The Pennsylvania State University

The Graduate School

College of Engineering

DESIGN OF AN AUTOMATIC SYSTEM BASED ON FLEXIBLE MICRO SPRING

ARRAYS FOR VIABLE CANCER CELL ENRICHMENT

A Thesis in

Bioengineering

by

Xin Zou

2017 Xin Zou

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Master of Science

May 2017

ii

The thesis of Xin Zou was reviewed and approved* by the following:

Siyang Zheng

Associate Professor of Bioengineering

Thesis Advisor

Nanyin Zhang

Associate Professor of Bioengineering

Pak Kin Wong

Professor of Bioengineering

William Hancock

Professor of Bioengineering

Chair of the Intercollege Graduate Program in Bioengineering

*Signatures are on file in the Graduate School

iii

ABSTRACT

Curing cancer has remained to be quite challenging although significant progresses have

been made over the past decades. It has been shown that more than 90% of cancer–related deaths

were caused by cancer metastasis, which involves dissemination and spread of circulating tumor

cells (CTCs). Thus the detection of CTCs has become a predictive cancer prognosis. In view of the

extremely low concentration of CTCs in blood, it is imperative to develop a high-efficiency

technology for enriching CTCs for cancer prognostic monitoring. With CTCs enrichment

technologies, bio-information of CTCs can be acquired, and more precious treatment time for

patients can be earned. In this thesis, we design a computer controlled automatic system for point-

of-care CTCs enrichment, based on a novel micro-structured membrane filter (flexible micro spring

array, FMSA). Compared to manual control, this automatic system not only accelerates enrichment

process but also increases experimental reliability and repeatability. Experimental results show that

this system can efficiently capture cancer cells spiked into healthy donor blood with high efficiency

and was sensitive enough for CTCs detection from patient blood samples.

iv

TABLE OF CONTENTS

List of Figures .......................................................................................................................... v

List of Tables ........................................................................................................................... vi

Acknowledgements .................................................................................................................. vii

Chapter 1 Introduction ............................................................................................................. 1

CTC Enrichment Techniques ........................................................................................... 2 Immuno-based Enrichment ...................................................................................... 3 Physical-based Enrichment ...................................................................................... 8

Summary .......................................................................................................................... 12 Project Outline ................................................................................................................. 13

Chapter 2 Computer-controlled Automatic CTCs Enrichment System ................................... 14

Instrument ........................................................................................................................ 14 Containers ................................................................................................................ 15 Selection Valves ....................................................................................................... 16 FMSA Device ........................................................................................................... 17 Fluidic Pumps and Collectors .................................................................................. 18 Controllable Power Source ....................................................................................... 18 Tubing and Adaptors ................................................................................................ 20

Filtration Procedure .......................................................................................................... 21 LabVIEW Program .......................................................................................................... 23

Pump Motion Section ............................................................................................... 25 Display Section ........................................................................................................ 31 Filtration Running/Stop Section ............................................................................... 32 Selection Valves Section .......................................................................................... 33

Chapter 3 Material and Methods .............................................................................................. 35

Cell Culture ...................................................................................................................... 35 Blood Sample Preparation................................................................................................ 35

MCF-7 Cancer Cells Spiked Experiment ................................................................. 35 Patient Sample .......................................................................................................... 36

FMSA O-ring Fabrication ................................................................................................ 36 Filtration Process .............................................................................................................. 37

Instrument Assembly................................................................................................ 37 Software Setting ....................................................................................................... 39 Filtration ................................................................................................................... 39

Immunofluorescent Detection .......................................................................................... 40

Chapter 4 Results ..................................................................................................................... 41

MCF-7 Spiked Experiments ............................................................................................. 41 Identification of MCF-7 Cancer Cells ...................................................................... 41

v

Effect of Spiked Cell Amount on Capture Efficiency .............................................. 43 Enrichment against Leukocytes ............................................................................... 45

Circulating Tumor Cells Detection .................................................................................. 45

Chapter 5 Conclusion and Future Work .................................................................................. 48

Conclusion ....................................................................................................................... 48 Future Work ..................................................................................................................... 49 References ........................................................................................................................ 50

vi

LIST OF FIGURES

Figure 1-1. Techniques for CTC enrichment grouped by capture principle. Immuno-

based enrichment techniques target CTCs or leukocytes by highly specific

immunoreactions. Physical-based enrichment techniques isolate CTCs based on

differences between CTCs and blood cells in size, deformability and electrical

properties. ......................................................................................................................... 3

Figure 1-2. Isolation of CTCs from whole blood using CTC-Chip. (Nagrath et al.5).

This chip captures CTCs by an array of microposts coated with Anti-EpCAM. (a)

The workstation setup. (b) The CTC-chip with microposts etched in silicon. (c)

Whole blood flowing through the device (d) SEM image pf a captured cancer cell. ...... 5

Figure 1-3. HB-Chip (Stott et al.10) improves capture performance of CTC-Chip.

Herringbone structure can generate microvortices that increases the interactions

between CTCs and surface. (A) Uniform blood flow through the device. (B)

Herringbone structure and parameters. (C) Flow example of HB-chip. (D) Flow

example of flat channel. (E) Flow visualization of HB-chip. (F) Flow visualization

of flat channel. .................................................................................................................. 6

Figure 1-4. Schematic diagram of a μ-MixMACS Chip for one-step CTC isolation

(Lee et al.12). Leukocytes can be captured by Anti-CD45 coated magnetic

nanoparticles, and collected by a magnetic sorter. ........................................................... 7

Figure 1-5. Track-etched membrane filter and parylene membrane filter. (A) ISET

membrane. Pores in ISET have random position and fusion problem. (B) 1st

generation membrane with round shape developed by Zheng et.al.21 (C) 1st

generation membrane with oval shape. ............................................................................ 9

Figure 1-6. 2nd generation membrane with 3D structure (Zheng et. al.22). (a)This two-

layer membrane can let red/white blood cells pass through from pores and gaps, but

trap CTCs at pores. The bottom layer provides trapped cells with support and sustain

cells viability shown by zoom in figure. (b) Design parameters of 2nd generation

membrane. ........................................................................................................................ 9

Figure 1-7. Diagram of 3rd generation membrane (Zheng et. al.23) (A)Blood cells can

pass through the membrane from pores on both layers, while CTCs are captured and

can migrate even proliferate on bottom layer. (B) Design parameters of 3rd

generation membrane. (C) An actual picture of 3rd generation membrane. ..................... 10

Figure 1-8. Structure of 4th generation membrane (flexible micro spring array,

FMSA) (Zheng et. al.3) Blood samples flow directionally through the image plane.

CTCs can be captured at gaps by this flexible structure. Scale bars are 20μm. ............... 11

Figure 2-1. Computer-controllable CTCs enrichment instrument. Two pumps and

selection valves are controlled by computer. Blood sample and PBS flow through

the FMSA in order, and cancer cells are enriched and washed on FMSA membrane. .... 15

vii

Figure 2-2. Diagram of a selection valve with four inlet ports. Every inlet of selection

valve is controlled by related actuator, and works independently. .................................. 16

Figure 2-3. Diagram of FMSA device. FMSA membrane is sandwiched by two PDMS

O-rings and placed inside a plastic jig with a clamp. ....................................................... 17

Figure 2-4. Circuit diagram of amplification circuit. 6V/12V voltage output can be

obtained by this circuit with 1V/2V input (G=6). ZL is the place for selection valves. ... 19

Figure 2-5. Filtration process. Red indicates blood, and blue indicates PBS. The whole

filtration process including 6 steps and two manual actions in step 3-2 and step 4-1. ..... 21

Figure 2-6. Auto-FMSA software front panel. Diameter of syringes, pumping rate and

serial port can be set in pump setting part. V1~V6 are volume of each step, which

are related to the volume of blood sample. T1~T6 and corresponding bars show the

progress of each step. pumping information can be sent by ‘Send’ button, and

filtration process can be triggered by ‘Run’ button.......................................................... 24

Figure 2-7. Software block diagram. Four sections in this manipulation VI have

independent function: ‘pump motion section’ and ‘selection valves section’ are used

to control pumps and selection valves; ‘filtration running/stop section’ controls the

start and stop of filtration process; ‘display section’ is responsible for front panel

display. ............................................................................................................................. 25

Figure 2-8. Block diagram of Pump motion section. Three Sub VIs are used in this

section, and two sequences of these three Sub VIs are set as filtration process. ............. 26

Figure 2-9. Block diagram of pump setting Sub VI. Syringe diameter can be set by this

VI. .................................................................................................................................... 29

Figure 2-10. Block diagram of run Sub VI. Pumping information including rate,

volume, and direction are set by this VI. ......................................................................... 30

Figure 2-11. Block diagram of pause Sub VI. Pausing time is set by this VI. ..................... 31

Figure 2-12. Block diagram of display section. Process bars and count down are

calculated and displayed by this part. .............................................................................. 32

Figure 2-13. Block diagram of filtration running/stop section. ‘Run’ button serves as

the trigger of filtration process, and can control two pumps at the same time. ................ 32

Figure 2-14. Block diagram of a part of selection valves section. Input voltage for

amplification circuit is generated by this section. ............................................................ 33

Figure 3-1. Picture of our assembled auto-FMSA instrument. .......................................... 38

Figure 4-1. MCF-7 cells captured by FMSA membrane. MCF-7 cells were stained

with Calcein AM (green) and DAPI (blue), leukocytes were stained with DAPI.

Scale bars are 20μm. ........................................................................................................ 42

viii

Figure 4-2. Filtration performance of MCF-7 spiked experiments. Spiked numbers

and output numbers are plotted. ....................................................................................... 44

Figure 4-3. Effect of spiked cell number on filtration efficiency. The mean capture

efficiency of each level and error bars are plotted. .......................................................... 44

Figure 4-4. Circulating tumor cell (in yellow circle) on FMSA membrane. CTCs are

stained with CK 8/18/19 (green) and DAPI (blue), while leukocytes are stained with

CD 45 (red) and DAPI (blue). Scale bars are 40μm. ....................................................... 46

ix

LIST OF TABLES

Table 1-1. Comparison of different CTCs enrichment techniques .................................... 12

Table 2-1. Adaptors and tubing ............................................................................................ 20

Table 2-2. filtration procedure .............................................................................................. 22

Table 2-3. Pump operation sequence.................................................................................... 27

Table 2-4. Command and relevant description ................................................................... 28

Table 2-5. Voltage sequence of analog output ..................................................................... 34

Table 3-1. Software setting parameters ............................................................................... 39

Table 4-1. Filtration results of MCF-7 spiked experiments. .............................................. 43

x

ACKNOWLEDGEMENTS

I would like to express my sincere thanks to my advisor, Dr. Siyang Zheng for providing

an opportunity to join and study in his research group when I came to the Penn State University

two years ago. I benefited from his invaluable guidance in both research and life, including giving

me different projects to work on, which enriched my basic knowledge in biomedical field, his

progressive advices on Auto-FMSA, his assistance on my reference letter, and his suggestions on

my future career life.

I also would like to thank all the members in our group, because their professional

dedication shaped a wonderful research environment in our group, which influenced me deeply and

encouraged me to keeping working when I faced difficulties. To Laura Ha, Sijie Hao, Wenlong

Zhang, Wenqing Li and Yiqui Xia, thank them all for their kind help of lab training, giving advices

on my experiments and technique support. To Yizhu Chen, Gong Cheng, Zhigang Wang, and

Hongzhang He, thank them all for their encouragement on my lab research and suggestions on my

daily life.

I would like to show my gratitude to all the professors for teaching me knowledge in these

two years, especially Dr. Pak Kin Wong, Dr. Ying Gu, Dr. William Hancock and Dr. Weihua Guan.

I also grateful to Eugene Gerber for helping me to build the box for Auto-FMSA system in this

thesis and Jenna Sieber for patient assistance on department affairs.

I would like to give my special acknowledgement to Dr. Jiamian Hu as my fiancé and a

research partner. Even we are not in the same department, his passion for research motivates me,

and without his accompany, my research and daily life would not have been so pleasant.

Finally, I want to thank my mother and father for their love and concern. I would never

have this wonderful opportunity of studying aboard without their support, and also their trust and

encouragement make me feel confident on doing research.

1

Chapter 1

Introduction

About 14.1 million new cases of cancer occurred globally in 2012, which caused about 8.2

million deaths or 14.6% of human deaths.1 Among these deaths, cancer metastasis is the main

reason. Until now, metastasis is still invincible because of its resistance to conventional cancer

therapies, despite significant achievements have been made in cancer diagnosis and treatment.

During the formation of primary tumor, some tumor cells detach from the primary tumor, circulate

through the blood stream, and then invade and proliferate in distant organs. These cells that finally

cause cancer metastasis are circulating tumor cells.2

It has been proved by CellSearch®, a validated CTC enrichment and enumeration

technology, that increased CTC numbers indicates worse patients’ physical condition. Also,

because of this technology, CTCs in the blood stream have been established as a cancer biomarker

of known prognostic value.3 As we said, CTCs detach from primary tumor and cause metastasis,

that means we may obtain information from both primary and metastatic tumors by analyzing CTCs

after enrichment. Further, CTC-based cancer early detection, non-invasive diagnosis and even CTC

analysis guided treatment can be realized and be able to fight for more time for patients suffering

from cancer.5

In this chapter, we will mainly focus on CTC enrichment techniques that have been

developed and successfully utilized in clinical samples.

2

CTC Enrichment Techniques

It is possible to obtain molecule information from CTCs once they are isolated. However,

the extremely low concentration of CTCs compared with leukocyte in blood becomes a major

challenge to researchers, and makes CTCs enrichment a doomed path to CTCs analysis. Till now,

numerous efforts have been made in this field, and many CTC enrichment techniques have been

developed and validated with blood samples from cancer patients.

A successful CTC enrichment technique should be sensitive enough to capture the rare

CTCs, as well as specific enough to select CTCs against blood cells (white blood cells and red

blood cells) from blood sample.4 When evaluating a CTC enrichment technique, apart from

enrichment efficiency and purity, whether the technique is repeatable, reliable, cost-effective, rapid,

capable of processing clinical volume of blood and able to maintain cell viability are also

considerable evaluation standards.

Among most of CTCs enrichment techniques that have been developed, techniques can be

grouped by capture principles. Immuno-based enrichment that uses controllable antibody to target

antigen expressed by interest cells and physical-based enrichment that isolates interested cells by

differences of physical properties are two main types of CTCs enrichment techniques (Figure 1-1).

On the other hand, if we focus on targeted cells, CTCs enrichment techniques can also be divided

as positive selection (target CTCs) and negative selection (target blood cells).

3

Figure 1-1. Techniques for CTC enrichment grouped by capture principle. Immuno-based enrichment

techniques target CTCs or leukocytes by highly specific immunoreactions. Physical-based enrichment

techniques isolate CTCs based on differences between CTCs and blood cells in size, deformability and

electrical properties.

All these CTCs enrichment techniques we are going to discuss in this chapter have two

levels of evaluation. First, cell line models were tested to evaluate performance, such as filtration

efficiency, purity, cell viability and processing speed. Then these techniques were validated by

clinical patient samples.

Immuno-based Enrichment

Immuno-based enrichment techniques use highly specific immunoreactions to isolate

CTCs or leukocyte from blood to achieve CTCs enrichment. Antibodies are usually functionalized

on magnetic beads or channel surface to target antigens present on interested cells.

4

Magnetic Beads

Magnetic beads can be functionalized with clinical relevant antibodies and then collected

with a magnet. EpCAM (epithelial cell adhesion molecule) usually serves as targeted antigen when

capture CTCs. EpCAM is a kind of protein that is frequently overexpressed by carcinomas of lung,

colorectal, breast, prostate, head and neck, and hepatic origin, but is absent from haematologic

cells.5

Currently, CellSearch (Veridex, LLC, Raritan, NJ) is the only one technology cleared by

US Food and Drug Administration (FDA) that enrich target cells expressing EpCAM from blood

sample with anti-EpCAM-coated magnetic beads.6 To distinguish CTCs from leukocyte, two

fluorescently labeled monoclonal antibodies: CD-45 (targets the leukocyte common antigen) and

CK (targets cytokeratin, a kind of filaments exist in epithelial cells) are used as extra identification

criteria. By using this technology, enumeration of CTCs from whole blood has been established as

a prognostic marker and predictor of patients’ future physical condition in metastatic breast,7

prostates,8 and colon cancers.9

Surface Functionalization

Nagrath et al. 5 developed a CTC-chip consists of an array of microposts coated with Anti-

EpCAM to enrich CTCs under precisely controlled laminar flow conditions without pre-labelling

process of samples (Figure 1-2). The capture efficiency of this CTC-chip was 65% and its capture

purity (the ratio of positive CK to positive CD45 cells) was around 50% when processing at a flow

speed of 2.5ml/h. In addition, this CTC-chip successfully isolated 7/7 patients with early-stage

prostate cancer.

5

Figure 1-2. Isolation of CTCs from whole blood using CTC-Chip. (Nagrath et al.5). This chip captures

CTCs by an array of microposts coated with Anti-EpCAM. (a) The workstation setup. (b) The CTC-chip

with microposts etched in silicon. (c) Whole blood flowing through the device (d) SEM image pf a captured

cancer cell.

To enhance the performance of CTC-chip, Stott et al.10 developed a new herringbone-chip

(HB-Chip) (Figure 1-3) The unique structure of herringbone increased the number of interactions

between target CTCs and antibody-coated chip surface because of the generation of microvortices.

This new design improved capture efficiency to 91.8% in cell-spiked experiments, and CTCs were

detected in 14/15 patients with prostate cancer.

6

Figure 1-3. HB-Chip (Stott et al.10) improves capture performance of CTC-Chip. Herringbone structure

can generate microvortices that increases the interactions between CTCs and surface. (A) Uniform blood

flow through the device. (B) Herringbone structure and parameters. (C) Flow example of HB-chip. (D) Flow

example of flat channel. (E) Flow visualization of HB-chip. (F) Flow visualization of flat channel.

Leukocyte Depletion

Immuno-based positive enrichment isolates CTCs with high purity using antibody-antigen

reaction, but relies on the expression of EpCAM, which however becomes its limitation. Several

studies have shown that the expression of EpCAM is heterogeneous among different cancer cells.

For example, Went et al.11 analyzed EpCAM expression on 3900 tissue samples of 134 different

histological tumor types and subtypes. According to their results, colon, pancreas and prostate have

strong positive tumors that express EpCAM, while most soft-tissue tumors and all lymphomas were

EpCAM negative. These negative or weak positive tumors will result in CTC number

7

underestimation, which is not our expectation. Therefore, to address this issue, negative selection

devices have been developed.

Lee et al.12 developed a fully integrated chip (μ-MixMACS Chip) with negative selection,

which used magnetic nanoparticles coated with anti-CD45 antibody to capture leukocytes and

remove them from blood sample. This chip comprises a micromixer that can generate

multivortexing flows to enhance mixing, and a magnetic sorter to remove magnet-coated cells

(Figure 1-4).

Figure 1-4. Schematic diagram of a μ-MixMACS Chip for one-step CTC isolation (Lee et al.12).

Leukocytes can be captured by Anti-CD45 coated magnetic nanoparticles, and collected by a magnetic

sorter.

Negative selection chips are capable of achieving high recovery efficiency, but result in

low purity at the same time. Also, blood samples used in most negative chips need preprocessing

to remove red blood cells using hemolysis buffer.

8

Physical-based Enrichment

Apart from biochemical differences, physical differences between tumor cells and blood

cells, including size, deformability, and electrical properties are exploited to isolate CTCs from

whole blood.

Microfiltration

CTCs normally have larger size and are stiffer than leukocytes, which allows cell trap

arrays or membrane filter to separate CTCs and leukocytes on the basis of their size and

deformability.13 Several membrane filters have been developed and applied in CTCs enrichment.

Vona et al.14 developed a polycarbonate membrane ISET (isolation by size of epithelial tumor cells)

fabricated by track-etched technology with 8μm diameter cylindrical pores (Figure 1-5 (A)). They

used ISET to enrich and count CTCs from blood samples from patients with liver cancer, and

demonstrated that the presence of CTCs is significantly associated with the presence of a diffuse

tumor.15 Since then, ISET has been used for different cancer studies, including melanoma16, lung

cancer17, prostate cancer18 and other cancers19. However, because of track etching, pores in ISET

usually have random position, low density, and fusion problems (Figure 1-5 (a)), which result in

low capture efficiency of 50%~60%.

Zheng et al. has been focused on size-based CTCs enrichment device for 10 years. Until

now, four generations of membrane filters have been developed with increasing enrichment

performance. All four generations of membrane filters were made from parylene membrane using

microfabrication technologies, which can be precisely controlled and are more cost effective

compared with track-etched polycarbonate filters.20

9

The 1st generation membrane has circular/oval shaped pores (Figure 1-5 (B & C)). This

membrane had capture efficiency of 89%, and enriched CTCs in 51 of 57 cancer patients while

CellSearch® was positive for only 26.21

Figure 1-5. Track-etched membrane filter and parylene membrane filter. (A) ISET membrane. Pores

in ISET have random position and fusion problem. (B) 1st generation membrane with round shape

developed by Zheng et.al.21 (C) 1st generation membrane with oval shape.

To enhance CTC viability, 2nd generation membrane with 3D structure was designed

(Figure 1-6). This membrane consists of two layers of parylene membrane with pores and gaps.

The pore positions of bottom membrane are shifted from that of top membrane, which permits

bottom membrane to support captured cells and sustain cell viability by reducing stress

concentration on cell membrane.22 To validate the viability of captured tumor cells on 2nd

generation membrane, captured cells were cultured on membrane for 2 weeks, and over 85% of

tumor cells had intact cell membrane.

Figure 1-6. 2nd generation membrane with 3D structure (Zheng et. al.22). (a)This two-layer membrane

can let red/white blood cells pass through from pores and gaps, but trap CTCs at pores. The bottom layer

provides trapped cells with support and sustain cells viability shown by zoom in figure. (b) Design

parameters of 2nd generation membrane.

10

Two-layer structure of the 2nd generation membrane is good for cell trapping, but restricts

cell proliferation and cell release after blood filtration.23 Therefore, a new separable bilayer

microfilter was designed (Figure 1-7). The bottom layer contains 8μm diameter holes and top layer

has 40μm holes, which make tumors trapped at the edge of large pores or gap between two layers.

After filtration, two layers of membrane can be easily separated using two pairs of tweezers, and

then expose captured tumor cells. Compared to 2nd generation membrane, 3rd designed allows

greater freedom for captured tumor cells to migrate and proliferate on the surface of the device,

which is desirable for CTCs analysis.

Figure 1-7. Diagram of 3rd generation membrane (Zheng et. al.23) (A)Blood cells can pass through the

membrane from pores on both layers, while CTCs are captured and can migrate even proliferate on bottom

layer. (B) Design parameters of 3rd generation membrane. (C) An actual picture of 3rd generation

membrane.

Finally, a flexible micro spring array (FMSA) device was developed, which enabled viable

enrichment of CTCs directly from whole blood without preprocessing.3 This new design enriched

CTCs on the basis of size and deformability because of its flexible structure (Figure 1-8). In

addition, porosity was maximized to prevent sample clogging, while operation pressure was

11

minimized for encouraging cell viability. The FMSA device can achieve 90% capture efficiency,

80% viability, and higher sensitivity when compared with CellSearch®, detecting more CTCs in

patient blood sample.3

Figure 1-8. Structure of 4th generation membrane (flexible micro spring array, FMSA) (Zheng et.

al.3) Blood samples flow directionally through the image plane. CTCs can be captured at gaps by this

flexible structure. Scale bars are 20μm.

Dielectrophoresis

The differences of dielectric properties between cancer and red/white blood cells were

exploited to separate cancer cells from normal blood cells since 1994.24 Cancer cells were attracted

by positive DEP, while normal blood cells flow past. This DEP separation technique can achieve

95% capture efficiency and high purity. To enhance cell throughput, Gupta et al.25 developed the

continuous flow ApoStreamTM device based on similar principle. In their experiment, capture

efficiency was greater than 70%, and cell viability was not affected by the ApoStreamTM device,

which maintained at >97% during the 7day culture period after separation process.

12

Summary

Both immune-based and physical-based CTCs enrichment techniques have their virtues

and limits. Antibody-antigen reaction ensures high level of purity, but at the same time, depends

on the express performance of antigen, which may extremely affect enrichment efficiency. On the

other hand, physical-based techniques have better efficiency, less processing time, but low purity.

Detailed characteristics of different enrichment techniques can be found in Table 1-1.

* Enrichment against leukocytes

Table 1-1. Comparison of different CTCs enrichment techniques)

Enrichment

techniques

Principle Selection

target

Efficiency Purity Speed

(ml/h)

Sampl

e

volum

e (ml)

Clinical sample

Anti-EpCAM-

coated

microspots5

Immune-

affinity

Positive ~65% 50% 2.5 0.9~5.

1

122/123Prostate

cancer

Herringbone

chip10

~91.85% 14% 2.5 4 14/15 prostate

cancer

μ-MixMACS

Chip12

Negative 84.73%~

90.67%

0.35%~

5.39%

18 >5 5/10 breast cancer

ISET Size-

based

Positive

- - - - various

Round pore

(1st generation)

>89% 10^7*

225 7.5 51/57 metastatic

caner

3D parylene

microfilter (2nd

generation)

86.5% 10^3* 12~20 - -

Separable

bilayer

microfiltration

device (3rd

generation)

78%~83% >10^3* - 7.5 Metastatic

colorectal cancer

FMSA (4th

generation)

>90% >10^4* - 7.5 Various cancer

ApoStreamTM Dielectro

phoresis

>70% 10^2* 7.5 - -

13

Project Outline

A computer-controlled automatic system including instrument and software was designed

and built for enriching cancer cells from whole blood based on FMSA membrane developed by

Dr. Siyang Zheng’s group. In this thesis, instrument structure, filtration procedure and software

coding are introduced in chapter 2. Detailed operation of cancer cell spiked experiment and

patient sample filtration are demonstrated in chapter 3. Chapter 4 shows filtration performance

results of these two experiments, and chapter 5 talks about conclusion and future work.

14

Chapter 2

Computer-controlled Automatic CTCs Enrichment System

The Flexible micro spring array (FMSA) developed by Dr. Siyang Zheng’s group has

realized extreme high (90%) capture efficiency and viability from whole blood in short time.3 The

whole filtration process in their experiment was conducted manually, which not only slows down

the diagnosis but also decreases experimental reliability and repeatability. Therefore, to address

this issue, a simple, friendly-use, automatic system (AUTO-FMSA) was developed. This system

sharply improves the performance of FMSA, and simplifies the filtration process at the mean time.

This system can enrich spiked MCF-7 cells from 4ml healthy donor blood with high efficiency

within 6 mins, and is sensitive enough for CTCs enrichment using blood sample from lung cancer

patient.

The automatic CTCs enrichment system consists of computer-controllable CTCs

enrichment instrument and software coded by LabVIEW. The whole system is very easy to be

assembled, and the setup process can be done within 5 mins before blood filtration. Every

electronical element in the instrument cooperates very well under the manipulation of software. In

addition, progress bars showed by software can visually display the filtration process, which helps

users to organize each step of operation without a hurry.

In this chapter, we will introduce the instrument, working principle and software coding of

our system in detail.

Instrument

As shown in Figure 2-1, CTCs enrichment instrument is made up of containers, tubing,

selection valves, FMSA device, fluidic pumps, syringes, and controllable power source. Among

15

them, fluidic pumps are directly controlled by software, while selection valves are controlled by

controllable power source.

Figure 2-1. Computer-controllable CTCs enrichment instrument. Two pumps and selection valves are

controlled by computer. Blood sample and PBS flow through the FMSA in order, and cancer cells are

enriched and washed on FMSA membrane.

Containers

As shown in Figure 2-1, both blood container (left) and PBS container (right) are BD plastic

syringes (slip tip) without plungers. Two pores with different sizes were drilled on the top board of

instrument box for containers to fit in, and flexible tubing was used for connection between

containers and adaptors on selection valves. Sizes for Blood container and PBS container are

different, that blood container is 10ml syringe, while PBS container is 30ml syringe, considering

different volume of blood and PBS is used in filtration process.

16

Selection Valves

Both two selection valves used in this instrument are 2-inlet-ports solenoid operated valves

(080T Series Flow Selection Valves) from Bio-Chem Fluidics, and can generate 4 different flow

paths between two containers and two collectors in total. For single selection valve, its two inlet

ports are controlled independently by separate actuators. When an inlet port is energized, the

solenoid retracts the armature which is attached to the flexible diaphragm (Figure 2-2). This raises

the diaphragm allowing fluid to flow between the common port and the energized inlet port. The

flow path will close when de-energized the corresponding inlet port.

Figure 2-2. Diagram of a selection valve with four inlet ports. Every inlet of selection valve is controlled

by related actuator, and works independently.

To energize selection valves, 12V is needed for excitation, and valves can keep working

under 6V after 100ms of excitation. Selection valves can become extremely hot or even get

damaged if they keep working under high voltage for long time according to Bio-Chem

introduction. Therefore, a controllable power source is needed to generate both 12V (excitation

voltage) and 6V (working voltage) to avoid extra heat.

17

FMSA Device

As shown in Figure 2-3, FMSA device is assembled by plastic jig, PDMS O-rings, FMSA

membrane and a clamp.

Figure 2-3. Diagram of FMSA device. FMSA membrane is sandwiched by two PDMS O-rings and placed

inside a plastic jig with a clamp.

Top and bottom parts of FMSA jig are symmetric. A cavity for O-ring and FMSA

membrane can be generated when top and bottom parts are assembled, and a clamp is used to

provide jig with tightness. Screws on the top and bottom parts were made in the same type as BD

Luer Lok syringe tip for easy connection.

PDMS O-rings were cut from PDMS sheet, and then drilled holes using a 10mm-square

shaped punch. FMSA membrane with 4.5μm-gap size was sandwiched by two PDMS O-rings.

18

Fluidic Pumps and Collectors

Two fluidic pumps in our system are NE-500 Programmable OEM Syringe Pumps from

New Era Pump System Inc., which come with data lines for data transmission between pumps and

computer (RS-232) to realize controllable motion. Two pumps form a pump network, and are

controlled by LabVIEW software.

Two 30ml BD plastic syringes were used as collectors. The syringe on the left pump is

blood collector, while the right one is waste (PBS) collector. After filtration, blood with small

amount of PBS collected in the left syringe can still be used for extra experiment, such as DNA

sequencing.

Controllable Power Source

As we just mentioned, two 2-way selection valves (four independent actuators in total)

need power source with dual voltage (12V/6V) output. A 4-channel analog voltage output module

(NI-9263) and an amplification circuit were used to reach this requirement.

NI-9263 is a simultaneously updating analog output module, and can be controlled by

LabVIEW through CompactDAQ (NI). The maximum analog voltage output of NI-9263 is 10V,

which is not enough to energize selection valves. Therefore, here we add an amplification circuit

using OPA547 a low cost, high-voltage/high-current operational amplifier, to amplify analog

voltage from 1V/2V to 6V/12V.

19

Figure 2-4. Circuit diagram of amplification circuit. 6V/12V voltage output can be obtained by this

circuit with 1V/2V input (G=6). ZL is the place for selection valves.

The circuit diagram is shown in Figure 2-4. In our case, voltage output is 6 times of voltage

input, which means amplification factor G equals to 6. Other parameters used in this circuit are

shown below.

G = 1 +𝑅2

𝑅1= 6 (Eq. 2.1)

𝑅1 = 5𝑅2 (Eq. 2.2)

𝑅1 = 10𝑘Ω, 𝑅2 = 2𝑘Ω

V+ =14V

V- =0V

20

Tubing and Adaptors

As shown in figure 2-1, multiple adaptors and tubing were used to connect different parts

of instrument, and also make easy assembly of whole instrument. Detailed information about these

adaptors and tubing can be found in the table below.

Object 1 Diameter

/Screw

Object 2 Diameter

/screw

Adaptor

/tubing

Model

Blood/PBS

container

(BD 30ml

syringe)

Slip tip Selection

valve 1

¼’’-28

female

PVC tubing

¼’’ ID, 5/16’’ OD

Adaptor 1

¼’’ ID, ¼’’-28 male

Selection

valve 1

¼’’-28

female

FMSA jig Luer-Lok

male

Adaptor 2

Luer-Lok female ,

¼’’-28 male

FMSA jig Luer-Lok

male

Selection

valve 2

¼’’-28

female

Adaptor 2 Luer-Lok female,

¼’’-28 male

Selection

valve 2

¼’’-28

female

Blood/PBS

collector

(BD 30ml

syringe)

Luer-Lok

female

Adaptor 3

Luer-Lok male, ¼’’-

28 male

Table 2-1. Adaptors and tubing

21

Filtration Procedure

During the filtration process, PBS was firstly used to rinse the system and remove air,

which can create a stable liquid environment to avoid generation of air bubbles when blood was

added. Then blood was loaded, filtered and recollected. Finally, PBS was used again to rinse the

system, especially the membrane. As shown in figure 2-4 and table 2-3, to conduct this process, we

used 6 steps in total, and both two pumps worked at 4ml/min during the whole process.

Figure 2-5. Filtration process. Red indicates blood, and blue indicates PBS. The whole filtration process

including 6 steps and two manual actions in step 3-2 and step 4-1.

22

Steps Pump1 Pump2 Action Purpose

0 Stop Stop Load PBS collector and

container with 8ml PBS.

Preparation

1 Stop Infuse Pump2 (right) infuses PBS

from PBS collector to PBS

container

Remove air in the right side

of system

2 Withdraw Stop Pump1 (left) withdraws PBS

from PBS container to blood

container

Remove air in the left-

bottom side of system

3 Stop Infuse Pump2 infuses PBS from PBS

collector to blood container;

When PBS reaches blood

container, add blood to blood

container.

Remove air in the left-top

side of system

4 Withdraw Stop Pump1 withdraws blood from

container to collector;

Blood get filtered by

membrane;

When blood volume is less

than 0.5ml , add small volume

of PBS into blood container

Dilute last amount of blood,

make blood get filtered as

much as possible

5 Withdraw Stop Pump1 withdraws PBS from

PBS container

Push blood to be collected,

and rinse the membrane

6 Stop Withdraw Pump1 withdraws PBS from

PBS container

Rinse membrane and

system

Table 2-2. Filtration procedure

23

In step1 and step3, we all chose to use infusion mode not withdrawn to remove air trapped

in the system, which means the flow direction of PBS is from bottom to top. In infusing mode,

liquid moves against its gravity, while in withdrawing mode, liquid moves in the same direction as

its gravity. Each part of flow path inside the system does not have the same diameter, for example,

the diameter of FMSA jig is much bigger than tubing. So, in step1, if PBS moves from top to

bottom, it is more likely to cause bubbles at liquid-air interface compared to infusing mode. Once

bubbles are generated, it’s very difficult to remove them in subsequent withdrawing steps. Bubbles

are fatal to cells. When bubbles occur on the membrane, cells can explode at bubble surface as they

go through the system because of strong surface tension. Therefore, in air exhaust process, we make

PBS move from bottom to top to avoid generation of bubbles.

LabVIEW Program

To obtain a smooth filtration process, accurate corporation between pumps and selection

valves was realized by our user-friendly LabVIEW (Figure 2-6) coded software.

24

Figure 2-6. Auto-FMSA software front panel. Diameter of syringes, pumping rate and serial port can be

set in pump setting part. V1~V6 are volume of each step, which are related to the volume of blood sample.

T1~T6 and corresponding bars show the progress of each step. pumping information can be sent by ‘Send’

button, and filtration process can be triggered by ‘Run’ button.

Four coding sections shown in block diagram (Figure 2-7) involving four Sub VIs compose

the final manipulation VI. Pumps are controlled by ‘Pump Motion Section’, while selection valves

are controlled by ‘Selection Valves Section’. ‘Display Section’ is responsible for software front

panel display, including progress bars, needed time and countdowns of each section. ‘Filtration

Running/Stop Section’ is the part to trigger or stop the filtration process.

25

Figure 2-7. Software block diagram. Four sections in this manipulation VI have independent function:

‘pump motion section’ and ‘selection valves section’ are used to control pumps and selection valves;

‘filtration running/stop section’ controls the start and stop of filtration process; ‘display section’ is

responsible for front panel display.

Pump Motion Section

This section controls the motion sequence of two pumps. To simplify the diagram and

realize modularization, three functional Sub VIs including ‘Pump setting Sub VI’, ‘Run Sub VI’

and ‘Pause Sub VI’ were integrated into this section. Filtration procedure demonstrated before in

Figure 2-4 and Table 2-3 can be obtained by two function sequences involving these 3 Sub VIs for

each pump. Each sequence started with Pump setting Sub VI, following by combinations of Run

Sub VIs and Pause Sub VIs as shown in Figure 2-11.

26

Figure 2-8. Block diagram of Pump motion section. Three Sub VIs are used in this section, and two

sequences of these three Sub VIs are set as filtration process.

27

Detailed operation sequence is shown in the table below.

Pump

Step

Pump1 (address 0) Pump2 (address 1)

Function Volume (ml) Time (s) Function Volume (ml) Time(s)

0 Setting Setting

1 Stop V1/rate INF V1

2 WDR V2 Stop V2/rate

3 Stop V3/rate INF V3

4 WDR V4 Stop V4/rate

5 WDR V5 stop V5/rate

6 Stop V6/rate WDR V6

Table 2-3. Pump operation sequence

Address Setting

Two pumps were used in our instrument, and formed a pump network. In the network, each

pump had its own address for VISA (Virtual Instrument Software Architecture) data transmission,

which needs to be set up before instrument assembly. To set the network address, each pump was

individually attached to computer and set with address command *ADR nn, where ‘nn’ is network

address (00 and 01 in our case). The set address was stored in the pump’s nonvolatile memory, so

that we didn’t need to set pump address every time before filtration. Once the command for address

setting was sent, the pump would only respond to commands sent to its set address.

28

Pump Commands

Before pumps can be operated, basic pumping data (syringe diameter and pumping rate)

and operation data (function type, pumping direction, volume, etc.) need to be set up in front panel.

In block diagram, commands used for controlling pumps consist of pump address, function name,

data and carriage return constant. Default volume, diameter and rate units in this system is milliliter

(ml), millimeter(mm), and milliliter per minute (ml/min). For example, 00 DIA 21.59 ↲ means

diameter of the syringe loaded on the pump with address 00 is 21.59mm. All commands used in

this software can be found in the table below.

Command Description

ADR nn Set pump network address

DIA <float> Set diameter

PHN nn Set program phase

FUN RAT Defines a pumping function with a fixed pumping rate.

RAT <float> [unit] Set pumping rate and unit.

VOL <float> Set volume to be dispensed, default unit is ml

DIR [INF|WDR] Set pumping direction as infuse or withdraw

FUN PAS nn Pause pumping for “nn” seconds. “nn” can range from 00 to 99 seconds.

RUN Start command

STP Stop command

Table 2-4. Command and relevant description

29

Pump Setting Sub VI

Pump setting Sub VI (Figure 2-9) was used to set syringe diameter for each pump. Apart

from two common input (VISA resource name and error), it also has an on/off input, a pump

address input and a diameter input. The commands connected by ‘Concatenate String Function’

used in this VI is nn DIA <diameter> ↲, and was written by ‘VISA Write Function’. The number

of diameter need to be converted from number to functional string.

Figure 2-9. Block diagram of pump setting Sub VI. Syringe diameter can be set by this VI.

Run Sub VI

In this Sub VI (Figure 2-10), pumping rate, pumping volume and direction were set.

Command ‘PHN’ was used to separate commands of each step and made the diagram more readable

and organized when all these Sub VIs were put together. Step number, pumping rate and pumping

volume were written using function string format.

30

Figure 2-10. Block diagram of run Sub VI. Pumping information including rate, volume, and direction

are set by this VI.

Commands in this Sub VI are:

<address> PHN <step number> ↲

<address> FUN RAT ↲

<address> RAT <rate> [unit] ↲

<address> VOL <volume> ↲

<address> DIR [direction] ↲

Pause Sub VI

This Sub VI (Figure 2-11) can make selected pump pause for needed time. During each

step, only one pump works (infuse or withdraw mode), and its running time should be exactly same

as the pause time of the static pump. Pause time of static pump can be calculated with the formula

shown below.

Pause time (s) = 𝑉𝑜𝑙𝑢𝑚𝑒 (𝑚𝑙)

𝑟𝑎𝑡𝑒 (𝑚𝑙/𝑚𝑖𝑛) × 60 (Eq. 2.3)

31

The maximum of pause time in this VI is 99 seconds. Multiple Sub VIs permit longer pause

time.

Figure 2-11. Block diagram of pause Sub VI. Pausing time is set by this VI.

Commands used in this Sub VI are:

<address> PHN <step number> ↲

<address> FUN PAS <time> ↲

Display Section

On the front panel, six process bars and a countdown indicator (Figure 2-6) were used to

give user an intuitive knowledge of filtration progress. In addition, they also acted as a guide for

users, especially in step3 and step 4, where extra manual actions were need. Detailed coding of this

section is shown in Figure 2-12.

32

Figure 2-12. Block diagram of display section. Process bars and count down are calculated and displayed

by this part.

Filtration Running/Stop Section

After filtration parameters and pump information were sent to pumps, ‘Run’ button shown

on the front panel served as a trigger of filtration process. As shown in Figure 2-13, ‘Run’ button

would keep comparing its current status and previous status. If the button status changed from ‘off’

to ‘on’, a running command (0 run * 1 run ↲) would be sent to pumps, otherwise a stop command

(0 stp * 1 stp ↲) would be sent. Here, ‘*’ means ‘and’.

Figure 2-13. Block diagram of filtration running/stop section. ‘Run’ button serves as the trigger of

filtration process, and can control two pumps at the same time.

33

Selection Valves Section

During the filtration process, without good cooperation between selection valves and

pumps, there was no possibility of achieving a smooth filtration process. As we mentioned before,

each actuator needs dual-voltage (12V/6V) to be energized and keep working. This dual-voltage

was generated by an analog voltage output module and an amplification circuit with amplification

factor equal to 6. According to the actual voltage measured from output, here we modified output

voltage of analog voltage module from 2V/1V to 2.1V/1.1V (Figure 2-14). 2.1V was first applied

to analog module for 1000ms, and then 1.1V was applied for 𝑇𝑛′(n is step number)

𝑇𝑛′ = 𝑇𝑛 − 1000𝑚𝑠 (Eq. 2.4)

Figure 2-14. Block diagram of a part of selection valves section. Input voltage for amplification circuit

is generated by this section.

In addition, a beep sound was used to mark the end of each step and served as manual

action guide as well. According to filtration procedure, voltage sequence that was sent to selection

valves was listed in the table below.

34

Analog Output

Step

P3

(PBS collector)

P2

(Blood collector)

P1

(PBS container)

P0

(Blood container)

Time

(s)

1

2.1 0 2.1 0 1

1.1 0 1.1 0 𝑇1′

2

0 2.1 2.1 0 1

0 1.1 1.1 0 𝑇2′

3

2.1 0 0 2.1 1

1.1 0 0 1.1 𝑇3′

4

0 2.1 0 2.1 1

0 1.1 0 1.1 𝑇4′

5

0 2.1 2.1 0 1

0 1.1 1.1 0 𝑇5′

6

2.1 0 2.1 0 1

1.1 0 1.1 0 𝑇6′

Default 0 0 0 0 _

Table 2-5. Voltage sequence of analog output

35

Chapter 3

Material and Methods

This chapter describes the materials and methods utilized in our experiments, and how we

used automatic CTCs enrichment system to conduct experiments with both cancer cells spiked

healthy donor blood samples and patient blood samples.

Cell Culture

To test enrichment efficiency of our system, human breast cancer cell line (MCF-7) was

spiked in healthy donor blood, and enriched. MCF-7 cancer cells were cultured in high-glucose

DMEM media with 10% feral bovine serum (FBS) and 0.1% insulin (insulin solution human) in

humidified incubators at 5% CO2 and 37°C.

Blood Sample Preparation

MCF-7 Cancer Cells Spiked Experiment

Healthy donor blood samples were provided by the Penn State General Clinical Research

Center. Samples were drawn into 10ml tubes, and processed within 6 hours. To identify MCF-7

cancer cells against leukocytes after filtration, MCF-7 cancer cells were pre-labeled with Calcein-

AM, a cell-permeant dye that can be used to determine cell viability in most eukaryotic cells. 5μl

Calcein-AM was added into 1ml MCF-7 cancer cells suspension and incubated for 10 minutes.

Then cell suspension was centrifuged at 1000rpm for 5 minutes. Supernatant was replaced by 1ml

PBS, and cell residue was mixed with PBS. This washing process was conducted for 3 times to

36

remove residue of Calcein-AM, which can avoid leukocytes being stained by Calcein-AM when

MCF-7 cancer cells were spiked into blood samples.

After washing, cell concentration was estimated using Glasstic® Slide (KOVA) after

several times of dilutions. 1μl cell suspension was added into a Glasstic® Slide, and enumerated

for three times. The average of these three numbers was considered as cell concentration (per

microliter). Five levels of cell amounts (1000, 200, 30, 15, 5) for spiking were tested by our system,

and at least 3 samples were processed for each level. For large cell amount, for example 1000 and

200, MCF-7 cells were diluted to 30~40 cells/ml, and then spiked in 4ml healthy donor blood with

needed volume. While for small cell amount (30, 15 and 5), MCF-7 cells were first diluted by

multiple times to the concentration (per microliter) near target concentration. Then 1μl cell

suspension was enumerated in 96 well plate and spiked into 4ml healthy donor blood. After that,

1μl PBS was added in the same well for enumerating the rest of cells. Finally, the difference

between two enumerated numbers was served as spiked cell amount.

Patient Sample

Patient samples were obtained with consent from small lung cancer patients at the Penn

State Hershey Medical Center. Preprocessing before filtration is not needed.

FMSA O-ring Fabrication

As shown in Figure 2-3, FMSA membrane was sandwiched by two square-shaped PDMS

O-rings. To fabricate PDMS O-rings, 26g elastomer and 2.6g curing agent (DOW Corning) were

mixed and poured into a 150mm-pertri dish (VWR) after being degassed. To obtain a PDMS sheet

with even thickness, petri dish was knocked with strength, and then stayed overnight on a flat table.

37

The thickness of PDMS sheet in the petri dish was slightly thicker than the inside cavity of FMSA

jig, which provided a good sealing for FMSA jig after being clamped. Single PDMS O-ring was

cut with knife, and a 10mm-square hole was punched to fit the efficient area of FMSA membrane.

Filtration Process

After preparing pre-labeled MCF-7 cells spiked blood samples or patient samples, the

whole filtration process can be finished in 15 minutes including instrument assembly, software

setting and actual filtration process (6 minutes for 4ml blood sample).

Instrument Assembly

Instrument including two containers for both PBS and blood, tubing, adaptors, FMSA

device, selection valves, collectors and pumps needs to be assembled before software setting and

filtration. The actual scene of assembled instrument is shown in Figure 3-1.

FMSA box were made from grey PVC sheet. Shelf and two slide doors (not shown in

Figure 3-1) were made from clear acrylic Sheet. Several holes were drilled on the back of the box

for data line to pass through. A small shelf with adjustable height was designed for selection valve

1.

38

Figure 3-1. Picture of our assembled auto-FMSA instrument.

Instrument assembly protocol:

1. Connect blood container (10ml BD syringe without plunger) and PBS container (30ml

BD syringe without plunger) to selection valve 1 with precut 7cm/10cm-tubing and

adaptors 1

2. Install FMSA jig top part on selection valve 1 and bottom part on selection valve 2

with adaptors 2

3. Load PBS collector (30ml BD syringe) with 8ml PBS, and mount it on selection valve

2 with adaptor 3.

4. Remove air inside blood container (30ml BD syringe), and mount it on selection valve

2 with Adaptor 3.

39

5. Install PBS container and blood container on pumps. Tight syringe clamps.

6. Clean precut FMSA membrane with IPA (Isopropyl Alcohol), and sandwich it by 2

PDMS O-rings.

7. Put O-ring-membrane on the bottom part of FMSA jig, and install the jig on the

selection valve 2. Clamp top and bottom part.

8. Load PBS container with 8ml PBS.

Software Setting

The blood sample volume used for filtration was 4ml. After calculation and some pre-

experiments, the most suitable volumes of each step and other information were set as the table

below.

Diameter

(mm)

Rate

(ml/min)

Serial

port

V1 (ml) V2 (ml) V3 (ml) V4 (ml) V5 (ml) V6 (ml)

21.59 4 COM10 4 2 2 10 3 3

Table 3-1. Software setting parameters

After setting all parameters, we clicked ‘Send’ button and waited for data transmission.

Then we clicked ‘Run’ button to trigger the filtration process.

Filtration

Filtration progress can be detected by progress bars, countdown and instrument itself. Only

two extra manual actions were need in the process. First, during Step 3, when PBS flowed from

bottom PBS collector and reached blood container, 4ml blood sample and 1ml PBS used to rinse

blood tube were added into blood container. Second, during Step 4 (withdrew blood sample from

40

blood container to blood collector), before countdown reached 110 (20 seconds before Step 5

started), small volume of PBS was added by several times to ensure the existence of liquid in blood

container and avoid generation of air bubbles in system before Step 5 started. So that, even small

volume of blood sample was left in the tubing that not filtrated by FMSA membrane, it was heavily

diluted by PBS. And we considered this as a systematic error.

Immunofluorescent Detection

When filtration ends, FMSA membrane with O-rings was took out from FMSA jig and

transferred to a clean petri dish. We fixed FMSA membrane with 4% paraformaldehyde (VWR)

for ten minutes, and washed it with PBS for three times. Then cells were permeablized in 0.3%

Triton X-100 (VWR) for ten minutes, and washed with PBS for three times.

Immunofluorescent detection methods used for patient samples and MCF-7 cancer cells

spiked blood samples were different.

For patient samples, cells were blocked in 5% goat serum (Sigma-Aldrich) at room

temperature for 1 hour. After that, cells were incubated in 100μl blocking buffer with 1% mouse

monoclonal anti-cytokeratin 8/18/19 and 0.1% monoclonal mouse anti-CD45 for another 1 hour at

room temperature. Afterwards, cells were washed by PBS for 3 times, and stained with1μg/ml of

4’, 6-diamidino-2-phenylindole (DAPI) (Invitrogen) for 10 minutes. While for MCF-7 cancer cells

spiked blood samples, cells were directly stained with DAPI for 10 minutes. Finally, FMSA

membrane was washed with PBS, mounted on a glass slide using ProLong® Diamond Antifade

Mountant (Molecular Probes), and covered by a cover slip. After being mounted, membrane needed

stay at room temperature overnight to make mountant dry before checking under microscope.

41

Chapter 4

Results

To test our system, healthy donor blood samples with different concentration of MCF-7

cancer cells were filtrated to demonstrate the effect of spiked cells amounts on enrichment

efficiency. Clinical samples from patients with small lung cancer were filtrated to verify the

capability of our system processing clinical samples.

MCF-7 Spiked Experiments

Identification of MCF-7 Cancer Cells

MCF-7 cancer cells were pre-labeled with Calcein-AM before filtration, fixed and stained

with DAPI on the FMSA membrane after filtration. During enumeration, the identification criterion

of MCF-7 cancer cells was set as dual-positive signal of DAPI (blue) and Calcein-AM (green)

(Figure 4-1).

42

Figure 4-1. MCF-7 cells captured by FMSA membrane. MCF-7 cells were stained with Calcein AM

(green) and DAPI (blue), leukocytes were stained with DAPI. Scale bars are 20μm.

As results shown above, MCF-7 cancer cells and leukocytes were trapped by FMSA

membrane. MCF-7 cancer cells had both green signal came from Calcein-AM and blue signal

marked by nucleus. While leukocytes only had blue signal.

43

Effect of Spiked Cell Amount on Capture Efficiency

We used five different spiked cell amounts, from 5 to 1000 to test capture efficiency of our

system (table 4-1). Capture efficiency of our system was defined as the ratio of MCF-7 cancer cells

retained on FMSA membrane after filtration to the number of MCF-7 cancer cells spiked in 4ml

healthy donor blood before filtration.

CE =𝑁𝑎𝑓𝑡𝑒𝑟

𝑁𝑏𝑒𝑓𝑜𝑟𝑒× 100% (Eq. 4.1)

According to the results shown in table 4-1 and figure 4-2, enrichment performance was

very steady and capture efficiency was very high (larger than 87%) when the number of spiked

cancer cells was larger than 30. Although the capture efficiency fluctuated widely (Figure 4-3)

when small amounts of cancer cells were spiked in blood samples, our system was still sensitive

enough to detect cancer cells against large amount of leukocytes. Detailed filtration results are

shown in Table 4-1 and Figure 4-3.

Level Input Output Capture efficiency mean

1

(1000)

1000 967 96.7% 95.8%

1000 916 91.6%

1000 990 99.0%

2

(200)

200 189 94.5% 92.7%

200 177 88.5%

200 190 95.0%

3

(30)

37 34 91.9% 83.5%

33 29 87.9%

23 18 78.3%

25 19 76.0%

4

(15)

18 12 66.7% 73.5%

13 10 76.9%

13 10 76.9%

5

(5)

3 1 33.3% 54.4%

5 4 80.0%

8 4 50.0%

Table 4-1. Filtration results of MCF-7 spiked experiments.

44

Figure 4-2. Filtration performance of MCF-7 spiked experiments. Spiked numbers and output numbers

are plotted.

Figure 4-3. Effect of spiked cell number on filtration efficiency. The mean capture efficiency of each

level and error bars are plotted.

0

0.5

1

1.5

2

2.5

3

3.5

0 0.5 1 1.5 2 2.5 3 3.5

log(

ou

tpu

t)

log(input)

Filtration Performance

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 0.5 1 1.5 2 2.5 3 3.5

Cap

ture

Eff

icie

ncy

log(input)

Effect of Spiked Cell Number on Capture Efficiency

45

Enrichment against Leukocytes

During filtration, erythrocytes were small enough to pass through the gaps between the

micro springs of FMSA device because of its disc-shape. However, leukocytes have a larger size

range, which has overlap with MCF-7 cancer cells. Therefore, leukocytes become the main concern

for filtration purity. Here, the enrichment factor was defined as the ratio of cancer cells to

leukocytes after filtration divided by the same ratio before enrichment3:

E =(

𝑛𝑇𝑛𝐿

)𝑎𝑓𝑡𝑒𝑟

(𝑛𝑇𝑛𝐿

)𝑏𝑒𝑓𝑜𝑟𝑒

= 𝐶𝐸 ×(𝑛𝐿)𝑏𝑒𝑓𝑜𝑟𝑒

(𝑛𝐿)𝑎𝑓𝑡𝑒𝑟≅

(𝑛𝐿)𝑏𝑒𝑓𝑜𝑟𝑒

(𝑛𝐿)𝑎𝑓𝑡𝑒𝑟 (Eq. 4.2)

Where E is the enrichment factor, 𝑛𝑇is the number of MCF-7 cancer cells and 𝑛𝐿 is the

number of leukocytes.

Images were acquired at five different locations on the FMSA membrane, and leukocytes

were enumerated using ImageJ. The number of leukocytes after filtration was then estimated by

the product of the average enumeration and the ration of membrane efficient area to image area.

The mean enrichment factor from 4ml MCF-7 cancer cells spiked blood sample experiments was

approximately 2.3 × 102 (n=3).

Circulating Tumor Cells Detection

Blood samples from patients with small lung cancer were used to verify the capability of

our system to enrich circulating tumor cells from peripheral blood. 4ml patient samples were

filtrated using our system and then stained as the protocol we described before in chapter 3. CTCs

were identified as large nucleated cells that stained positively for cytokeratin 8/18/19 (green) and

negatively for CD45 (red) (Figure 4-4). While leukocytes had smaller size and were stained

positively for CD 45.

46

Figure 4-4. Circulating tumor cell (in yellow circle) on FMSA membrane. CTCs are stained with CK

8/18/19 (green) and DAPI (blue), while leukocytes are stained with CD 45 (red) and DAPI (blue). Scale

bars are 40μm.

As shown above, in DAPI channel, a circulating tumor cell in yellow circle is very

conspicuous because of its large nucleus. CK signal is not very strong, so the concentration of CK

8/18/19 used in staining still needs to be regulated. From two 4ml patient samples, at least 5 CTCs

47

were detected by our device in each sample, which verified that our system was sensitive enough

to detect CTCs from peripheral blood.

48

Chapter 5 Conclusion and Future Work

Conclusion

An automatic CTCs enrichment system based on flexible micro spring array (FMSA) was

developed and built. The geometry of FMSA membrane device permits the enrichment of large

cancer cells but let small blood cells pass through at the same time. This system can be controlled

by a user-friendly software coded by LabVIEW, and conduct 4ml-blood sample filtration within 6

minutes. Every element in our system works smoothly during the filtration process under the

control of software. New users can be trained to use our system by a demonstration, and can easily

organize each step of operation with guidance provided by front panel of our software.

To test performance of our system, we did two level of experiments. The first level was

cancer cells spiked experiment and the second was patient sample experiment. In cell spiked

experiments, different amounts of pre-labeled MCF-7 cancer cells were spiked in 4ml healthy

donor blood samples to test the capture efficiency of our system under different situation. While

for patient sample experiments, 4ml blood samples from patients with small lung cancer were used

to prove the possibility of our system to detect CTCs from peripheral blood. From the results we

showed in chapter 4, our system can enrich spiked cancer cells with high efficiency, and is sensitive

enough to detect CTCs from patient samples.

49

Future Work

Only two patient samples were filtrated by our system, because patient samples are limited.

More patient samples need to be filtrated by our system to provide more CTCs data and verify our

system. The staining process still has much room for improvement, because CK 8/18/19 signal was

very weak in our experiments, which made cell nucleus size the first identification criteria of CTCs.

Also, the CD 45 signal was not ideal. In addition, for patient sample experiments, we didn’t know

the enrichment performance of our system. So a comparison between our system and CellSearch®

using patient samples can be conducted in the future to demonstrate how sensitive our system is.

For software part, so far the parameters correspond to 4ml blood sample. More parameter

sets can be pre-tested to meet different requirements, such as different sample volume, higher cell

viability and multiple cell types, and be integrated into software function mode. By doing so, users

can simply choose enrichment mode without caring about parameter settings.

For instrument part, to make CTC enrichment process more integrated, fix process and

staining process are considered to be operated by system. Selection valves currently used in our

instrument have two inlets and a common outlet. If we use selection valves with multiple inlets and

add more solution fluidic paths, cells retained on the membrane can be fixed and stained in the

FMSA jig without being taken out. However, the main problem is how to wash system completely,

because residues caused by fix or staining solution can clog selection valves and affect future

experiments.

50

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