Gemcitabine Sensitivity on

Podocalyxin Knockdown Pancreatic Cell Line SW1990

by

Yuan-Hsiao Billy Huang

A thesis submitted to Johns Hopkins University in conformity with the requirements

for the degree of Master of Chemical Biomolecular Engineering

Baltimore, Maryland

November, 2014

© 2014 Yuan-Hsiao Billy Huang

All Rights Reserved

ii

Abstract

In this study, we tested cell survival following treatments of Gemcitabine (GE)

in scrambled control and Podocalyxin (PODXL) knockdown pancreatic cancer cell

line SW1990. After testing several major chemotherapeutic drugs with MTT cell

survival assay, GE, brand name Gemzar, was shown to have a much higher cytotoxic

effect on PODXL knockdown cells than control SW1990 pancreatic cancer cells.

Upon observation, we suggested that knocking down PODXL could potentially

reduce the resistance and/or increase the sensitivity of SW1990 to GE. With this

hypothesis, we proceeded to test the expression of NF-κB targeted genes, which are

indicator of cell survival mostly related to cell apoptosis, in both scrambled control

and PODXL knockdown cells with and without GE treatments. The seven specific

genes analyzed were cyclin D1, c-myc, bcl-2, bcl-xL, c-IAP1, cox-2 and MMP9.

Apoptosis-related gene expressions were analyzed by reverse transcription-

polymerase chain reaction (rt-PCR). Our results showed that five out of seven of the

anti apoptotic genes were down regulated in PODXL knockdown and two out of

seven of the anti apoptotic genes were significantly down regulated in PODXL

knockdown following GE treatment. Combined data supports our hypothesis that

PODXL knockdown cells are more sensitive to GE as a result of apoptosis induction.

ACKNOWLEDGEMENTS

The excellent technical support by Dr. Jack Shih-Hsun Chen is greatly acknowledged.

iii

Table of Content

Introduction ............................................................................................................................... 1

Experimental Methods ............................................................................................................ 7

Result ............................................................................................................................................ 9

Discussion ................................................................................................................................ 15

Reference .................................................................................................................................. 19

Curriculum Vitae .................................................................................................................... 22

1

INTRODUCTION

Pancreatic cancer is one of the most deadly diseases in the world. Due to

its resistance to most cytotoxic drugs, pancreatic cancer patients’ five-year

relative survival rate is as low as 3 percent. [1] Such low percentage could be the

result of suboptimal drug delivery, poor drug efficacy, insufficient cellular uptake,

and chemotherapeutic resistance. In this study, a surprising increase in cell

death was observed following treatment of GE in pancreatic cancer cell line

SW1990 upon the knockdown of a transmembrane protein, PODXL.

PODXL is a transmembrane protein that is expressed in many types of

human cells such as platelets and vascular endothelial cells. [2] First discovered

in kidney, PODXL’s main function is the keep the unitary filtration barrier open.

[3] It is upregulated in numerous types of cancers such as breast, colon, and

pancreatic cancers. [4] PODXL has been identified as a functional E- and L-

selectin ligand expressed by metastatic pancreatic cancer. [5] Data also shown

that metastatic pancreatic cancer cells overexpress PODXL compared with

nonmalignant pancreatic epithelial cells. [5] Yet, PODXL’s connection with GE

resistance is still largely unexplored.

GE is a nucleoside analogue used as an anti-neoplastic drug. It causes

apoptosis by replacing cytidine during DNA replication or by binding to

ribonucleotide reductase (RNR). [6] Once transported through cellular

2

membrane, GE is phosphorylated into its active diphosphate and triphosphate

form, which competes with the natural deoxycytidine-triphosphate analog for

incorporation in DNA and RNA. This metabolism inhibits cytidine triphosphate

synthase and terminates DNA synthesis. GE is currently the first-line agent for

advanced pancreatic treatment. However, low protein binding of less than 10%

as revealed by pharmacokinetic data low response rate of 20% indicate the need

for improvement in overall GE performance. [7]

GE response in pancreatic cancer involves many intracellular pathways.

In this study we specifically focused on the end terminus of the

Phosphatidylinositol-3 kinase/AKT/NF-κB axis, the NF-κB targeted genes.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is anti-

apoptotic transcription factor that promotes cancer growth. In short, GE exerts

its cytotoxic effect in pancreatic cancer cells in vitro and in vivo by inhibiting NF-

κB and its targeted genes cyclin D1, c-myc, bcl-2, bcl-xL, c-IAP1, cox-2 and MMP-9,

which promotes cell death. [8,9,10]

In this work we focused on the expression of seven targeted genes of

control and PODXL knockdown cells before and after treatment with 10 μM GE

for 72 hours. Each of the seven genes listed influence the survival of pancreatic

cancer cells, but the knowledge of the seven NF-κB targeted genes is not yet fully

understood and their complete functions are still under investigation. Since this

work mainly focuses on the NF-κB gene expressions, we provide a

3

comprehensive review of each gene with special regards to how they affect

apoptosis.

NF-ΚB: cyclin D1, c-myc, bcl-2, bcl-xL, c-IAP1, cox-2 and MMP-9

Cyclin D1 is a protein kinase that regulates cell cycle. It is overexpressed

gene in breast carcinoma. [11] Overexpression of cyclin D1 promotes growth in

pancreatic tumor cancer cells and cyclin D1-overexpressing cells exhibit

significantly reduced chemosensitivity to cisplatin. [12] This suggests that

overexpression of cyclin D1 could cause chemoresistance in pancreatic cancer

due to its roles in promoting cell proliferation and inhibiting drug-induced

apoptosis.

C-myc is another regulatory gene that regulates cell proliferation. In many

cancers, mutated version of c-myc causes c-myc to be persistently expressed,

leading to disrupted expression of many other downstream genes associated

with cell proliferation. [13] C-myc is overexpressed in pancreatic cancers and

renders them resistance to cisplatin. [14] Overexpressing c-myc could interfere

with the process of apoptosis and leads to unregulated cell growth.

Bcl-2 is an anti-apoptotic protein in pancreatic cancer and many other

tumors. [15] Bcl-2 is an integral membrane protein located on the outer

4

membrane of mitochondria that prevents apoptosis by blocking the release of

cytochrome c, an initiation factor of apoptosis [16] In a multiple myeloma

chemotherapy study, bcl-2 overexpression is associated to resistance to

paclitaxel, but not GE. [17] Paclitaxel induces cell arrest at G2/M phase whereas

GE induces cell arrest at S phase of the cell cycle. In this particular study,

paclitaxel induced a down regulation of bcl-2 whereas treatment with GE did not

affect bcl-2 expression. This suggests that bcl-2 might be irrelevant to GE

resistance in pancreatic cancer. Although bcl-2 might not be directly influenced

by GE treatment, its antiapoptotic character is noteworthy.

B-cell lymphoma-extra large (bcl-xL) is also a member of the bcl-2 family.

Like bcl-2, bcl-xL is a transmembrane protein in the mitochondria, which is also a

pro-survival protein that prevents the release of mitochondrial substances, such

as many proteases, and eventually prevents apoptosis. bcl-xL protein level is a

very useful tool to determine the apoptotic index. In a recent study analyzing the

expression of Bcl-2, Bcl-xL, and Bax in pancreatic ductal carcinoma and their

correlation to the extent of apoptosis, an upregulation of all the apoptotic

regulatory molecules, especially bcl-xL, was observed. (The apoptotic index was

determined by terminal deoxynucleotidyl transferase-mediated nick end

labeling (TUNEL) essay) [18] Therefore bcl-xL expression could be used as an

indicator of relative apoptotic rate in this GE study.

5

Baculoviral IAP repeat-containing protein 2 (c-IAP1), like bcl-2 and bcl-xL,

is a prosurvival protein that inhibits apoptosis by interfering with the activation

of caspases, which are a family of cysteine proteases that play essential role in

apoptosis. In both neoplastic and non-neoplastic pancreatic cancer cells,

overexpression of c-IAP1 is observed in the early stage of cancer progression. [19]

In particular, c-IAP1 plays a role in the inhibition of TNF-alpha-induced apoptosis

by inducing NF-κB. [19] Therefore its expression is expected to decrease in

PODXL knockdown cells since they became more sensitive to GE.

Cyclooxygenase-2 (cox-2) is an enzyme responsible for inflammation and

pain, which converts arachidonic acid to prostaglandin during inflammation.

Prostaglandin E2, a prostaglandin derivative, can stimulate cancer progression.

[20] It is upregulated in pancreatic ductal adenocarcinomas (PDAC). [21]

However, the mechanism of how cox-2 promotes PDAC is unclear. Some has

suggested that deregulation of the cox-2/PGE2 pathway appears to affect

colorectal tumorgenesis by promoting tumor maintenance and progression, and

encouraging metastatic spread. [22] In relation to apoptosis, inhibition of cox-2

in a hepatocellular carcinoma study induces apoptosis signaling, activation of

caspases, and apoptosis originating from mitochondria. [23]

At last, matrix metallopeptidase 9 (MMP-9) is a protease that is involved

in the breakdown of the extracellular matrix in normal physiological processes

and plays a major role in pancreatic cancer growth and metastasis. [24] MMP-9 is

6

more associated with cancer cell invasiveness rather than apoptosis. However,

MMP-9 causes the recruitment of pericytes and pericytes invasion in primary

tumor development during the degradation of extracellular matrix. [25]

Pericytes induced the antiapoptotic protein Bcl-w in tumor endothelial cells both

in vivo and in vitro thereby protecting tumor cells from cytotoxic elements. [26]

Therefore, indirectly, MMP-9 may regulate apoptosis either through the

generation of extracellular matrix molecules or transactivation of cell surface

receptors. [25] Data also suggests the anti-apoptotic roles of a numbers of MMPs

in vitro including MMP-7, MMP-9, MMP-10 and MMP-15. [27,28,29] These MMPs

could also be related to apoptosis resistance in tumor cells by assisting or

enhancing the early stages of tumor dissemination. [30,31] Therefore a decrease

in MMP-9 expression may be expected in PODXL knockdown cells against GE.

7

Table 1 summarizes the above information of each gene on apoptosis and

the predicted expression upon GE treatment to the more sensitive PODXL

knockdown pancreatic cancer cells.

NF-κB

Genes

cyclin

D1

c-myc bcl-2 bcl-xl c-IAP1 cox-2 MMP-9

Apoptotic

Effect

_ _ _ _ _ _ _*

Expected

Expression

in GE-

PODXL

Condition

TABLE 1: SUMMARY OF NF-ΚB TARGETED GENES EFFECTS ON APOPTOSIS. A NEGATIVE MARK INDICATES ANTI-

APOPTOTIC AND THE ASTERISK INDICATES INDIRECT EFFECT. THE DOWNWARD ARROW INDICATES A DOWN

REGULATION OF THE GENE EXPRESSION. GE-PODXL REFERS TO THE PODXL KNOCKDOWN CELLS TREATING

WITH GE FOR 24 HOURS.

EXPERIMENTAL METHODS

CELL CULTURE:

The pancreatic adenocarcinoma cell lines SW1990 was obtained from the

American Type Culture Collection (Manassas, VA) and Pa03C was a generous gift

from Dr. Anirban Maitra (Johns Hopkins School of Medicine, Baltimore, MD, USA).

[5] SW1990 and Pa03c-PODXL knockdown cell line was provided by Dr. Matt

Dallas. [5] SW1990 cells were cultured in DMEM supplemented with 10% FBS

and 50 μg/ml gentamicin. Cells were harvested via mild trypsinization (0.25%

trypsin/EDTA·4Na for 5 min at 37°C) and incubated at 37°C for 2 h to regenerate

surface glycoproteins. [5]

8

CELL SURVIVAL ASSAY:

SW1990/Pa03c control and PODXL knockdown cells (5,000/well) were

seeded in 96-well plates in duplicates and allowed to adhere overnight. After

overnight incubation, the cells were treated with various concentrations of GE of

0, 0.1, 1, 10, and 100 μM for 72 hours. Cell survival was measured by a standard

MTT assay.

MTT ASSAY:

Cell viability was evaluated using 3-[4,5-dimethylthiazol-2-yl]-3,5-

diphenyl tetrazolium bromide (MTT) test. After the treatment period as

indicated above, the incubation media were removed and exchanged with 100 µl

of fresh media containing MTT (0.2 mg/ml), and cells were incubated at 37°C for

3 hours. The media were removed, and formazan crystals were solubilized with

150 µl of DMSO. Absorbance at 570 nm was measured in triplicate using a

microplate reader. [32]

Reverse Transcription Polymerase Chain Reaction (rt-PCR):

Control and PODXL knockdown cells and control and PODXL knockdown

cells treated with GE for 24 hours were prepared in 6-well plates. Instead of 72

hours, we treated cells with GE for 24 hours to test gene expressions before

9

major cell death occurred. Upon 100% confluency, cells were washed with cold

PBS twice before extracting the mRNA with Trizol. Extracted mRNAs were

prepared at sample size of 5 μg in triplicate along with other necessary reagents

(RNAse, primers, etc.). The reagents are iTaqTM Universal Supermixes from Bio-

Rad. Primers’ sequences were obtained from NCBI. Primers were obtained from

Invitrogen. GAPDH was used as a reference to all genes.

RESULT

SW-PODXL KNOCK DOWN CELLS ARE 13% MORE SENSITIVE TO GEMCITABINE THAN

CONTROL CELLS

To investigate the sensitivity of human pancreatic cancer cell line

SW1990 and its PODXL knockdown to anticancer drugs, we tested

chemotherapeutic agent GE for pancreatic cancer in a dosage dependent manner.

Cells were incubated for 72 hours to a range of each agent’s concentrations going

from 0.1 to 100 μM. Among all the different conditions, PODXL knockdown cells

exhibited increased sensitive to GE compared to the control cells. Shown in

Figure 1, differences in cytotoxic effect were observed in different

concentrations of GE treatment. There is 10 to 22 percent cell death differences

in different drug concentrations. Figure 2 is the supplementary data of another

pancreatic cell line Pa03c for the generalization purpose.

10

FIGURE 1: PANCREATIC TUMOR CELLS SW 1990 SENSITIVITY TO GE. CELL SURVIVAL ASSAY WAS PERFORMED

USING MTT METHOD. CELLS WERE TREATED FOR 72 HOURS WITH INCREASING CONCENTRATION OF GE (0.1 –

100 UM) CELL VIABILITY WAS PRESENTED AS A PERCENTAGE OF UNTREATED CELL CONTROL VALUES. BLUE

DOT MARK REPRESENTS SCRAMBLE CONTROL WHILE THE TRIANGLE MARK REPRESENTS THE PODOCALYXIN

KNOCKDOWN CELL LINE.

FIGURE 2: PANCREATIC TUMOR CELLS PA03C SENSITIVITY TO GE. CONDITIONS SAME AS FIGURE 1.

0

25

50

75

100

0 0.1 1 10 100

% C

ell

Su

rviv

al

Gemcitabine (uM)

SW-SC

SW-POD

0

25

50

75

100

0 0.1 1 10 100

% C

ell

Su

rviv

al

Gemcitabine (μM )

E3-SC

E3-POD

11

NF-ΚB GENE EXPRESSIONS

We’ve shown that PODXL knock down is more sensitive to GE than

control SW1990. In the following figures we present the NF-κB genes

expressions in four conditions: SW control (SW SC), SW PODXL Knockdown (SW

KD), SW control treated with GE (SW GE SC), and SW PODXL Knockdown treated

with GE (SW GE KD). Tumor cells were treated with GE for 24 hours. NF-κB

targeted genes are anti-apoptotic; therefore a more GE-sensitive cell line would

expect lower NF-κB gene expressions. Our data agrees with the prediction except

for a little deviation. The Results are shown as follows.

FIGURE 3 CYCLIN D1 GENE EXPRESSIONS ON SW1990 PANCREATIC CANCER CELLS. FROM LEFT TO RIGHT:

CONTROL, PODXL KNOCKDOWN, CONTROL WITH GE TREATMENT, AND PODXL KNOCKDOWN WITH GE

TREATMENT.

12

FIGURE 4 C-MYC GENE EXPRESSIONS ON SW1990 PANCREATIC CANCER CELLS. FROM LEFT TO RIGHT: CONTROL,

PODXL KNOCKDOWN, CONTROL WITH GE TREATMENT, AND PODXL KNOCKDOWN WITH GE TREATMENT.

FIGURE 5 BCL-2 GENE EXPRESSIONS ON SW1990 PANCREATIC CANCER CELLS. FROM LEFT TO RIGHT: CONTROL,

PODXL KNOCKDOWN, CONTROL WITH GE TREATMENT, AND PODXL KNOCKDOWN WITH GE TREATMENT.

13

FIGURE 6 BCL-XL GENE EXPRESSIONS ON SW1990 PANCREATIC CANCER CELLS. FROM LEFT TO RIGHT: CONTROL,

PODXL KNOCKDOWN, CONTROL WITH GE TREATMENT, AND PODXL KNOCKDOWN WITH GE TREATMENT.

FIGURE 7 CIAP1 GENE EXPRESSIONS ON SW1990 PANCREATIC CANCER CELLS. FROM LEFT TO RIGHT: CONTROL,

PODXL KNOCKDOWN, CONTROL WITH GE TREATMENT, AND PODXL KNOCKDOWN WITH GE TREATMENT.

14

FIGURE 8 COX2 GENE EXPRESSIONS ON SW1990 PANCREATIC CANCER CELLS. FROM LEFT TO RIGHT: CONTROL,

PODXL KNOCKDOWN, CONTROL WITH GE TREATMENT, AND PODXL KNOCKDOWN WITH GE TREATMENT.

FIGURE 9 MMP9 GENE EXPRESSIONS ON SW1990 PANCREATIC CANCER CELLS. FROM LEFT TO RIGHT: CONTROL,

PODXL KNOCKDOWN, CONTROL WITH GE TREATMENT, AND PODXL KNOCKDOWN WITH GE TREATMENT.

15

Cyclin D1 gene overexpresses in SW GE SC but had insignificant difference

in expressions on other three conditions. C-Myc gene expresses the same in both

control and gemcitabine treated cells, but greatly reduced in both PODXL

knockdown conditions. Bcl2 gene expressions are very interesting because it

decreased 1 fold in SW GE KD, which supports the argument that PODXL KD

increases the sensitivity of SW cell line to GE. Bcl-xl gene expressions decrease

for 2 folds in SW KD, but are also lowered in both GE conditions. Ciap1 gene

expressions have no significant difference in GE treated and GE non-treated but

show a consistent decrease in the KD conditions. Cox-2 expression is significantly

lowered in SW GE KD, while still appears to have a consistent reduction in the KD

condition compared to SC condition. MMP-9 gene expression decreases in both

KD conditions though it has no direct apoptotic effect. Since all the NF-κB genes

are pro survival to cell apoptosis, downregulated gene expressions in the SW GE

KD condition would be favorable to our prediction.

DISCUSSION

The focus of this work is to discover the reason of increased sensitivity of

PODXL knockdown pancreatic cancer cells to GE in mRNA level. PODXL is a

transmembrane protein involved in hematopoietic cell homing, kidney

morphogenesis, breast cancer progression, and epithelial cell polarization. [33] It

is also a selectin binding ligand on pancreatic cancer cells. On the other hand, GE

16

is taken up by cells through the aid of human equilibrative nucleoside

transporter 1 and 2 (hENT1, 2) and eventually incorporates into DNA to cause

apoptosis through series of phosphorylation. We also know that GE resistance is

connected to PI3K/Akt/ NF-κB pathways. There seems to be very little

connection between PODXL and GE. However, in a very recent astrocytoma study,

PODXL overexpression was shown to promote astrocytoma cell survival against

chemotherapeutic drug temozolomide by significantly increased

phosphorylation of Akt. [34] Interestingly, our work shows that down regulation

of PODXL inhibits cell survival against gemcitabine. Instead of being a nucleoside

analogue like gemcitabine, temozolomide damages the DNA by methylation and

causes apoptosis. Though those are two separate studies with different cells and

drugs, both indicate the significance of PODXL in cancer cell survival. Combining

our result with the astrocytoma and temozolomide study, it is logical to suggest

that PODXL expression influences both NF-κB and Akt in gemcitabine resistance.

Further investigation is required to confirm and generalize this statement.

Another early study suggested that NF-κB activity grants resistance to

pancreatic cancer cells while PI3K/Akt seem to be less important against

gemcitabine. [35] In this study they alter the gemcitabine dosage from 0.4 to 20

μM treating five pancreatic cell lines and observed that NF-κB induction was

dose-dependent while PI3K/Akt activity was dose-independent. In our NF-κB

gene expression results, bcl-2, bcl-xL, and cox-2 showed reduction upon PODXL

knockdown with GE treatment compare to control. We randomly tested

gemcitabine concentration of 10 μM and the differences in gene expressions

17

among different conditions were significant. A GE dosage and time dependent

assay on PODXL knockdown would be a plus to our work. For example, assessing

NF-κB gene expressions by altering the GE concentration profile with fixed

treatment time and vise versa. (Alter time, fixed concentration). Further work is

required to confirm the correlation between GE concentration and

chemoresistance due to the PI3K/Akt mechanism.

So far we have discussed what is happening inside the cell, now lets look

at the uptake efficiency of GE and the possible correlation among hENTs and

PODXL and GE. GE moves through cell membrane by the aid of hENT1 and

hENT2. High expression of hENT1 in stage I to VI pancreatic patients showed

improved overall survival upon treatment of GE. [36] However, a recent study

reported that inhibition of hENT1 transporter does not affect the cytotoxicity of

gemcitabine on resistant pancreatic cancer cells. Additionally, the common

proposals for chemoresistance of most nucleoside analogs are the lack of

intracellular transport proteins and the dysregulation of intracellular enzymes.

[32] Therefore it is reasonable to suggest that PODXL expression affects GE

resistance by interrupting the intracellular signaling pathways but a test for the

cellular uptake of GE through the cell membrane could further confirm whether

GE cellular uptake is affected upon PODXL knockdown.

In conclusion, we’ve shown downregulation of PODXL promotes cell

death against gemcitabine and affects the mRNA expression of NF-κB targeted

18

genes. Future investigation on the Akt/PI3K signaling pathway would give a

more complete picture of GE resistance in pancreatic cancer. In fighting

chemoresistance, blocking key proteins could improve response to a common

chemotherapy drug. In a paclitaxel study, it has been reported that stabilizing

microtubules by blocking certain proteins could enhance the cytotoxic response.

[37] We do not yet know the mechanism behind the increased cytotoxic effect on

GE-PODXL, but the increased cytotoxic effect of GE-PODXL itself is already a very

good start. Besides investigating the signaling pathways of this increased

cytotoxic effect, an in vitro cytotoxicity test of GE on control SW1990 with

pharmacological inhibition of PODXL would strengthen our work. Eventually this

would lead to higher drug efficacy, improved IC50, and better overall survival

rate to the gemcitabine chemo treatment in pancreatic cancer

19

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CURRICULUM VITAE

Billy Huang

857-233-6171 | billyhuang14@gmail.com

110 River Dr. #2205, Jersey City, NJ, 07310

Education

Johns Hopkins University, Baltimore, MD Dec 2014

Master of Science in Chemical & Biomolecular Engineering,

Concentration in Molecular and Cellular Biotechnology

GPA: 3.7/4.0 Johns Hopkins University, Baltimore, MD May 2013

Bachelor of Science in Chemical & Biomolecular Engineering

Concentration in Molecular and Cellular Biotechnology

Design & Research Experience

JHU Cell Engineering and Molecular Biology Lab, Baltimore, MD May 2011- Dec 2014

Graduate Researcher

Specializes in cell-based assay development for biomarker identification and mechanism of drug resistance in pancreatic cancer cells and gene targeting and profiling

Designed and led a team project of a group of 3 on investigation of drug sensitivity on Podocalyxin knockdown cell line

Manages undergraduate research team and optimized overall lab process workflow by implementing an integrated lab equipment rotation system across multiple laboratories

Project in Design: Pharmacokinetics/ Pharmacodynamics, Baltimore, MD Sep 2013 – May 2014

Project Leader

Developed a pharmacology model that predicts the relationship between smoking and drinking behaviors for heavy smoker/drinker

Collaborated with Biomedical Engineering department to improve current type II diabetes treatments in weekly progress review meetings and PowerPoint presentations

Biochemical Engineer Senior Product Design Project, Baltimore, MD Sep 2012 - May 2013

Project Leader

Designed and transformed a snail slime-based product that targeted a niche market into an oral gel treatment for periodontal disease and preventative treatment with a larger addressable market

Conducted market analysis and developed business proposal to pitch to potential investors

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Co-authored 60 page report detailing initial design concepts, quality control methods, product efficacy data, and targeted indication

Institute of Nanobiotechnology (INBT), Baltimore, MD Sep 2010 - May 2011

Research Assistance

Assisted in improving and modifying the microfluidic cell-based assays for protein-cell binding study, gene analysis in live cells

Organized and coordinated administrative duties (e.g., lab equipment rotation schedule) to facilitate more efficient laboratory activities

Activities

Johns Hopkins Business and Consulting Club Sep 2013 - Present

2013 Mini Case Competition Participant Johns Hopkins University Symphony Orchestra Sep 2009- Dec 2012

First Violinist, Second Stand 10+ concert performances

Representative, Taiwanese Student Association Sep 2009 - Present

Hosted 20+ intercultural events with average 50+ participants Introduced and executed new growth strategy in 2010 resulting in significant expansion

of the association Technical Skills & Languages

Technical Skills: MATLAB, JAVA, ImageJ

Language: Fluent in Mandarin Chinese; Proficient in Taiwanese