poster #4967 label-free collection of prostate circulating...
TRANSCRIPT
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1. Introduction
Vortex Biosciences, Inc. – vortexbiosciences.com – [email protected]
4. Conclusions & Future Directions
3. Results
2. Methods and Workflow
5. Acknowledgements & References
Poster #4967
[1] http://www.cancer.org/cancer/prostatecancer/. [2] H.I. Scher et al., Circulating Tumor Cell Number as a Prognostic
Marker in Progressive Castration-Resistant Prostate Cancer: Use in Clinical Practice and Clinical Trials. Lancet Oncol.,
2009. [3] J. Che et al., Classification of Large Circulating Tumor Cell Isolated with Ultra-high Throughput Microfluidic
Vortex Technology. Oncotarget, 2016.
Jemal A el al., 2008. CA: A Cancer Journal for
Clinicians. CA Cancer J Clin. 2008;58(2):71-96.
• Prostate cancer (PC) is the most common cancer diagnosed
worldwide in men. About 4/5 PC are diagnosed at early stage (I
and II), with nearly 100% of 5-year survival rate. The 5-year
survival rate for distant stage IV (M1) prostate cancer decreases
to 28% [1].
• Better markers for early detection of PC, progression, therapeutic
resistance and minimal residual disease are still needed.
• Patient Cohort: 23 patients with metastatic prostate cancer
(mPC), resistant to castration.
3. Wash: RBCs and
WBCs are washed
away, while CTCs
remain in vortices.
4. Release: Cells
are released off-chip
in a well-plate.
1. Prime: Chambers
are filled with PBS.
2. Infuse: 10X diluted
blood is injected into
the chip.WBCCTC
• CTC isolation using a label-free microfluidic device that utilizes inertia and laminar microvortices (Vortex technology): CTCs
(>13um), larger and more deformable, are trapped in vortices while smaller cells (red and white blood cells) pass through [3].
• Cells are identified and enumerated using standardized classification criteria [3].
We thank the nurses and all donors for their help towards the blood collection. We acknowledge
a research agreement from Vortex Biosciences for E.P., R.K. and D.D.C. (UCLA), and for M.T.
and S.S.J. (Stanford).
① Performance with prostate cell lines
② Patient samples: CTC enumeration & staining pattern
ALL
Prostate
Patients
Healthy
Age
Matched
• Using a label-free CTC enrichment method (Vortex technology) and enumeration, 80% of
metastatic castration-resistant PC samples were positive for CTCs using a threshold derived from
age-matched healthy controls (>3.37 CTCs / 7.5 mL). Purity ranged from 25 to 316.9 WBCs / 7.5
mL.
• Large variation in CK and PSA expression observed between CTCs. Vim/Ncad staining helps
characterize further CK- CTCs.
• Processing the sample through the chip multiple times increases capture efficiency significantly.
• Limitations of using cell lines as model to evaluate performance of CTC enrichment technologies.
Next?
• Genomic profiling of CTCs and mutational analysis of PTEN, AR, TP53 genes.
• Morphological analysis of the CTCs to identify potential patterns.
• Additional studies to understand the difference in atypical cells collected between age-
matched and younger healthy donors.
• Enumeration of CTCs from prostate cancer patients with early-stage disease.
Label-free Collection of Prostate Circulating Tumor Cells (CTCs) Using Microfluidic Vortex Technology
1 UCLA Department of Bioengineering, USA. 2 Vortex Biosciences Inc., USA. 3 Department of Surgery, Stanford University, USA. 4 Department of Medicine, Stanford University, USA. 5 UCLA Medical Center, Division of Dermatology, USA. 6 Institute of Urologic Oncology, UCLA David Geffen School of Medicine, USA.
Pao E.1, Renier C.2, Lemaire C.2, Che J.1,2, Matsumoto M.1, Triboulet M.3, Srivinas S.4, Jeffrey S.S.3, Kulkarni R. P.5, Rettig M.6, Sollier-Christen E.2, Di Carlo D.1
• CTCs play a role in cancer metastasis.
• CTCs are extremely rare but are minimally invasive
biomarkers for patient prognosis, disease
monitoring, personalized medicine, and
biological studies [2].
Current technologies:
Cell affinity
antibody capture
• cost reagents
• cancer-specific
Filtration
trapping in pores
• pore clogging
• cell recovery
Active forces
dielectric, acoustic
• complexity
• cost, speed
• efficiency
Inertial fluidics
continuous flow
• purity
• multi-steps
• Label-free, compatible with various downstream assays
• No sample preparation, fast processing
• High capture, purity, cell viability
Common needs
Order Characteristic Classification
1 Jagged shape, dark fill, or dark outline in BF Dust
2 CD45+ / DAPI+ / CK- WBC
3 Lobed nucleus WBC
4 CK+ and/or PSA+ / CD45- / DAPI+ CTC
5 CK+ / CD45+ / DAPI+ Favor WBC
6 Nucleus < 9 μm WBC
7 Nucleus > 9 μm, N/C < 0.60 WBC
8 Nucleus > 9 μm, N/C > 0.60 CTC
>> Recycling the blood sample
increases Capture Efficiency from
24.5% (Cycle 1) to up to 50% (from
Cycle 5) at the expense of Purity
(74% to 55%).
Pan-CK = Cytokeratin. PSA = Prostate-Specific Antigen. N/C = Nuclear / Cytoplasm Ratio. BF = Brightfield.
5. Staining: Cells are fixed
with 4% PFA, stained for
DAPI, Pan-CK, PSA and
CD45.
80%
Ce
ll N
um
be
r
Ce
ll N
um
be
r
Cells Diameter (m)Cells Diameter (m)
Control Cells Prostate CTCs
CK
+
VN
C -
CK
–
VN
C +
WB
C
BF+DAPI
CK
+
VN
C +
RBC
CT
Cs
WB
Cs
DAPI PSA CK BF DAPI CD45BF
③ Patient samples: Markers of EMT
Vim/NCADCD45 CK
PC3 WBC
Vim/NCAD
CD45
CK
DAPI
• Staining for Vimentin and N-cadherin (markers for Epithelial to Mesenchymal
Transition, EMT) identifies some DAPI only cells as CTCs going through EMT.
Healthy
Threshold
=
Mean + 2 STDV
WBCsCTCs
Ce
lls
/ 7
.5 m
L b
loo
d
CT
Cs
/ 7
.5 m
L b
loo
d
Healthy
< 30
CT
Cs
/ m
L b
loo
d
0
20
40
60
80
100
0
20
40
60
80
100
cycle 1 cycle 2 cycle 3 cycle 4 cycle 5 cycle 6
Ca
ptu
re P
uri
ty (
%)
Ca
ptu
re E
ffic
ien
cy (
%)
Cumulative Efficiency
Efficiency per Cycle
Cumulative Purity
• LNCaP prostate cancer cells were
spiked in healthy blood and processed
through Vortex HT chip (N=3).
• Patients have variable expression patterns of CK / PSA. For Patient P1, 82% of the
CTCs express PSA only, while for Patient P4, 94% of the CTCs express CK only.
• 80% of mPC samples ‘positive’ for CTCs
using age-matched healthy threshold
(>3.37 CTCs / 7.5 mL).
• Patients:
0 – 93.7 CTCs / 7.5 mL
25 – 316.9 WBCs / 7.5 mL
• HD, age-matched: 1.25 – 2.5 CTCs / 7.5 mL
• HD, < 30 yrs.-old: 0 CTCs / 7.5 mL
• After Vortex collection, LNCaP cells are
proliferating for up to 7 days.
Da
y 1
Da
y 4
Da
y 7
• Harvesting of LNCaP on 2
different days (different
confluency) provides cells with
very different size range, i.e.
different cell recovery (N=1).
>> Limitation of using cell
lines as a model to evaluate
performance of CTC
technologies.
DAY 2DAY 1
• Controls:
5 healthy donors,
age-matched,
5 healthy donors,
< 30 years old.
P4
P1