices and arrays. Analytical Chemistry 73, 3193-3197 (2001).Nagrath, S, LV Sequist, et. al. “Isolation of rare circulating tumour cells in cancer patients
by microchip technology.” Nature. 450: 1235-1239. 2007.Sieuwerts, A. M. et al. Anti-Epithelial Cell Adhesion Molecule Antibodies and the
Detection of Circulating Normal-Like Breast Tumor Cells. Journal of the National Cancer Institute 101, 61-66 (2009).
Thomas, W. Research Projects: Catch Bonds.<https://faculty.washington.edu/wendyt/research.html>. 2009.
Went, P. T. et al. Frequent EpCam protein expression in human carcinomas. Human Pathology 35, 122-128 (2004).
Zen K, Liu D-Q, Guo Y-L, Wang C, Shan J, et al. (2008) CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration. PLoS ONE 3(3): e1826.