Download Early Cancer Diagnosis and Treatment through the Detection of

Early Cancer Diagnosis and Treatment through the Detection of
Circulating Tumor Cells using Drop-based Microfluidics
Neil Davey, Montgomery Blair High School, Silver Spring, MD
Objective I: Early Cancer Diagnosis
through Circulating Tumor Cell Detection
Results I: Detection of CTC Genes
by Fluorescence
Results II: Detection and Isolation of
Full CTCs
•  Cancer is a leading cause of death worldwide; 580,350 cancerrelated deaths and 1.6 million new cancer cases in 2013 in US alone
•  Low survival rates due to lack of early detection; traditional diagnosis
involves tumor biopsy: invasive, dangerous, and often arbitrary
•  Demand for an early-stage, non-invasive detection of cancer cells
from the blood to significantly reduce cancer mortality
•  Circulating Tumor Cells (CTCs) are shed from primary tumor and
circulate in the bloodstream; seeding of CTCs creates secondary
tumors, which cause a vast majority of cancer-related deaths
•  CTCs contain genetic abnormalities of cells within the original tumor
masses, and may reveal information about the cancer’s progression
•  Estimated market size for CTC detection from blood is $7.9 billion
•  Two prostate cancer (PC) cell lines were used, LNCaP and PC3
•  Known number of PC cells mixed with 500,000 RBC and 5,000 WBC,
to mimic CTC detection from iChip product
•  cDNA was purified from this cell mix and 16 primers and fluorophores
were tested in the drop-based platform to detect PC cells
•  8 of 16 primers – AR, KLK3, FOLH1, AMACR, KRT8, KRT18, and
KRT19 gene loci in PC cells - resulted in most effective amplification
In addition to cDNA, full cells can be encapsulated into microfluidic
drops. This would preserve genomic information of individual cells within
the drops. To test this, a mixture containing a known number of PC cells,
500,000 RBC, and 5,000 WBC was encapsulated. Cells were lysed only
after encapsulation into drops, within which PCR was then performed.
Single-cell PCR results: Roughly all cells that were present in the sample
were detected in each case
•  Negative control sample showed no bright drops; no false-positive signal
•  Capture efficiency ranged from 75-80% (best reported to date)
•  Cell suspension prepared to ensure that one drop contains at most one cell
•  Forked microfluidic device was used to sort cells by dielectrophoresis after
PCR to achieve a pure CTC sample
The Challenge and Current Technology
•  CTCs are very diverse and extremely rare; Only 1-10 CTCs among >1
billion RBC and >1 million WBC in 1 mL of blood
•  Due to their heterogeneity, no universal marker exists to identify CTCs
originating from various cancers
Discussion
Limitations:
Selection of CTCs by immunostaining
based only on known surface markers
is tedious and has limited sensitivity
Summary of amplification results: 16 primers from 5 different cell-specific
markers were tested for each cell type (LNCaP, PC3, and WBC)
LNCaP (+)
PC3 (+)
WBC (-)
Still left with a complex mix of
~505,000 cells from which CTCs must
be detected
*Ozkumur, E., et al.,
Science Translational
Medicine, 2013.
5(179): p. 179.
Fluorescence images showing amplification in
LNCaP and PC3 cells, but not in WBC, using KRT8.
Multiplex amplification to
increase the CTC
detection sensitivity
Example of image analysis of the fluorescent drops using LabVIEW
software. A clear correlation was observed between the number of input
CTC cells and frequency of fluorescent drops (red circles in graphs).
Reproducible CTC Gene Detection
λ = 0.0064%
λ = 0.00054%
λ = 0.000039%
Negative Control
•  As more primers are identified for the amplification of PC genes, they
can be incorporated into the PCR to increase the scope of detection
•  Sample enriching steps similar to those described in the CTC iChip
can be integrated upstream my device
•  My platform can easily be adapted for the detection of CTCs from a
broad range of cancers (lung, breast, etc.), provided specific primers
Objective II: Drop-based Microfluidics for
Individualized Cancer Therapies
Subsequent to sorting and isolating CTCs, these cells can be sequenced
or characterized to give insight upon possible treatment strategies for the
individual patient’s cancer. As a test case, I investigated KRAS mutations
in colorectal cancer (CRC). 30-40% of all CRC cases are associated with
mutations in the gene KRAS.
•  Current CRC drugs target the epidermal growth factor receptor (EGFR)
•  Patients with a mutation in codons 12 or 13 of the KRAS gene are
resistant to CRC drug therapy
•  There are 12 unique mutations possible in these two codons of KRAS
•  Urgent need of a test that predicts CRC patient response to EGFRtargeted therapy by investigating KRAS mutations
Hypothesis: Could a drop-based microfluidics platform allow for sensitive
KRAS mutation detection in CRC cells?
Method III: Peptide Nucleic Acid Clamp
12 unique primers were synthesized for each of the 12 possible KRAS
mutations. Frequency of each specific mutation was determined by separating
drops into 12 clusters, using varying concentration of dyes to bar-code each.
Discussion
•  Current methods of detecting mutations in KRAS can identify 1 mutant in
100 genes; My results showed a detection sensitivity of 1 mutant in
100,000 genes (1,000-fold increase in sensitivity)
•  This approach can facilitate personalized therapies for colorectal cancer
•  Combination of CTC isolation platform with a cancer cell characterization
technique similar to KRAS mutation detection platform would allow for early
cancer detection and treatment
Conclusions
Breakthroughs Achieved:
(1) Microfluidics diagnostic platform that detects and isolates pure individual CTCs
(2) Novel technique for detecting gene mutations - allows for personalized therapies
Future Goal: To create a universal microfluidic platform for the rapid early diagnosis
and treatment of all cancers
Relevant Applications to Biotechnology
Future Potentials:
CRC cells have both wild-type and mutant KRAS genes. Genomic DNA was (1) Integrated CTC isolation and characterization for cancer detection and treatment
purified from CRC cells and then encapsulated. In order to detect KRAS
(2) Genome sequencing of isolated CTCs for targeted drug therapy
mutations, the wild-type must be masked. Peptide nucleic acid (PNA) was
(3) Improved early diagnosis and significantly reduced cancer mortality
used to mask the wild-type KRAS gene during PCR. PNA binds to a
(4) Reduced economic burden due to cancer ($263 billion annual cost in the US)
complementary DNA strand and blocks DNA replication and fluorescence.
(5) Using a drop-based microfluidics platform coupled with PCR for the detection
and isolation of stem cells or pathogens (parasites, bacteria, etc.) from the blood
(6) Rapid, low-cost disease screening employing microfluidics technology
Method II:
With full cells
Method I: With DNA
My Innovation Workflow in microfluidic devices
Advantages: Unprecedented sensitivity and specificity in CTC detection; High
throughput (Drop-making frequency: 2000 Hz; Detection and sorting frequency:
500 Hz); Allows for the isolation of single CTC from a complex mix
•  My platform for single-cell PCR is successful in isolating pure CTCs
•  Stronger signal and downstream applications from single-cell PCR are
major advantages of this method
•  Breakthrough microfluidic technique known as drop-based PCR for the
quantitative detection of rare CTC genes and cells for diagnosis
•  By isolating a pure sample of CTCs, these cells can be characterized
and their genomes can be sequenced, shedding light upon the patient’s
cancer, and allowing for individualized, targeted drug therapy
In the presence of PNA, the KRAS gene in the wild-type CRC cell line (HT29)
was masked, while the KRAS gene in the mutant CRC cell line (SW480) was
not. In the absence of PNA, both wild-type and mutant cell lines showed
amplification. An agarose gel electrophoresis confirmed this result. As low as
one mutant in presence of 100,000 wild-type genes was detected.
Future Work
Methods I & II: Drop-based Microfluidics
•  Microfluidics is the precise control and manipulation of fluids in micronsized capillaries (~10-50 µm), and can be used to make emulsions
•  Polymerase chain reaction (PCR) within microfluidic drops using
fluorescence can detect rare cells with great sensitivity
•  I conceived the idea to combine microfluidics with PCR to detect CTCs
by examining cancer-specific gene expression
•  Can overcome limitations of immunostaining-based detection
Hypothesis: Could PCR in microfluidic drops allow for the detection and
isolation of rare CTCs from the blood sample? Results III: KRAS Mutation Detection and
Characterization
Acknowledgements
Samples containing cDNA from the equivalent of 50, 5, and 0.5 cells showed
approximate 10-fold decreases in the number of amplification-positive drops,
from 0.0064% to 0.00054% to 0.000039%. No bright drops were seen in the
negative control healthy blood sample, indicating no false-positive signal.
I would like to thank my mentor Dr. Huidan Zhang, post-doctoral fellow at the
Harvard School of Engineering of Applied Sciences (SEAS), for guiding me
through the research process. I would also like to thank Professor David Weitz at
Harvard SEAS for permitting me to work in his lab this past summer.