0

100

200

300 Control Cells Cycle 1 Cycle 2

0

100

200

300 Control Cells Cycle 1 Cycle 2

1. Introduction

Vortex Biosciences, Inc. – vortexbiosciences.com – info@vortexbiosciences.com

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